An improved synthesis of a selective αvβ3-integrin antagonist cyclo(-RGDfK-)

Article abstract of DOI:10.1016/S0040-4039(00)01060-1

The cyclic pentapeptide cyclo(-Arg-Gly-Asp-D-Phe-Lys-) is a highly potent and selective inhibitor for the αvβ3 integrin. A related compound, cyclo(-Arg-Gly'Asp-D-Phe-Val-), is a promising anticancer drug candidate; it inhibits angiogenesis and induces apoptosis in vascular cells. We have developed an improved solid-phase synthesis based on Kessler's procedure to afford cyclo(-RGDfK-) peptide in high purity and high yield in a multi-gram scale. This improved synthesis is environmentally friendly and may be generally applicable to other related cyclic peptide syntheses. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01060-1

Tetrahedron Letters 41 (2000) 6295±6298
An improved synthesis of a selective avb3-integrin
antagonist cyclo(-RGDfK-)
Xuedong Dai, Zhuang Su and Jun O. Liu*
Center for Cancer Research, Departments of Biology and Chemistry, Massachusetts Institute of Technology,
40 Ames Street, Cambridge, MA 02139, USA
Received 2 May 2000; revised 20 June 2000; accepted 21 June 2000
Abstract
The cyclic pentapeptide cyclo(-Arg-Gly-Asp-d-Phe-Lys-) is a highly potent and selective inhibitor for the
avb3 integrin. A related compound, cyclo(-Arg-Gly-Asp-d-Phe-Val-), is a promising anticancer drug
candidate; it inhibits angiogenesis and induces apoptosis in vascular cells. We have developed an improved
solid-phase synthesis based on Kessler's procedure to a€ord cyclo(-RGDfK-) peptide in high purity and
high yield in a multi-gram scale. This improved synthesis is environmentally friendly and may be generally
applicable to other related cyclic peptide syntheses. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: avb3-integrin antagonist; cyclic RGD peptide; solid-phase synthesis.
The integrins are a class of cell surface adhesion proteins that play important roles in cell±cell
and cell±matrix interactions.1 They are involved in diverse physiological processes. In particular,
it has been shown that the avb3 and avb5 subgroups of integrins are required for tumor-induced
angiogenesis. An inhibition of the binding of the avb3 and avb5 integrins to their native ligands by
antibodies or cyclic peptides interferes with angiogenesis and induces tumor regression.2
Antagonists of avb3 are undergoing clinical trials as potential cancer therapeutic agents.2a
Integrins interact with their ligand proteins mainly through a tripeptide motif consisting of
Arg-Gly-Asp (RGD). A number of RGD-containing peptides have been designed, synthesized
and tested for their ability to serve as antagonists of integrins. Of the antagonists synthesized to
date,3,4 RGDfV (1)3a and its methylated analog by Kessler and co-workers are the most selective
for avb3.4
* Corresponding author.
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01060-1

6296
In a search for novel anti-angiogenic agents, we needed to obtain the cyclic pentapeptide
cRGDfK (2). First designed and synthesized by Kessler's group,3a cRGDfK binds to avb3 with
high potency and selectivity. As the ®fth amino acid has little in¯uence on the activity, the pri-
mary amino group of the lysine in cRGDfK o€ers an ideal site for further functionalization of
this cyclic peptide. Indeed, the amino group on the lysine side chain has been coupled with
¯uorescein, and the resulting conjugate was shown to be enriched in retina undergoing
neovascularization.5
The original synthesis of cRGDfK employed the Fmoc solid-phase chemistry to build a linear,
protected RGDfK peptide, followed by cleavage from the solid resin, cyclization of the linear
peptide, and removal of the protecting groups.3a Despite its high eciency, several disadvantages
remain: (1) After the cleavage of the linear peptide from the resin, the cyclization of the acid
terminal of glycine and the a-amino terminal of lysine was performed via in situ activation using
diphenylphosphoryl azide (DPPA), requiring aqueous work-up; (2) the removal of the guanidine-
protecting group from arginine, the 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) group,
necessitated the use of a mixture of several toxic and malodorous reagents, including
tri¯uoroacetic acid (TFA), phenol, thioanisole, and ethanedithiol; and (3) puri®cation of the ®nal
product requires HPLC separation.
We set out to improve the original synthesis in all three aspects: (1) We decided to use 1-propane-
phosphonic acid cyclic anhydride (T3P) for the cyclization of the linear peptide. This reagent is
highly e€ective as a coupling reagent in peptide synthesis.6 The product should be easily puri®ed
by silica gel chromatography, eliminating the need for the toxic DPPA; (2) we decided to change
the protecting group of arginine from Mtr to 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pbf),
which requires much milder deprotection conditions (1:19 water:tri¯uoroacetic acid (TFA)).7 An
added advantage is that all the other protecting groups can be removed under the same condition;
and (3) as the ®nal deprotection step does not involve the aforementioned toxic and high-boiling
reagents, puri®cation of the ®nal product by HPLC is unnecessary. Here we report the improved
synthesis of cRGDfK (Scheme 1).
Similar to the literature procedure, the synthesis of the linear peptide started with the
incorporation of glycine onto o-chlorotrityl chloride resin by mixing the resin (16.72 g, 1.35
mmol/g, 22.57 mmol) with a solution of Fmoc-Gly-OH (16.78 g, 56.44 mmol) and N,N-di-
isopropylethylamine (DIEA, 9.04 mL, 51.90 mmol) in dry dichloromethane (DCM) in a 500 mL
peptide synthesis vessel (ChemGlass). After shaking the reaction mixture for 2.5 h using a Burrell
Wrist-Action shaker, 9.0 mL of DIEA and 50 mL of methanol were added to cap the unreacted
sites of the resin. After 30 min, the resin was washed with dimethylformamide (DMF, 2Â), DCM
(2Â), methanol (2Â), and diethylether (2Â) and dried in vacuo. The loading of the resin was

6297
Scheme 1. Improved synthesis of cRGDfK
calculated as 17.60 mmol Fmoc-glycine. The Fmoc-protecting group was then removed by shaking
with a 20% solution of piperidine in DMF twice. The deprotection reaction was monitored by
ninhydrin test. The deprotected resin was washed with DMF six times.
To the resin was added a solution of Fmoc-Arg(Pbf)-OH acids (25.00 g, 38.53 mmol), 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetra¯uoroborate (TBTU, 10.16 g, 31.64 mmol), 1-
hydroxybenzotriazole (HOBt, 4.28 g, 31.67 mmol), and DIEA (19.68 mL, 112.98 mmol) in DMF.
The mixture was shaken for 1.5 h. The resulting resin was washed with DMF six times, deprotected
by shaking with a 20% solution of piperidine in DMF twice, and washed with DMF for an addi-
tional six times. Subsequently, Fmoc-Lys(Boc)-OH, Fmoc-d-Phe-OH, and Fmoc-Asp(OtBu)-OH
were coupled to the resin in the same manner.
The linear RGDfK peptide was cleaved from the resin without a€ecting other protecting
groups by treating with 250 mL of a mixture of acetic acid, 2,2,2-tri¯uoroethane (TFE), and
DCM (1:1:3) for 1 h at room temperature. The resin was washed twice with 250 mL of the same
mixture and then with DCM three times. The eluents were combined and concentrated. The
excess acetic acid was azeotroped o€ with toluene.
The head-to-tail cyclization was performed by slowly adding a solution of the linear peptide
acetate salt in 200 mL of DCM to a solution of 50% 1-propanephosphonic acid cyclic anhydride
(T3P) in EtOAc (54.24 mL), triethyl amine (TEA, 63.2 mL), and DMAP (200 mg) in 8 L of
DCM. After stirring overnight, the starting material could not be detected by RP-HPLC. The
reaction mixture was concentrated and puri®ed by ¯ash chromatography (methanol:ethyl acetate,
1:10) to a€ord 17.23 g (MW=1012.23, 96.7%, 17.02 mmol, relative to the amount of Gly coupled
to the resin) of the protected cyclic peptide as light yellow solid.8
The protecting groups of the above cyclic peptide (16.05 g) were removed with a mixture of
water and tri¯uoroacetic acid (TFA) (1:19). TFA was removed by azeotroping with toluene.
Trituration of a DCM solution of the product with diethyl ether followed by ®ltration yielded
.
cRGDfK 2TFA salt (MW=831.72, 10.81 g, 82.0%) as a light yellow powder. The overall yield
(79.3% relative to the amount of Gly coupled to the resin) is signi®cantly higher than the reported
yield (44%).3a The cyclic peptide salt obtained this way is pure and shows only one peak in

6298
reverse phase high-performance liquid chromatography (RP-HPLC). ESI mass spectrum and
NMR data of the compound were identical to those previously reported.3a
In conclusion, we have developed an improved multi-gram-scale solid-phase synthesis of the
cRGDfK. In comparison with the original synthesis, the current procedure not only eliminates
the use of several toxic reagents, but also signi®cantly improves the yield of the synthesis. These
improvements may ®nd applications in practical, large-scale synthesis of cRGDfV and related
cyclic peptides.
References
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Pignatelli, M., Eds.; Harwood Academic Publishers: Chur, 1995. (c) Hynes, R. O. Cell 1992, 69, 11±25.
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8. Retention time tR=38 min on a reverse phase C-18 column. Elution with a linear gradient over 60 min of bu€er A
and bu€er B from 49:1 to 1:49 (bu€er A=4 mM ammonium acetate and 0.1% formic acid in water; bu€er B=4
mM ammonium acetate, 80% acetonitrile and 0.1% formic acid in water). Flow rate: 50 mL/min. Detection
wavelength: 220 nm. ESI MS for C49H73N9O12S, calculated: 1011.5; found: 1011.7.