Unified flexible total synthesis of chlorofusin and artificial Click mimics as antagonists against p53-HDM2 interactions

Article abstract of DOI:10.1016/j.tetlet.2014.09.028

A practical HPLC-free total synthesis of chlorofusin has been successfully accomplished in this work. The new synthesis showed great flexibility and convenience in generating artificial natural product-like mimics through Click chemistry, and enabled us to further investigate the biological importance of the chromophore and the spiro-aminal functionality of the natural product. The entire skeleton is believed to be essential for satisfactory biological activities of both natural chlorofusin and unnatural mimics. Two artificial Click hybrids were found to exhibit improved inhibitory activity against p53-HDM2 bindings over the natural product.

Full text of DOI:10.1016/j.tetlet.2014.09.028

Tetrahedron Letters 55 (2014) 6055–6059
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Unified flexible total synthesis of chlorofusin and artificial Click
mimics as antagonists against p53–HDM2 interactions
⇑
Hai-Bo Qiu, Xin-Ya Chen, Qing Li, Wen-Jian Qian, Shun-Ming Yu, Gong-Li Tang, Zhu-Jun Yao
State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A practical HPLC-free total synthesis of chlorofusin has been successfully accomplished in this work. The
new synthesis showed great flexibility and convenience in generating artificial natural product-like
mimics through Click chemistry, and enabled us to further investigate the biological importance of the
chromophore and the spiro-aminal functionality of the natural product. The entire skeleton is believed
to be essential for satisfactory biological activities of both natural chlorofusin and unnatural mimics.
Two artificial Click hybrids were found to exhibit improved inhibitory activity against p53–HDM2 bind-
ings over the natural product.
Received 13 August 2014
Accepted 6 September 2014
Available online 16 September 2014
Keywords:
Chlorofusin
Artificial mimic
Total synthesis
p53–MDM2 interaction
Inhibitory activity
Ó 2014 Elsevier Ltd. All rights reserved.
Chlorofusin (1, Fig. 1) was discovered from fungal strain Micro-
dochium caespitosum as a novel secondary metabolite, and found to
exhibit low micromolar inhibitory ability (antagonist) by direct
(CH2)6CH3
O
H3C(H2C)6
BnO2C
O
O
D-Ada 8
N
H
OH
HN
N
H
OH
D-Leu 7
L-Orn 9
HN
O
binding to the N-terminal domain of MDM2 (IC50 4.6
lM, KD
O
HN
O
4.7
l
M).1 The structure of chlorofusin was initially proposed to
BocHN
O
HO
L-Thr 6
O
HN
HO
N
NH
9
L-Thr 1
NH
contain a densely functionalized azaphilone-derived chromophore
linked through the terminal amine of ornithine to a 27-membered
nonacyclopeptide (composed of nine amino acid residues). The
absolute stereochemistry of the cyclopeptide part was confirmed
through the total synthesis by Boger2 and Searcey3 independently
in 2003. In 2007, we reassigned the relative stereochemistry of the
chromophore part and completed an enantioselective total synthe-
sis of the (4S,8R,9S)-diastereomer of chlorofusin using an asym-
metric oxidative dearomatization reaction.4 Shortly thereafter,
Boger’s group accomplished the total synthesis of natural chlorofu-
sin and its seven chromophore diastereomers and reassigned the
absolute stereochemistry of the natural chromophore as 4R,8S,9R
(as shown in Fig. 1).5
1
HO
8
D-L eu 5
2
NH
O
O
7
O
NH
O
O
3
O
NH
Cl
O
6
5
O
NH
O
O
L-Ala 2
O
O
4
HN
O
O
HN
N
H
NH2
N
H
NH2
O
D-Asn 4
O
O
L-Asn 3
2
I
NH2
chlorofusin (1)
NH2
+
Cl
OH
O
O
O
Cl
O
OH
Cbz2N
CO2Bn
O
O
O
O
4
N
NH
O
O
Cbz2N
CO2Bn
5
3
Our previous total synthesis of the (4S,8R,9S)-diastereomer of
chlorofusin was disclosed in a convergent fashion, via mild N-
insertion of the entire cyclic peptide-amine to an optically pure
azaphilone by asymmetric synthesis followed by elaboration of
the spiro-aminal functionality in one pot with the aid of reverse
phase HPLC purification at the final step.4a Accordingly, a semi-pre-
parative chiral HPLC separation of the racemic azaphilone was
applied in Boger’s total syntheses of chlorofusin and its seven
Figure 1. Retrosynthetic analysis of chlorofusin (1).
chromophore diastereomers.5 Furthermore, their biological assess-
ment of chlorofusin and its chromophore diastereomers, as well as
the synthetic fragments, indicated that the stereochemistry of the
chromophore showed slight effects on the inhibitory activity
against p53–MDM2 interactions, and molecular integrity was
found to be essential for the bioactivity.6 In order to facilitate fur-
ther investigation of chlorofusin with various biomedical consider-
ations, an economic synthesis route toward the natural product
⇑
Corresponding author. Tel./fax: +86 21 54925123.
E-mail address: yaoz@sioc.ac.cn (Z.-J. Yao).
http://dx.doi.org/10.1016/j.tetlet.2014.09.028
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

6056
H.-B. Qiu et al. / Tetrahedron Letters 55 (2014) 6055–6059
and its biologically interesting chromophore diastereomers would
be of importance. It is of extreme value to establish an inexpensive
and time-saving HPLC-free approach to acquire enantiopure mate-
rials containing the C-4 quaternary stereocenter in the early stage
of total synthesis. Furthermore, suitable chemistry for late-stage
generation of the spiro N,O-aminal functionality and the hydroxyl
group on the chromophore is also crucial to deliver enantiopure
chlorofusin and its single diastereomers. Clearly, chlorofusin is an
unusual natural ‘hybrid’ containing two biogenetically different
subunits, one of which is the polyketide-characterized chromo-
phore and the other is its cyclopeptide portion.1a Though such a
natural hybrid has been achieved by a mild biomimetic reaction
between azaphilone and cyclopeptide-amine,4 proper alternatives
of the natural hydrocarbon linkage between these two functional-
ities might be a workable protocol for discovering chlorofusin-like
bioactive molecules. Based on the above thoughts, we wish to
report herein our recent results on the development of a new flex-
ible HPLC-free total synthesis of natural chlorofusin through a con-
vergent [chromophore + one amino acid + octapeptide]-assembly
protocol (Fig. 1) and its further application to the synthesis
of several natural product-like artificial hybrids through mild
Huisgen-[3+2] chemistry,7 as well as their inhibitory activity
against p53–HDM2 bindings.
ambient temperature4b,10 afforded two silica gel flash chromatog-
raphy-separable diastereomeric vinylogous -pyridones 10 and
c
11 in equal amounts. Determination of the absolute stereochemis-
try of the C-4 carbon of each single product was carried out by the
circular dichroism (CD) measurement11 and compared with the
reference data.5 The chiral auxiliary was then removed with (i-Pr)3-
SiH in TFA and water, and replaced with an N-allyl in the presence
of allyl bromide and K2CO3 and a catalytic amount of Pd(PPh3)4 in
DMF, affording stable enantiopure intermediate 13. The above pro-
cedure, as expected, showed great advantages of scalability, stable
products, high yields, and ease of operation and reduplication, and
was capable to support our further synthesis.
Using vinylogous c-pyridone 13, we further established a ste-
reodivergent entrance to all four enantiopure chromophore diaste-
reomers (Scheme 2). Treatment of acetate (R)-13 with K2CO3 in
MeOH followed by intramolecular iodoetherification and in situ
hydrolysis in one pot afforded a mixture of four spiro-aminal prod-
ucts (14) with adjacent hydroxyl functionality in high yields. Dess–
Martin oxidation of the mixture of four diastereomers afforded two
silica-gel chromatographically separable diastereomeric ketones
15 and 16. Reduction of the individual ketones, in parallel, with
NaBH3CN in methanol and chromatographic separation on silica
gel afforded four single fully functionalized chromophores 17–20
in satisfactory yields. The relative stereochemistry of these diaste-
reomers was determined by comparison of their 1H NMR spectros-
copies with the reference analogs, which were confirmed by our
previous X-ray single crystal analyses.4c
Using (4R,8S,9R)-chromophore diastereomers 19 (with natural
configurations), a number of protocols were attempted to conju-
gate the chromophore part with the cyclopeptide, including cross
metathesis, Tsuji–Trost N-allylation, and intermolecular SN2 reac-
tion. Unfortunately, all these efforts only succeeded in the model
reactions with single amino acid derivatives and failed to link with
the whole cyclopeptide part. A stepwise strategy was then taken
into our consideration, by which the chromophore derivative was
linked at first with a single amino acid derivative and then coupled
Construction of the enantiopure chromophore is one crucial
task in the total synthesis of chlorofusin. It is noteworthy here that
few enantioselective methodologies are currently available to pro-
vide both individual R and S enantiomers of the C-4 quaternary ste-
reocenter.4a,8 However, our previous study found that the
commercially available (R)- or (S)-a-methyl benzylamine could
efficiently discriminate the racemic material of azaphilone through
the unique azaphilone-amination reaction under mild condi-
tions.4b This transformation was successfully applied to our
current synthesis (Scheme 1). Silver(I)-catalyzed oxo-alkyne
cycloisomerization of o-alkynylbenzaldehyde 6 in TFA and CH2Cl2
followed by IBX oxidation4,9 was slightly optimized and scaled
up (to 10 g) to provide sufficient quantity of rac-7 in one-pot
(80% yield) for the following synthesis. Subsequent butyrylation
and selective chlorination afforded azaphilone rac-9. Dehydrative
amination of azaphilone 9 with commercially available (R)-(+)-4-
Cl
Cl
OH
AcO
N
O
O
O
O
O
methoxyl-a-methylbenzylamine in CH3CN and aq. NaHCO3 at
O
8
a, b
O
9
4
N
O
(4R)-
O
14
13
AcO
inseparable diastereomers
AcO
AcO
O
O
c
O
O
HO
a, b
c
15 : 16 = 3 : 1
O
O
Cl
O
HO
Cl
O
O
CHO
O
O
O
O
O
O
rac-7
O
OH
O
9
N
9
N
6
rac-8
+
4
4
Cl
AcO
O
O
O
O
O
O
d
4
N
(4R, 9S)-15
(4R, 9R)-16
19 20
= 3 : 1
OAc
f
O
Cl
d
17 18
:
d
= 3 : 1
:
AcO
N
Cl
(4R)-10
O
O
O
O
O
Cl
OH
Cl
OH
O
e
+
O
O
R1
4
O
O
OMe
O
O
O
8
O
O
O
O
Cl
9
O
AcO
N
4
N
N
1
O
O
O
rac-9
12
(4R)- (R =H)
O
g
4
O
O
1
13
(4R)- (R =allyl)
(4R, 8R, 9S)-17
(4R, 8S, 9R)-19
(4R)-10 : (4S)-11 = 1 : 1
O
+
Cl
+
Cl
OH
OH
O
O
(4S)-11
O
O
O
O
OMe
N
N
Scheme 1. Reagents and conditions: (a) AgNO3, TFA, ClCH2CH2Cl; (b) IBX, n-Bu4NI,
O
O
20
(4R, 8R, 9R)-
80% (two steps); (c) butyryl chloride, pyridine, DMAP, CH2Cl2, 76%; (d) SO2Cl2,
(4R, 8S, 9S)-18
CH2Cl2, 69%; (e) (R)-(+)-4-methoxyl-
CH3CN, 90%; (f) (i-Pr)3SiH, TFA/H2O (10:1), 80%; (g) allyl bromide, KFÁAl2O3,
Pd(PPh3)4, DMF, 85%. IBX = 2-iodoxybenzoic acid, DMAP = 4-
dimethylaminopyridine.
a-methylbenzylamine, aq NaHCO3 (sat.),
Scheme 2. Reagents and conditions: (a) K2CO3, MeOH, 80%; (b) I2, NaHCO3, n-
Bu4NI, CH3CH/H2O (10:1), 91%; (c) DMP, NaHCO3, CH2Cl2, 90%; (d) NaBH3CN,
CeCl3Á7H2O, MeOH, 90%. DMP = Dess–Martin periodinane.

H.-B. Qiu et al. / Tetrahedron Letters 55 (2014) 6055–6059
6057
bindings.3,6,13 So, it is reasonable to deduce that the structural
integrity of chlorofusin is required to maintain its bioactivity. Fur-
thermore, Boger’s pioneer SAR study on chlorofusin mentioned
that stereochemistry of the chlorofusin chromophore showed little
effects on the inhibition of p53–MDM2 binding.6 Obviously, our
newly established total synthesis and multi-functionalized syn-
thetic intermediates enabled us to further explore potential artifi-
cial chlorofusin-like hybrids for biological assessment.
To probe the effects of the C8/C9 stereochemistries and spiro-
aminal moiety of the chlorofusin chromophore on the inhibitory
activity, two diastereomeric products 27a and 27b4a were prepared
at first by a mild reaction of azaphilone rac-24 with cyclopeptide
amine 254d in CH3CN and aq NaHCO3, followed by removal of N-
Trt groups under mild acid conditions (Scheme 4).
Utilizing the available intermediates, replacement of the linear
hydrocarbon between the chromophore and cyclic peptide of chlo-
rofusin with an easily accessible heterocycle with Cu(I)-catalyzed
azide-alkyne cycloaddition (CuAAC) reaction would be a conve-
nient approach to generate potentially useful artificial mimics of
chlorofusin. The required azido-cyclopeptides were synthesized
through a [1+8]-fragment coupling strategy using the same octa-
peptide 22 in the synthesis of chlorofusin. We envisioned that
adjusting the length of the ‘linker’ between the chromophore and
cyclopeptide might affect the bioactivity. Therefore, serine
(three-carbon length) and glutamic acid (five-carbon length) were
chosen as the linking units. Incorporation of an azido group into
commercially available serine and glutamic acid derivatives was
carried out in several routine steps, affording two azido-containing
amino acid derivatives 29 and 30, respectively.14 In the meantime,
N-propargylation of (4R,8S,9R)-chromophore enamine 31 (with
natural configurations) was carried out by treatment with propar-
gyl bromide and K2CO3 in DMF, affording terminal alkyne 32 in 94%
yield (Scheme 5). With both N-propargylated chromophore 32 and
azido-containing cyclopeptides 29 and 30 in hand, Click generation
of artificial mimics was accomplished smoothly in the presence of
catalytic amount of CuSO4Á5H2O and sodium ascorbate in a mix-
ture of t-BuOH and H2O, giving the corresponding 1,2,3-triazole
33 and 34, respectively.
Cl
O
OH
O
Cl
O
OH
O
O
O
O
O
O
O
N
a, b
N
R1
N
CO2R3
CO2Bn
NCbz2
I
R2
19
(4R,8S,9R)-
20
21 (R1 = R2 = Cbz; R3 = Bn)
c
(R1 = R2 = R3 = H)
21B (R1 = Cbz; R2 = R3 = H)
21A
e
d
22
H3C(H2C)6
BnO2C
NHCbz
O
Cl OH
O
O
O
O
N
H
N
HN
O
O
OH
O
HN
O
HO
NH
NH
O
O
23
f, g
O
NH
O
O
HN
N
H
O
NH2
H2N
O
H3C(H2C)6
O
Cl OH
O
O
O
8
HN
O
N
H
OH
9
4
N
HN
HO
O
O
O
HN
O
NH
NH
O
O
O
NH
O
O
(4R,8S,9R)-chlorofusin 1
( )
HN
N
H
NH2
O
NH2
Scheme 3. Reagents and conditions: (a) Pd(PPh3)4 (10 mol %), NDMBA, CH2Cl2,
microwave; (b) 20, KFÁAl2O3, DMF, 62% (two steps); (c) H2, Pd/C, THF/DMF (2:1); (d)
Cbz-OSu, NaHCO3, THF/H2O (1:1); (e) octapeptide amine 22, EDCI, HOBt, DIPEA,
CH2Cl2/DMF (1:1), 83% (three step); (f) H2, 10% Pd/C, THF/DMF (2:1); (g) EDCI, HOAt,
NaHCO3, DMF, 35% (two steps). NDMBA = 1,3-dimethylbarbituric acid, EDCI = 1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, HOBt = 1-hydroxy-
benzotriazole,
DIPEA = N,N-diisopropylethylamine,
HOAt = 1-hydroxy-7-
azabenzotriazole.
with the octapeptide and afforded the entire linear precursor for
final macrocyclization (Scheme 3). Removal of the N-allyl group
of 19 was carried out with 10 mol % Pd(PPh3)4 and 1,3-dimethyl-
barbituric acid in CH2Cl2 under microwave irradiation conditions.
The newly born enamine was immediately subjected to the reac-
tion under mild basic conditions with iodo-ornithine derivative
20, in which the amino group was fully protected with two N-Cbzs
(to avoid the formation of proline derivative), affording compound
21 in 62% yield. Global deprotection of two N-Cbz groups and one
benzyl ester of 21 was carried out by hydrogenation in the pres-
ence of 10% Pd/C. The free amino group was then masked with
Cbz-OSu in DMF and aq. NaHCO3. Condensation of ornithine-
attached chromophore (the acid 21B) with octapeptide amine
2212 was accomplished with EDCI, HOBt, and DIPEA in CH2Cl2–
DMF, affording the entire linear precursor 23 in high yield (83%
for three steps from 21). After removal of both N-Cbz and benzyl
ester groups of 23 by hydrogenolysis in THF–DMF, final macrocyc-
lization was carried out with EDCI and HOAt in the presence of
NaHCO3 in DMF, providing natural (4R,8S,9R)-chlorofusin (1, 35%
yield in two steps), whose spectroscopic data well matched with
those reported.1,5
O
H
O
N
O
NHR2
N
H
NH
H3C(H2C)6
Cl
NH
HO
O
O
HO
O
HO
O
O
O
O
O
NH
O
+
O
H
N
HN
N
H
N
H
O
rac-24
O
NHR2
R1HN
O
1
2
25
R =Cbz; R =Trt
a
26 R1=H; R2=Trt
b
27a : 27b = 1 : 1
(CH2)6CH3
O
O
Cl
HO
O
O
N
O
8
HN
9
N
H
4
HN
O
O
OH
O
c
HN
O
HO
NH
NH
O
O
(4S)-27a R=Trt
28a
O
NH
O
O
(4S)-
R=H
HN
N
H
NHR
To our knowledge, there is no apparent mechanism disclosed to
elucidate how chlorofusin plays a role in the inhibition of p53–
MDM2 binding. Several subunits of the cyclic peptide and the chro-
mophore of chlorofusin were previously examined and found to
exhibit little or no inhibitory activity against p53–MDM2
(4R)-27b
R=Trt
R=H
O
c
28b
(4R)-
NHR
Scheme 4. Reagents and conditions: (a) H2, Pd/C, MeOH, AcOH; (b) aq NaHCO3,
CH3CN, 80%; (c) (i-Pr)3SiH, TFA/H2O (10:1), 72% for 28a and 54% for 28b.

6058
H.-B. Qiu et al. / Tetrahedron Letters 55 (2014) 6055–6059
Cl
O
OH
activity by comparison with the natural product, though it is still
Cl
O
OH
O
O
O
O
O
O
difficult to explain whether the spatial distance between the chro-
mophore and the cyclic peptide or the newly introduced 1,2,3-tri-
azole makes certain contribution to the binding. Besides the
competitive p53–HDM2 binding assay, we also measured KD values
of (4R,8S,9R) and (4S,8R,9S)-chlorofusin and two 1,2,3-triazole-
linked mimics with HDM2 protein by SPR methods. They were
found to be in approximate agreement with their IC50 values.
O
O
a
NH
N
31
32
N3
n
NH
O
O
O
HN
O
O
NH
OH
HN
(CH2)6CH3
In summary, we have successfully completed
a practical
H2N
HN
HPLC-free convergent total synthesis of chlorofusin (1) in this
work. The newly accomplished flexible synthesis enabled us to fur-
ther synthesize several unnatural Click hybrids starting from the
common azaphilone intermediates. Biological assessment revealed
that two chlorofusin-like mimics 33 and 34 having a 1,2,3-triazole
linkage exhibited improved inhibitory activities against p53–
HDM2 interactions over the natural product. Furthermore, the
developed methodology, especially that for the stereodivergent
synthesis of single enantiopure diastereomeric azaphilones, could
be potentially applicable to the total synthesis of other seven chro-
mophore diastereomers of chlorofusin, as well as more diverse
unnatural mimics for the development of novel p53–HDM2
antagonists.
b
O
OH
HN
O
HN
O
O
NH
O
HN
O
29
30
n = 1
n = 2
(CH2)6CH3
O
H2N
O
HN
N
N
N
N
H
O
HN
O
OH
N
n
O
HN
O
HO
NH
O
O
HO
NH
O
O
Cl
O
NH
O
O
O
O
HN
N
H
NH2
33
34 (n=3)
(n=1)
O
Acknowledgments
NH2
We thank Dr. Pingzhao Zhang (Fudan University) for kindly pro-
viding the HDM2 full length plasmid. Grants from the Ministry of
Science and Technology of the People’s Republic of China (Nos.
2010CB833202 and 2013AA092903) and the National Natural Sci-
ence Foundation of China (Nos. 21032002 and 21472087) are
greatly appreciated.
Scheme 5. Reagents and conditions: (a) propargyl bromide, K2CO3, DMF, 94%; (b)
29 or 30, CuSO4Á5H2O, NaAsc, t-BuOH/H2O (2:1), 65% for 33, 50% for 34. NaAsc = (+)-
sodium L-ascorbate.
Table 1
Inhibitory activity and affinity analysis for binding of chlorofusin and its mimics to
HDM2
Supplementary data
a
b
Compound
IC50
(
lM)
KD
(
lM)
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2014.0
9.028.
(4R,8S,9R)-chlorofusin (1)
(4S,8R,9S)-chlorofusin
(4S)-28a
(4R)-28b
33
20.0
23.1
51.3
21.1
16.9
7.6
45.9
97
NDc
NDc
24.5
50
References and notes
34
1. (a) Duncan, S. J.; Gruschow, S.; Williams, D. H.; McNicholas, C.; Purewal, R.;
Hajek, M.; Gerlitz, M.; Martin, S.; Wrigley, S. K.; Moore, M. J. Am. Chem. Soc.
2001, 123, 554–560; (b) Duncan, S. J.; Williams, D. H.; Ainsworth, M.; Martin,
S.; Ford, R.; Wrigley, S. K. Tetrahedron Lett. 2002, 43, 1075–1078; (c) Duncan, S.
J.; Cooper, M. A.; Williams, D. H. Chem. Commun. 2003, 316–317.
2. Desai, P.; Pfeiffer, S. S.; Boger, D. L. Org. Lett. 2003, 5, 5047–5050.
3. Malkinson, J. P.; Zloh, M.; Kadom, M.; Errington, R.; Smith, P. J.; Searcey, M. Org.
Lett. 2003, 5, 5051–5054.
a
IC50 concentration to inhibit the binding of wild-type p53 to HDM2 (11–124).
KD (affinity analysis) constants were measured by Biacore SPR (surface plasmon
b
resonance) system.
c
ND = not determined.
4. (a) Qian, W.-J.; Wei, W.-G.; Zhang, Y.-X.; Yao, Z.-J. J. Am. Chem. Soc. 2007, 129,
6400–6401; (The proposal on the absolute stereochemistry of the
chromophore of natural chlorofusin in Ref. 4a has been proven to be
incorrect, and a revision has been made by Refs. 5a,b.) (b) Wei, W.-G.; Yao,
Z.-J. J. Org. Chem. 2005, 70, 4585–4590; (c) Wei, W.-G.; Qian, W.-J.; Zhang, Y.-X.;
Yao, Z.-J. Tetrahedron Lett. 2006, 47, 4171–4174; (d) Wang, Y.-L.; Qian, W.-J.;
Wei, W.-G.; Zhang, Y.; Yao, Z.-J. Tetrahedron 2010, 66, 3427–3432.
5. (a) Lee, S. Y.; Clark, R. C.; Boger, D. L. J. Am. Chem. Soc. 2007, 129, 9860–9861; (b)
Clark, R. C.; Lee, S. Y.; Boger, D. L. J. Am. Chem. Soc. 2008, 130, 12355–12369; (c)
Clark, R. C.; Lee, S. Y.; Searcey, M.; Boger, D. L. Nat. Prod. Rep. 2009, 26, 465–477.
6. Clark, R. C.; Lee, S. Y.; Hwang, I.; Searcey, M.; Boger, D. L. Tetrahedron Lett. 2009,
50, 3151–3153.
The synthetic samples of natural chlorofusin (1) and our previ-
ously synthesized (4S,8R,9S)-chlorofusin,4a aminal-deleted deriva-
tives 28a and 28b, and Click hybrids 33 and 34 were then
evaluated by ELISA,15 together with several synthetic intermedi-
ates (Table 1). The results showed that the stereochemistry of
the chromophore plays an inappreciable role in inhibitory activity
(IC50 20.0 lM for natural chlorofusin, and IC50 23.1 lM for
(4S,8R,9S)-chlorofusin), similar to previous observation by Boger’s
group.6 Interestingly, removal of spiro-aminal functionality does
not significantly affect the inhibitory activity (compounds 28a
and 28b, two times difference) either. It is noteworthy that the
activity of the chromophore-linear peptide precursor 23 (IC50
7. (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004–
2021; (b) Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128–1137;
(c) Bock, V. D.; Hiemstra, H.; van Maarseveen, J. H. Eur. J. Org. Chem. 2005, 51–
68; (d) Lutz, J.-F. Angew. Chem., Int. Ed. 2007, 46, 1018–1025.
8. (a) Stark, L. M.; Pekari, K.; Sorensen, E. J. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
12064–12066; (b) Zhu, J. L.; Grigoriadis, N. P.; Lee, J. P.; Porco, J. A., Jr. J. Am.
Chem. Soc. 2005, 127, 9342–9343; (c) Zhu, J.; Porco, J. A., Jr. Org. Lett. 2006, 8,
5169–5171; (d) Germain, A. R.; Bruggemeyer, D. M.; Zhu, J. L.; Genet, C.;
O’Brien, P.; Porco, J. A., Jr. J. Org. Chem. 2011, 76, 2577–2584.
9. (a) Wei, W.-G.; Yao, Z.-J. Tetrahedron 2003, 59, 6621–6625; (b) Wei, W.-G.;
Zhang, Y.-X.; Yao, Z.-J. Tetrahedron 2005, 61, 11882–11886.
10. Powell, A. D. G.; Robertson, A.; Whalley, W. B. Chem. Soc. Spec. Publ. 1956,
5, 27.
329
(IC50 >1000
(IC50 804 M) and 30 (IC50 897
mation of the macrocycle might be essential for competitively
docking to a preferred position upon human MDM2 (HDM2) pro-
tein.16 More importantly, the two Click mimics (compounds 33
and 34) show up to nearly 3 times improvement of the inhibitory
l
M) has a promotion compared to the chromophores 19
M) and 32 (IC50 997 M) or the cyclic peptides 29
M) alone. We speculated that for-
l
l
l
l

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6059
11. The absolute stereochemistry of the C-4 carbon of single diastereomers was
determined by the circular dichroism (CD) spectroscopy (see the Supporting
information for details).
12. Synthesis of octapeptide 22 was accomplished utilizing a [2+3+3]-fragment
coupling strategy (See the Supporting information for details).
13. Woon, E. C. Y.; Arcieri, M.; Wilderspin, A. F.; Malkinson, J. P.; Searcey, M. J. Org.
Chem. 2007, 72, 5146–5151.
14. See the Supporting information for details of the synthesis of two azido-
containing amino acid derivatives 29 and 30.
15. Sakurai, K.; Chung, H. S.; Kahne, D. J. Am. Chem. Soc. 2004, 126, 16288–16289.
16. Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.; Levine, A. J.;
Pavletich, N. P. Science 1996, 274, 948–953.