Two-step hantzsch based macrocyclization approach for the synthesis of thiazole-containing cyclopeptides

Article abstract of DOI:10.1021/jo1016822

Macrocyclization via an efficient high-yielding solid-phase intramolecular thioalkylation reaction is described. The reaction of S-nucleophiles with newly generated N-terminal 4-chloromethyl thiazoles led to the desired macrocyclization products 5 in high

Full text of DOI:10.1021/jo1016822

pubs.acs.org/joc
include intramolecular nucleophilic substitutions,1a,4 intramo-
Two-Step Hantzsch Based Macrocyclization
Approach for the Synthesis of Thiazole-Containing
Cyclopeptides§
lecular amide formations,1c,5 disulfide formations,6 intramolec-
ular Suzuki reactions,7 ring-closing metathesis reactions,8 and
SNAr displacement reactions.9 Of particular interest, thioalkyl-
ation reactions offer a facile and versatile approach to the
synthesis of cyclic peptides.4,10 Examples of described macro-
cyclizations via thioalkylation include the reaction of the thiol
group of a C-terminal cysteine with N-terminal acetyl bromide
or N-terminal benzyl bromide.4,10 A conceptually different
approach, wherein thioalkylation proceeds via Michael addi-
tion of a thiolate anion to an R,β-unsaturated ester, has been
reported for the synthesis of cyclic thioether dipeptides.11
Herein, we describe an innovative thioalkylation ap-
proach toward the generation of macrocyclic peptides fol-
lowing the intramolecular nucleophilic substitution (SN2) of
N-terminus 4-chloro methyl thiazole peptides with the thiol
group of cysteine. The final products are not entirely pep-
tidic, and the described newly generated macrocyclic com-
pounds contain the thiazole ring, a pharmacophore present
in many natural and synthetic products with a wide range of
pharmacological activities that can be well illustrated by the
large numbers of naturally occurring thiazole-containing
macrocyclic compounds12 and drugs in the market contain-
ing this function group.13
Adel Nefzi,* Sergey Arutyunyan, and Jason E. Fenwick
Torrey Pines Institute for Molecular Studies,
11350 SW Village Parkway, Port Saint Lucie,
Florida 34987, United States
adeln@tpims.org
Received August 27, 2010
Macrocyclization via an efficient high-yielding solid-phase
intramolecular thioalkylation reaction is described. The
reaction of S-nucleophiles with newly generated N-terminal
4-chloromethyl thiazoles led to the desired macrocyclization
products 5 in high purities and good overall yields.
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Macrocycles are known for their broad range of activities
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proaches on the solid-phase synthesis of macrocyclic compounds
§ Dedicated to Dr. Richard Houghten for his 65th birthday.
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DOI: 10.1021/jo1016822
r
Published on Web 10/29/2010
J. Org. Chem. 2010, 75, 7939–7941 7939
2010 American Chemical Society

Nefzi et al.
JOCNote
SCHEME 1a
aReagents 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.
We have shown the feasibility of the proposed approach
by the parallel synthesis of different thiazole-containing macro-
cyclic peptides. Starting from resin-bound orthogonally pro-
tected Fmoc-Cys-(Trt)-OH 1, the thiomethyl thiazolyl macro-
cyclic peptidomimetics 5 were synthesized following stepwise
Fmoc deprotection14 and standard repetitive Fmoc-amino-acid
couplings yielding the linear tripeptide 2. The resulting N-termi-
nal free amine was treated with Fmoc-isothiocyanate. Follow-
ing Fmoc deprotection, the thioureas were treated with 1,3-
FIGURE 1
dichloroacetone to afford following Hantzsch’s cycloconden-
sation15 the resulting resin-bound chloro methyl thiazolyl
of β-turn-promoting structures such as glycine, proline, or a
D-amino acid.16 Using the approach outlined in Scheme 1, we
first tested our approach by performing the parallel synthesis
of various thiazole-containing cyclic tetrapeptides and pen-
tapeptides from all L-amino acids (Figure 1).
peptide 4. The Trt group was deprotected in the presence of
5% TFA in DCM, and the resin was treated with a solution of
Cs2CO3 in DMF to undergo an SN2 intramolecular cycliza-
tion. The resin was cleaved with HF/anisole, and the desired
thiazolyl thioether cyclic peptides 5 were obtained in good yield
and high purity. An Ellman test for free thiols was performed
on all samples. No free thiol was present in the crude material.
The identity of the final products was confirmed by LC-MS
and NMR spectroscopy.
Many reagents and techniques have been developed to
facilitate the synthesis of cyclic peptides, for which the yield-
limiting step is generally the cyclization reaction. Particu-
larly, the cyclization of tetra-, penta-, and hexapeptides in the
all-L configuration can be problematic, especially in the absence
We selected different amino acids for each of the position
of diversities R1, R2, and R3 for the synthesis of 11 tetrapep-
tides (5a-5k). Thus, we choose Phe, Tyr, Ser, and Val for the
position R1, Phe, Tyr, and Pro for the position R2 and Tyr,
Pro, and Lys for the position R3. Similarly, we selected three
amino acids for each of the position of diversities R1 (Tyr,
Phe, Asp), R3 (Tyr, Pro, Arg), and two amino acids for
each of the position of diversities R2 (Tyr, Gly) and R4
(Tyr, Pro) for the synthesis of nine pentapeptides (5l-5t).
As shown in Table 1, high purities were obtained for all
compounds. In all cases, the intramolecular thioalkylation
reaction led to the desired cyclic monomers with negligible
traces of dimerization. The NMR data show a clear singlet
at 6.4 ppm that is specific to the proton on C-5 of the
aminothiazole ring. The presented methodology is not
limited to cyclic tetrapeptides and pentapeptides. It was
successfully used for the synthesis of cyclic hexapeptides
5u-5v and heptapeptide 5w.
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Orlando, FL, 1985; Vol. 37, pp 1-109. (b) Olson, G. L.; Bolin, D. R.; Bonner,
M. P.; Bos, M.; Cook, C. M.; Fry, D. C.; Graves, B. J.; Hatada, M.; Hill,
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We have presented a new method for macrocyclization
reaction via thioalkylation. The reaction of S-nucleophiles
with newly generated N-terminal 4-chloromethyl thiazoles
led to the desired macrocyclization products in high purities
and good overall yields. The presented approach can be
extended toward the synthesis of macrocyclic libraries where
7940 J. Org. Chem. Vol. 75, No. 22, 2010

Nefzi et al.
JOCNote
TABLE 1. Synthesis of Thiazole-Containing Cyclic Tetrapeptides and
Pentapeptides
was coupled using the conventional reagents hydroxybenzotriazole
(HOBt, 1.07 g, 7.93 mmol) and diisopropylcarbodiimide (DIC,
1.16 mL, 7.93 mmol) in 300 mL of anhydrous DMF over-
night at room temperature. Completion of the coupling was
monitored by the ninhydrin test.
General Procedure for the Solid-Phase Synthesis of Resin-
Bound Linear 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 20% piperidine in
DMF (2 Â10 min). The resin was then washed with 15 mL of
DMF (3Â) and 15 mL of DCM (3Â). L-Fmoc-Phe-OH (6 equiv,
0.267 g, 11.04 mmol) was coupled in the presence of hydroxy-
benzotriazole (HOBt, 6 equiv, 0.094 g, 11.04 mmol) and
diisopropylcarbodiimide (DIC, 6 equiv, 0.101 mL, 11.04 mmol)
in 15 mL of 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 ninhy-
drin test. The Fmoc group was deprotected with 15 mL of 20%
piperidine in DMF (2Â10 min) and followed by the coupling of
L-Fmoc-Phe-OH (6 equiv, 0.267 g, 11.04 mmol) using the same
reaction conditions. The Fmoc group was deprotected, and the
resin-bound tripeptide was coupled to L-Fmoc-Tyr(tBu)-OH 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 of anhydrous DMF 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 anhydrous DMF overnight at 70 °C to afford following
Hantzsch’s cyclocondensation the resulting resin-bound chloro
methyl thiazolyl peptide 4a. The Trt group was deprotected 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 of DMF at room temperature to undergo an SN2 intra-
molecular thioalkylation. The resin was cleaved 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 peptides
5a was purified by preparative reverse-phase HPLC.
entry
peptide
Tyr-Phe-Phe
Pro-Phe-Phe
Tyr-Tyr-Phe
Pro-Tyr-Phe
Tyr-Phe-Tyr
Pro-Phe-Tyr
Tyr-Tyr-Tyr
Pro-Tyr-Tyr
puritya (%)
yieldb
5a
5b
5c
5d
5e
5f
5g
5h
5i
88
85
88
89
87
76
83
89
85
86
82
86
90
80
83
82
85
80
78
84
82
80
82
45
38
43
41
40
29
38
46
47
43
40
38
39
31
37
31
35
34
35
43
40
37
39
Tyr-Pro-Ser
Tyr-Tyr-Ser
Lys-Tyr-Val
5j
5k
5l
Tyr-Tyr-Tyr-Phe
Pro-Tyr-Tyr-Phe
Tyr-Pro-Tyr-Phe
Pro-Pro-Tyr-Phe
Tyr-Tyr-Tyr-Tyr
Pro-Tyr-Tyr-Tyr
Tyr-Pro-Tyr-Tyr
Pro-Pro-Tyr-Tyr
Tyr-Arg-Gly-Asp
Arg-G ly-Asp-Tyr-Tyr
Phe-Tyr-Val-Ser-Ala
Phe-Ala-Pro-Tyr-Ser-Phe
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
aPurity of crude products. The products were run on a Vydac column
with a gradient of 5% to 95% formic acid in ACN in 7 min. The purity
was estimated on analytical traces at λ = 214 and 254 nm. bThe yields are
based on the weight of the purified products and are relative to the initial
loading of the resin. (The purity of the purified compounds is higher than
95% for all compounds).
the cysteine residue can be placed anywhere in the peptide
sequence, allowing for extension of the peptide beyond the
cyclic link. As part of our drug discovery program, we are in
the process of preparing a variety of thiazole-containing macro-
cyclic libraries. The synthesis and the screening results will be
reported elsewhere.
Acknowledgment. The authors would like to thank the
State of Florida Funding, NIH (1R03DA025850-01A1,
A.N.; 5P41GM081261-03, Houghten; 3P41GM079590-03S1,
Houghten) for financial support.
Experimental Section
Synthesis of Resin-Bound Cysteine. A 100 mg sample of p-meth-
ylbenzhydrylamine hydrochloride (MBHA HCl) resin (CHEM-
3
IMPEX INTERNATIONAL 1.15 mequiv/g, 100-200 mesh,
1% DVB) was contained within a sealed polypropylene mesh bag.
Twenty-three bags (23 Â 100 mg resin, 2.64 mmol) were put
in a polyethylene bottle. Following the neutralization of resin
with 500 mL of 5% diisopropylethylamine (DIEA) in dichloro-
methane (DCM), L-Fmoc-Cys(Trt)-OH (3 equiv, 4.64 g, 7.93 mmol)
Supporting Information Available: Experimental details and
analytical data including NMR data and spectra; HPLC chro-
matograms and mass spectra for all reported cyclic peptides;
HPLC and LC-MS of additional cyclic tetrapeptides and
pentapeptides not reported in the paper. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 75, No. 22, 2010 7941