Solid-phase synthesis of chlorofusin analogues

Article abstract of DOI:10.1021/jo070450a

(Chemical Equation Presented) We report an efficient and versatile solid-phase synthesis through which two series of chlorofusin analogues, one bearing varying chromophores and the other with various amino acid substitutions in the cyclic peptide, were synthesized. These peptides were prepared using a strategy involving side-chain immobilization, on-resin cyclization, and postcyclization modification. The success of these syntheses demonstrates the broad utility of the method. Both series of analogues were evaluated for their inhibitory activity against the p53/MDM2 interaction but were shown to be inactive in the concentration range tested. This suggests that the full chromophore structure may be required for activity.

Full text of DOI:10.1021/jo070450a

Solid-Phase Synthesis of Chlorofusin Analogues
Esther C. Y. Woon,† Mariangela Arcieri, Andrew F. Wilderspin, John P. Malkinson, and
Mark Searcey*,†
Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, UniVersity of London,
29-39 Brunswick Square, London WC1N 1AX
m.searcey@uea.ac.uk
ReceiVed March 5, 2007
We report an efficient and versatile solid-phase synthesis through which two series of chlorofusin analogues,
one bearing varying chromophores and the other with various amino acid substitutions in the cyclic
peptide, were synthesized. These peptides were prepared using a strategy involving side-chain
immobilization, on-resin cyclization, and postcyclization modification. The success of these syntheses
demonstrates the broad utility of the method. Both series of analogues were evaluated for their inhibitory
activity against the p53/MDM2 interaction but were shown to be inactive in the concentration range
tested. This suggests that the full chromophore structure may be required for activity.
Introduction
p53/MDM2 inhibitors initially focused on short peptides
mimicking the primary structure of p5310-12 in order to validate
the target and identify key interactions between the two proteins.
More recently, small molecules, such as derivatives of chal-
cones,13 benzodiazepinediones,14 and particularly cis-imidazo-
lines15 have moved toward demonstrating clinical utility as
inhibitors of this key protein-protein interaction.
The tumor-suppressor protein p53 plays a crucial role in the
development of cancer,1-3 with around 50% of all tumors
containing a mutated, inactivated form.4 However in about 7%
of tumors where wild type p53 is retained, its function is
compromised by an overexpression of MDM2 (its main negative
regulator).5 An inhibition of p53/MDM2 binding in these tumor
cells would reactivate the p53 pathway, leading to cell-cycle
arrest or apoptosis, hence providing a promising strategy for
anticancer chemotherapy.6-9 Research efforts in the quest for
(8) Wasylyk, C.; Salvi, R.; Argentini, M.; Dureuil, C.; Delumeau, I.;
Abecassis, J.; Debusche, L.; Wasylyk, B. Oncogene 1999, 18, 1921-1934.
(9) Che`ne, P.; Fuchs, J.; Bohn, J.; Garcia-Echeverria, C.; Furet, P.;
Fabbro, D. J. Mol. Biol. 2000, 299, 245-253.
(10) Garc´ıa-Echeverr´ıa, C.; Che`ne, P.; Blommers, M. J. J.; Furet, P. J.
Med. Chem. 2000, 43, 3205-3208.
* Author to whom correspondence should be addressed. Tel +44-1603592026,
fax +44-1603592003.
(11) Sakurai, K.; Chung, H. S.; Kahne, D. J. Am. Chem. Soc. 2004, 126,
16288-16289.
† Current address: School of Chemical Sciences & Pharmacy, University of
East Anglia, Norwich NR4 7TJ.
(12) Bo¨ttger, V.; Bo¨ttger, A.; Howard, S. F.; Picksley, S. M.; Che`ne, P.;
Garc´ıa-Echeverr´ıa, C.; Hochkeppel, H. K.; Lane, D. P. Oncogene 1996, 2,
2141-2147.
(1) Vogelstein, B.; Lane, D.; Levine, A. J. Nature 2000, 408, 307-310.
(2) Levine, A. J. Cell 1997, 88, 323-331.
(3) Vousden, K. H.; Lu, X. Nat. ReV. Cancer 2002, 2, 594-604.
(4) Hainaut, P.; Hollstein, M. AdV. Cancer Res. 2000, 77, 81-137.
(5) Mom, J.; Jung, D.; Wilczynski, S.; Niland, J. Nucleic Acids Res. 1998,
26, 3453-3459.
(6) Mendrysa, S. M.; McElwee, M. K.; Michalowski, J.; O’Leary, K.
A.; Young, K. M.; Perry, M. E. Mol. Cell Biol. 2003, 23, 462-472.
(7) Bo¨ttger, A.; Bo¨ttger, V.; Sparks, A.; Liu, W. L.; Howard, S. F.; Lane,
D. P. Curr. Biol. 1997, 7, 860-869.
(13) Stoll, R.; Renner, C.; Hansen, S.; Palme, S.; Klein, C.; Belling, A.;
Zeslawski, W.; Kamionka, M.; Rehm, T.; Muhlhahn, P.; Schumacher, R.;
Hesse, F.; Kaluza, B.; Voelter, W.; Engh, R. A.; Holak, T. A. Biochemistry
2001, 40, 336-344.
(14) Parks, D. J.; LaFrance, L. V.; Calvo, R. R.; Milkiewicz, K. L.; Gupta,
V.; Lattanze, J.; Ramachandren, K.; Carver, T. E.; Petrella, E. C.; Cummings,
M. D.; Maguire, D.; Grasberger, B. L.; Lu, T. Bioorg. Med. Chem. Lett.
2005, 15, 765-770.
10.1021/jo070450a CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/09/2007
5146
J. Org. Chem. 2007, 72, 5146-5151

Inhibitors of p53/MDM2 Interaction
FIGURE 1. The structure and relative stereochemistry of chlorofusin.16
Chlorofusin is the first natural product to be identified as an
inhibitor of the p53/MDM2 interaction.16 It is a secondary
metabolite isolated from the fermentation of Microdochium
caespitosum, with an IC50 for the inhibition of MDM2 binding
to p53 of 4.6 µM. To date, its molecular structure remains
largely unexploited for drug discovery, and very little is known
about its actual binding interaction with MDM2, other than the
fact that it binds to the N-terminus.17 In order to study the
structure-activity relationship between chlorofusin and MDM2,
there is a requirement for the development of versatile synthetic
methods for the generation of a library of chlorofusin analogues.
The synthesis of the chlorofusin peptide was disclosed simul-
taneously by two groups in 2003,18,19 using both solution and
solid-phase methods to verify the natural product structure.
Herein, we describe the synthesis of a number of exemplar
chlorofusin analogues containing either variations in the orni-
thine side-chain or “point mutations” in the peptide ring, using
the side-chain immobilization methodology, and demonstrate
the synthetic utility of this approach as a general route to
chlorofusin analogues.
FIGURE 2. The structures of proposed analogues 2a-c.
Analogue Design
Based on current spectroscopic data,16 the chemical structure
and relative stereochemistry of chlorofusin is believed to consist
of two main components: a highly functionalized azaphilone
chromophore and a cyclic peptide composed of nine amino acid
residues 1. The nitrogen atom bonded to the δ-carbon of the
side-chain of Orn9 is directly incorporated into the bicyclic core
of the chromophore, linking the two components together
(Figure 1). The structure of the cyclic peptide was established
by synthesis using both solid- and solution-phase methods and
demonstrated S and R absolute stereochemistry for Asn3 and
Asn4, respectively.18,19 The solid-phase route involved side-
chain immobilization of a protected aspartic acid Via a Rink
amide linker that would generate the â-carboxamide of Asn3
FIGURE 3. The structures of proposed analogues 3a-e.
on release from the resin. This approach to the synthesis is
amenable to the rapid parallel synthesis of analogues of the
cyclic peptide containing single point mutations. Studies of
peptides analogous to the N-terminal domain of the p53 protein
established that p53 binds to MDM2 with an R-helical confor-
mation, forming three critical contacts through the side-chains
of Phe19, Trp23, and Leu26.20 Recent studies of â-hairpin
constructs demonstrated that cyclodecapeptides could be con-
structed based upon these observations with extremely high
binding affinities for MDM2.21 We suspected that Ala2, Orn9,
and D-Ade8 of the chlorofusin peptide may interact in a similar
manner with the p53 binding site on MDM2. In order to test
the utility of the solid-phase synthesis, a small subset of
compounds containing modifications at Orn9 and Ala2 (2a-c,
Figure 2) were synthesized.
(15) Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.;
Filipovic, Z.; Kong, N.; Kammlott, U.; Lukacs, C.; Klein, C.; Fotouhi, N.
R.; Liu, E. A. Science 2004, 303, 844-848.
(16) Duncan, S. J.; Gru¨schow, 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.
To explore the role of the chromophore in p53/MDM2
binding, analogues of the structure 3a-e (Figure 3) were
(17) Duncan, S. J.; Cooper, M. A.; Williams, D. H. Chem. Commun.
2003, 316-317.
(18) Pankaj, D.; Pfeiffer, S. S.; Boger, D. L. Org. Lett. 2003, 5, 5047-
5050.
(20) Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.;
Levine, A. J.; Pavletich, N. P. Science 1996, 274, 948-953.
(21) Fasan, R.; Dias, R. L. A.; Moehle, K.; Zerbe, O.; Vrijbloed, J. W.;
Obrecht, D.; Robinson, J. A. Angew. Chem., Int. Ed. 2004, 43, 2109-2112.
(19) Malkinson, J. P.; Zloh, M.; Kadom, M.; Errington, R.; Smith, P. J.;
Searcey, M. Org. Lett. 2003, 5, 5051-5054.
J. Org. Chem, Vol. 72, No. 14, 2007 5147

Woon et al.
SCHEME 1. Synthesis of 1 and Analogues 2a-c
proposed. In this design, the chromophore is replaced with a
simplified substituted benzamide ring, which serves to mimic
any possible interactions involving the 4-, 5-, 6-, and 7-positions
of the azaphilone ring with MDM2, while retaining the planar
conformation of the cyclohexane-1,3-dione ring. With an
orthogonal Mtt protecting group on the ornithine side-chain, it
is possible to carry out the synthesis entirely on solid phase
and deprotect and react selectively at this position prior to
cleavage from the resin. This route also has potential in parallel
synthesis.
Fmoc-based chain extension (i.e., incorporating Fmoc-Ala-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Orn(Boc)-OH, Fmoc-D-Ade-OH,
Fmoc-D-Leu-OH, Fmoc-Thr(tBu)-OH, Fmoc-D-Leu-OH and
then Fmoc-D-Asn(Trt)-OH employing cycles of Fmoc depro-
tection with 20% v/v piperidine in DMF and HBTU/HOBt/
DIPEA-mediated coupling). The completion of each coupling
cycle was monitored after 30 min of agitation by means of the
Kaiser test.22 The linear peptide chain 5 thus assembled was
then subjected to intramolecular head-to-tail cyclization on-resin
to furnish the macrocycle 6. This was achieved by first
deprotecting the NR-Fmoc protecting group of Asn4 and the
R-carboxyl Dmab protecting group of Asn3 using 20% piperi-
dine in DMF and 2% hydrazine monohydrate in DMF,
respectively, followed by coupling with a 5-fold excess of DIC
and HOBt in DMF. Subsequent treatment of the resin with 95%
TFA led to global deprotection and cleavage of the peptide from
the solid support. The crude product was then dissolved in 50%
acetic acid and lyophilized before purification by semiprepara-
tive reverse-phase HPLC using a gradient of acetonitrile in water
to furnish cyclic peptide 1 in 27% yield. The above solid-phase
strategy was then applied to the synthesis of analogues 2a-c
(Scheme 1). These targets have specific point mutations applied
Results and Discussion
The synthesis of cyclic peptides posed a synthetic challenge
as a much-strained conformation is imposed on a linear peptide
through cyclization. Our lab has recently developed an innova-
tive side-chain immobilization solid-phase strategy for the
synthesis of the chlorofusin peptide 1 (Scheme 1, Route A).19
Essentially, the synthesis involved three key steps: (1) attach-
ment to the solid support Via the side-chain â-carboxyl of Fmoc-
Asp-ODmab, (2) linear chain formation, and (3) on-resin head-
to-tail cyclization through amide bond formation between the
R-carboxyl group of Asn3 and the R-amino group of D-Asn4.
To initiate the synthesis, NR-Fmoc-Asp-ODmab was im-
mobilized onto a Rink amide MBHA resin Via its carboxylic
acid side-chain using HBTU/HOBt activation in the presence
of DIPEA in DMF. Selective NR-Fmoc deprotection of the resin-
bound aspartic acid residue 4 was followed by standard NR-
(22) Kaiser, E. T.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 84, 595-598.
(23) Wu¨thrich, K. NMR of the Proteins and Nucleic Acids; Wiley: New
York, 1986.
(24) Koppitz, M.; Huenges, M.; Gratias, R.; Kessler, H. HelV. Chim.
Acta 1997, 80, 1280-1300.
5148 J. Org. Chem., Vol. 72, No. 14, 2007

Inhibitors of p53/MDM2 Interaction
Accordingly, incorporating the chromophore on-resin should
circumvent the problem of side -chain interactions as they all
remain protected. In addition, the peptide, being still attached
to the hydrophobic solid support, will presumably be prevented
from assuming the conformation required for hydrogen-bond
formation with Orn9. Hence, the original strategy used for the
synthesis of 1 was modified to incorporate an on-resin,
postcyclization coupling step. The principal advantage of this
approach is that the entire synthesis is completed on-resin,
without the need for further reaction in solution and necessitating
only a single purification by preparative HPLC. A potential
drawback, however, is that any resin-bound reactive or nucleo-
philic byproduct remaining after cyclization may also be
acylated, resulting in a more complex mixture of similar (cyclic)
peptides that may be more difficult to remove during purifica-
tion. The attachment of the chromophore was intentionally
carried out after cyclization (as opposed to prior to), as we
desired to use intermediate 9 as a template through which a
library of chlorofusin analogues could be rapidly assembled.
Hence analogues 3a-e were prepared by first immobilizing
Fmoc-Asp-ODmab on Rink amide resin (Scheme 2). The linear
peptide was then assembled as previously, incorporating Fmoc-
Orn(Mtt)-OH instead of Fmoc-Orn(Boc)-OH. The Mtt group
(4-methyltrityl) was chosen as it can be removed selectively
and rapidly by very mild acidolysis using 1% TFA in DCM,
leaving the peptide-resin linkage and the rest of the protecting
groups, namely OtBu and CONHTrt, unaffected. After on-resin
cyclization, orthogonal deprotection of Mtt and selective
coupling of the resulting free amino group with benzoic acids
10a-e, the peptides were released from the solid support with
simultaneous side-chain deprotection. Purification of the crude
products with semipreparative reverse-phase HPLC gave the
corresponding target compounds 3a-e in 14-27% overall yield.
In all cases, the desired compounds were the major products,
hence demonstrating the high efficiency and utility of the
synthesis.
FIGURE 4. The solution structure of chlorofusin peptide 1 determined
by NMR.19 This view shows the folding of Orn side-chain into the
hydrophilic core, forming two intramolecular hydrogen bonds: one with
the hydroxyl group of Thr6 and the other with the carbonyl oxygen of
Ala2.
to the cyclic peptide 1 to mimic the binding interactions between
p53 and MDM2. Their preparations were very similar to that
of 1, and they took advantage of the on-resin cyclization
technique which provided a pseudo-dilution effect that helped
to minimize (cyclo)oligomerization. It also allowed the use of
excess reagents which is important in driving the lactam ring
formation toward completion, improving both the rate of reaction
and the yield. The appropriate sequence modifications were
made during the assembly of the linear peptides, replacing either
Ala2 with Phe2 (2a) or Orn9 with Trp9 (2b), or making both
substitutions simultaneously (2c). All three syntheses proceeded
smoothly to give the desired compounds 2a-c in good yield
(13-24%), with >95% purity.
1
Complete H NMR resonance assignments for all peptides
were determined Via standard homonuclear and hetero-
nuclear two-dimensional NMR techniques23 (Tables 1-3, Sup-
porting Information). Their chemical shifts compare well with
that of chlorofusin and previously reported spectra of 1. Their
mass spectral analyses and HPLC retention times are also
reported.
In adapting the same tactic to the synthesis of analogues
3a-e, two approaches were considered. First we examined the
possibility of coupling 1 directly with the chromophore using
solution synthesis. The advantage of this method is that it
involves using relatively mild reaction conditions compared to
those employed in Fmoc-solid-phase chemistry, in particular
the basic and strongly acidic conditions required for Fmoc-
deprotection and peptide release respectively. The latter would
not be suitable for chromophores with acid- or base-sensitive
functional groups. To investigate this method, 1 was treated
with 3-chlorobenzoic acid using various coupling conditions,
such as stirring in acetonitrile at room-temperature both in the
presence and absence of CSA, microwave irradiation at various
temperatures in DMF, or employing a mixture of solvents.
However, all attempts were unsuccessful (data not shown).
Apparently, factors such as the presence of unprotected side
chains could have resulted in the failure of the reaction. Careful
examination of the solution structure of the chlorofusin peptide
1,19 previously determined using NMR spectroscopy, also
suggests the possibility of the Orn9 side-chain folding into the
hydrophilic core, forming two putative intramolecular hydrogen-
bonds with the hydroxyl group of Thr6 (2.77 Å) and the
carbonyl of Ala2 (1.75 Å) (Figure 4). This could potentially
reduce the availability of its amino group for reaction, thus
further hindering the reaction.
Biological Results. Preliminary biological evaluation of these
analogues was conducted through an ELISA competition assay
adapted from the method described by Kahne et al.11 It in-
volved a measurement of the ability of various analogues to
disrupt the interaction between HDM2 (17-125) and biotiny-
lated SGSG-p53 peptide (17-27), which was immobilized on
a streptavidin-coated 96-well plate. The experimental pro-
cedure for the assay is given in Supporting Information. The
control p53 peptide (SQETFSDLWKLLPEN) produces an
IC50 value of 4.16 µM in this assay, comparing well with
literature values. All the compounds tested had little or no
p53/MDM2 inhibitory activity, with IC50 values outside the
solubility range in this assay. Chlorofusin has been reported to
have an IC50 of 4.6 µM in a similar assay. The overall reduction
in potency compared to the parent compound (chlorofusin)
suggests that interactions involving regions other than those
replicated by the benzamide core, in particular, the cyclic ketal
and the 3-hydroxyl group of the azaphilone are likely to be
important for potency.
J. Org. Chem, Vol. 72, No. 14, 2007 5149

Woon et al.
SCHEME 2. Synthesis of Analogues 3a-e
(860 mg, 0.46 mmol). The loaded resin was then split into four
equal portions (200 mg, 0.11 mmol) and transferred into separate
vessels A, B, C, and D for the assembly of 1, 2a, 2b, and 2c,
respectively.
Conclusions
We describe, herein, an efficient and versatile solid-phase
strategy through which a series of chlorofusin analogues, bearing
various cyclic peptides 2 and varying chromophores 3, were
generated. These syntheses demonstrate the broad utility of the
side-chain immobilization strategy previously devised for the
formation of the chlorofusin peptide. We have also reported on
the in Vitro activities of these analogues on p53/MDM2
interaction. It appears that both the cyclic peptide and the full
chromophore structure are essential for p53/MDM2 inhibitory
activity.
Each portion was allowed to swell in DMF for 30 min and was
then thoroughly washed with DMF (4 × 20 mL). Following NR-
Fmoc deprotection, the resin portions in vessels B and D were
coupled with Fmoc-Phe-OH (213 mg, 0.55 mmol, 5 equiv), while
those in vessels A and C were coupled with Fmoc-Ala-OH
(171 mg, 0.55 mmol, 5 equiv). All four were then deprotected and
coupled with Fmoc-Thr(tBu)-OH (219 mg, 0.55 mmol, 5 equiv),
after which chain extension was continued for A and B using Fmoc-
Orn(Boc)-OH (250 mg, 0.55 mmol, 5 equiv), while C and D using
Fmoc-Trp(Boc)-OH (290 mg, 0.55 mmol, 5 equiv). Subsequent
couplings were the same for all four and were performed in this
order: Fmoc-D-Ade-OH (225 mg, 1.65 mmol, 5 equiv), Fmoc-D-
Leu-OH (194 mg, 0.55 mmol, 5 equiv), Fmoc-Thr(tBu)-OH
(219 mg, 0.55 mmol, 5 equiv), Fmoc-D-Leu-OH (194 mg,
0.55 mmol, 5 equiv), and then Fmoc-D-Asn(Trt)-OH (328 mg,
0.55 mmol, 5 equiv). All couplings were carried out in the presence
of HBTU (204 mg, 0.54 mmol, 4.9 equiv), HOBt (84 mg, 0.55
mmol, 5 equiv), and DIPEA (142 mg, 1.10 mmol, 10 equiv). Prior
to cyclization, the NR-Fmoc group of the terminal amino acid (Asn4)
was removed with 20% v/v piperidine in DMF (2 × 10 min). This
was followed by the removal of the R-carboxyl Dmab protection
group of Asn3, using 2% hydrazine monohydrate in DMF (3 ×
5 min). The resin was then washed with 5% DIPEA in DMF (4 ×
20 mL) and treated with DIC (69 mg, 0.55 mmol, 5 equiv) and
HOBt (84 mg, 0.55 mmol, 5 equiv) in a minimal volume of DMF
(2 × 24 h). The resin was then washed thoroughly with DMF,
DCM, and then MeOH/DCM (1:1) and dried in Vacuo over KOH
to constant weight.
Experimental Section
Chlorofusin Peptide 1 and Analogues 2a-c. Rink amide
MBHA resin (700 mg, 0.46 mmol, substitution ) 0.66 mmol/g)
was suspended in DMF (15 mL) in a peptide synthesis vessel. The
vessel was gently agitated to remove air bubbles, and the resin
was allowed to swell for 30 min. Vacuum was then applied to
remove DMF, and the NR-Fmoc group was removed by treatment
with 20% v/v piperidine in DMF (2 × 10 min). The resin was then
thoroughly washed with DMF (4 × 20 mL) and treated with a DMF
solution of Fmoc-Asp-ODmab (1.54 g, 2.31 mmol, 5 equiv), the
latter being activated just before addition with HBTU (859 mg,
2.26 mmol, 4.9 equiv), HOBt (353 mg, 2.31 mmol, 5 equiv), and
DIPEA (596 mg, 4.62 mmol, 10 equiv). After 30 min of agita-
tion at room temperature, a small sample of resin was analyzed
for free amines using the Kaiser test. Upon completion of the
coupling reaction, as indicated by a negative color test, the resin
was thoroughly washed with DMF, DCM, and then MeOH/
DCM (1:1), and dried in Vacuo over KOH to a constant weight
5150 J. Org. Chem., Vol. 72, No. 14, 2007

Inhibitors of p53/MDM2 Interaction
Simultaneous side-chain deprotection and release of the peptides
from the solid support was achieved by stirring with a cleavage
mixture containing TFA/H2O/TIPS (95:2.5:2.5), 10-25 mL/g resin
at room temperature for 3 h, after which the TFA was removed
under reduced pressure. The crude peptides were precipitated and
washed with cold anhydrous Et2O, extracted into 50% acetic acid,
and lyophilized. Purification of the crude peptides 3a-e was carried
out using semipreparative reverse-phase HPLC performed on a
Waters Prep LC C18 column (100 mm × 25 mm). Separation was
achieved using a linear gradient of solvent A (water + 0.02% TFA)
and solvent B (90% MeCN + 9.98% water + 0.02% TFA), eluting
at a flow rate of 5 mL/min and monitoring at 214 nm: 0% B to
40% B over 60 min and then 40% B to 60% B over 60 min. In all
cases, a purity of >95% was obtained as determined by analytical
reverse-phase HPLC, which was performed on a Vydac C4 Protein
column (250 mm × 4.6 mm), eluting at a flow rate of 1.2 mL/min
using a linear gradient of 0% B to 60% B over 120 min.
1 (15 mg, 27% yield): MALDI-TOF MS m/z: 1011.4 [M +
H]+ (calcd for C46H82N12O13, 1010.6124); HPLC retention time )
18.583 min.
The dried resin was then split into five equal portions and
transferred into separate peptide synthesis vessels. Each portion was
allowed to swell in anhydrous DCM for 30 min and thoroughly
washed with anhydrous DCM (5 × 20 mL). A mixture of TFA/
DCM/TIPS (1:94:5) was then added to selectively remove the Mtt
protecting group from Orn9. After standing for 2 min, the mixture
was removed Via vacuum and this step was repeated a few times
until a colorless solution was obtained, indicating the absence of
remaining Mtt protecting groups (a pronounced yellow color on
addition of the 1% TFA mixture indicates the liberation of trityl
cations). The resin portions were then separately coupled with 5
equiv of one of the following benzoic acids: 3-chlorobenzoic acid,
3-chloro-4-methoxybenzoic acid, 5-chloro-2-methoxybenzoic acid,
2,4-dimethoxybenzoic acid, and 3-hydroxybenzoic acid to give
resin-bound peptides 11a, 11b, 11c, 11d, and 11e, respectively.
The benzoic acids were activated with HBTU (4.9 equiv), HOBt
(5 equiv), and DIPEA (10 equiv) just before addition. The resin-
bound peptides 11 were then washed, dried, removed from the solid
support (with simultaneous side-chain deprotection), and purified
as described by HPLC (0% B to 100% B over 20 min, and then
100% B for 5 min, and then 100% B to 0% B over 7 min).
2a (15 mg, 13% yield): MALDI-TOF MS m/z: 1087.9 [M +
H]+ (calcd for C52H86N12O13, 1086.6437); HPLC retention time )
19.400 min.
3a (14 mg, 22% yield): MALDI-TOF MS m/z: 1171.8 [M +
Na]+ (calcd for C53H85ClN12O14, 1148.5997); HPLC retention time
) 30.150 min.
3b (10 mg, 15% yield): MALDI-TOF MS m/z: 1202.0 [M +
Na]+ (calcd for C54H87ClN12O15, 1178.6102); HPLC retention time
) 30.067 min.
2b (19 mg, 16% yield): ES+ MS m/z: 1105.7 [M + Na]+ (calcd
for C52H82N12O13, 1082.6124); HPLC retention time ) 22.183 min.
2c (31 mg, 24% yield): ES+ MS m/z: 1181.7 [M + Na]+ (calcd
for C58H86N12O13, 1158.6437); HPLC retention time ) 23.733 min.
1
For H NMR data, see Table 1 and 3, Supporting Information.
3c (11 mg, 17% yield); MALDI-TOF MS m/z: 1201.6 [M +
Na]+ (calcd for C54H87ClN12O15, 1178.6102); HPLC retention time
) 30.500 min.
3d (9 mg, 14% yield): MALDI-TOF MS m/z: 1175.7 [M +
H]+, 1197.7 [M + Na]+ (calcd for C55H90N12O16, 1174.6598):
HPLC retention time ) 29.450 min.
3e (11 mg, 18% yield): MALDI-TOF MS m/z: 1154.3 [M +
Na]+ (calcd for C53H86N12O15, 1130.6336); HPLC retention time
) 27.750 min.
For 1H NMR data, see Tables 1 and 2, Supporting Information.
Chlorofusin Analogues 3a-e. Fmoc-Asp-ODmab (1.10 g,
1.65mmol, 5 equiv) was immobilized onto a Rink amide MBHA
resin (500 mg, 0.33 mmol, substitution ) 0.66 mmol/g) as described
above. Upon completion of the coupling reaction, as indicated by
a negative color test, the resin was thoroughly washed with DMF
(4 × 20 mL) and the above coupling procedure was repeated with
the following amino acids, in this order: Fmoc-Ala-OH (514 mg,
1.65 mmol, 5 equiv), Fmoc-Thr(tBu)-OH (656 mg, 1.65 mmol,
5 equiv), Fmoc-Orn(Mtt)-OH (1.01 g, 1.65 mmol, 5 equiv), Fmoc-
D-Ade-OH (675 mg, 1.65 mmol, 5 equiv), Fmoc-D-Leu-OH
(583 mg, 1.65 mmol, 5 equiv), Fmoc-Thr(tBu)-OH (656 mg, 1.65
mmol, 5 equiv), Fmoc-D-Leu-OH (583 mg, 1.65 mmol, 5 equiv),
and then Fmoc-D-Asn(Trt)-OH (985 mg, 1.65 mmol, 5 equiv). Prior
to cyclization, the NR-Fmoc group of the terminal amino acid (Asn4)
was removed with 20% v/v piperidine in DMF (2 × 10 min). This
was followed by the removal of the R-carboxyl Dmab protecting
group of Asn3 using 2% v/v hydrazine monohydrate in DMF (3 ×
5 min). The resin was then washed with 5% DIPEA in DMF (4 ×
20 mL) and treated with DIC (208 mg, 1.65 mmol, 5 equiv) and
HOBt (252 mg, 1.65 mmol, 5 equiv) in a minimal volume of DMF
(2 × 24 h). After cyclization, the resin was washed thoroughly
with DMF, DCM, and then MeOH/DCM (1:1) and dried in Vacuo
over KOH to constant weight.
Acknowledgment. We thank Professor J. A. Robinson
(Institute of Organic Chemistry, University of Zu¨rich) for
providing the clone for the production of HDM2(17-125), and
we acknowledge the Association for International Cancer
Research (04-394) for funding.
Supporting Information Available: The NMR data and
experimental procedure for the biological assay. This material is
available free of charge Via the Internet at http://pubs.acs.org.
JO070450A
J. Org. Chem, Vol. 72, No. 14, 2007 5151