Synthesis of novel Gn-RH analogues using Ugi-4MCR

Article abstract of DOI:10.1016/j.bmcl.2008.11.111

An efficient method for the synthesis of some Gn-RH analogues based on Ugi reaction has been developed. Four-component reaction of N- and C-terminus peptides, aromatic aldehydes and isocyanides affords novel Gn-RH analogues derived from triptorelin and go

Full text of DOI:10.1016/j.bmcl.2008.11.111

Bioorganic & Medicinal Chemistry Letters 19 (2009) 887–890
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel Gn-RH analogues using Ugi-4MCR
b
Armin Arabanian a, Mahdieh Mohammadnejad a, Saeed Balalaie a, , Jürgen H. Gross
*
a Peptide Chemistry Research Group, K. N. Toosi University of Technology, PO Box 15875-4416, Tehran, Iran
b Organisch-Chemisches Institut der Universität Heidelberg, Im Neuenheimer Feld 270, 69120 Heidelberg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient method for the synthesis of some Gn-RH analogues based on Ugi reaction has been devel-
oped. Four-component reaction of N- and C-terminus peptides, aromatic aldehydes and isocyanides
affords novel Gn-RH analogues derived from triptorelin and gonadorelin. All of the products were purified
using preparative HPLC and the structures were assigned according to MALDI-mass spectrometry data.
Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
Received 23 October 2008
Revised 26 November 2008
Accepted 26 November 2008
Available online 6 December 2008
Keywords:
Gn-RH analogues
4-MCR Ugi reaction
Triptorelin and gonadorelin analogues
Ligation
Gonadotropin-releasing hormone (Gn-RH) is secreted from the
hypothalamus and its action on the pituitary gland then leads to
the release of luteinizing hormone (LH) and follicle-stimulating hor-
mone (FSH). Both of these hormones then act on the ovaries and are
responsible for the pro-fertility effects of Gn-RH, primarily through
the release of steroidal hormones. Gn-RH was first isolated from por-
cine hypothalamus and was shown to be a decapeptide (I). Gn-RH
plays a key role in the biology of reproduction. Similar peptides have
been isolated and sequenced from other species such as chicken, sal-
mon and lamprey.1,2
rally occurring proteins. Another method for the coupling of two
peptide segments is Staudinger reaction.14
It was shown that the existence of more amide bonds and aryl or
alkyl groups in the structure of peptides could affect the hydropho-
bicity of the peptide and biological activities.15 We hoped to develop
a general and practical method to synthesize novel Gn-RH analogues
contained additional amide bond plus aryl or alkyl groups located in
the middle position of the peptides. The best way for our design is 4-
MCR Ugi reaction. MCRs applications in all areas of applied chemis-
try are very popular because, they offer a wealth of products, while
requiringonlya minimumof effort. MCR isapplyingforthesynthesis
of complex molecules in one-pot reactions with high bond-forming
efficiency (BFE). Meanwhile, a wide variation among these starting
materials opens up versatile opportunities for the synthesis of com-
pound libraries. Among many types of MCRs, the most useful are iso-
cyanide based MCRs (IMCRs).16,17 It has been known that Ugi-4CR
provides an elegant way to prepare natural products. The Ugi four-
component reaction (Ugi-4CR) was used as an efficient method for
the construction of peptide and cyclopeptide backbones.18
According to this high potential, herein, we present an efficient
method for the synthesis of novel Gn-RH analogues using Ugi four-
component reaction from C-terminus heptapeptides, N-terminus
tripeptide, aromatic aldehydes and isocyanides (Scheme 1).
The reaction resulted new analogues of Gn-RH with additional
peptide backbones between Leu-7 and Arg-8. It seems the presence
of aryl and alkyl groups in Gn-RH analogues could increase hydro-
phobic properties of the peptides. It was shown that the existence
of Arg-8 is critical for high-affinity interaction of Gn-RH analogues
with receptors.19 In all our synthetic peptides, the active sites for
the interactions with receptors are remained.
pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
ðIÞ
The bulky hydrophobic amino acid residues in position 6 appear
to be very important for the high potency of the analogues. In this
way, substitution and addition of hydrophobicity at position 6 in
Gn-RH analogues is an interesting subject. It was shown that the
substitution could affect the biological activities of synthetic pep-
tides. Results from a number of ongoing studies on prostate cancer
patients using goserelin, buserelin, triptorelin and leuprolide have
recently been published. Synthesis of novel Gn-RH has attracted
particular interest.3–9
There are some methods for the coupling of peptide segments;
among the techniques employed for this purpose native chemical
ligation is the most useful method. The native chemical ligation
(NCL) reaction10–13 is a powerful method to join two unprotected
peptides in aqueous solution. In the native chemical ligation an
unprotected C-terminal peptide thioester reacts with an unpro-
tected N-terminal cysteine residue. The requirement for the rare
amino acid cysteine limits the applicability in the synthesis of natu-
Two different heptapeptides were used for the synthesis of Gn-
RH analogues. In cases 5a–d, there were Gly at the position 6 in
* Corresponding author. Tel.: +98 21 2288 6575; fax: +98 21 2285 3650.
E-mail address: balalaie@kntu.ac.ir (S. Balalaie).
0960-894X/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.111

888
A. Arabanian et al. / Bioorg. Med. Chem. Lett. 19 (2009) 887–890
60:40. The ratio of diastereomers was obtained according to the
HPLC chromatogram (Fig. 1).
p-Glu-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-[A.A]-Leu-COOH
2
This research concerned the design and synthesis of C-terminus
heptapeptides and N-terminus tripeptide as starting materials. The
C-terminus heptapeptides 2 were synthesized using known Fmoc
solid phase peptide synthesis strategy.20,21
_
C
O
+
N
4
R
+
Ar
H
1
Meanwhile, TBTU was used as the coupling reagent22 for the
synthesis of C-terminus heptapeptides and N-terminus tripeptide.
The tripeptide 3 was synthesized in solution phase and with Boc/Z
strategy. The details for the synthesis of N-terminus tripeptide are
shown in Scheme 2.
H2N-Arg(Pbf)-Pro-Gly-CONH2
3
1) MeOH, rt
2) Reagent K
The synthesis of tripeptide 3 started with the coupling reaction
of Boc-Pro-OH with Gly-NH2ꢀHCl in presence of TBTU as coupling
reagent and base to afford the protected dipeptide 6. Acidification
of compound 6 with Acetic acid/hydrochloric acid (1 M) is an
established method for the Boc-deprotection to provide the depro-
tected dipeptide 7. After removal of the Boc group, coupling reac-
O
Ar
N
R
p-Glu-His-Trp-Ser-Tyr-[A.A]-Leu
N
O
5
Arg-Pro-Gly-CONH2
tion of dipeptide
7 with Z-Arg(Pbf)-OH provides protected
[A.A] = Gly, D-Trp
tripeptide 8, and finally Z-deprotection was done using hydrogena-
tion in the presence of Pd/C successfully.23
Ar = 4-Pyridyl, 4-CN-C6H4
R = t-Bu, Cy-Hexyl
The generally accepted mechanism of the Ugi four-component
coupling (Ugi-4CC) reaction involves four elementary steps, the
last of which is irreversible. In the first step, the aldehyde con-
denses with an amino group of N-terminus tripeptide to form an
imine that could be converted and produces iminium salt in the
presence of C-terminus heptapeptides. Next, the isocyanide is
added to the iminium salt to produce a nitrilium ion. Then, a reac-
Scheme 1.
gonadorelin analogues sequence and D-Trp in triptorelin analogues
5e–h. Using Ugi-4MCR a new stereocenter was created in the prod-
ucts. In Table 1 the yields and diastereomeric ratio of products are
shown. For example in compound 5f the diastereomeric ratio was
tive O-acyl iminolate is formed via the
a-addition of carboxylate
anion to the nitrilium ion. The final step involves the O- to N-acyl
transfer (Mumm rearrangement) to afford the novel Gn-RH ana-
logues (Scheme 3).
Table 1
Synthesis of novel Gn-RH analogues via Ugi-4MCR
The products were purified using preparative HPLC and their
structures were confirmed using MALDI and ESI Mass spectroscopy
methods and also amino acid analysis data.24 In Figure 2 MALDI-
Mass spectrum of compound 5f is shown.
The prepared peptide has more amide bonds, and it seems that
it could be more stable against proteases and it can be considered
as a better candidate for therapeutic purposes. The presence of
aryl, cyclohexyl and alkyl groups in the structure of the products
mimic the peptide backbone and increase hydrophobic properties
of the molecule and it seems it can affect the biological activity
Entry
A.A.
Ar
R
Yielda (%)
d.r.b
5a
5b
5c
5d
5e
5f
Gly
Gly
Gly
Gly
D-Trp
D-Trp
D-Trp
D-Trp
4-CN-C6H4
4-Pyridyl
4-Pyridyl
4-CN-C6H4
4-CN-C6H4
4-Pyridyl
4-Pyridyl
4-CN-C6H4
t-Bu
t-Bu
cy-Hexyl
cy-Hexyl
t-Bu
t-Bu
cy-Hexyl
cy-Hexyl
89
75
88
82
80
69
86
84
30/70
55/45
80/20
75/25
50/50
60/40
50/50
70/30
5g
5h
a
Isolated yields.
Diastereomeric ratio.
b
Figure 1. HPLC chromatogram of compound 5f.

A. Arabanian et al. / Bioorg. Med. Chem. Lett. 19 (2009) 887–890
889
TBTU, HOBt, DIPEA
Boc-Pro-Gly-NH2
Boc-Pro-OH + Gly-NH2.HCl
EtOAc, rt
6
HCl/AcOH (1M)
rt, 1 h
Z-Arg(Pbf)-OH
H2 , Pd (C)
MeOH
H-Pro-Gly-NH2.HCl
3
Z-Arg(Pbf)-Pro-Gly-NH2
TBTU, HOBt, DIPEA
EtOAc, rt
8
7
Scheme 2. Synthesis of N-terminus tripeptide.
-
O
Peptide 2
O
Peptide 2
O
Ar
H
N
-
+
R
O
H
O
R
N
C
Peptide 2
R
+
N
N
O
Peptide 1
N
Peptide 1
H
+
Ar
H
Peptide 1
N
Ar
H
Scheme 3. Proposed mechanism for the synthesis of Gn-RH analogues 5.
Figure 2. MALDI-MS of compound 5f.
of the peptide. Investigations concerning biological activity are
underway.
Engineering Co. for kind cooperation. A part of this research work
was supported by research council of K. N. Toosi University of
Technology which is gratefully acknowledged.
The results indicate that the current method could be used to
synthesize some novel Gn-RH analogues using 4-MCR Ugi reaction.
Because the current method can link two peptide segments in mild
conditions via the Ugi type reaction as the key step, we named in
the Ugi-ligation.
References and notes
1. Millar, R. P.; Lu, Z.-L.; Pawson, A. J.; Flanagan, C. A.; Morgan, K.; Maudsley, S. R.
Endocr. Rev. 2004, 25, 235.
2. Padula, A. M. Anim. Reprod. Sci. 2005, 50, 115.
3. Maudsley, S. Cancer Res. 2004, 64, 7533.
4. Palmon, A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4994.
5. Azad, N. Endocrinology 1991, 128, 1679.
6. Engel, J. B.; Strutbers R. S. In GnRH Analogs in Human Reproduction; Lunenfeld,
B., Ed.; Taylor & Francis: Wiltshire, UK, 2005, pp 35–52.
7. Grundker, C. Eur. J. Endocrinol. 2002, 46, 1.
In conclusion, we created novel synthetic Gn-RH analogues via
Ugi four-component reaction of C-terminus heptapeptides, N-ter-
minus tripeptide, aromatic aldehydes, and isocyanides at room
temperature. In this research, triptorelin and gonadorelin ana-
logues were synthesized with good yields.
8. Kovacs, M. Peptides 2007, 28, 821.
Acknowledgments
9. Ripka, A. S.; Rich, D. H. Curr. Opin. Chem. Biol. 1998, 2, 441.
10. Hase, C.; Rohde, H.; Seitz, O. Angew. Chem., Int. Ed. 2008, 47, 6807.
11. Dose, C.; Seitz, O. Org. Lett. 2005, 7, 4365.
We thank National Research Institute for Science Policy (NRISP,
Iran) for financial support. We also thank Tofigh Daru Research &
12. Mende, F.; Seitz, O. Angew. Chem., Int. Ed. 2007, 46, 4577.

890
A. Arabanian et al. / Bioorg. Med. Chem. Lett. 19 (2009) 887–890
13. Hasse, C.; Seitz, O. Angew. Chem., Int. Ed. 2008, 47, 1553.
14. Nepomniaschiy, N.; Brik, A. J. Pept. Sci. 2008, 14, 67.
15. Muir, T. W.; Sondhi, D.; Cole, P. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6705.
16. Dömling, A. Chem. Rev. 2006, 106, 17.
17. Hebach, C.; Kazmaier, U. Chem. Commun. 2003, 5, 596.
18. (a) Vercillo, O. E.; Andrade, C. K.; Wessjohann, L. A. Org. Lett. 2008, 10, 205; (b)
Waki, M.; Meienhofer, J. J. Am. Chem. Soc. 1977, 99, 6075.
19. Mamputha, S.; Lu, Z.-L.; Roeske, R. W.; Millar, R. P.; Katz, A. A.; Flanagan, C. A.
Mol. Endocrinol. 2007, 21, 281.
completion of the reaction, the solution was washed with citric acid (20%) and
sodium carbonate (3%) and iso-hexane was added to the residue to afford
crystals, which were collected by filtration (yield = 67%). For Boc-deprotection,
dipeptide 6 (4.10 g, 15.0 mmol) was dissolved in AcOH/HCl (45 ml) and the
mixture was stirred for1 h, and thenMTBE(65 ml)was added and theprecipitate
was collected via filtration (yield = 99%). Dipeptide H-pro-Gly-NH2 (7) (3.12 g,
15 mmol) was dissolved in EtOAc (75 ml), Z-Arg(Pbf)-OH (7.2 g, 12.8 mmol),
TBTU (4.37 g, 13.6 mmol), HOBt (1.96 g, 12.8 mmol) and DIPEA (6.33 ml,
37.0 mmol) was added to the solution. The solution was stirred overnight at
room temperature. The work-up was done according to last procedure
(yield = 62%). Z-Arg(Pbf)-Pro-Gly-NH2 (8) (5.58 g, 7.8 mmol) in MeOH (40 ml)
was debenzylated using H2 over Pd/C catalyst. After removal of Pd/C and the
solvent, the residue was collected (yield = 99%). The overall yield for the
synthesis tripeptide 3 was 41%.
20. (a) Yang, Y.-Y.; Ficht, S.; Brick, A.; Wong, C.-H. J. Am. Chem. Soc. 2007, 129, 7690;
(b) Fiels, C.-B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161; (c) Barlos, K.;
Chatzi, O.; Gatos, D.; Stavropulos, G. Int. J. Pept. Protein Res. 1991, 37, 513.
21. General procedure for the synthesis of heptapeptides pGlu-His(Trt)-Trp(Boc)-
Ser(tBu)-Tyr(tBu)-[A.A.]-Leu-COOH (2): Synthesis was carried out using 2-
chlorotrityl chloride resin (1.0 mmol/g) following standard Fmoc strategy.
Fmoc-Leu-OH (3.54 g, 10 mmol) was attached to the 2-CTC resin (5.0 g) with
DIPEA (6.85 ml, 40 mmol) in anhydrous DCM: DMF (50 ml, 1:1) at room
temperature for 2 h. After filtration, the remaining trityl chloride groups were
capped by a solution of DCM/MeOH/DIPEA (17:2:1, 120 ml) for 30 min. The
resin was filtered and washed thoroughly with DCM (1ꢁ 20 ml), DMF (4ꢁ
20 ml) and MeOH (5ꢁ 20 ml). The loading capacity was determined by weight
after drying the resin under vacuum and was 1.0. The resin-bound Fmoc-amino
acid was washed with DMF (3ꢁ 20 ml) and treated with 25% piperidine in DMF
(65 ml) for 30 min and the resin was washed with DMF (3ꢁ 20 ml).20 Then a
solution of Fmoc-AꢀA-OH (7.5 mmol), TBTU (2.40 g, 7.5 mmol), DIPEA (3.0 ml,
17.5 mmol) in 30 ml DMF was added to the resin-bound free amine and shaken
for 1 h at room temperature. After completion of coupling, resin was washed
with DMF (4ꢁ 20 ml) and DCM (1ꢁ 20 ml). The coupling was repeated as the
same methods for other amino acids of their sequences. In all cases for the
presence or absence of free primary amino groups, Kaiser Test was used. Fmoc
determination was done using UV spectroscopy method. After completion of
couplings, resin was washed with DMF (4ꢁ 20 ml), DCM (1ꢁ 20 ml). The
produced heptapeptides 2 were cleaved from resin by treatment of TFA (1%) in
DCM (275 ml) and neutralization with pyridine (4%) in MeOH (85 ml). The
solvent was removed under reduced pressure and precipitated in water. The
yields were 90% (A.A. = Gly) and 84% (A.A. = D-Trp).
24. General procedure for the synthesis of Gn-RH analogues (5): The tripeptide 3
(0.58 g, 1.0 mmol) was added to a solution of aldehydes 1 (1.0 mmol) in
methanol (6 ml) and the reaction was stirred at room temperature for 30 min.
Then C-terminus heptapeptides 2 (1.0 mmol) was added and stirring was
continued for 15 min, followed by addition of isocyanide derivatives
4
(1.0 mmol), the solution was stirred for 4 days at room temperature. Water
(90 ml) was added and after 2 h, the precipitate was filtered and washed with
saturated NaHCO3 and water. The final deprotection of the protected peptides
was performed by reacting with reagent K (20 ml/g peptide) for 2 h at room
temperature. The final peptides 5 were dried under vacuum at 40 °C (yields: 69–
88%). Further purification was done using Prep-HPLC with column (ODS-C18
,
120 ꢁ 20 mm) and UV detector (k = 210 nm). The elution solvent was ACN/
10 mM NaH2PO4 buffer. The MALDI-MS spectral data for the compound 5a–h are
as follows: 5a: C68H89N19O14
[M]+ = 1396.69070, [M+H]+ = 1397.69403,
C66H89N19O14, Calcd 1372.5570, MS (MALDI), found m/z: [M]+ = 1372.69085,
,
Calcd 1396.5790, MS (MALDI), found m/z:
[M+Na]+ = 1419.67617;
5b:
[M+H]+ = 1373.69439, [M+Na]+ = 1395.67646; 5c: C68H91N19O14
,
Calcd
1398.5950, MS (MALDI), found m/z: [M]+ = 1398.70666, [M+H]+ = 1399.71012,
[M+Na]+ = 1421.69224; 5d: C70H91N19O14, Calcd 1422.6170, MS (MALDI), found
m/z: [M]+ = 1422.70648, [M+H]+ = 1423.70989, [M+Na]+
=
1445.69214; 5e:
C77H96N20O14, Calcd 1525.7410, MS (MALDI), found m/z: [M]+ = 1525.74863,
[M+H]+ = 1526.75211, [M+Na]+ = 1548.73371; 5f: C75H96N20O14
,
Calcd
22. Han, S.; Kim, Y. Tetrahedron 2004, 60, 2447.
1501.7190, MS (MALDI), found m/z: [M]+ = 1501.74854, [M+H]+ = 1502.75188,
[M+Na]+ = 1524.73378; 5g: C77H98N20O14, Calcd 1527.7570, MS (MALDI), found
23. Procedure for the synthesis of tripeptide H2N-Arg(Pbf)-Pro-Gly-NH2 (3): To a
mixture of Boc-Pro-OH (4.43 g, 20.6 mmol) and Gly-NH2ꢀHCl (3.18 g, 29.0 mmol)
in 150 ml EtOAc were added TBTU (7.26 g, 22.6 mmol), HOBt (3.15 g, 20.6 mmol)
and DIPEA (10.50 ml, 61.0 mmol) and the mixture was stirred overnight. After
m/z: [M]+ = 1527.76467, [M+H]+
=
1528.76772, [M+Na]+ = 1550.74992; 5h:
C79H98N20O14, Calcd 1551.7790, MS (MALDI), found m/z: [M]+ = 1551.76465,
[M+H]+ = 1552.76791, [M+Na]+ = 1574.75116.