Synthesis of the cyclic nonapeptide of chlorofusin using a convergent [3+3+3]-fragment coupling strategy

Article abstract of DOI:10.1016/j.tet.2010.03.032

A [3+3+3]-fragment coupling strategy was successfully applied in the synthesis of the nonacyclopeptide of chlorofusin, a potent natural antagonist against p53-MDM2 interactions. The accomplished convergent synthesis includes parallel syntheses of three tripeptides and their sequential assembly, and macrocyclization of the linear precursor to the required 27-membered nonacyclopeptide.

Full text of DOI:10.1016/j.tet.2010.03.032

Tetrahedron 66 (2010) 3427–3432
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of the cyclic nonapeptide of chlorofusin using a convergent
[3þ3þ3]-fragment coupling strategy
,
,
a,
*
Yan-Li Wang a b, Wen-Jian Qian a, Wan-Guo Wei a, Yue Zhang b , Zhu-Jun Yao
*
a State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road,
Shanghai 200032, China
b Department of Chemistry and Pharmaceutical Engineering, Hebei University of Technology and Science, 70 East Yuhua Road, Shijiazhuang, Hebei 050018, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A [3þ3þ3]-fragment coupling strategy was successfully applied in the synthesis of the nonacyclopeptide
of chlorofusin, a potent natural antagonist against p53–MDM2 interactions. The accomplished conver-
gent synthesis includes parallel syntheses of three tripeptides and their sequential assembly, and
macrocyclization of the linear precursor to the required 27-membered nonacyclopeptide.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 27 January 2010
Received in revised form 6 March 2010
Accepted 9 March 2010
Available online 12 March 2010
1. Introduction
D-ADA 8
(CH2)6CH3
14
12
O
O
Cl
OH
The tumour suppressor p53 induces cell death by apoptosis in
response to various stress conditions, such as oncogene activation
and DNA damage, and therefore plays a crucial role in the
prevention of cancers.1–4 The overexpression of MDM2 causes
uncontrolled cell proliferation in a variety of human tumours,5–8
and the binding of p53 with MDM2 has been found to result in
the high risk of a number of human cancers.9 As a new chemo-
therapy of cancers, discovery of potent small-molecule antagonists
against p53-MDM2 interactions has attracted great attentions in
medicinal chemistry in recent years. A number of small-molecular
heterocyclic compounds have been successfully designed and
developed with potent inhibitory activities against p53–MDM2
interactions, and thus showing great potentials for future drug
development.10,11
O
D-Leu 7
7
2
O
O
11
N
H
10
8
1
O
HN
6
3
5
9
17
4
O
HN
O
L-Thr
N
15
OH
6
16
18
13
L-Orn 9
O
HN
1
HO
NH
L-Thr
O
NH
O
O
D-Leu 5
O
NH
O
O
L-Ala 2
HN
N
H
NH2
O
D-Asn 4
L- Asn 3
Chlorofusin (1)
NH2
Figure 1. The revised structure of chlorofusin (1).
Chlorofusin (1, Fig. 1), a unique nonacyclopeptide isolated from
the fermentation broth of the fungal strain Microdochium
caespitosum, has been found to inhibit the p53–MDM2 interaction
The cyclic peptide portion of chlorofusin (1, Fig. 1) was firstly
determined by the corresponding spectroscopic studies and
chemical degradation methods.12 It includes two
-threonines, one
-alanine, one - and one -asparagine, two -leucines, one -2-
aminodecanoic acid, and one -ornithine. The two asparagine
residues, Asn3 and Asn4, were suggested to have opposite stereo-
chemistries ( - and -form, respectively), but their order in the
structure could not be determined at that moment. In 2003, Boger’s
group17 and Searcey’s group18 synthesized the cyclic peptide por-
tions of chlorofusin independently, and finally resulted in the as-
with an IC50¼4.6
mM and a KD¼4.7
m
M.12,14 It thus represents a new
L
lead structure of novel antagonists against the p53–MDM2 protein–
protein interactions.13 The structure of chlorofusin was initially
proposed to contain a novel densely functionalized chromophore
and a 27-membered nonacyclopeptide, through the linkage be-
tween its chromophore with a terminal amine of ornithine of the
cyclopeptide.12 Two total syntheses of chlorofusin and its enan-
tiopure diastereomers have been accomplished in recent years,
including revisions of relative and absolute configurations of its
chromophore part.15,16
L
L
D
D
D
L
L
D
signment of
L-Asn3 and
D-Asn4. In 2007, Nakata’s group
successfully applied an [8þ1]-fragment coupling strategy to the
synthesis of the cyclic peptide.19 Very recently, Boger’s group
reported their second synthesis of the cyclic peptide using
a [2þ2þ2þ3]-fragment assembly strategy.20
* Corresponding authors. Tel.: þ86 21 5492 5133; fax: þ86 21 6416 6128; e-mail
address: yaoz@sioc.ac.cn.
0040-4020/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.03.032

3428
Y.-L. Wang et al. / Tetrahedron 66 (2010) 3427–3432
2. Results and discussion
(Scheme 1). Removal of the N-Cbz protecting group of 8 was carried
out by hydrogenolysis in the presence of 10% Pd–C, affording
2.1. Retrosynthesis
quantitative yield of amine 9. Further coupling of peptide amine 9
with L-Z-Asn(Trt)–OH (10) with EDCI and HOBt in DCM followed by
The first total synthesis of enantiopure (4S,8R,9S)-chlorofusin
and other two diastereomers were successfully accomplished by
our group in 2007.15 To support our ongoing project on the second
generation total synthesis of chlorofusin and its all seven chro-
mophore diastereomers, a scalable synthesis of the cyclic peptide
portion was studied. Herein, we report our synthesis of the cyclic
peptide of chlorofusin, which is featured with a [3þ3þ3]-fragment
coupling strategy (Fig. 2). Such an equal-cut retrosynthesis would
result in a new convergent route to the cyclic peptide 2 and lead to
high synthetic efficiency.
ester hydrolysis with LiOH in THF–H2O afforded the first tripeptide
3 in 81% yield (two steps).
O
RHN
OMe
EDCI, HOBt,
N
H
O
D-Z-Asn(Trt)-OH (6)
iPr2NEt, CH2Cl2
O
+
.
87%
7
D-Leu-OMe HCl ( )
NHTrt
H2, Pd-C
100%
8
R = Cbz
9 R = H
coupling
O
O
H
NHTrt
O
N
O
NHR2
O
H3C(H2C)6
O
N
H
NH
H
10
L-Z-Asn(Trt)-OH (
)
N
OR
NH
HO
O
O
CbzHN
N
H
NHTrt
HO
O
O
EDCI, HOBt, CH2Cl2
89%
O
O
NH
O
11
O
11
H
N
HN
LiOH.H2O
THF-H2O, 90%
R = Me
R = H
N
H
N
H
3
O
O
NHR2
coupling
Scheme 1.
coupling and
macrocyclization
NHR1
R1 = R2 = H
2
2a R1 = Cbz, R2 = Trt
2.3. Synthesis of tripeptide 4
[3+3+3]-fragment
coupling
Preparation of the unusual
D-2-aminodecanic acid (D-Ada)
O
started from the Scho¨llkopf reagent 1221 (Scheme 2). Deprotona-
tion of 12 with n-BuLi followed by alkylation with 1-iodooctane in
THF at ꢀ78 ꢁC afforded 13 in 94% yield. Hydrolysis of 13 with 0.5 M
NHTrt
O
O
H
CbzHN
N
N
H
OH
aqueous HCl at 25 ꢁC for 24 h afforded
titative yield.
D-Ada–OMe (15) in a quan-
O
NHTrt
3
O
OH
(CH2)6CH3
n-BuLi, THF, -78 o
then C8H17I, 94%
C
O
N
N
H
OMe
N
OMe
N
BocHN
CbzHN
N
H
CO2Me
MeO
O
MeO
N
(CH2)7CH3
12
13
4
O
0.5 M HCl, rt H2N
24h, quant.
O
H
OMe
(CH2)7CH3
N
OH
BocHN
N
H
O
O
15
OH
5
Scheme 2.
Figure 2. Retrosynthesis of the cyclic peptide 2.
Coupling of N-Boc–L-Thr–OH (14) with D-Leu–OMe (7) in the
presence of EDCI and HOBt in DCM followed by ester hydrolysis
provided dipeptide 16 in 77% yield (Scheme 3). Further condensa-
tion of dipeptide-acid 16 with newly prepared amine 15 with EDCI
and HOBt in DCM afforded the second tripeptide 4 in 81% yield.
According to this design, the cyclopeptide 2 could be divided
into three parallel tripeptides 3, 4 and 5. Compared to the dipeptide
intermediates, tripeptides are usually more stable, and are able to
avoid the production of undesired piperazine-2,5-dione derivatives
via self-cyclization during their couplings. After sequential cou-
plings of these three fragments, the final amide-bond formation
2.4. Synthesis of tripeptide 5
between
L-Orn9 and D-Ada8 was devised for the head-to-tail
macrocyclization.
Coupling of N-Boc–L-Thr–OH (14) with L-Ala–OMe (18) was
carried out with EDCI and HOBt in DCM, affording dipeptide 19 in
80% yield (Scheme 4). Deprotection of N-Boc group of 19 with 2 M
2.2. Synthesis of tripeptide 3
HCl in EtOAc followed by coupling with N-Boc–L-Orn(Z)–OH (21)
First of all, coupling of
OMe$HCl (7) with EDCI and HOBt in the presence of N,N-diiso-
propylethylamine (DIEA) afforded dipeptide in 87% yield
D
-Z-Asn(Trt)–OH (6) with
D
-Leu–
with EDCI and HOBt in DCM afforded tripeptide 22 in 81% yield in
two steps. Hydrolysis of 22 with LiOH$H2O in THF–H2O afforded the
third tripeptide (acid) 5 in 83% yield.
8

Y.-L. Wang et al. / Tetrahedron 66 (2010) 3427–3432
3429
O
O
OH
O
N
EDCI, HOBt,
D
-Leu-OMe.HCl (7)
H
NHTrt
OH
(CH2)6CH3
O
i-Pr2NEt
O
O
H
BocHN
OR
+
H
N
N
CbzHN
RHN
85%
N-Boc-
L
-Thr-OH (14)
O
N
H
CO2Me
+
N
H
O
NHTrt
LiOH.H2O
29
R = Me
R = H
O
2M HCl in
4 R = Boc
16
THF-H2O, 90%
3
23 R = H.HCl
EtOAc, quant.
OH
O
N
N
(CH2)6CH3
CO2Me
O
H
NHTrt
D
-Ada-OMe (15)
(CH2)6CH3
CO2Me
OH
O
N
O
EDCI, HOBt
O
BocHN
H
EDCI, HOBt
DIPEA, DCM
H
H
RHN
N
N
O
N
H
81%
N
H
H
O
80%
O
4
NHTrt
O
Scheme 3.
24
25
R = Cbz
R = H
H2, Pd-C
100%
O
H
H3C(H2C)6
O
O
N
NHTrt
O
N
NH
EDCI, HOBt,
H
L
-Ala-OMe.HCl (18)
RHN
OMe
O
NH
O
i-Pr2NEt
N
H
HO
O
O
+
5
N-Boc-Orn(Z)-Thr-Ala-OH ( )
OMe
O
HO
NH
N-Boc-
L-Thr-OH (14)
80%
OH
O
O
EDCI, HOBt, CH2Cl2
81%
H
N
BocHN
19 R = Boc
20 R = H
2M HCl in EtOAc
95%
N
H
N
H
O
O
NHTrt
CbzHN
26
21
N-Boc-L-Orn(Z)-OH ( ),
CbzHN
EDCI, HOBt, i-Pr2NEt
O
H
N
Scheme 5.
OR
O
BocHN
N
H
85%
O
OH
LiOH.H2O
THF-H2O, 83%
22
R = Me
R = H
5
Scheme 4.
O
H
N
H3C(H2C)6
O
O
N
NH
NHTrt
NH
H
O
O
O
2.5. Synthesis of the linear nonapeptide
HO
OR1
HO
O
NH
O
O
With all three tripeptides in hand, we turned our attention to
their couplings and the final macrocyclization. The N-Boc protect-
ing group of tripeptide 4 was removed with 2 M HCl in EtOAc,
affording the corresponding peptide amine 23 as a hydrochloric
acid salt (Scheme 5). Condensation of tripeptide acid 3 and tri-
peptide amine 23 was achieved by treatment with EDCI and HOBt
in the presence of DIEA in DCM, providing hexapeptide 24 in 80%
yield. The N-Cbz group of 24 was then removed by hydrogenolysis
in MeOH in the presence of 10% Pd–C. The resulting crude amine 25
was immediately used in the subsequent coupling with tripeptide
acid 5 in the presence of EDCI and HOBt in DCM, providing the
needed nonapeptide 26 with protecting groups.
H
N
R2HN
N
H
N
H
O
O
NHTrt
1
R
= Me, R2 = Boc
= H, R2 = Boc
26
CbzHN
1
27
28
R
2M HCl, EtOAc
R1 = H, R2 = H.HCl
O
H
NHR2
O
N
O
N
H
NH
H3C(H2C)6
O
NH
HO
O
O
HO
O
O
NH
EDCI, HOAt, DIEA
O
H
N
HN
CH2Cl2
60% (three steps)
N
H
N
H
2.6. Macrocyclization
O
O
NHR2
R1HN
H2, Pd/C; then
R
R
1 = Cbz, R2 = Trt
Hydrolysis of nonapeptide 26 with LiOH$H2O in THF–H2O fol-
lowed by treatment with 2 M HCl in EtOAc afforded the linear
head–tail free amino acid 28 (Scheme 6). Macrocyclization of the
linear precursor 28 was carried out with EDCI and HOAt in the
presence of DIEA in DCM, affording cyclopeptide 2a (60% yields in
three steps). Global deprotection of N-Cbz and N-Trt groups of
cyclopeptide 2a was accomplished by hydrogenolysis in the
presence of 10% Pd–C in MeOH and subsequent treatment with
TFA–H2O–iPr3SiH (v/v/v¼95:2.5:2.5), providing the target non-
acyclopeptide 2 in 90% yield. NMR spectra and other physical
characterizations of the synthetic sample of 2 obtained in this work
are identical to those reported in the reference.19
2a
1 = R2 = H
TFA/H2O/(i-Pr)3SiH
2
(95 : 2.5 : 2.5)
90%
Scheme 6.
3. Conclusion
In summary, a new synthesis of the cyclic nonapeptide of
chlorofusin has been accomplished in 20 longest linear steps and
9% overall yield. Successful application of new convergent
[3þ3þ3]-fragment assembly strategy and macrocylization at the

3430
Y.-L. Wang et al. / Tetrahedron 66 (2010) 3427–3432
26
amide-bond position between Orn9 and Ada8 under mild condi-
tions makes this synthesis very efficient and capable in material
accumulation, and thus facilitates the further total synthesis of
chlorofusin and other stereoisomers.
a white foam (6.1 g, 89%). [
a
]
ꢀ1.30 (c 1.80, CHCl3); IR (KBr): nmax
3059, 1734, 1668, 1493 cmꢀD1
;
1H NMR (acetone-d6, 400 Hz, rt):
d
8.06 (2H, d, J¼2.8 Hz), 7.87 (1H, d, J¼8.0 Hz), 7.48 (1H, d, J¼7.6 Hz),
7.33 (1H, d, J¼2.4 Hz), 7.25–7.19 (30H, m), 6.63 (1H, d, J¼7.6 Hz),
5.03 (2H, q, J¼12.4 Hz), 4.75–4.70 (1H, m), 4.42–4.33 (2H, m),
3.00–2.79 (3H, m), 2.77–2.72 (2H, m), 1.65–1.58 (1H, m), 1.46–1.42
(2H, t, J¼6.4 Hz), 0.81 (3H, d, J¼5.1 Hz), 0.79 (3H, d, J¼5.4 Hz); 13C
4. Experimental
4.1. General
NMR (acetone-d6, 75 Hz, rt): d 173.4, 171.8, 171.7, 170.8, 170.2, 157.0,
145.8, 145.7, 137.6, 129.7, 129.6, 129.1, 128.7, 128.6, 128.5, 127.7, 71.1,
71.0, 67.1, 53.3, 52.1, 51.8, 50.6, 40.7, 39.0, 38.6, 25.3, 23.1, 22.0.
HRMS (ESI, m/z) calcd for C61H61N5NaO8 [MþNaþ] 1014.4412, found
1014.4418.
All reactions were conducted using oven-dried glassware. All
the temperatures were uncorrected. IR spectra were recorded on an
FT-IR instrument. 1H NMR spectra were recorded at 300 MHz or
400 MHz, and 13C NMR spectra were recorded at 75 MHz or
100 MHz. Reference peaks for chloroform in 1H NMR and 13C NMR
spectra were set at 7.27 ppm and 77.2 ppm, respectively. For DMSO-
d6, the reference peaks in 1H NMR and 13C NMR spectra were set at
2.50 ppm and 39.5 ppm. For acetone-d6, the reference peaks in 1H
NMR and 13C NMR spectra were set at 2.05 ppm, and 29.9 ppm and
206.7 ppm, respectively. Flash column chromatographies were
performed on silica gel H (10–40 mesh). Petroleum ether and ethyl
acetate were obtained from commercial suppliers and used with-
out further distillations.
4.4. N-Boc–L-Thr-D-Leu–OMe (29)
A mixture of N-Boc–
25 mmol) and EDCI (4.8 g, 25 mmol) in dry DCM (200 mL) was
stirred at 0 ꢁC for 0.5 h. Then,
-Leu–OMe$HCl (7, 3.73 g, 21 mmol)
L-Thr–OH (14, 5 g, 23 mmol), HOBt (3.4 g,
D
and DIEA (3.24 g, 25 mmol) were added successively at 0 ꢁC. The
reaction mixture was allowed to warm to rt and stirred until
completion. Saturated aqueous NaHCO3 (50 mL) was added to the
reaction mixture. The whole mixture was extracted with EtOAc
(100 mLꢂ3). The combined EtOAc extracts were washed succes-
sively with water, aqueous 1 N HCl (40 mLꢂ2), water and brine
(40 mL), dried (MgSO4) and concentrated in vacuo. The residue was
4.2. N-Cbz–D-Asn(Trt)-D-Leu–OMe (8)
A solution of
(60 mL) was treated with HOBt (0.9 mg, 6.6 mmol) and EDCI (1.3 g,
6.6 mmol) under stirring at 0 ꢁC for 0.5 h. Then,
-Leu–OMe$HCl
D-Z-Asn(Trt)–OH (6, 3.1 g, 6.1 mmol) in dry DCM
chromatographied (CH2Cl2/MeOH¼200:1 to 100:1) to afford 29 as
27
a white foam (6.1 g, 85%). [
a
]
D
7.13 (c 1.50, CHCl3); IR (KBr): nmax
D
3308, 2966, 1748, 1662 cmꢀ1; 1H NMR (DMSO-d6, 400 Hz, rt):
d 8.12
(7, 1.0 g, 5.5 mmol) and DIEA (0.9 g, 6.6 mmol) were added suc-
cessively at 0 ꢁC. The reaction mixture was allowed to warm to rt
and stirred until completion. Saturated aqueous NaHCO3 (50 mL)
was added to quench the reaction. The whole mixture was
extracted with EtOAc (100 mLꢂ3). The combined EtOAc extracts
were washed successively with water, aqueous 1 N HCl (40 mLꢂ2),
water and brine (40 mL), dried (MgSO4) and concentrated in vacuo.
(1H, d, J¼8.0 Hz), 6.26 (1H, d, J¼8.0 Hz), 4.69 (1H, d, J¼5.6 Hz), 4.30–
4.25 (1H, m), 3.89–3.84 (2H, m), 3.62 (2H, m), 1.62–1.58 (2H, m),
1.49–1.47 (1H, m), 1.39 (9H, s), 1.02 (3H, d, J¼6.0 Hz), 0.88 (3H, d,
J¼6.4 Hz), 0.82 (3H, d, J¼6.4 Hz); 13C NMR (acetone-d6, 100 Hz, rt):
d
172.9, 170.5, 155.2, 78.1, 67.0, 60.0, 51.7, 50.2, 28.1, 24.1, 22.8, 21.1,
19.9. HRMS (ESI, m/z) calcd for C16H30N2NaO6 [MþNaþ] 538.3463,
found 538.3468.
The residue was chromatographied (CH2Cl2/MeOH¼200:1 to
25
100:1) to afford 8 as a white foam (3.06 g, 87%). [
a
]
2.75 (c 1.90,
D
CHCl3); IR (KBr): nmax 3307, 3058, 2955, 1659 cmꢀ1
;
1H NMR (ace-
8.04 (1H, s), 7.56 (1H, d, J¼7.6 Hz), 7.37
4.5. N-Boc–L-Thr-D-Leu-D-Ada–OMe (4)
tone-d6, 400 Hz, rt):
d
(5H, m), 7.26–7.22 (15H, m), 6.69 (1H, d, J¼8.0 Hz), 5.08 (2H, s), 4.53
(1H, q, J¼6.4 Hz), 4.45 (1H, q, J¼7.2 Hz), 3.65 (3H, s), 2.95–2.89
(1H, m), 2.82–2.75 (1H, m), 1.70–1.66 (1H, m), 1.55 (2H, t, J¼6.8 Hz),
0.89 (3H, d, J¼4.0 Hz), 0.87 (3H, d, J¼4.0 Hz); 13C NMR (acetone-d6,
A solution of 29 (1.8 g, 5.2 mmol) in THF (40 mL) and H2O
(10 mL) was treated with LiOH$H2O (440 mg, 10.4 mmol) at 0 ꢁC for
1 h until 1 N aqueous HCl (10 mL) was added. The mixture was
extracted with EtOAc (20 mLꢂ5). The combined EtOAc extracts
were dried (MgSO4) and concentrated in vacuo to provide acid 16 as
a white foam (1.55 g, 90%).
To a solution of 16 (0.55 g, 1.6 mmol) in dry DCM (20 mL) was
added HOBt (0.24 g, 1.8 mmol) and EDCI (0.34 g, 1.8 mmol) at
0 ꢁC. After stirring for 0.5 h,
D-Ada–OMe (15, 0.3 g, 1.5 mmol) was
100 Hz, rt):
d 173.5, 172.2, 170.3, 156.7, 145.7, 137.7, 129.6, 129.0,
128.5, 128.2, 127.2, 71.0, 66.8, 52.4, 52.2, 51.6, 41.0, 39.3, 25.2, 23.1,
21.9. HRMS (ESI, m/z) calcd for C38H41N3NaO6 [MþNaþ] 658.2893,
found 658.2888.
4.3. N-Cbz–
L
-Asn(Trt)-
D
-Asn(Trt)-
D
-Leu–OMe (11)
added at 0 ꢁC. The reaction mixture was allowed to warm to rt
and stirred until completion. Saturated aqueous NaHCO3 (10 mL)
was added to quench the reaction. The mixture was extracted
with EtOAc (20 mLꢂ3). The combined EtOAc extracts were
washed successively with water, aqueous 1 M HCl (10 mLꢂ2),
water and brine (20 mL), dried (MgSO4) and concentrated in
A suspension of 8 (3.06 g, 4.8 mmol) and 10% Pd–C (300 mg) in
MeOH (50 mL) was stirred under H2 (1 atm) at rt for 4 h. The solid
was removed by filtration through a pad of Celite and washed with
EtOAc (20 mL). The filtrate was concentrated in vacuo to afford 9
(
D
-Asn(Trt)-
A solution of
(60 mL) was treated with HOBt (0.9 mg, 6.6 mmol) and EDCI
(1.26 g, 6.6 mmol) under stirring at 0 ꢁC for 0.5 h. Then,
-Asn(Trt)-
-Leu–OMe (9, 2.75 g, 5.5 mmol) was added and the mixture was
D
-Leu–OMe) as a white foam (2.75 g, 100%).
vacuo. The residue was chromatographied (CH2Cl2/MeOH¼200:1
27
L-Z-Asn(Trt)–OH (10, 3.1 g, 6.0 mmol) in dry DCM
to 50:1) to afford 4 as a white foam (0.77 g, 81%). [
a
]
8.57
D
(c 1.50, CHCl3); IR (KBr): nmax 2959, 2930, 1747, 1647 cmꢀ1
;
1H
8.13 (1H, d, J¼7.2 Hz), 7.92 (1H, d,
D
NMR (DMSO-d6, 400 Hz, rt):
d
D
J¼8.4 Hz), 6.41 (1H, d, J¼7.2 Hz), 4.77 (1H, d, J¼5.6 Hz), 4.33–4.32
(1H, m), 4.18–4.16 (1H, m), 3.87–3.81 (2H, m), 3.60 (3H, d,
J¼11.2 Hz), 1.66–1.60 (3H, m), 1.49–1.43 (2H, m), 1.38 (9H, s), 1.23
(12H, m), 1.02 (3H, d, J¼6.0 Hz), 0.88–8.82 (9H, m); 13C NMR
stirred at 0 ꢁC for 0.5 h. The reaction mixture was allowed to warm
to rt, and stirred until completion. Saturated aqueous NaHCO3
(40 mL) was added, and the whole mixture was extracted with
EtOAc (80 mLꢂ3). The combined EtOAc extracts were washed se-
quentially with water, aqueous 1 N HCl (30 mLꢂ2), water and brine
(40 mL), dried (MgSO4) and concentrated in vacuo. The residue was
chromatographied (CH2Cl2/MeOH¼200:1 to 150:1) to afford 11 as
(acetone-d6, 100 Hz, rt):
d 173.2, 173.1, 171.9, 156.5, 79.5, 68.2,
61.2, 53.1, 52.5, 41.5, 32.5, 32.4, 29.9, 28.5, 26.3, 25.3, 23.2, 21.8,
19.8, 14.3. HRMS (ESI, m/z) calcd for C26H49N3NaO7 [MþNaþ]
538.3463, found 538.3468.

Y.-L. Wang et al. / Tetrahedron 66 (2010) 3427–3432
3431
4.6. N-Boc–
L
-Thr-
L
-Ala–OMe (19)
To a solution of tripeptide acid 3 (802 mg, 0.82 mmol) in dry
DCM (10 mL) was added HOBt (122 mg, 0.9 mmol) and EDCI
(173 mg, 0.9 mmol) at 0 ꢁC. The mixture was stirred at 0 ꢁC for 0.5 h,
and then the amine hydrochloride 23 (339 mg, 0.75 mmol) and
DIEA (121 mg, 0.9 mmol) were added successively. After being
stirred at 0 ꢁC for 0.5 h, the reaction mixture was allowed to warm
to rt and stirred until completion. Saturated aqueous NaHCO3
(20 mL) was added to quench the reaction. The mixture was
extracted with EtOAc (30 mLꢂ3). The combined EtOAc extracts
were washed successively with water, aqueous 1 N HCl (20 mLꢂ2),
water and brine (40 mL), dried (MgSO4) and concentrated in vacuo.
The residue was purified by column chromatography on silica gel
(CH2Cl2/MeOH¼200:1 to 100:1) to afford hexapeptide 22 as a white
A mixture of N-Boc–
(2.3 g, 17 mmol) and EDCI (3.3 g, 17 mmol) in dry DCM (160 mL)
was stirred at 0 ꢁC for 0.5 h. Then,
-Ala–OMe$HCl (18, 2.6 g,
14.1 mmol) and DIEA (3.0 mL, 17 mmol) were added successively at
0 ꢁC. The reaction mixture was allowed to warm to rt and stirred
until completion. Saturated aqueous NaHCO3 (50 mL) was added to
quench the reaction. The mixture was extracted with EtOAc
(50 mLꢂ3). The combined EtOAc extracts were washed sequentially
with water, aqueous 1 N HCl (20 mLꢂ2), water and brine (20 mL),
dried (MgSO4) and concentrated in vacuo. The residue was chro-
L-Thr–OH (14, 3.4 g, 15.5 mmol), HOBt
L
matographied (petroleum ether/EtOAc¼20:1 to 10:1) to afford 19
26
as a white foam (3.8 g, 80%). [
a
]
ꢀ49.2 (c 1.95, CHCl3); IR (KBr):
foam (825 mg, 80%). [
2955, 2926, 1650, 1529, 1448 cmꢀ1; 1H NMR (DMSO-d6, 400 Hz, rt):
8.62–8.47 (2H, m), 8.20–8.18 (1H, m), 7.88–7.65 (4H, m), 7.37–7.32
a]
26 10.8 (c 3.15, CHCl3); IR (KBr): nmax 3300,
D
D
nmax 3445, 2250, 2124, 1733 cmꢀ1
rt):
;
1H NMR (DMSO-d6, 400 MHz,
d
8.18 (1H, d, J¼7.2 Hz), 6.35 (1H, d, J¼8.0 Hz), 4.73 (1H, d,
d
J¼5.6 Hz), 4.32–4.25 (1H, m), 3.85 (2H, s), 3.61 (3H, s), 1.39 (9H, s),
(5H, m), 7.20 (31H, m), 5.12–4.98 (2H, m),4.90–4.75 (1H, m),
4.58–4.49 (1H, m), 4.35–4.12 (5H, m), 3.92–3.91 (1H, m), 3.64 (3H,
d, J¼12.0 Hz), 2.76–2.60 (3H, m), 2.44–2.35 (1H, m), 1.64–1.46 (8H,
m), 1.22 (12H, s), 0.98 (3H, d, J¼6.0 Hz), 0.85–0.80 (15H, m); 13C
1.28 (3H, d, J¼7.6 Hz), 1.05 (3H, d, J¼6.0 Hz); 13C NMR (acetone-d6,
100 Hz, rt): d 173.8, 171.1, 156.5, 79.4, 68.1, 59.9, 52.4, 48.8, 19.2, 17.9.
HRMS (ESI, m/z) calcd for C13H24N2NaO6 [MþNaþ] 327.1532, found
327.1527.
NMR (MeOH-d4, 100 Hz, rt): d 173.9, 173.4, 173.3, 173.1, 172.9, 172.2,
172.1, 171.8, 171.4, 170.5, 170.4, 157.4, 145.8, 145.5, 137.5, 129.7, 129.2,
128.7, 128.4, 127.4, 71.2, 68.0, 67.6, 61.3, 60.5, 54.9, 54.5, 53.5, 53.3,
52.9, 52.2, 52.1, 41.3, 41.1, 40.5, 39.9, 39.2, 38.7, 38.6, 38.3, 38.0, 32.5,
32.1, 26.3, 25.3, 25.1, 23.9, 23.3, 21.8, 21.3, 20.8, 20.2, 19.3, 14.4.
HRMS (ESI, m/z) calcd for C81H98N8NaO12 [MþNaþ] 1397.7202,
found 1397.7196.
4.7. N-Boc–L-Orn(Cbz)-L-Thr-L-Ala–OMe (22)
Dipeptide 19 (4.5 g, 14.8 mmol) was treated with 2 M HCl in
EtOAc (50 mL) at rt for 1 h. The mixture was concentrated in vacuo
to afford 20 as white foam, which was directly used in the next step
without further purification.
To a solution of N-Boc–L-Orn(Z)–OH (21, 6.0 g, 16.3 mmol) in dry
4.9. N-Boc–
L
-Orn(Cbz)-L-Thr-L-Ala-L-Asn(Trt)-D-Asn(Trt)-D-
DCM (150 mL) was added HOBt (2.4 g, 17.8 mmol) and EDCI (3.4 g,
17.8 mmol) at 0 ꢁC. After the mixture was stirred at 0 ꢁC for 0.5 h,
the above freshly prepared dipeptide amine 20 (4.3 g, 14.8 mmol)
and DIEA (3.1 mL, 17.8 mmol) were added successively at 0 ꢁC. The
reaction mixture was stirred at 0 ꢁC for 0.5 h, and then allowed to
warm to rt and stirred until completion. Saturated aqueous NaHCO3
(20 mL) was added to quench the reaction. The mixture was
extracted with EtOAc (40 mLꢂ3). The combined EtOAc extracts
were washed sequentially with water, aqueous 1 N HCl (10 mLꢂ2),
water and brine (20 mL), dried (MgSO4) and concentrated in vacuo.
Leu- -Thr- -Leu-D
L
D
-Ada–OMe (26)
A suspension of 24 (0.99 g, 0.72 mmol) and 10% Pd–C (200 mg)
in MeOH (10 mL) was stirred under H2 (1 atm) at rt for 4 h. The solid
was removed by filtration through a pad of Celite and washed with
MeOH (10 mL). The filtrate and washings were concentrated in
vacuo to afford peptide amine 25 as a white paste (893 mg, 100%).
A solution of 22 (1.7 g, 3.1 mmol) in THF (20 mL) and H2O (5 mL)
was treated with LiOH monohydrate (258 mg, 6.2 mmol) at 0 ꢁC for
2 h until 1 M aqueous HCl (10 mL) was added. The mixture was
extracted with EtOAc (5 mLꢂ5). The combined EtOAc extracts were
dried (MgSO4) and concentrated in vacuo to provide tripeptide acid
5 as a white foam.
To a solution of tripeptide acid 5 (780 mg, 1.45 mmol) in dry
DCM (20 mL) was added HOBt (213 mg, 1.58 mmol) and EDCI
(304 mg, 1.58 mmol) at 0 ꢁC. After being stirred for 0.5 h, the pep-
tide amine 25 (1.6 g, 1.32 mmol) and DIEA (204 mg, 1.58 mmol)
were added at the same temperature. The reaction mixture was
allowed to warm to rt and stirred until completion. Saturated
aqueous NaHCO3 (20 mL) was added to quench the reaction. The
mixture was extracted with EtOAc (50 mLꢂ3). The combined EtOAc
extracts were washed successively with water, aqueous 1 M HCl
(20ꢂ2 mL), water and brine (20 mL), dried (MgSO4) and concen-
trated in vacuo. The residue was purified by column chromatog-
Purification by column chromatography (CH2Cl2/MeOH¼200:1 to
26
100:1) afforded 22 as a white foam (7.0 g, 85%). [
a]
ꢀ30.5 (c 2.00,
D
CHCl3); IR (KBr): nmax 3502, 2917,1704,1249 cmꢀ1; 1H NMR (DMSO-
d6, 300 MHz, rt):
d
8.11 (1H, d, J¼6.4 Hz), 7.57 (1H, d, J¼8.4 Hz),
7.38–7.30 (5H, m), 7.22 (1H, t, J¼5.6 Hz), 7.03 (1H, d, J¼7.6 Hz), 5.00
(2H, s), 4.83 (1H, d, J¼4.4 Hz), 4.30–4.26 (1H, m), 4.20–4.17 (1H, m),
3.95–3.92 (2H, m), 3.60 (3H, s), 2.97 (2H, d, J¼6.0 Hz), 1.63 (1H, m),
1.49–1.42 (9H, s), 1.28 (3H, d, J¼7.6 Hz), 1.05 (3H, d, J¼6.4 Hz); 13C
NMR (acetone-d6, 100 Hz, rt):
d 172.8, 172.3, 172.0, 169.8, 156.1,
155.5, 137.2, 128.3, 127.7, 78.2, 66.6, 65.1, 60.5, 57.7, 54.2, 51.8, 47.6,
28.9, 28.2, 26.0, 19.6, 17.0, 14.0. HRMS (ESI, m/z) calcd for
C26H40N4NaO9 [MþNaþ] 575.2688, found 575.2693.
4.8. N-Cbz-L-Asn(Trt)-D-Asn(Trt)-D-Leu-L-Thr-D-Leu-D-Ada–
OMe (24)
raphy (CH2Cl2/MeOH¼200:1 to 100:1) on silica gel to afford the
30
linear nonapeptide 26 as a white foam (1.88 g, 81%). [
a]
ꢀ11.8
D
Tripeptide N-Boc–
L-Thr-D-Leu-D-Ada–OMe (4, 465 mg, 0.9 mmol)
(c 3.20, CHCl3); IR (KBr): nmax 3300, 2955, 2926, 1650, 1529,
1448 cmꢀ1; 1H NMR (DMSO-d6, 400 Hz, rt):
8.58 (2H, d, J¼9.2 Hz),
wastreatedwith2 MHClinEtOAc(5 mL)atrtfor2 h.Themixturewas
concentrated in vacuo to afford the peptide amine 23 as white foam,
which was directly used in the next step without further purification.
A solution of 11 (2.6 g, 2.6 mmol) in THF (40 mL) and H2O
(10 mL) was treated with LiOH$H2O (110 mg, 2.6 mmol) and stirred
at 0 ꢁC for 2 h until 1 N aqueous HCl (10 mL) was added. The mix-
ture was extracted with EtOAc (10 mLꢂ5). The combined EtOAc
extracts were dried (MgSO4) and concentrated in vacuo. The crude
tripeptide acid 3 was provided as white foam, and directly used in
the next step without further purification.
d
8.22 (2H, m), 7.91 (1H, d, J¼6.4 Hz), 7.80 (1H, d, J¼8.4 Hz), 7.73
(1H, d, J¼7.2 Hz), 7.66 (1H, d, J¼8.0 Hz), 7.35–7.21 (5H, m), 7.20–7.14
(31H, m), 7.08 (1H, d, J¼7.6 Hz), 4.99 (2H, s), 4.96 (1H, d, J¼4.4 Hz),
4.73 (2H, d, J¼5.6 Hz), 4.57–4.56 (1H, m), 4.50–4.49 (1H, m),
4.39–4.31 (3H, m), 4.26–4.24 (1H, m), 4.16–4.10 (2H, m), 4.02–4.00
(1H, m), 3.95–3.89 (2H, m), 3.56 (3H, s), 2.96 (2H, d, J¼5.2 Hz),
2.72–2.59 (4H, m), 1.63–1.45 (12H, m), 1.38 (9H, s), 1.23 (15H, m),
1.01 (3H, d, J¼6.0 Hz), 0.96 (3H, d, J¼6.4 Hz), 0.85–0.79 (15H, m);
13C NMR (MeOH-d4, 100 Hz, rt):
d 174.8, 174.3, 173.9, 173.4, 173.2,

3432
Y.-L. Wang et al. / Tetrahedron 66 (2010) 3427–3432
173.0, 172.4, 172.0, 171.6, 170.3, 170.2, 157.5, 157.3, 145.9, 145.7, 138.3,
129.8, 129.7, 129.1, 128.6, 128.5, 128.4, 128.3, 127.4, 127.3, 80 .3, 71.2,
71.1, 68.3, 68.2, 68.1, 67.2, 67.1, 66.4, 61.1, 60.1, 60.0, 56.3, 53.9, 53.2,
52.1, 50.7, 41.3, 41.1, 41.0, 40.1, 38.6, 32.5, 32.2, 29.9, 27.0, 26.2, 25.2,
25.1, 23.5, 23.3, 22.0, 21.3, 20.6, 20.2, 20.1, 16.7,14.4. HRMS (ESI, m/z)
calcd for C98H128N12NaO18 [MþNaþ] 1783.9362, found 1783.9367.
removed under reduced pressure. The residue was washed with
30
Et2O (2 mLꢂ5) to afford 2 (6.0 mg, 99%) as a colourless solid. [
a]
þ5.13 (c 2.07, MeOH); IR (KBr): nmax 3334, 2944, 2832, 1032 cmꢀD1
;
1H NMR (acetone-d6, 400 Hz, rt):
d 9.09–9.04 (1H, m), 8.79 (1H, m),
8.67 (1H, s), 7.80–7.72 (5H, m), 7.50 (1H, d, J¼8.8 Hz), 7.25 (1H, m),
7.09 (2H, m), 6.95 (3H, d, J¼22 Hz), 6.70–6.66 (1H, m), 5.32–5.31
(1H, m), 4.99 (1H, m), 4.76 (1H, m), 4.58 (1H, m), 4.47–4.43 (1H, m),
4.37 (1H, m), 4.18–4.16 (1H, m), 4.00–3.98 (5H, m), 3.63 (1H, s), 2.91
(1H, d, J¼12.4 Hz), 2.77–2.67 (3H, m), 2.57 (1H, m), 2.39–2.33 (1H,
m), 1.77 (5H, m), 1.64–1.50 (6H, m), 1.37 (3H, m), 1.24 (13H, m),
1.14(3H, d, J¼5.6 Hz), 1.08 (3H, s), 0.91–0.75 (15H, m); 13C NMR
4.10. Cyclo(-L-Orn(Cbz)-
L-Thr-L-Ala-L-Asn(Trt)-D-Asn(Trt)-D-
Leu- -Thr- -Leu-D
L
D
-Ada-) (2a)
To a solution of 26 (330 mg, 0.187 mmol) in THF (2 mL) and H2O
(0.5 mL) was added LiOH monohydrate (24 mg, 0.57 mmol) at 0 ꢁC.
The reaction mixture was stirred at 0 ꢁC for 2 h, and then quenched
with the addition of 1 M aqueous HCl (10 mL). The mixture was
extracted with EtOAc (5 mLꢂ5). The combined EtOAc extracts were
dried (MgSO4), and concentrated in vacuo to provide peptide acid
27 as a white foam, which was directly used in the next step
without further purification.
(acetone-d6, 75 Hz, rt): d 174.1, 173.1, 173.0, 172.6, 172.4, 172.3, 172.0,
171.8, 171.5, 171.4, 170.4, 158.5, 158.1, 119.2, 115.2, 111.3, 65.0, 62.2,
54.0, 52.8, 52.1, 51.0, 50.8, 49.2, 40.3, 40.1, 39.8, 39.5, 39.2, 39.0, 38.7,
38.4, 37.4, 36.2, 31.3, 30.0, 28.6, 28.5, 27.8, 26.0, 24.2, 24.0, 23.3,
23.2, 22.9, 22.1, 20.6, 20.3, 20.2, 16.4, 13.9. HRMS (ESI, m/z) calcd for
C46H82N12NaO13 [MþNaþ] 1033.6022, found 1033.6049.
The above freshly prepared peptide acid 27 was treated with
2 M HCl in EtOAc (1 mL). The resulting solution was stirred at rt for
2 h. The mixture was concentrated in vacuo to afford the crude
linear amino acid 28 as a white foam, which was directly used in the
next step.
To a stirred solution of HOBt (120 mg, 0.9 mmol), EDCI (172 mg,
0.9 mmol) and DIEA (116 mg, 0.9 mmol) in dry DCM (20 mL) was
added 28 (309 mg, 0.187 mmol) in DMF (10 mL) via syringe pump
at 0 ꢁC. The reaction mixture was allowed to warm to rt and stirred
until completion. Saturated aqueous NaHCO3 (10 mL) was added to
quench the reaction. The mixture was extracted with EtOAc
(20 mLꢂ3). The combined EtOAc extracts were washed successively
with H2O, aqueous 1 M HCl (10 mLꢂ2), H2O and brine (10 mL),
dried (MgSO4) and concentrated in vacuo. The residue was purified
Acknowledgements
This project was financially supported by MOST
(2010CB833200), MOH (2009ZX09501), NSFC (20621062), CAS
(KJCX2-YW-H08), and Shanghai Municipal Committee of Science
and Technology (08JC1422800).
Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2010.03.032.
References and notes
by column chromatography on silica gel (CH2Cl2/MeOH¼200:1 to
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2. Vogelstein, B.; Lane, D.; Levine, A. J. Nature 2000, 408, 307–310.
3. Review: Vousden, K. H. Cell 2000, 103, 691–694.
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30
50:1) to afford cyclopeptide 2a as a white foam (186 mg, 61%). [a]
33.9 (c 1.80, CHCl3); IR (KBr): nmax 3436, 2918, 2249, 1333 cmꢀ1; 1DH
NMR (DMSO-d6, 400 Hz, rt): d 9.36 (1H, s), 8.89 (1H, s), 8.65 (1H, s),
8.20 (1H, s), 7.84 (2H, t, J¼1.2 Hz), 7.35–7.00 (37H, m), 6.88 (1H, m),
6.87 (1H, d, J¼8.4 Hz), 6.32 (1H, m), 5.35 (1H, d, J¼3.2 Hz), 4.99
(2H, s), 4.83–4.74 (1H, m), 4.52 (1H, d, J¼9.2 Hz), 4.15 (3H, m),
3.94–3.79 (4H, m), 3.72 (1H, s), 3.30–3.22 (2H, m), 3.00–2.90 (3H,
m), 2.38 (1H, d, J¼15.6 Hz), 2.04–2.03 (1H, m), 1.87–1.79 (3H, m),
1.61–1.46 (5H, m), 1.33–1.24 (21H, m), 0.98 (3H, d, J¼5.2 Hz),
0.86–0.74 (12H, m), 0.31 (3H, d, J¼5.2 Hz); 13C NMR (MeOH-d4,
6. Freedman, D. A.; Levine, A. J. Cancer Res. 1999, 59, 1–7.
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10. Review: Zhang, R.; Wang, H. Curr. Pharm. Des. 2000, 6, 393–416.
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75 Hz, rt):
d
177.0, 175.6, 175.5, 172.6, 175.3, 175.0, 174.5, 174.4, 174.0,
173.4,171.1,170.0,158.6, 146.0, 145.7,138.3, 130.1,129.9, 129.4, 128.9,
128.8, 127.8, 72 .1, 71.6, 71.5, 67.3, 67.1, 66.9, 66.6, 64.1, 55.4, 55.0,
53.7, 52.6, 51.0, 50.6, 41.0, 40.9, 40.5, 39.6, 33.0, 31.6, 30.7, 30.4, 30.3,
30.1, 27.8, 26.9, 26.0, 24.4, 23.8, 23.7, 21.3, 21.2, 20.9, 20.7, 16.9, 15.4,
14.5. HRMS (ESI, m/z) calcd for C92H116N12NaO5 [MþNaþ]
1651.8575, found 1651.8581.
4.11. Cyclo(-L-Orn-L-Thr-L-Ala-L-Asn-D-Asn-D-Leu-L-Thr-D-Leu-
D-Ada-) (2)
A suspension of 2a (124 mg, 0.076 mol) and 10% Pd–C (60 mg) in
dry MeOH (2 mL) and AcOH (0.5 mL) was stirred under H2 atmo-
sphere (1 atm) at rt for 4 h. The solid was filtered and the filtrate
was evaporated to afford the crude product (114 mg). A portion of
this product (10 mg) was then treated with a mixture of TFA–
H2O–iPr3SiH (0.26 mL, 95:2.5:2.5) at rt for 3 h. The solvents were
20. Lee, S. Y.; Boger, D. L. Tetrahedron 2009, 65, 3281–3284.
21. (a) Schollkopf, U.; Groth, U.; Deng, C. Angew. Chem., Int. Ed. Engl. 1981, 20, 798–
799; (b) Schollkopf, U. Tetrahedron 1983, 39, 2085–2091; (c) Chen, J.; Corbin, S.
P.; Holman, N. J. Org. Process Res. Dev. 2005, 9, 185–187.