Improved synthesis of rupintrivir

Article abstract of DOI:10.1007/s11426-011-4478-5

An improved synthesis of rupintrivir (AG7088) was accomplished using three amino acids (l-glutamic acid, d-4-fluorophenylalanine, and l-valine) as the building blocks. The key fragment ketomethylene dipeptide isostere was constructed with the valine derivative and phenylpropionic acid derivative, followed by coupling with a lactam derivative and an isoxazole acid chloride to provide AG7088 totally in eight steps.

Full text of DOI:10.1007/s11426-011-4478-5

SCIENCE CHINA
Chemistry
June 2012 Vol.55 No.6: 1101–1107
doi: 10.1007/s11426-011-4478-5
• ARTICLES •
Improved synthesis of rupintrivir
LIN DaiZong1†, QIAN WangKe2†, HILGENFELD Rolf1,3, JIANG HuaLiang1,
CHEN KaiXian1 & LIU Hong1*
1State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 201203, China
2School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China
3Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Luebeck, 23538 Luebeck, Germany
Received August 31, 2011; accepted October 12, 2011; published online January 5, 2012
An improved synthesis of rupintrivir (AG7088) was accomplished using three amino acids (L-glutamic acid,
D-4-fluorophenylalanine, and L-valine) as the building blocks. The key fragment ketomethylene dipeptide isostere was con-
structed with the valine derivative and phenylpropionic acid derivative, followed by coupling with a lactam derivative and an
isoxazole acid chloride to provide AG7088 totally in eight steps.
rupintrivir, AG7088, synthesis
chymotrypsin-like protease (3Cpro), which is required for
1 Introduction
proteolytic processing of the large polyprotein translated
from the viral RNA genome and is thus essential for the
propagation of the virus [6]. As the safety of AG7088 has
been confirmed, the development of AG7088 will be a
shortcut for HFMD therapy. In addition, AG7088 derivative
have also been shown to have antiviral activity against se-
vere acute respiratory syndrome (SARS) [7–9]. Although
AG7088 possesses good antiviral activity and has received
much attention, there is limited information available in the
literature concerning methods for its synthesis. Therefore,
an efficient synthesis method for AG7088 is important and
will benefit the development of antiviral drugs of this type.
In this paper, we provide details of investigation and im-
provement of a new synthetic route for the preparation of
AG7088.
As shown in the retrosynthetic analysis outlined in Fig-
ure 1, AG7088 was prepared from three fragments: the lac-
tam derivative (A), the ketomethylene dipeptide isostere (B),
and an isoxazole acid chloride (C). The original synthetic
route, reported by Dragovich et al., was coupling the ke-
toacid (B) with the lactam derivative (A) and the isoxazole
acid chloride (C). Using this approach, AG7088 was syn-
Over 100 deaths were reported in the outbreaks of hand,
foot and mouth disease (HFMD) over the past two years in
China, and thousands of people were admitted to hospitals
with moderate to severe symptoms of this disease. HFMD,
commonly caused by coxsackievirus A16 (CVA16) or en-
terovirus 71 (EV71), can proceed to serious complications
such as meningitis, which can be fatal to small infants and
children [1]. Unfortunately, there is no specific treatment
for HFMD; therefore, there is an urgent need to discover
effective drugs against this viral infection. AG7088 was
developed originally to combat human rhinovirus (HRV),
which is the major cause of the common cold. In Phase-II
clinical trials, AG7088 demonstrated moderate antiviral and
clinical efficacy. Because of a lack of efficacy in natural
infection studies, further development was stopped. How-
ever, AG7088 has received attention again in recent years,
because it is now considered a potent inhibitor of EV71
[2–5]. The compound has been shown to inhibit the viral
*Corresponding author (email: hliu@mail.shcnc.ac.cn)
†These authors contributed equally to this work
© Science China Press and Springer-Verlag Berlin Heidelberg 2012
chem.scichina.com www.springerlink.com

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Lin DZ, et al. Sci China Chem June (2012) Vol.55 No.6
Figure 1 Retrosynthetic analysis of AG7088 (rupintrivir).
thesized with a total yield of about 0.6% in 13 steps [10, 11].
In this route, the lactam derivative (A) was obtained using
natural glutamic acid within 13 steps [10], and the key build-
ing block ketoacid (B) was obtained through a lactone (D)
using diastereoselective alkylation of ()-pseudoephedrine-
derived amide with p-fluorobenzyl bromide and subsequent
lactonization was promoted by N-bromosuccinimide (NBS)
as the key steps [11]. Then Tian et al. improved the synthe-
sis of the lactam derivative (A) and simplified the reaction
step to 8 steps [12]. Ma et al. improved the synthesis of the
ketoacid (B) via the chiral alkylation of the lactone (E),
avoiding use of the chiral catalyst as the key step. Based on
Tian’s work, they obtained AG7088 using L-valine as the
starting material in about 7% yield in 16 steps [13, 14].
Meanwhile, Chng et al. reported a synthetic method of
AG7088 using the isoxazole acid (C) as the starting mate-
rial; however, the use of toxic reagents and the harsh reac-
tion condition of this method might limit its synthetic
usefulness [15]. Shie et al. reported an alternative route to
synthesize the ketoacid (B) and obtained AG7088 using
Cbz-L-valine as the starting material in 15.3% yield and 12
steps [16]. However, the harsh reaction conditions and
inconvenient chromatographic fractionation were disad-
vantageous. Based on Shie’s work, we improved the syn-
thesis of the ketoacid (B) from two parts: the 2-substituted
propionic acid B1 and the valine derivative B2. In this
improved route, the synthesis of part B1 was accomplished
using commercially available 4-fluorophenylalanine as the
material in one step and the product was easily purified.
An SN2 reaction of part B1 and part B2 provided the ke-
toacid (B) without using chiral ligands or low temperature,
and was much more convenient than chiral synthesis. Then
we linked the ketoacid (B) with the lactam derivative (A)
and the isoxazole acid chloride (C) to provide the final
product AG7088 in 3 steps, which was much shorter than
the one reported Shie et al. Finally, we obtained AG7088
in about 12% yield in 8 steps using Cbz-L-valine as the
starting material.
2 Results and discussion
After a thorough retrosynthetic analysis, we designed a
synthetic route for AG7088 (rupintrivir) preparation
(Scheme 1). In this route, we started with the dianionic al-
kylation of N-Boc glutamic acid dimethyl ester 1 with io-
doacetonitrile. As expected, this alkylation occurred in a
highly stereoselective manner, giving 2 as the exclusive
product (72% yield). In the following step, the cyano group
in 2 was subjected to hydrogenation to generate the inter-
mediate 3. Then, the in situ cyclization of intermediate 3
afforded the lactam 4 in 40% yield (from 1). The ester
group in 4 was then reduced to the corresponding alcohol.
The oxidization of the alcohol product 5 by Dess-Martin
periodinane, followed by the Wittig reaction with an appro-
priate reagent, gave rise to the conjugated ester 7 in 65%

Lin DZ, et al. Sci China Chem June (2012) Vol.55 No.6
1103
Scheme 1 Synthesis of AG7088.
yield (from 4). The designed lactam derivative 8 (Fragment
A) was generated by removal of the protecting group of 7.
Compound 10 (Part B1) was prepared from D-4-fluoro-
phenylalanine via bromination and methyl esterification.
Meanwhile, the valine-derived malonate 11 was obtained
via Claisen condensation. The SN2 reaction with bromoace-
tate 10 and the valine-derived malonate 11 was followed by
removal of the tert-butyloxycarbonyl (Boc) group to pro-
vide the ketoester 13. The direct conversion of the ben-
zyloxycarbonyl (Cbz) group to the Boc group in 14 was
accomplished by catalytic hydrogenation of 13 in the pres-
ence of di-tert-butyl carbonate, which was followed by hy-

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Lin DZ, et al. Sci China Chem June (2012) Vol.55 No.6
drolysis to give the key ketoacid 15 (Fragment B). In the
final step, AG7088 was prepared by coupling the ketoacid
15 with the lactam derivative 8 (at the right side initially)
and then the isoxazole acid chloride (Fragment C) on the
left side at the end.
The reaction was quenched with water (30 mL). The sus-
pension was extracted with ethyl acetate. The organic layers
were combined, dried (MgSO4), and filtered. The light
brown filtrate was concentrated and purified by silica gel
column chromatography (petroleum ether/ethyl acetate =
4/1) to give the product 4 as colorless oil. 1H NMR (CDCl3)
 6.02 (1H, br), 5.49 (1H, d, J = 7.8 Hz), 4.27–4.33 (1H, m),
3.72 (3H, s), 3.31–3.36 (2H, m), 2.40–2.49 (2H, m),
2.06–2.16 (1H, m), 1.77–1.89 (2H, m), 1.41 (9H, s). ESI-MS
(m/z): 287 (M + H)+.
3 Conclusion
In summary, we have improved the synthetic route for
AG7088 production using an easy and practical method.
This route has many merits compared to previous methods,
such as the non-usage of chiral catalysts, fewer reaction
steps, low-cost starting materials, and convenient chroma-
tographic fractionation.
tert-Butyl (S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-
ylcarbamate 6
The complex 4 (1.0 g, 3.49 mmol) was dissolved in metha-
nol (40 mL), then NaBH4 (0.53 g, 14 mmol) was added un-
der ambient conditions. The reaction mixture was stirred at
room temperature for 2 h. Then the reaction was quenched
with water (30 mL). The suspension was extracted with
ethyl acetate. The organic layers were combined, dried, and
filtered. The filtrate was evaporated to dryness and then
dissolved in dichloromethane, then Dess-Martin periodinane
(1.48 g, 3.49 mmol) and NaHCO3 (0.37 g, 3.49 mmol) were
added. The resulting mixture was stirred at room tempera-
ture for 1 h. The mixture was concentrated and purified by
column chromatography on silica gel (dichloromethane/
methanol = 100/1) to give the product 6 as a white solid. 1H
NMR (CDCl3)  9.73 (1H, s), 6.02 (1H, br), 5.48 (1H, d, J =
7.8 Hz), 4.36–4.25 (1H, m), 3.38–3.32 (2H, m), 2.50–2.44
(2H, m), 2.11–2.03 (1H, m), 1.88–1.76 (2H, m), 1.43 (9H,
s). ESI-MS (m/z): 279 (M + Na)+.
4 Experimental
(2S,4R)-Dimethyl 2-(tert-butoxycarbonylamino)-4-(cyanome-
thyl)pentanedioate 2
To a solution of N-Boc-L-glutamic acid dimethyl ester (1,
6.0 g, 21.8 mmol) in THF (60 mL) was added dropwise a
solution of lithium bis(trimethylsilyl)amide in THF (47 mL,
1 M) at 78 °C under an argon atmosphere. The resulting
dark mixture was stirred at 78 °C for 1 h. At the same time,
iodoacetonitrile (3.72 g, 2.33 mol) was added dropwise to
the dianion solution over a period of 1 h while maintaining
the temperature below 70 °C. The reaction mixture was
stirred at 78 °C for additional 2 h and the disappearance of
the starting material (1) was confirmed by TLC analysis.
The reaction was quenched with methanol (3 mL) and a
pre-cooled acetic acid in THF solution (2.7 mL HOAc/20
mL THF) was added. After stirring for 30 min, the cooling
bath was removed. The reaction mixture was allowed to
warm up to room temperature and then poured into a brine
solution (40 mL). The layers were separated, and the organ-
ic layer was concentrated to afford dark brown oil. The
crude residue was purified by flash column chromatography
(petroleum ether/ethyl acetate = 4/1) to give product 2 as
colorless oil. 1H NMR (CDCl3, 400 MHz) δ 5.23 (1H, d, J =
9.0 Hz), 4.43–4.36 (1H, m), 3.77(1H, s), 3.76 (1H, s),
2.89–2.69 (3H, m), 2.20–2.14 (2H, m), 1.45 (9H, s). ESI-MS
(m/z): 315 (M + H)+.
(S,E)-Ethyl 4-(tert-butoxycarbonylamino)-5-((S)-2-oxopyrro-
lidin-3-yl)pent-2-enoate 7
To a solution of triethyl phosphonoacetate (0.44 g, 1.95
mmol) in anhydrous THF, at 78 °C under an argon atmos-
phere, was added sodium hydride (78 mg, 1.95 mmol). The
suspension was stirred for 15 min at the same temperature,
and a solution of 6 (0.5 g, 1.95 mmol) in anhydrous THF
was added dropwise. The reaction was gradually warmed to
50 °C and stirred for 2 h and upon completion was
quenched by the addition of saturated aqueous NH4Cl. The
mixture was diluted with diethyl ether (100 mL) and the
water layer was extracted with diethyl ether. The combined
organic layer was dried over MgSO4, and the solvent was
evaporated under reduced pressure. The residue was puri-
fied by flash column chromatography on silica gel (petro-
(S)-Methyl 2-(tert-butoxycarbonylamino)-3-((S)-2-oxopyrro-
lidin-3-yl)propanoate 4
leum ether/ethyl acetate = 1/1) to afford 7 as white foam.
1
[]22 6.7 (c 1.0 g/100 mL, CHCl3) HNMR (CDCl3, 400
In a hydrogenation flask was placed compound 2 (5.0 g,
15.9 mmol), 5 mL of chloroform and 60 mL of methanol
before the addition of PtO2. The resulting mixture was
pressurized to hydrogen and mechanically stirred at room
temperature for 12 h. Then the mixture was filtered over
Celite. NaOAc (8.46 g, 31.8 mmol) was added to the filtrate
before the resulting mixture was stirred at 60 °C for 12 h.
D
MHz)  6.86 (1H, dd, J = 15.6, 5.2 Hz), 6.47 (1H, br), 5.95
(1H, dd, J = 15.6, 1.6 Hz), 5.37 (1H, d, J = 8.4 Hz),
4.37–4.26 (1H, m), 4.19 (2H, q, J = 7.2 Hz), 3.37–3.32 (2H,
m), 2.53–2.47 (2H, m), 2.05–1.98 (2H, m), 1.85–1.79 (1H,
m), 1.62–1.56 (1H, m), 1.44 (9H, s), 1.28 (3H, t, J = 7.2 Hz);
ESI-MS (m/z): 327 (M + H)+.

Lin DZ, et al. Sci China Chem June (2012) Vol.55 No.6
1105
(R)-2-Bromo-3-(4-fluorophenyl)propanoic acid 9
(3R)-1-tert-Butyl 4-methyl 2-((S)-2-(benzyloxycarbonyla-
mino)-3-methylbutanoyl) -3-(4-fluorobenzyl)succinate 12
D-2-Amino-3-(4-fluorophenyl)propanoic acid (5.0 g, 27.3
mmol) was dispersed in 48%HBr (60 mL) and cooled to
0 °C with an ice bath. NaNO2 (2.18 g, 32 mmol) was added
by portion. The reaction was stirred for 1 h at 0 °C, then
slowly warmed to room temperature, and stirred for another
10 h. The aqueous solution was extracted with 400 mL of
EtOAc (4×100 mL), washed with 100 mL of brine and dried
over Na2SO4. The solvent was evaporated and the crude
material was purified on silica eluting with mixtures of
hexane/EtOAc (2:1) to afford the product 9 (5.57 g, 83%) as
To a solution of 11 (2.16 g, 6.18 mmol) in DMF (10 mL)
was added compound 10 (1.77 g, 6.80 mmol), K2CO3 (2.56
g, 18.54 mmol), potassium iodide (216 mg, 10%), and the
reaction mixture was then stirred for 8 h at 80 °C until the
disappearance of the starting material 10. The reaction mix-
ture was poured into 60 mL water. The aqueous solution
was extracted with 100 mL of EtOAc (2 × 50 mL) and
washed with 100 mL of saturated brine (2 × 50 mL) and
dried over Na2SO4. The solvent was evaporated and the
crude material was purified on silica eluting with mixtures
of hexane/EtOAc (4:1) to afford the product 12 (1.96 g,
1
colorless oil. H NMR (CDCl3, 400 MHz):  11.1 (1H, s),
7.18–7.15 (2H, m), 7.03–6.97 (2H, m), 4.34–4.31 (1H, m),
3.48–3.42 (1H, dd, J = 14.0 Hz, 8.4 Hz), 3.25-3.21 (1H, dd,
J = 14.4 Hz, 7.2 Hz) ; ESI-MS (m/z): 245 (M  H)+.
60%) as a white solid. []22 +6.6 (c 1.0 g/100 mL, CHCl3)
D
1H NMR (CDCl3, 400 MHz):  7.39–7.33 (7H, m), 7.03–6.92
(2H, m), 5.50–5.48 (1H, d, J = 8.4 Hz), 5.14 (2H, s), 4.54–
4.52 (1H, m), 4.14–4.10 (1H, m), 3.74 (3H, s), 3.68–3.66
(1H, m), 3.65–3.63 (1H, m), 3.16–3.08 (1H, m), 2.26–2.22
(1H, m), 1.42 (9H, s), 0.98–0.96 (3H, d, J = 6.8 Hz), 0.88–
0.86 (3H, d, J = 6.8 Hz); ESI-MS (m/z): 552 (M + Na)+.
(R)-Methyl 2-bromo-3-(4-fluorophenyl)propanoate 10
Compound 9 (4.0 g) was dissolved in MeOH (30 mL), and
1 mL concentrated sulfuric acid was added. The reaction
was stirred for 2 h at 70 °C. Then MeOH was evaporated,
and 10 mL water was added, followed by extraction with
100 mL EtOAc, washing with 50 mL of saturated brine (2 ×
25 mL) and drying over Na2SO4. The solvent was evapo-
rated and the crude material was purified on silica eluting
with mixtures of hexane/EtOAc (8:1) to afford the product
(2R,5S)-Methyl 5-(benzyloxycarbonylamino)-2-(4-fluoroben-
zyl)-6-methyl-4-oxo heptanoate 13
To a mixture solution (TFA:CH2Cl2=3:1, 4 mL) was added
compound 12 (430 mg, 0.81 mmol), and the reaction was
stirred for 4 h at 30 °C. Then the solvent was evaporated,
and 10 mL water was added, followed by extraction with 50
mL EtOAc, washing with 50 mL of saturated brine (2 × 25
mL) and drying over Na2SO4. The solvent was evaporated
and the crude material was purified on silica, eluting with
1
10 (3.93 g, 93%) as colorless oil. H NMR (CDCl3, 400
MHz):  7.20–7.16 (2H, m), 7.02–6.97 (2H, m), 4.38–4.34
(1H, m), 3.73 (3H, s), 3.46–3.40 (1H, dd, J = 14.0 Hz, 8.4
Hz), 3.24–3.19 (1H, dd, J = 14.4 Hz, 7.2 Hz). ESI-MS (m/z):
261 (M + H)+.
mixtures of hexane/EtOAc (2:1) to afford the product 13
1
(262 mg, 75%). []22 +13.6 (c 0.15 g/100 mL, CHCl3) H
(S)-tert-Butyl 4-(benzyloxycarbonyl)amino-5-methyl-3-oxo-
hexanoate 11
D
NMR (CDCl3, 400 MHz):  7.38–7.32 (6H, m), 7.24–7.21
(1H, m), 7.03–6.96 (2H, m), 5.42–5.40 (1H, d, J =8.4Hz),
5.09 (2H, s), 4.32–4.25 (1H, m), 3.65 (3H, s), 3.63–3.61
(1H, m), 3.58–3.56 (1H, m), 2.48–2.40 (2H, m), 2.36–2.30
(1H, m), 2.26–2.23 (1H, m), 0.98–0.96 (3H, d, J = 6.8 Hz),
0.74–72 (3H, d, J = 6.8 Hz); ESI-MS (m/z): 452 (M + Na)+.
Cbz-L-Valine (3.0 g, 11.9 mmol) was dissolved in 15 mL of
dry THF. To this solution was added 1,1′-carbonyldiimida-
zole (2.13 g, 13.1 mmol) with stirring under nitrogen at
room temperature. Diisopropylamine lithium (LDA, 2 M
THF solution, 3.3 equiv) under nitrogen was diluted with 10
mL of THF and cooled to 78 °C. To the LDA solution was
added tert-butyl acetate (4.87 g, 42 mmol). After 1 h the
Boc-amino acid/imidazole solution was cooled to 78 °C
and added to the enolate solution under nitrogen. The reac-
tion was allowed to stir for 2 h at 78 °C, then quenched
with 2 mL water and allowed to warm to room temperature.
The aqueous solution was extracted with 200 mL of EtOAc
(2 × 100 mL), and washed with 50 mL of brine, and dried
over Na2SO4. The solvent was evaporated and the crude
material was purified on silica eluting with mixtures of
hexane/EtOAc (4:1) to afford the product 11 (2.08 g, 60%)
(2R,5S)-Methyl 5-(tert-butoxycarbonylamino)-2-(4-fluoro-
benzyl)-6-methyl-4- oxoheptanoate 14
Pd/C (17 mg, 5%) was added to a solution of compound 13
(340 mg, 0.79 mmol) in MeOH (20 mL) at 25 °C. The mix-
ture was stirred overnight and filtered. (Boc)2O (151 mg,
0.87 mmol) and Et3N (0.13 mL) were added to the filtrate.
The reaction was stirred for another 2 h. Then the solvent
was evaporated, and 10 mL water was added, followed by
extraction with 50 mL EtOAc, washing with 50 mL of sat-
urated brine (2 × 25 mL) and drying over Na2SO4. The sol-
vent was evaporated and the crude material was purified on
silica eluting with mixtures of hexane/EtOAc (2:1) to afford
the product 14 (244 mg, 78%). 1H NMR (CDCl3, 400 MHz):
 7.20–7.16 (2H, m), 7.02–6.97 (2H, m), 5–5.44 (1H, m),
4.33–4.27 (1H, m), 3.67 (3H, s), 3.61–3.59 (1H, m), 3.57–
1
as yellow oil. H NMR (CDCl3, 400 MHz):  7.35 (5H, s),
5.48–5.46 (1H, d, J = 8.4 Hz), 5.09 (2H, s), 4.46–4.42 (1H,
dd, J = 8.8 Hz, 4.0 Hz), 3.43 (2H, s), 2.28–2.24 (1H, m),
1.44 (9H, s), 1.04–1.02 (3H, d, J = 6.8 Hz), 0.81–0.79 (3H,
d, J = 6.8 Hz); ESI-MS (m/z): 348 (M  H)+.

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Lin DZ, et al. Sci China Chem June (2012) Vol.55 No.6
3.54 (1H, m), 2.46–2.41 (2H, m), 2.35–2.30 (1H, m), 2.25–
2.23 (1H, m), 1.40 (9H, s), 1.01–0.99 (3H, d, J = 6.8 Hz),
0.78–0.76 (3H, d, J = 6.8 Hz); ESI-MS (m/z): 418 (M + H)+.
crude intermediate was resolved in 5 mL CH2Cl2, and
5-methylisoxazole-3-carbonyl chloride (20 mg, 0.14 mmol)
and Et3N (0.04 mL, 0.18 mmol) were added. The mixture
was stirred for another 10 h at room temperature. The reac-
tion mixture was diluted with 20 mL CH2Cl2, washed with
50 mL of saturated brine (2 × 25 mL), and dried over NaSO4.
The solvent was evaporated and the crude material was puri-
fied on silica eluting with mixtures of CH2Cl2/MeOH (40:1)
to afford the product AG7088 (49 mg, 71%). []24D +31.7 (c
(2R,5S)-5-(tert-Butoxycarbonylamino)-2-(4-fluorobenzyl)-6-
methyl-4-oxoheptanic acid 15
10% NaOH/H2O (5 mL) was added to a solution of com-
pound 14 (240 mg, 0.61 mmol) in ethanol (5 mL). The reac-
tion was stirred for 1 h at room temperature. Then 2M HCl
was added to the reaction solution until pH 1. Then the so-
lution was extracted with 100 mL of EtOAc (2 × 100 mL)
and washed with 50 mL of brine and dried over Na2SO4.
The solvent was evaporated and the crude material was pu-
rified on silica, eluting with mixtures of hexane/EtOAc (2:1)
to afford the product 15 (232 mg, 90%) as a white solid. 1H
NMR (CDCl3, 400 MHz):  7.18–7.15 (2H, m), 7.03–6.98
(2H, m), 5.43–5.41 (1H, m), 4.29–4.24 (1H, m), 3.60–3.57
(1H, m), 3.55–3.53 (1H, m), 2.49–2.43 (2H, m), 2.33–2.20
(2H, m), 1.41 (9H, s), 0.98–0.96 (3H, d, J = 6.8 Hz), 0.76–
0.74 (3H, d, J = 6.8 Hz); ESI-MS (m/z): 380 (M  H)+.
1
0.2 g/100 mL, CHCl3). H NMR (CDCl3, 400 MHz): 
7.35–7.33 (1H, d, J = 8.7 Hz), 7.10–7.06 (2H, m), 6.94–
6.92 (2H, m), 6.60–6.57 (1H, dd, J = 11.7, 5.2 Hz), 6.47
(1H, s), 6.08 (1H, m), 5.43–5.40 (1H, dd, J = 11.6, 1.4 Hz),
4.65–4.63 (1H, dd, J = 8.7, 4.4 Hz), 4.48–4.39 (1H, m),
4.17–4.14 (2H, q, J = 6.8 Hz), 3.32–3.19 (2H, m), 3.08–3.00
(1H, m), 2.96–2.91 (1H, m), 2.89–2.80 (1H, m), 2.73–2.54
(3H, m), 2.51 (3H, s), 2.26–2.02 (3H, m), 1.87–1.74 (1H,
m), 1.73–1.62 (1H, m), 1.49–1.41 (1H, m), 1.28–1.23 (3H,
m), 0.94–0.92 (3H, d, J = 6.8 Hz), 0.78–0.76 (3H, d, J = 6.8
Hz). 13CNMR (CDCl3, 400 MHz):  206.7, 175.2, 173.1,
166.8, 163.4, 162.1, 160.0, 158.1, 148.2, 135.4, 131.6,
122.5, 115.7, 113.6, 102.1, 64.3, 61.5, 50.2, 44.7, 42.5, 41.2,
39.1, 35.3, 31.6, 29.4, 20.1, 18.2, 14.8, 12.6. ESI-MS (m/z):
599 (M + H)+.
(S,E)-Ethyl 4-((2R,5S)-5-(tert-butoxycarbonylamino)-2-(4-
fluorobenzyl)-6-methyl-4-oxoheptanamido)-5-((S)-2-oxopy-
rrolidin-3-yl)pent-2-enoate 16
Compound 15 (260 mg, 0.68 mmol) was dissolved in 10 mL
of dry CH2Cl2. To this solution was added 1.1 equiv of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride (EDCI), and the mixture was stirred for 0.5 h at room
temperature. Then compound 8 (195 mg, 0.68 mmol) and
Et3N (0.20 mL, 1.36 mmol) were added to the reaction. The
reaction was stirred for another 6 h. The reaction mixture
was poured into 10 mL water. The aqueous solution was
extracted with 50 mL of CH2Cl2 (2 × 25 mL) and washed
with 50 mL of saturated brine (2 × 25 mL) and dried over
Na2SO4. The solvent was evaporated and the crude material
was purified on silica, eluting with a mixture of CH2Cl2/
We gratefully acknowledge financial support from the National Natural
Science Foundation of China (20872153, 21021063, 20720102040 and
81025017), the National Basic Research Program of China grant
(2009CB918502), the Chinese Academy of Sciences (XDA01040305), and the
SILVER project of the European Commission (contract HEALTH-F3-2010-
260644). HR is supported by a Chinese Academy of Sciences Visiting Pro-
fessorship for Senior International Scientists (2010T1S6).
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MeOH (80:1) to afford the product 16 (252 mg, 63%). H
NMR (CDCl3, 400 MHz):  7.36–7.34 (1H, d, J = 8.7 Hz),
7.08–7.06 (2H, m), 6.93–6.91 (2H, m), 6.61–6.58 (1H, dd, J =
11.7, 5.2 Hz), 6.06 (1H, m), 5.40–5.37 (1H, dd, J = 11.6, 1.4
Hz), 4.62–4.60 (1H, m), 4.51–4.38 (1H, m), 4.16–4.13 (2H,
q, J = 6.8 Hz), 3.30–3.24 (2H, m), 3.11–3.04 (1H, m), 3.00–
2.95 (1H, m), 2.88–2.81 (1H, m), 2.72–2.57 (3H, m), 2.19–
2.02 (3H, m), 1.87–1.80 (1H, m), 1.78–1.62 (1H, m), 1.51–
1.44 (1H, m), 1.42 (9H, s), 1.23–1.20 (3H, m), 0.90–0.88
(3H, d, J = 6.8 Hz), 0.79–0.77 (3H, d, J = 6.8 Hz); ESI-MS
(m/z): 590 (M + H)+.
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(S,E)-Ethyl 4-((2R,5S)-2-(4-fluorobenzyl)-6-methyl-5-(5-methy-
lisoxazole-3-carboxamido)-4-oxoheptanamido)-5-((S)-2-oxo-
pyrrolidin-3-yl)pent-2-enoate AG7088
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The compound 16 (70 mg, 0.12 mmol) was dissolved in 6
mL of dry CH2Cl2. To this solution was added 2 mL of TFA,
and the mixture was stirred for 2 h at room temperature.
Then the reaction mixture was evaporated in vacuum. The

Lin DZ, et al. Sci China Chem June (2012) Vol.55 No.6
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