N-terminus 4-chloromethyl thiazole peptide as a macrocyclization tool in the synthesis of cyclic peptides: Application to the synthesis of conformationally constrained RGD-containing integrin ligands

Article abstract of DOI:10.1016/j.tetlet.2010.12.043

The synthesis of conformationally constrained RGD-containing integrin ligands via an efficient solid-phase intramolecular thioalkylation reaction is described. The reaction of S-nucleophiles with newly generated N-terminal 4-chloromethyl thiazoles leads t

Full text of DOI:10.1016/j.tetlet.2010.12.043

Tetrahedron Letters 52 (2011) 817–819
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
N-terminus 4-chloromethyl thiazole peptide as a macrocyclization tool in
the synthesis of cyclic peptides: application to the synthesis of
conformationally constrained RGD-containing integrin ligands
⇑
Adel Nefzi , Jason E. Fenwick
Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port Saint Lucie, FL 34987, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of conformationally constrained RGD-containing integrin ligands via an efficient solid-
phase intramolecular thioalkylation reaction is described. The reaction of S-nucleophiles with newly gen-
erated N-terminal 4-chloromethyl thiazoles leads to the desired cyclic RGD products 5 in high purities
and good overall yields.
Received 4 November 2010
Revised 7 December 2010
Accepted 9 December 2010
Available online 15 December 2010
Ó 2010 Elsevier Ltd. All rights reserved.
The RGD sequence is a well-known binding motif for specific
transmembrane proteins and is involved in cellular adhesion to
the extracellular matrix (ECM).1 There is a continuous interest in
the development of RGD-based therapeutics that function either
as agonists to promote the interaction of cells and tissues with arti-
ficial matrices or as antagonists to control the nature of cell–cell
and cell–ECM interactions. Many reviews have reported general
and specific topics related to the field of integrin.2
Of particular interest, thioalkylation reactions offer a facile and
versatile approach to the solid-phase synthesis of cyclic pep-
tides.16,17 Examples of macrocyclizations via thioalkylation include
the reaction of the thiol group of a C-terminal cysteine with an
N-terminal acetyl bromide or N-terminal benzyl bromide.16,17
A
conceptually different approach, wherein thioalkylation proceeds
via Michael addition of a thiolate anion to an a,b-unsaturated ester,
has been reported for the synthesis of cyclic thioether dipeptides,18
and more recently thioalkylation using thiol–ene click chemistry
was applied for the generation of RGD analogs.19
Cyclic RGD peptides have been developed for various applica-
tions including fibrinogen receptor antagonists,3 selective
aVb3
integrin antagonists for the treatment of human tumor metastasis
and tumor-induced angiogenesis,4 phagocytosis of cells
undergoing apoptosis, bone remodeling, and osteoporosis, diabetic
retinopathy, tumor imaging and targeting2a, and acute renal fail-
ure.4 RGD-containing cyclic peptides were used to target tetraphe-
nylchlorin as novel photosensitizers for selective photodynamic
activity.5 The cyclization of RGD-containing peptides restricts the
flexibility of peptides and, therefore, offers the possibility to
present the RGD sequence in a specific conformation to provide en-
hanced selectivity of the ligands toward the receptors. Further-
more cyclic peptides are often more stable to peptidases, and
therefore they can have improved pharmacokinetic profiles and
represent promising lead compounds for further development.6
Reported approaches on the solid-phase synthesis of RGD-con-
taining cyclic peptides include intramolecular Heck reaction.7
Nucleophilic substitution,8 intramolecular amide formation,9
disulfide formation,10 intramolecular Suzuki reactions,11 SNAr dis-
placement reactions involving haloaryl electrophiles,12 intramolec-
ular urea bond formation,13 ring closing metathesis reaction,14 and
more recently click chemistry.15
Due to the potential clinical advantages of cyclic peptides, and
the importance of the RGD motif for integrin-recognition, there
remains an unmet need to develop rapid and efficient synthetic
strategies for the combinatorial generation of RGD-containing
cyclic peptides. Herein, we describe an efficient approach toward
the generation of a new class of cyclic RGD peptides by an intramo-
lecular thioalkylation reaction by the halogen displacement of
N-terminus 4-chloro methyl thiazole peptide with the thiol group
of cysteine.
As outlined in Scheme 1, we performed the parallel synthesis of
different thiazolyl containing RGD cyclic peptides. Starting from
p-methylbenzhydrylamine hydrochloride (MBHAÁHCl) resin-
bound orthogonally protected Fmoc-Cys-(Trt)-OH 1, the thio-
methyl thiazolyl macrocyclic peptidomimetics 5 were synthesized
following stepwise Fmoc deprotection and standard repetitive
Fmoc-amino-acid couplings20 yielding the linear RGD peptide 2.
The resulting N-terminal free amine was treated with Fmoc-isothi-
ocyanate. Following Fmoc deprotection, the thiourea was treated
with 1,3-dichloroacetone (Hantzsch’s cyclocondensation21) to af-
ford the resulting resin-bound chloromethyl thiazolyl peptide 4.
The Trt group was removed with 5% TFA in DCM and the resin-
bound peptide was treated with a solution of Cs2CO3 in DMF to
effect an SN2 intramolecular thioalkylation reaction. The resin
was treated with HF/anisole and the desired thiazolyl thioether
⇑
Corresponding author.
E-mail address: adeln@tpims.org (A. Nefzi).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.12.043

818
A. Nefzi, J. E. Fenwick / Tetrahedron Letters 52 (2011) 817–819
O
O
S
a
b, c
H
RGD analog
2
H2N
N
FmocHN
1
N
H
N
H
S
Trt
Trt
O
O
S
d
H
N
H
N
RGD analog
3
RGD analog
4
HN
S
HN
N
H
N
H
S
N
NH2
S
Trt
Trt
Cl
RGD analog
e, f, g
O
HN
HN
N
NH2
S
S
5
Scheme 1. Reagents and conditions: (a) solid-phase peptide synthesis using Fmoc chemistry; (b) FmocNCS (6 equiv) in DMF (0.3 M), rt, overnight; (c) 20% piperidine/DMF;
(d) 1,3-dichloroacetone (10 equiv) in DMF (0.3 M), 70 °C, overnight; (e) TFA/(But)3SiH/DCM (5:5:90), 30 min; (f) Cs2CO3 in DMF overnight; (g) HF/anisole, 0 °C, 90 min.
Table 1
RGD-containing cyclic peptides
5b–5g, and hexapeptides 5h–5z (Table 1). All peptides were ob-
tained in good yield and purity. The final products were purified
by preparative HPLC and the identity of the products was con-
firmed by LC–MS and NMR spectroscopy. The NMR data show a
RGD analog
O
HN
HN
clear singlet between 6.4 and 6.8 ppm which is specific to the pro-
ton on C-5 of the aminothiazole ring.22
N
NH2
S
S
5
Acknowledgments
RGD analog
Observed MW
Puritya (%)
Yieldb
The authors would like to thank the State of Florida Funding,
NIH (1R03DA025850-01A1, Nefzi), NIH (5P41GM081261-03,
Houghten), and NIH (3P41GM079590-03S1, Houghten) for their
financial support.
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
-Arg-Gly-Asp-
544.6 (MH+)
707.9 (MH+)
691.7 (MH+)
657.7 (MH+)
707.7 (MH+)
691.7 (MH+)
657.7 (MH+)
870.9 (MH+)
854.9 (MH+)
820.9 (MH+)
854.9 (MH+)
838.9 (MH+)
804.9 (MH+)
820.9 (MH+)
804.9 (MH+)
770.9 (MH+)
870.9 (MH+)
854.9 (MH+)
820.9 (MH+)
854.9 (MH+)
838.9 (MH+)
804.9 (MH+)
820.9 (MH+)
804.9 (MH+)
769.9 (MH+)
81
83
80
76
84
82
84
82
85
78
86
88
85
79
82
84
80
83
81
77
79
80
74
81
82
45
42
41
38
43
44
42
40
47
41
43
45
42
40
39
43
41
44
42
39
41
43
35
40
44
-Arg-Gly-Asp-Tyr-
-Arg-Gly-Asp-Phe-
-Arg-Gly-Asp-Leu-
-Tyr-Arg-Gly-Asp-
-Phe-Arg-Gly-Asp-
-Leu-Arg-Gly-Asp-
References and notes
-Arg-Gly-Asp-Tyr-Tyr-
-Arg-Gly-Asp-Phe-Tyr-
-Arg-Gly-Asp-Leu-Tyr-
-Arg-Gly-Asp-Tyr-Phe-
-Arg-Gly-Asp-Phe-Phe-
-Arg-Gly-Asp-Leu-Phe-
-Arg-Gly-Asp-Tyr-Leu-
-Arg-Gly-Asp-Phe-Leu-
-Arg-Gly-Asp-Leu-Leu-
-Tyr-Tyr-Arg-Gly-Asp-
-Phe-Tyr-Arg-Gly-Asp-
-Leu-Tyr-Arg-Gly-Asp-
-Leu-Phe-Arg-Gly-Asp-
-Phe-Phe-Arg-Gly-Asp-
-Leu-Phe-Arg-Gly-Asp-
-Tyr-Leu-Arg-Gly-Asp-
-Phe-Leu-Arg-Gly-Asp-
-Leu-Leu-Arg-Gly-Asp-
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22. General procedure for the solid-phase synthesis of cyclic peptide 5a: One bag of
resin 1 (100 mg, 0.115 mmol) was put into a small polyethylene bottle and the
Fmoc group was deprotected with 15 mL of a solution of 20% piperidine in DMF
(2 Â 10 min). The resin was then washed with 15 mL DMF (3Â) and 15 mL
DCM (3Â).
L-Fmoc-Asp(Bzl)-OH (6 equiv, 0.307 g, 0.69 mmol) was coupled in
the presence of hydroxybenzotriazole (HOBt, 6 equiv, 0.094 g, 0.69 mmol) and
diisopropylcarbodiimide (DIC, 6 equiv, 0.101 mL, 0.69 mmol) in 15 mL
anhydrous DMF for 2 h at room temperature. The resin-bound dipeptide was
washed with DMF (3Â) and DCM (3Â). Completion of the coupling was
monitored by the ninhydrin test. The Fmoc group was deprotected with 15 mL
20% piperidine in DMF (2 Â 10 min) and followed by the coupling of
Gly-OH (6 equiv, 0.205 g, 0.69 mmol) using the same reaction conditions. The
Fmoc group was deprotected and the resin-bound tripeptide was coupled to
L-Fmoc-
L-
Fmoc-Arg(Pmc)-OH (6 equiv, 0.457 g, 0.69 mmol) in the same conditions to
yield following Fmoc deprotection the corresponding resin-bound protected
linear peptide 2a. The resulting N-terminal free amine of resin-bound linear
peptide 2a was treated with Fmoc-isothiocyanate (6 equiv, 0.193 g,
11.04 mmol) in 15 mL DMF anhydrous overnight at room temperature.
Following Fmoc deprotection with a solution of 20% piperidine in DMF, the
resin-bound N-terminal thiourea was treated with 1,3-dichloroacetone
(10 equiv, 0.145 g, 18.4 mmol) in DMF anhydrous overnight at 70 °C to afford
following Hantzsch’s cyclocondensation the resulting resin-bound chloro
methyl thiazolyl peptide 4a. The Trt group was removed in the presence of
TFA/(But)3SiH/DCM (5:5:90) for 30 min. The resin was washed with DCM (5Â)
and DIEA/DCM (5:95) and was treated overnight with a solution of Cs2CO3
(10 equiv, 0.325 g) in 15 mL DMF at room temperature to undergo an SN2
intramolecular thioalkylation. The resin was treated with HF/anisole for
90 min at 0 °C, and the desired thiazolyl thioether cyclic peptides 5a was
obtained following extraction with 95% acetic acid in water and lyophilization
as a white powder (61.9 mg). The cyclic peptide 5a was purified by preparative
reverse-phase HPLC. Compound 5a: 1H NMR (500 MHz, DMSO-d6). d ppm 8.78
(br s, 1H), 8.37 (br s, 1H), 8.30 (br s, 1H), 7.42 (br s, 1H), 7.32 (s, 1H), 7.09 (s,
1H), 6.41 (s, 1H), 4.47 (dd, J = 13.3 Hz, J = 5.5 Hz, 1H), 4.12 (dd, J = 12.7 Hz,
J = 7.6 Hz, 1H), 3.93 (m, 2H), 3.64 (d, J = 13.3 Hz, 1H), 3.53 (d, J = 13.5 Hz, 1H),
3.14 (m, 1H), 3.05 (dd, J = 13.6 Hz, J = 8.6 Hz, 1H), 2.97 (m, 1H), 2.84 (dd,
J = 13.6 Hz, J = 4.7 Hz 1H), 2.38 (m, 2H), 2.02 (m, 1H), 1.88 (m, 1H), 1.51 (m, 2H).