Efficient synthesis of novel conjugated 1,3,4-oxadiazole-peptides

Article abstract of DOI:10.1039/c7nj04720g

We were interested in the design and synthesis of novel bioisosteric analogues of leuprolide acetate containing the oxadiazole moiety at the C- or N-terminal of the peptide. An efficient approach for the synthesis of 2-amino-1,3,4-oxadiazoles through the reaction of hydrazide with ammonium thiocyanate and desulfurization reaction of the thiosemicarbazides using different coupling reagents was employed. These compounds are bioisosteres of the amide bond. Furthermore, the coupling of 2-amino-1,3,4-oxadiazoles at the C-terminal of leuprolide analogues was carried out, using a coupling reagent in the solution phase. On the other hand, the addition of a 2-amino-1,3,4-oxadiazole to the N-terminal of the peptide sequence was carried out through the reaction of the 2-amino-1,3,4-oxadiazole with succinic anhydride that led to the formation of a carboxylic acid moiety. Addition of the synthesized oxadiazole containing carboxylic acid to the peptide sequence was performed using a coupling reagent and on the surface of the resin. The synthesized peptides containing the oxadiazole moiety at the C- or N-terminal of the peptide sequence are peptidomimetics of leuprolide acetate. All of the synthesized peptides were purified using preparative HPLC and their structures were confirmed using HR-MS (ESI).

Full text of DOI:10.1039/c7nj04720g

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Efficient synthesis of novel conjugated 1,3,4-oxadiazole-peptides
Farhad Golmohammadi,a Saeed Balalaie,*a,b Fatima Hamdan,a Shokoofeh Maghari a
a Peptide Chemistry Research Center, K. N. Toosi University of Technology, P. O. Box 15875ꢀ4416,
Tehran, Iran, balalaie@kntu.ac.ir, Tel:+98ꢀ21ꢀ23064226, Fax: +98ꢀ21ꢀ22889403
b Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
Abstract
We were interested in the design and synthesis of novel bioisosteric analogues of leuprolide
acetate containing oxadiazole moiety at Cꢀ or Nꢀterminal of the peptide. An efficient approach
for the synthesis of 2ꢀaminoꢀ1,3,4ꢀoxadiazoles through the reaction of hydrazide and ammonium
thiocyanate and desulfurization reaction of the thiosemicarbazides via different coupling reagents
was employed. These compounds are bioisosteres of the amide bond. Furthermore, the coupling
of 2ꢀ aminoꢀ1,3,4ꢀ oxadiazoles at the Cꢀterminal of leuprolide analogues was carried out, using
coupling reagent in solution phase. On the other hand, the addition of 2ꢀ aminoꢀ1,3,4ꢀ oxadiazole
to the Nꢀterminal of the peptide sequence was done through the reaction of 2ꢀ aminoꢀ 1,3,4ꢀ
oxadiazole with succinic anhydride that led to the formation of a carboxylic acid moiety.
Addition of the synthesized oxadiazole containing carboxylic acid to the peptide sequence was
made using coupling reagent and on the surface of the resin. The synthesized peptides containing
oxadiazole moiety at the Cꢀ or Nꢀterminal of the peptide sequence are peptidomimetics of
leuprolide acetate. All of the synthesized peptides were purified using preparative HPLC and
their structures were confirmed using HRꢀMS (ESI).
Keywords: 2ꢀAminoꢀ1,3,4ꢀoxadiazoles, 2ꢀAminoꢀ1,3,4ꢀoxadiazoles containing carboxylic acid,
Peptidomimetic, Leuprolide acetate as antiꢀcancer peptide, Leuprolide acetate analogues
Dedicated to Prof. Norbert Sewald on the occasion of his birthday.
Introduction
1

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The resurgence of peptides as key compounds in a variety of fields ranging from drug discovery
and chemical biology to medical applications has been made by improved methods for their
synthesis.1ꢀ5 Over the years, peptides have been evolving as promising therapeutic agents in the
treatment of extended diseases such as cancer, diabetes and cardiovascular diseases. Meantime,
the application of peptides in a variety of other therapeutic areas is growing rapidly.6
Despite the high potential of peptides as therapeutic agents, there are problems connected with
the use of natural peptides, due to the low stability against proteolysis, resulting in a short
duration of in vivo activity and rapid metabolism. 7
Nevertheless, synthesizing of novel peptides with different modifications to improve their
activities and overcome their limitations has been a big challenge for scientists. There are
different approaches to improve the activity of peptides such as: a) cyclization of linear peptides,
8
b) chemical modifications including the incorporation of Dꢀamino acids,9 c) changing the
sequence of the peptide,10 d) synthesis of cyclopeptides or linear peptides containing bioactive
heterocyclic backbones,11 e) synthesis of pseudopeptides. 12
Bioisosteres are chemical substituents that are used to replace some important functional groups
such as: carboxylic acid, amide bond etc. affording similar biological properties. The reason
behind using bioisosteres is to obtain new analogues with better properties. Such replacements
are the toolbox of medicinal chemists to optimize their lead structures with respect to
lipophilicity, solubility, activity, selectivity, absorption, metabolism, lack of toxic and other side
effects. Amide isosteres are generally introduced to modulate polarity and bioavailability. 13
Peptidomimetic approach contains the building block structures that can be used as bioisosteric
replacement of the amide bond in the peptide backbone to generate compounds with improved
pharmacokinetic profiles. In this approach, amide bond bioisosteres can affect the active
conformation of the peptide. 14
1,3,4ꢀOxadiazoles are known amide bioisosteres and have drawn attention in drug design. 15 2ꢀ
Aminoꢀ1,3,4ꢀoxadiazoles also display a wide range of biological activities. 16 There are different
reported approaches for the synthesis of oxadiazoles.17 Efficient syntheses of 2,5ꢀdisubstituted
1,3,4ꢀoxadiazoles from isothiocyanates and hydrazides through cyclodesulfurization in the
presence of TBTU as an uronium coupling reagent was developed in our group.18
Studies of peptides (cyclic or linear) containing heterocycles within their backbones, have
demonstrated that heterocyclic scaffolds can influence the conformational properties and
2

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biological activity of the peptide.19 Leuprolide acetate is a synthetic nonapeptide (Figure 1) that
is a potent gonadotropinꢀreleasing hormone receptor (GnꢀRH) agonist used for diverse clinical
applications, including the treatment of prostate cancer. 20
Figure 1. The peptide sequence of leuprolide acetate
Herein, we report the synthesis of leuprolide acetate and their conjugated 1,3,4ꢀoxadiazoleꢀ
peptides at the Cꢀor Nꢀterminal of the peptide. Oxadiazole skeleton replaced the ethyl amide
group at the Cꢀterminal to access the conjugated 1,3,4ꢀoxadiazoleꢀpeptides (2a-e). On the other
hand, 2ꢀaminoꢀ1,3,4ꢀoxadiazole was coupled to the Nꢀterminal through modifying its amine
group to a carboxylic acid moiety via reaction with succinic anhydride thus affording the
conjugated 1,3,4ꢀoxadiazoleꢀpeptides (3a-e) (Scheme 1).
Scheme 1. Synthesis of leuprolide analogues containing oxadiazole moiety
Results and discussion:
3

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To achieve the conjugated oxadiazoleꢀpeptide, at first the linear protected peptide (Fmocꢀ
His(Trt)ꢀTrp(Boc)ꢀSer(tBu) ꢀTyr(tBu)ꢀDꢀLeuꢀLeuꢀArg(Pbf)ꢀProꢀ2ꢀCTC) was synthesized on the
surface of the 2ꢀchloroꢀtritylchloride (2ꢀCTC) resin through the standard solid phase peptide
21
synthesis (SPPS) strategy. At first, FmocꢀProꢀOH was added to the surface of resin using
diisopropylethylamine (DIPEA) and after capping with acetic anhydride other Fmocꢀprotected
amino acids were added to the peptide sequence using TBTU as coupling reagent and also HOBt
was used to prevent racemization. Deprotection of the Fmoc group was achieved by treatment of
the resin with 25% piperidine in DMF. After adding the nine amino acids, the desired fully
protected nonapeptide on the surface of resin 1 was obtained. The nonapeptide on the surface of
resin can be used to access leuprolide acetate and other targeted conjugated 1,3,4ꢀoxadiazoleꢀ
peptides. To access leuprolide acetate, the following steps were made a) the supported peptide 1
was cleaved using 1% TFA and compound 4 was formed. b) Then, ethylamidation was
accomplished in solution phase using ethylammonium chloride and TBTU as coupling reagent in
the presence of DIEA as base affording compound 5. c) The final deprotection was done using
reagent K (TFA/TES/Water 95:2.5:2.5). d) The purification of the peptide was done using
preparative HPLC (Column Cꢀ18, Eurospher 100, 7 ꢁm). The structure of the desired leuprolide
acetate was approved using HRꢀMS (ESI). (Scheme 2)
4

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Scheme 2. General procedure for the synthesis of Leuprolide
To access the conjugated 1,3,4ꢀoxadiazolesꢀpeptide, protected nonapeptide 1 and 2ꢀaminoꢀ1,3,4ꢀ
oxadiazole can be used. There are extended methods for the synthesis of 2ꢀaminoꢀ1,3,4ꢀ
oxadiazoles but some of them have limitations such as byproduct formation, handling of harsh
and toxic reagents and longtime reaction. 22 Recently, trimethylsilyl isothiocyanate (TMSNCS)
was used as a suitable reagent for the synthesis of 2ꢀaminoꢀ1,3,4ꢀoxadiazoles through
23
thiosemicarbazide. Here, we present a practical method for the synthesis of 2ꢀaminoꢀ1,3,4ꢀ
oxadiazoles through the reaction of aryl hydrazides with ammonium thiocyanate instead of
TMSNCS that led to the desired thiosemicarbazide intermediate (8a-e) and its cyclization
accompanied by desulfurization was done using coupling reagents such as N,N'ꢀ
diisopropylcarbodiimide (DIC) or TBTU to afford compounds (9a-e). (Scheme 3)
5

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Scheme 3. Synthesis of (9a-e) through cyclization of thiosemicarbazide (8a-e) using coupling reagent
The aforementioned reaction was studied with different coupling reagents such as DCC, DIC,
and TBTU. The optimized reaction condition with different solvents such as DMF, MeOH, and
CH3CN was studied. The best results were achieved using DIC in acetonitrile as solvent (Entry 6
in Table 1).
Table 1. The optimization reaction condition for the synthesis of 9a
Entry
Coupling reagents
Solvent
DMF
Time (h)
22
Yield %
65
DIC
1
2
DCC
DMF
26
40
TBTU
TBTU
TBTU
DIC
DMF
CH3CN
MeOH
CH3CN
MeOH
14
24
24
24
24
65
3
4
5
6
7
ꢀ
ꢀ
70
DIC
Trace
The structure and yields of the synthesized 2ꢀaminoꢀ1,3,4ꢀoxadiazoles (9a-e) are shown in Figure
2.
6

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Figure 2. Structure of the synthesized 2ꢀaminoꢀ1,3,4ꢀoxadiazoles 9a-e
The possible mechanism for the synthesis of 2ꢀamino oxadiazoles (9a-e) is shown in Scheme 4.
This conversion involves the initial nucleophilic addition reaction of the sulfur of
thiosemicarbazide with the electrophilic center of the carbodiimide to form the desired
intermediate (A), then the subsequent cyclization of intermediate (A) followed by the elimination
of the diisopropylthiourea group leads to the desired product (Scheme 4).
Scheme 4. The proposed mechanism for the synthesis of 2ꢀaminoꢀ1,3,4ꢀoxadiazole (9a-e)
Meanwhile, in another try to synthesize 2ꢀaminoꢀ1,3,4ꢀoxadiazole 9f, at first phenylalanine
methyl ester hydrochloride was transformed to the Bocꢀphenylalanine methyl ester derivative
and its further reaction with hydrazine hydrate led to Bocꢀphenylalanine hydrazide 6f that was
7

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reacted with phenylisothiocyanate 7b to give thiosemicarbazide 8f the protected phenylalanine
oxadiazole (B) was formed using TBTU in DMF, and after Boc deprotection with TFA (50%)
chiral oxadiazole 9f was obtained as shown in Scheme 5. The selection of this approach was
related to the solubility of the starting material 6f.
Scheme 5. General method for the synthesis of compound 9f
The protected peptide sequence (1) (PyrꢀHis(Trt)ꢀTrp(Boc)ꢀSer(tBu)ꢀTyr(tBu)ꢀDꢀLeuꢀLeuꢀ
Arg(Pbf)ꢀProꢀOꢀ2ꢀCTC Resin) was synthesized using SPPS approach. After cleavage of the
peptide from the resin surface, 2ꢀamino oxadiazole derivatives (9a-d,9f) were added to the
peptide sequence 1 at the Cꢀterminal using TBTU as a coupling reagent in the presence of
DIPEA (Scheme 6).
8

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Scheme 6. Synthetic procedure for adding oxadiazole moiety to the Cꢀterminal of peptide sequence
The structures of the synthesized peptide analogs were confirmed using high resolution mass
+
spectrometry (HRMSꢀESI). For example, the distinguished peak of compound 2e of [M+H]
was 1444.7265 that confirmed the attachment of the peptide to the oxadiazole moiety.
On the other hand, adding the 2ꢀaminoꢀ1,3,4ꢀoxadiazole derivative to the peptide sequence at the
Nꢀterminal requires a carboxylic acid moiety. The reaction of the 2ꢀaminoꢀ1,3,4ꢀoxadiazole with
succinic anhydride as a linker to form the desired carboxylic acid oxadiazole derivatives is used
(Scheme 7). 24 Figure 3 summarizes the obtained oxadiazole backbone containing carboxylic acid
moiety 11a-e.
O
N
N
O
N
O
N
H
N
O
HN
O
N
O
H
MeOH,rt
NH2
O
OH
9f
11d
Scheme 7. Synthesis of oxadiazole backbone containing carboxylic acid 11d
The structures of the synthesized compounds were confirmed using spectroscopic data that is
enclosed in the supporting information.
9

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Figure 3. Structure of oxadiazole backbone containing carboxylic acid products 11a-e
In another try, protected octapeptide (NH2ꢀHis(Trt)ꢀTrp(Boc)ꢀSer(tBu)ꢀTyr(tBu)ꢀDꢀLeuꢀLeuꢀ
Arg(Pbf)ꢀProꢀOꢀ2ꢀCTC resin) 13 was synthesized using SPPS approach, then oxadiazoles
containing carboxylic acid moiety 11a-e were added to the Nꢀterminal of the peptide sequence
using TBTU as coupling reagent in the presence of DIEA as base and finally after cleavage using
1% TFA the conjugated oxadiazoleꢀoctapeptides 14a-e were formed. In this case, functionalized
oxadiazole was attached to the Nꢀterminal of the synthesized peptide instead of the pyroglutamic
acid moiety. After the cleavage of the peptide analog from the resin, amidation of the Cꢀterminal
was achieved using ethylammonium chloride and also TBTU as coupling reagent in the presence
of DIEA. Then final deprotection was done using reagent K and desired conjugated 1,3,4ꢀ
oxidiazoleꢀlinkerꢀpeptides 3a-e were formed. The procedure for the synthesis of the Nꢀterminal
oxadiaolylꢀpeptide analogs is shown in Scheme 8.
10

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Scheme 8. Synthetic procedure for the synthesis of conjugated oxadiazoleꢀoctapeptide 3a-e
The structures of the synthesized peptide analogues were confirmed using high resolution mass
+
spectrometry HRMS (ESI). For example, the distinguished peak of compound 3d of [M+H]
was 1460.75853 that confirm attaching of the peptide to the oxadiazole moiety.
In conclusion, we have described the design and synthesis of novel conjugated oxadiazolyl
peptides, which represent the peptidomimetic structures of leuprolide acetate. We have
established a strategy for the incorporation of the oxadiazolyl moiety onto the peptide backbone
at Cꢀ or ꢀ N ꢀ terminal. Further studies to assemble the biological properties of the formed
products are ongoing and will be reported in due course.
Experimental section:
General Information. Commercially available materials were used without further purification.
2ꢀClꢀTrt resin and the amino acids were purchased from Iris. N,NꢀDiisopropylethylamine (DIEA)
from SigmaꢀAldrich. Organic Solvents (DMF, DCM, MeOH and CH3CN) were purchased from
Merck. RPꢀHPLC quality acetonitrile and water were used for RPꢀHPLC analyses and
purification. Melting points were recorded using an electrothermal capillary melting point
11

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apparatus and were uncorrected. Fourier transform infrared (FTꢀIR) spectra were recorded as
1
KBr pellets using an ABB FTꢀIR FTLA 2000 spectrophotometer. The H NMR (300 MHz) and
13C NMR (75 MHz) spectra were recorded on BRUKER NMRꢀSpectrometer. Chemical shifts
were reported in parts per million (δ) using TMS as internal standard and DMSO (d6) as the
solvent, and coupling constants were expressed in Hertz. High resolution mass spectra were
recorded on MassꢀESIꢀPOS (Apex QeꢀFTꢀICR instrument) spectrometer.
Peptides were synthesized on an AAPTEC FocusꢀXi instrument using standard Fmoc solidꢀphase
peptide synthesis chemistry. The amino acids were used: FmocꢀProꢀOH, FmocꢀArg(pbf)ꢀOH,
FmocꢀLeuꢀOH, FmocꢀDꢀLeuꢀOH, FmocꢀTyr(tBu)ꢀOH, FmocꢀSer(tBu)ꢀOH, FmocꢀTrp(Boc)ꢀ
OH, FmocꢀHis(Trt)ꢀOH, HꢀPyrꢀOH
General procedure for the synthesis of thiosemicarbazide intermediate (8a-e):
To a solution of ammonium thiocyanate 7a (0.19 g, 2.5 mmol) in water (2ml), hydrochloric acid
1M (2ml) was added. The reaction mixture was magnetically stirred at 110ꢀ130 °C for 45 min to
in situ produce isothiocyanic acid intermediate. Then arylhydrazide (1 mmol) was added to the
reaction mixture with continued stirring and heated for 5h. The progress of the reaction was
monitored by TLC (nꢀhexane: EtOAc: MeOH 3:1:1). After the completion of the reaction, ice
was added to reaction mixture and the resulting precipitate of thiosemicarbazide was collected by
filtration, washed with water and dried.
2-Benzoylhydrazine-1-carbothioamide (8a):
1
mp: 179ꢀ181ᵒC; IR (KBr, cmꢀ1) ν = 1545, 1701, 3388,3526 cmꢀ1; HꢀNMR (300 MHz, DMSOꢀ
d6): δ(ppm) 3.35 (brs, NH2, exchange with water), 7.42ꢀ7.68 (m, 5H, 3HꢀAr, NH2), 7.89 (d, 2H, J
= 7.7, HꢀAr), 9.34 (s, 1H, NH), 10.38 (s, 1H, NH); 13CꢀNMR (75 MHz, DMSOꢀd6): δ(ppm)
127.9, 128.2, 131.8, 132.5, 165.9, 182.1.
General procedure for the synthesis of 2-amino-oxadiazoles 9a-e:
A oneꢀnecked flask was charged with DIC (Coupling reagent) (0.24 ml, 1.5 mmol),
thiosemicarbazide (1mmol), diisopropylethylamine (0.27 ml, 1.5 mmol) and CH3CN (5 ml). The
reaction mixture was stirred under reflux for 24h. The progress of the reaction was monitored by
TLC (nꢀhexane: EtOAc 3:1). After completion of the reaction, the product was isolated by
12

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filtration and the precipitate was washed with MeOH then dried. For further purification, the
product was recrystallized in EtOH to afford of desired products (9a-e) in 52%ꢀ83% yields.
5-phenyl-1,3,4-oxadiazol-2-amine (9a):
ᵒ
White powder (0.113 g, 0.7 mmol, yield 70%). mp: 242ꢀ243 C; IR (KBr, cmꢀ1) ν = 1680, 3196
cmꢀ1; 1HꢀNMR (300 MHz, DMSOꢀd6): δ(ppm) 7.25 (s, 2H, NH2), 7.42ꢀ7.58 (m, 3H, HꢀAr), 7.75ꢀ
13
7.80 (m, 2H, HꢀAr); CꢀNMR (75 MHz, DMSOꢀd6): δ(ppm) 124.4, 125.1, 129.2, 130.3, 157.3,
163.9.
General procedure for the synthesis of 1,3,4 –oxadizaole (11a-e) using succinic anhydride:
1 mmol of 2ꢀamino oxadiazole (9b-f) and succinic anhydride (0.1 g, 1 mmol) were combined in
MeOH (5 ml), in a 20ꢀml roundꢀbottom flask at room temperature and stirred for 10 h. The
progress of the reaction was monitored using TLC (nꢀHexane: EtOAc 1:1). After completion the
reaction, the solvent was evaporated under vacuum and the resultant precipitate was washed with
diisopropylether and filtered. The yields of products (11a-e) were 68%ꢀ96%.
4-Oxo-4-((5-(p-tolyl)-1,3,4-oxadiazol-2-yl) amino) butanoic acid (11a):
White powder (0.259 g, 0.94 mmol, yield 94%). mp: 209ꢀ213 ᵒC; IR (KBr, cmꢀ1) ν = 1655, 1734,
1
3448 cmꢀ1; HꢀNMR (300 MHz, DMSOꢀd6): δ(ppm) = 2.34 (s, 3H, ꢀCH3), 2.46ꢀ2.49 (m, 4H, ꢀ
CH2), 7.12 (s, 1H, NH), 7.31 (d, 2H, J = 8.1 Hz, HꢀAr), 7.67 (d, 2H, J = 8.1 Hz, HꢀAr), 12.0 (brs,
13
1H, ꢀCOOH); CꢀNMR (75 MHz, DMSOꢀd6): δ(ppm) = 21.1, 28.5, 28.7, 121.7, 125.1, 129.8,
140.3, 157.5, 163.7, 172.7, 173.5.
General Method for the synthesis of leuprolide analogues (2a-e) by coupling oxadiazole (9a-
d,9f) to the peptide sequence (4) at C-terminal:
To a solution of nonapeptide (4) (1.2 g, 0.64 mmol) in DMF (1.8 ml), NMM (0.39 ml, 3.2 mmol)
was added and stirred for 15 min. Then TBTU (0.306 g, 0.96 mmol) was added and The reaction
mixture was magnetically stirred for 30 min to activate the carboxylic acid group at Cꢀterminal,
after that 1.3 mmol of oxadiazole (9a-d,9f) was added to the reaction mixture. The mixture was
stirred for 24 h at room temperature and the progress of reaction was monitored using TLC
(EtOAc: MeOH: H2O 10:2:1). The desired Cꢀterminal amidated unprotected nonapeptides (10a-
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e) was precipitated in water and dried. Final deprotection of the peptides (10a-e) was done by the
same procedure mention previously to give leuprolide analogues (2a-e), followed further
purification was done using PrepꢀHPLC by the same procedure of purification.
(2a)
HPLC analysis found that peptide (2a) was obtained in 94%< purity (Rt: 45.497 min). mp: 107ꢀ
110°C; HRꢀMass (ESI): C69H92N18O13m/z = [M+ (CH3CN+H2O) +H]+ Found 1434.6868, Calc.
1434.6867; [M+ (CH3CN+H2O) +2H] +/2 Found 717.8472, Calc. 717.8472.
(2b)
HPLC analysis found that peptide (2b) was obtained in 96%< purity (Rt: 32.883 min). mp: 182ꢀ
+
184°C; HRꢀMass (ESI): C66H86N18O13m/z =[MꢀC8H7N2O+H] For Found 1196.6205, Calc.
1196.6203; [MꢀC8H7N2O +2H]+/2 Found 598.8142, Calc. 598.8143.
(2c)
HPLC analysis found that peptide (2c) was obtained in 95%< purity (Rt: 46.048 min). mp: 112ꢀ
114 °C; HRꢀMass (ESI): C66H86N18O14m/z = [M+HOAc+H2O+H]+ Found. 1434.6869, Calc.
1434.6871; [M+HOAc+H2O+2H]+/2 Found 717.8474, Calc. 717.8472.
(2d)
HPLC analysis found that peptide (2d) was obtained in 97%< purity (Rt: 32.258 min). mp: 214ꢀ
+
218°C; HRꢀMass (ESI): C65H83ClN18O13m/z =[MꢀC8H4ClN2O+H] Found 1182.6048, Calc.
1182.6050; [MꢀC8H4ClN2O +2H]+/2 Found 591.8063, Calc. 591.8065.
(2e)
HPLC analysis found that peptide (2e) was obtained in 96%< purity (Rt: 39.063 min). mp: 94ꢀ
96°C; HRꢀMass (ESI): C73H93N19O13m/z =[M+H] +Found 1444.7265, Calc. 1444.7264;
[M+2H]/2+ Found 722.8669, Calc. 722.8668.
General procedure for the synthesis of leuprolide analogues (3a-e) by attaching
functionalized oxadiazole 11a-e to the N-terminal of the peptidyl-resin sequence (12):
14

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After synthesis of the peptidylꢀresin sequence (12) as mentioned previously, pyroglutamic acid
was replaced with the functionalized oxadiazole (11a-e). In this way at first deprotection of the
FmocꢀHis was made, and then a solution of oxadiazole (11a-e) (2 mmol), TBTU (0.64 g, 2.0
mmol), DIPEA (0.6 ml, 3.5 mmol) in 10 ml DMF was added to the resinꢀbound free amine (13)
and shaken for 2 h at room temperature. Completion of the coupling was monitored by the
Kaiser test. The resin was washed with DMF (3×10ml). Cleavage, ethyl amidation and final
deprotection of the peptide were done by the same procedure mention previously to give
leuprolide analogs (3a-e), followed further purification was done using PrepꢀHPLC by the same
procedure of purification.
(3a)
HPLC analysis found that peptide (3a) was obtained in 95%< purity (Rt: 34.77 min). mp: 142ꢀ
+
145°C; HRꢀMass (ESI): C67H90N18O13m/z = [MꢀC9H7N3O+H]
1185.6049; [MꢀC9H7N3O+2H] +/2 Found 593.3061, Calc. 593.3062.
(3b)
Found 1185.6048, Calc.
HPLC analysis found that peptide (3b) was obtained in 96%< purity (Rt: 34.428 min). mp: 119ꢀ
+
121°C; HRꢀMass (ESI): C67H90N18O14m/z = [MꢀC8H7N2O+H] Found 1212.6524, Calc.
1212.6525; [MꢀC8H7N2O+2H] +/2 Found 606.8296, Calc. 606.8295.
(3c)
HPLC analysis found that peptide (3c) was obtained in 94%< purity (Rt: 35.398 min). mp: 121ꢀ
123°C; HRꢀMass (ESI): C66H87N18ClO14m/z = [MꢀC8H5ClN3O+H]+ Found 1185.6050, Calc.
1185.6051; [MꢀC8H5ClN3O+2H]+/2 Found 593.3062, Calc. 593.3061.
(3d)
HPLC analysis found that peptide (3d) was obtained in 97%< purity (Rt: 43.682 min). mp: 104ꢀ
106°C; HRꢀMass (ESI): C74H97N19O13m/z =[M+H] +Found 1460.7585, Calc. 1460.7585; [M+2H]
+/2 Found 730.8824, Calc. 730.8824.
(3e)
HPLC analysis found that peptide (3e) was obtained in 96%< purity (Rt: 35.988 min). mp: 108ꢀ
110°C; HRꢀMass (ESI): C66H87N19O15m/z = [MꢀC8H5N3O3+H]+ Found 1185.6047, Calc.
1185.6046; [MꢀC8H5N3O3+2H]+/2 Found 593.3061, Calc. 593.3060.
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Acknowledgments:
We thank the Iran National Science Foundation (INSF) (Grant No.960003234) and National
Institute for Medical Research Development (NIMAD) (Grant No. 943185) for their financial
support.
References and notes:
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Efficient synthesis of novel conjugated 1,3,4-oxadiazole-peptides
Farhad Golmohammadi,a Saeed Balalaie,*a,b Fatima Hamdan,a Shokoofeh Maghari a
Table of Content:
In this study, we developed an efficient approach for the synthesis of 2-amino-1,3,4-oxadiazoles
that are bioisostere of amide functional group. The synthesized oxadiazoles were conjugated to
octa-, and nonapeptides through C- or N- terminus as precursors of leuprolide acetate. The
synthesized compounds are peptidomimetics of leuprolide acetate.