Synthesis and conformational study of novel pyrazine-based pseudopeptides bearing amidoxime, amidoxime ester and 1,2,4-oxadiazole units

Article abstract of DOI:10.1016/j.tet.2016.04.069

An efficient synthesis of pyrazine-based amidoximes bearing amino acid residues is reported. Esterification of amidoximes with further microwave-assisted cyclization into α-amino acid-derived 1,2,4-oxadiazoles has been developed. Conformational analysis o

Full text of DOI:10.1016/j.tet.2016.04.069

Accepted Manuscript
Synthesis and conformational study of novel pyrazine-based pseudopeptides bearing
amidoxime, amidoxime ester and 1,2,4-oxadiazole units
Olga V. Ovdiichuk, Olga V. Hordiyenko, Axelle Arrault
PII:
S0040-4020(16)30346-5
10.1016/j.tet.2016.04.069
TET 27712
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 11 February 2016
Revised Date: 12 April 2016
Accepted Date: 22 April 2016
Please cite this article as: Ovdiichuk OV, Hordiyenko OV, Arrault A, Synthesis and conformational study
of novel pyrazine-based pseudopeptides bearing amidoxime, amidoxime ester and 1,2,4-oxadiazole
units, Tetrahedron (2016), doi: 10.1016/j.tet.2016.04.069.
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Synthesis and Conformational Study of Novel
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Pyrazine-Based Pseudopeptides Bearing Amidoxime,
Amidoxime Ester and 1,2,4-Oxadiazole Units
Olga V. Ovdiichuka,b, Olga V. Hordiyenkoa,* and Axelle Arraultb
aTaras Shevchenko National University of Kyiv, Chemistry Department, 64/13 Volodymyrs’ka str., Kyiv, 01601,
Ukraine
bLaboratoire de Chimie Physique Macromoléculaire, ENSIC, Université de Lorraine, 1 rue Grandville, BP 451,
54001 Nancy, France
R
R
CO2Me
O R
O
HN CO2Me
O
N
HN CO2Me
N
N
N
N
N
N
N
N
N
N
NH CO2Me
OH
N
O
N
O
O
NHBoc
R'
up to 95% yield
N
N
NHBoc
R'
OH
O
NH2
N
O
NH2
trans/cis:
from 54/46 to 76/24
up to 100% yield
up to 93% yield

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Synthesis and Conformational Study of Novel Pyrazine-Based Pseudopeptides
Bearing Amidoxime, Amidoxime Ester and 1,2,4-Oxadiazole Units
Olga V. Ovdiichuka,b, Olga V. Hordiyenkoa,* and Axelle Arraultb
aTaras Shevchenko National University of Kyiv, Chemistry Department, 64/13 Volodymyrs’ka str., Kyiv, 01601, Ukraine
bLaboratoire de Chimie Physique Macromoléculaire, ENSIC, Université de Lorraine, 1 rue Grandville, BP 451, 54001 Nancy, France
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
An efficient synthesis of pyrazine-based amidoximes bearing amino acid residues is reported.
Esterification of amidoximes with further microwave-assisted cyclisation into α-amino acid-
derived 1,2,4-oxadiazoles has been developed. Conformational analysis of the obtained
pseudopeptides by NMR and FT-IR spectroscopy revealed intramolecular interactions due to the
influence of a pyrazine ring and prolyl amide bond isomerization. The content of trans/cis
conformers depending on the substitution at the pyrazine moiety for Pro-containing derivatives
has been estimated.
Available online
Keywords:
Pyrazine
Coupling
2009 Elsevier Ltd. All rights reserved
.
Amidoximes
1,2,4-oxadiazoles
Peptidomimetics
trans/cis conformers
1. Introduction
amidoxime function. On the other hand, esterification and/or
condensation of amidoximes presents a route towards the novel
double prodrugs of amidines.7 Therefore, the synthesis and
characterization of novel amidoximes, esterified amidoximes and
1,2,4-oxadiazoles are topics of our great interest.
Heterocycle-containing amino acids have become important
precursors in the design of peptidomimetics. The pyrazine ring
can improve the binding affinity and bioavailability of potential
drugs. A number of compounds containing a pyrazine moiety
(VE-821, Fig. 1) are in late stage clinical trials or have reached
the market (bortezomib, Fig. 1). In addition, an amidoxime group
introduced into the structure can be employed as an amidine
replacement, an excellent mimic of the arginine side chain.1 The
prodrug approach “Amidoximes instead of amidines” has a
growing interest due to enhanced physicochemical properties and
oral bioavailabilities.2 Certain amidoximes have been
demonstrated to be reduced by the mARC-containing enzyme
system to the corresponding amidines,3 this approach was
successfully applied to the thrombin inhibitor ximelagatran (Fig.
1), an amidoxime prodrug.4 Furthermore, the synthesis of
amidoximes has attracted attention because of their use as
precursors in the preparation of 1,2,4-oxadiazoles that have wide
range of applications in medicinal chemistry.5
N
N
NH2
O
OH
B
O
N
N
NH
N
O
OH
HN
H
O
S
O
VE-821
H2N
bortezomib
N
OH
O
O
O
O
NH
N
N
H
ximelagatran
Fig. 1. Selected examples of biologically active pyrazine and amidoxime
compounds.
Building on our experience with the synthesis of nicotinic acid
based pseudopeptides,8 in this paper, we describe the synthesis of
novel pyrazine based pseudopeptides containing 1,2,4-
oxadiazolylpyrazines with an amino acid substituted pyrazine
core. Compounds were designed to possess o-substitution and to
be considered as non-peptidic turn structures. The extra nitrogen
atom introduced gives rise to the potential for the pyrazine ring to
provide a site for H-bonding.
1,2,4-oxadiazoles have long been known as efficient
amide/ester bioisosteres, an efficient peptide bond replacement in
constructing
peptidomimetics.6
These
1,2,4-oxadiazole-
containing surrogates cannot be cleaved by enzymes and
therefore prolong availability of molecules. Therefore, we were
interested in introducing the 1,2,4-oxadiazole ring into pyrazine-
based pseudopeptides by transformation of the present
———
* Corresponding author. Tel.: +38-044-239-3315; fax: +38-044-235-1273; e-mail: ov_hordiyenko@univ.kiev.ua.

2
Tetrahedron
ACCEPTED MANUSCRIPT
2. Results and discussion
O
N
CO2Me
CN
N
CO2H
N
CO2H
CN
Cl OMe
NaOH
2.1. Synthesis of pyrazine-based pseudopeptides
TEA, DCM
2 h, 0 °C
MeOH
3 h, rt
N
N
CONH2
N
Recently, amino acid derived pyrazine carboxamides have
been synthesized from pyrazine-2,3-dicarboxylic anhydride and
amino acids in refluxing toluene, and the conversion was
accompanied by decarboxylation along with pyrazine-2,3-
dicarboximide formation in some cases.9 Our studies began with
the synthesis of amino acid derived pyrazine-2-carboxamides
with amidoxime groups at position 3. The synthesis carried out in
two steps utilizing the strategy elaborated before for the pyridine
series8: coupling of 3-cyanopyrazine-2-carboxylic acid (3) with
several methyl esters of L-α-amino acids was followed by further
reaction of the obtained nitriles with hydroxylamine
hydrochloride. Starting acid 3 was synthesized from readily
available 3-carbamoylpyrazine-2-carboxylic acid (1) by treatment
with methyl chloroformate10 followed by selective hydrolysis of
the ester group with 1M NaOH to give the corresponding acid 3
in 71% overall yield (Scheme 1).
2
1
3
Scheme 1. Synthesis of starting 3-cyanopyrazine-2-carboxylic
acid 3.
Esters of (2S)-N-(3-cyanopyrazin-2-yl)carbonyl substituted
amino acids 4 were prepared by coupling of 3-cyanopyrazine-2-
carboxylic acid (3) with commercially available methyl esters of
L-alanine, L-phenylalanine and L-proline amino acids according
to a previously developed protocol8 (Table 1). As an activating
agent,
N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDCI) in the presence of 1-hydroxybenzotriazole
(HOBt) was used.11 As a solvent, tetrahydrofuran instead of
dichloromethane was taken due to the low solubility of the
starting material in dichloromethane.
Table 1
Synthesis of Ala-, Phe-, Pro-containing pseudodipeptides 4-6a
O
R
O
O
R
O
N
(ii)
N
N
N
N
(i)
N
N
R
X
N
CO2Me
OH
OH
N
X
N
CO2Me
N
+
CO2Me
N
N
NH2
6a-c
(E) H
3
4a-c
5a,b
Entry
1
X
R
4 yield (%)b
5 yield (%)b
O
6 yield (%)b
O
O
N
N
N
CO2Me
NH
N
N
CO2Me
N
NH
N
NH
Me (a)
CO2Me
N
N
OH
N
4a 23%
NH2
H
H
5a 28%
5b 65%
6a 84%
O
N
N
O
O
N
N
N
CO2Me
2
NH
CH2Ph (b)
N
N
NH
CO2Me
CO2Me
NH
N
N
N
OH
NH2
4b 3%
6b 63%
CO2Me
N
CO2Me
N
N
N
O
3
-
N
O
N
N
(c)
N
OH
N
NH2
4c 49%
6c 93%
aReaction conditions: (i) 3 (5.0 mmol), H-L-Ala-OMe (a), or H-L-Phe-OMe (b), or H-L-Pro-OMe (c) (5.0 mmol), Et3N (10.0 mmol),
HOBt (5.0 mmol), EDCI (5.05 mmol), THF (30 mL), 18 h; (ii) 4 and/or 5 (2.0 mmol), hydroxylamine hydrochloride (4.0 mmol), Et3N
(4.0 mmol), MeOH (15 mL), 18 h.
bIsolated yields.
1
Along with the desired methyl esters of amino acid 4a,b a
tautomeric pyrrolopyrazine products 5a,b were produced.
Separation and purification of derivatives 4 and 5 was achieved
by column chromatography and allowed us to isolate the open
4a,b and cyclic 5a,b products in different ratios (Table 1). When
alanine was used, the content of both products 4a and 5a was
approximately the same while in the case of phenylalanine the
cyclic form 5b predominated. The coupling with proline methyl
ester necessarily afforded only the open-chain pyrazine derivative
4c in 49% yield.
analogy to the pyridine counterparts, the H NMR spectra of the
coupling products 4, 5 revealed NH proton resonances at δ = 8.26
and 8.20 ppm for the open-chain compounds 4a,b, and a
downfield shift of δ = 9.51 and 9.46 ppm for the tautomeric
cyclic derivatives 5a,b. Their values are very close to the values
of those for the pyridine series 9.34 (L-Ala) and 9.29 (L-Phe)
ppm.8 Therefore, one could conclude by analogy with [8] that
pyrrolopyrazines 5a,b adopt the E configuration at the C=N
exocyclic bond and syn orientation of the NH proton to the
heterocyclic ring.
The IR spectra of 4a-c showed a characteristic cyano group
Amidoximes 6 could be prepared efficiently from single
compounds 4 or 5 as well as from the mixture of 4 and 5 in a
absorption band at 2240, 2237 and 2239 cm-1, respectively. By

3
reaction with hydroxylamine hydrochloride (Table 1). Thus, as in
the case of the pyridine analogues, the products 6 were formed as
a result of addition to nitriles 4 as well as pyrrolidine ring
opening of pyrrolopyrazines 5.
conjugation of amidoximes with L-valine amino acid can
ACCEPTED MANUSCRIPT
enhance water solubility and bioavailability of the molecules,
thus the latter was employed as one of the carboxylic residues.7a
L-phenylalanine amino acid was introduced into proline
derivative 6c in order to investigate a propensity for cis amide
bonds which is high when Pro is preceded by an aromatic
residue.14 This is considered as a stabilizing Ar-Pro interaction in
the cis conformation. Hence, the amidoxime 6c was reacted with
Boc-protected phenylalanine to produce Phe-amidoxime ester 7c
in quantitative yield. Indeed, the population of the molecules in
the cis conformation in 7c was slightly increased (29%) relative
to that in 6c (26%).
As a part of our ongoing program to develop non-peptidic turn
structures, we then turned our attention to 1,2,4-oxadiazoles. It is
known that 1,2,4-oxadiazoles can generally be formed by the
reaction of an amidoxime with a carboxylic acid or an acid
chloride.12a The application of microwave energy can be
employed to shorten the reaction time and/or increase product
purities and yields.12b The synthesis of chiral 1,2,4-oxadiazoles
from amino acids has been reported before using long reaction
times at high temperature13a-c or under MW irradiation.13d,e
Heating esters 7a-c under microwave irradiation afforded the
1,2,4-oxadiazoles 8a-c in high yields (Table 2). Different
solvents, temperatures and prolonged heating were explored in
order to provide the best conversion. Phenylalanine derivative 7b
was selected as a model compound. The results are summarized
in Table 3. The reaction in dichloromethane at 150 °C for 25 min
under microwave heating afforded the highest conversion in the
shortest time as judged by LC/MS analysis (entry 3).
We describe herein an easy microwave assisted synthetic
route for the preparation of a new variety of chiral α-amino acid
derived 1,2,4-oxadiazoles. The following two-step reaction led to
the desired 1,2,4-oxadiazoles 8 which were submitted to the ring
formation under microwave irradiation of the intermediate
acylated amidoximes 7 (Table 2, 3). Firstly, Boc-protected L-
valine was esterified with the corresponding amidoximes 6a,b
using DCC/4-DMAP in anhydrous acetonitrile to obtain O-acyl
amidoximes 7a,b in good yields. Recently, it has been shown that
Table 2
Synthesis of 1,2,4-oxadiazole pseudopeptides 8
R
R
O
R'
X
CO2Me
O
O
R
X
N
CO2Me
BocHN CO2H
N
N
N
N
N
N
MW
O
N
X
N
CO2Me
OH
NHBoc
R'
NHBoc
DCC, 4-DMAP
CH3CN, rt
150 °C
25 min
DCM
N
O
NH2
R'
NH2
O
6a-c
7a-c
8a-c
Entry
1
X
R
R’
7 yield (%)a
8 yield (%)a
HN CO2Me
HN CO2Me
N
N
N
N
O
N
O
N
O
NH
Me (a)
i-Pr
NHBoc
NHBoc
O
N
O
NH2
8a 75%
8b 94%
7a 84%
7b 67%
HN CO2Me
O
HN CO2Me
2
3
NH
CH2Ph (b)
i-Pr
N
N
N
N
O
N
O
NHBoc
N
NHBoc
O
N
O
NH2
CO2Me
CO2Me
O
N
N
N
N
N
N
O
N
O
N
NHBoc
CH2Ph
N
NHBoc
O
(c)
N
O
NH2
7c quantitative
8c 76%
aIsolated yields.
Table 3
Optimization of microwave assisted condensation conditions
for 7b as a model compound
To the best of our knowledge, the synthesis of 1,2,4-
oxadiazolylpyrazines with amino acid substituted pyrazines and a
chirally substituted 1,2,4-oxadiazole ring is unprecedented. The
only examples of pyrazines bearing proline and a 1,2,4-
oxadiazole unit have been described recently.15
Entry
Solvent
T, °C
Time, min
Conversion of
7b, %a
85
1
2
3
4
CH3CN
THF
DCM
DMF
160
150
150
150
50
50
25
60
2.2. Structural and thermodynamic studies of proline derivatives
77
96
49
The singular behavior of proline in peptide and protein folding
represents another way to introduce different turn motives into
the amino acid backbone due to its N-terminal extremity that is
involved in a cycle. However, unlike the majority of natural
aConversion is determined by crude LC/MS.

4
Tetrahedron
ACCEPTED MANUSCRIPT
amino acids that adopt the trans configuration of peptidic bond,
increased cis content along the Xaa-Pro bond is significant since
both the cis and trans isomers are energetically similar.16
Fig. 2. Trans/cis conformational states of proline-containing derivatives
4c, 6c, 7c and 8c.
In this context, we wish to report the effect of substitution
with proline amino acid on the conformational behavior of the
products. Information concerning the conformation adopted by
compounds 4c, 6c, 7c and 8c in solution was obtained by 1D, 2D
NMR and FT-IR absorption techniques. The 1H, 13C NMR
spectra of 4c, pseudopeptides 6c, 7c and 1,2,4-oxadiazole 8c
revealed two sets of resonances at room temperature
corresponding to the cis and trans amide bond rotamers. The
major contributions into cis-trans equilibrium mixture have been
clearly determined by NMR spectroscopy to be from the trans
conformers. The assignments were made based on the 13C
chemical shifts of the α- and δ-carbons in CDCl3.17 In the Pro
derivatives, the α-carbon signal of the trans rotamer is shielded
relative to that of the cis due to the syn position to the carbonyl
oxygen of the amides. The signal of the δ-carbon, however,
should be more shielded in the cis isomer. Indeed, in the trans
conformers of 4c, 6c, 7c, 8c, the chemical shifts of the α-carbon
range from 59.3 to 60.2 ppm, while in the cis form range from
61.1 to 61.4 ppm (Table 4). The δ resonances of the trans
conformers vary from 48.3 to 49.5 ppm and lie within the range
47.2 - 48.4 ppm for the cis, respectively. Furthermore, the β-
carbon signal of the trans isomer appears upfield from the cis
isomer and the γ-carbon of the trans rotamer is downshifted
relative to that of cis rotamer.
In the cases of 6c and 7c, we observed a general tendency to
adopt the stabilized trans conformation probably due to the
presence of an n → π* interaction which involves the oxygen
lone pair of the amide carbonyl and the antibonding orbital of the
ester C=O (Figure 2). The difference in strength of this
interaction accounts for an increase in Kt/c (Table 5). The trans
conformation of 8c is stabilized due to the H-bonding of the Phe
NHBoc and Pro amide bond, although an n → π* interaction may
also contribute to its stabilization.
Furthermore we investigated the impact of temperature (in the
range 25 – 90 °C) on the trans/cis equilibrium constant (Kt/c).
The Kt/c values were measured by determining the relative
concentrations of each isomer by integration of all well-resolved
1
proton signals in each H NMR spectrum in DMSO-d6. The
resulting data are illustrated in Van’t Hoff analysis (Figure 3,
Table 5). All derivatives have
a slight positive slope
demonstrating temperature dependence. For the Pro derivative
4c, a reduction in the magnitude of Kt/c was observed,
demonstrating less enthalpic preference for the trans
conformation.
In most cases the content of the trans isomer was about 71-
76% while cis was 24-29%. However, when 4c was studied, a
decreased trans/cis ratio to 54/46 was observed. We assumed the
presence of a noncovalent intramolecular n → π* interaction
between the N of the pyrazine ring and Cco of the pyrrolidine ring
for all Pro-based derivatives (Fig. 2, structure I).14,18 However,
there is an increased amount of the cis conformation for cyano
compound 4c, as the trans product is stabilized by a relatevely
weaker interaction between the more electronegative oxygen and
the carbonyl (Fig. 2, structure II). Thus, a stronger n → π*
interaction in the cis isomer of 4c causes the difference in Kt/c
values (Table 5).
Fig. 3. Van’t Hoff plots for 4c, 6c, 7c, 8c in DMSO-d6.
O
O
O
O
Although differences in entropy favor the cis isomer, the trans
isomer in all cases is favored by enthalpy. The equivalent
constants decrease with increasing temperature in the order
7c˃8c˃6c˃4c (Table 5).
N
N
N
N
MeO
N
OMe
OMe
OMe
N
N
N
N
N
N
O
N
O
N
O
N
O
N
Boc
HN
R
O
N
Bn
O
H2N
(4c)
IV (8c)
cis- : I trans- : II
III
R = H (6c)
R = Phe-residue (7c)
Table 4
Selected carbon chemical shifts in the pyrrolidine ring of proline derivatives in CDCl3
Entry
Product
Cα
Cβ
Cγ
trans
25.6
25.2
25.4
25.5
Cδ
cis
trans
60.2
59.3
59.4
59.5
cis
trans
29.4
30.2
30.2
30.1
cis
cis
trans
49.5
48.3
48.7
48.8
1
2
3
4
4c
6c
7c
8c
61.2
61.1
61.4
61.2
31.8
31.5
31.5
31.7
22.6
23.5
23.5
23.3
48.4
47.3
47.2
47.4

5
interactions. A small shoulder of the CO-stretching band is
ACCEPTED MANUSCRIPT
observed at 1615 cm-1 in CHCl3 solution that presumably
corresponds to the weak H-bonded CO of amide bond together
with an intensive CO amide band at 1650 cm-1, which is free
from intramolecular interaction. In comparison, the amide CO
bands of 8c at 1667 and 1649 cm-1 in DMSO correspond to the
free carbonyl stretching vibrations of the cis and trans forms.
This result provides support that the NHBoc of the phenylalanine
is H-bonded with the amide carbonyl group of pyrazinyl-Pro
amide bond. This conclusion is also supported by the NH signal
shifting in the trans isomer of 8c compared with the free NH of
7c (ꢀδ NHBoc = +0.48 ppm) and a NOESY experiment for 8c in
solution in CDCl3 where a NOE correlation is observed for the
NHBoc proton when the Pro-OMe resonance was saturated (Fig.
4).
Table 5
Thermodynamic parameters for compounds 4c, 6c, 7c, 8c.
Additionaly the trans/cis equilibrium constant at 300K is
given
Proline
derivative DMSO-d6
Kt/c
ꢀH0
ꢀS0
ꢀG0
300K
(kJ mol-1) (J mol-1 K-1) (kJ mol-1)
(CDCl3)
1.30 (1.17) -1.24
2.73 (2.85) -3.43
3.37 (2.45) -4.60
3.25 (3.17) -3.51
4c
6c
7c
8c
-1.84
-2.97
-5.06
-1.84
-0.69
-2.53
-3.08
-2.96
2.3. Conformational behavior studies of pyrazine-based
pseudopeptides 7 and 8
The titration of the NH proton shift of pseudopeptide 8c
revealed that DMSO-d6 as a strong hydrogen-bonding solvent
produces a significant downfield shifting (∆δ = 2.22/2.67 ppm).
However, the initial steps of titration reveal low downfield shifts
∆δ = 0.36 ppm (5% DMSO-d6) and ∆δ = 0.80 ppm (10% DMSO-
d6). This could be evidence of a weakly H-bonded Phe NHBoc
proton at low concentrations of DMSO-d6, that become solvent-
exposed as a result of conformational change at polar solvent
higher concentration.19
Considering that o-substitution in the structures 7 and 8 could
introduce an intramolecular hydrogen-bonding site forming turn-
like structures, the conformational tendencies were investigated
1
by FT-IR absorption and H NMR experiments. Firstly, FT-IR
spectra in CHCl3 of 7a-c (Figs. 1-6 in SI) were studied, and the
presence of non-hydrogen-bonded amide N-H bands above 3400
cm-1 demonstrate that the Boc and ester carbonyl groups are not
H-bonded. IR-spectra of 8a,b exibit NH stretching vibrations at
3397 cm-1 and 3395 cm-1, respectively. It may be due to
involvement of the NH amidic protons and the pyrazine moiety
in an NH - π interaction (Fig. 4). This is in agreement with the 1H
NMR data showing the downfield shifting ꢀδ NHamide = +0.23
ppm on going from 7a,b to 8a,b (Table 6). It is also notable that
this interaction is little affected by solvation (up to 20% of
DMSO-d6) (ꢀδ NHamide = +0.43 and +0.39 ppm; Fig. 14 in SI).
In addition, all compounds 4, 5 and 6 were compared with
similar ones from the pyridine series that were studied previously
(Table 7, 8).8 The coupling reaction proceeded easily for pyridine
analogues in contrast to pyrazine ones besause of a low solubility
of the latter that led to decreased yields (Table 7). The
EDCI/HOBt protocol was applied in both cases with the
difference in solvents, DCM was used for the coupling with 2-
cyanonicotinic acid and THF was found to be the best solvent for
3-cyanopyrazine-2-carboxylic acid (3). The ratio of isolated
isomeric Ala-containing open form 4a and cyclic pyrrolopyrazine
5a vary as in the case of cyanonicotinic acid. But when Phe was
coupled, we succeed in isolation of a small portion of open form
4b along with a majoring pyrrolopyrazine 5b unlike for
cyanonicotinic acid where only the cyclic pyrrolopyridine
analogue of 5b was obtained.
O
R
H
OMe
H
N
N
N
N
CO2Me
N
N
N
NOE
Boc
N
O
N
O
N
NHBoc
R'
N
O
O
(8 a,b)
(8c)
Fig. 4. Intramolecular interactions in oxadiazole derivatives 8a-c.
1
The comparison of NH proton signals in the H NMR spectra
Oxadiazole derivative 8c in its FT-IR spectrum exibits bands
due to both free NH (3438 cm-1) and hydrogen-bonded NH (3362
cm-1) stretching vibrations over the concentration of 10 mM; and
further dilution confirms the absence of solute-solute
of the pyridine and pyrazine derivatives 5 revealed a low field
shift above 1 ppm for the pyrazine compounds due to an NH - π
interaction that was absent in the case of pyridine core.8
Table 6
Сhemical shifts and solvent sensitivity of the NH protons in 7a-c and 8a-c
δ NH (ppm)
∆δ (ppm) = δ (DMSO-d6) - δ (CDCl3)
7a
7b
7c
8a
8b
8c
8a
8b
8c
(trans/cis)
5.15/5.05
-
(trans/cis)
5.63/5.17
-
(trans/cis)
2.22/2.67
-
NHBoc
NH-amide
5.17
7.82
5.15
7.72
5.20
8.05
5.19
7.95
2.59
1.17
2.58
1.28

6
Tetrahedron
ACCEPTED MANUSCRIPT
Table 7
Comparison of compounds 4, 5 (a,b) with those obtained from 2-cyanonicotinic acid8
Entry
Pyrazine derivative
νmax CN
(cm-1)
2240
δ NH
(ppm)
8.26b
yielda, % Pyridine derivative
(THF)
23
νmax CN
(cm-1)
2240
δ NH
(ppm)
7.15d
yielda, %
(DCM)
15
1
H
H
N
CO2Me
N
CO2Me
N
N
O
O
CN
N
CN
4a
2
2237
8.20c
3
-
-
-
e
e
e
Ph
Ph
H
H
N
CO2Me
N
CO2Me
N
O
O
N
N
CN
O
N
CN
O
4b
5a
3
4
-
-
9.51
9.46
28
68
-
-
9.34
9.29
58
76
N
N
N
N
N
CO2Me
CO2Me
N
H
H
H
O
N
N
O
N
N
N
N
Ph
Ph
CO2Me
CO2Me
N
H
5b
a Isolated yield. b d, J = 7.4 Hz. c d, J = 7.9 Hz. d d, J = 5.7 Hz. e Not isolated.
The general tendency for pyrrolidine ring opening of 5 by
hydroxylamine to afford the open-chain amidoximes 6 was
observed; the same structures were obtained from the
corresponding cyano esters 4 similar to the formation of pyridine
based amidoximes.
conditions in DCM and with a reduced reaction time that
afforded high product yields. Conformational behavior and the
steric effects of pyrazine substitution at position 2 on the
cis/trans equilibrium of proline-pyrazine pseudopeptides was
estimated demonstrating a general tendency for trans isomer
predomination. The increased fraction of the cis isomer up to
46% for Pro-containing 3-cyanopyrazinic acid due to the
stabilizing effect of the n → π* interaction between the pyrazine
nitrogen and Cco of the pyrrolidine ring on the cis/trans
equilibrium was observed. Thus, the pyrazine moiety in some
cases can be used as a structural motif to stabilize the cis
conformation of Pro-pseudopeptides.
Finally, the comparison of the corresponding Pro-based
derivatives was performed (Table 8). Although the decreased
yield of 4c was observed, the increased content of the cis rotamer
was estimated compared to the pyridine analogue. The
hypothesis that the presence of the extra nitrogen atom of the
pyrazine ring favors the cis conformation for cyano derivatives
has already been discussed. In the event, an approximately
similar trans/cis ratio with a little increase of the trans isomer
from pyridine to pyrazine based Pro-amidoximes was observed.
4. Experimental section
All reactions were carried out in dry solvents. Dry THF was
obtained by distillation over sodium and benzophenone; MeCN
was purchased in anhydrous form. Reagents were obtained from
commercial sources and were used without further purification.
All microwave reactions were performed using a CEM Focused
MicrowaveTM Synthesis System, Model Discover, in septa
capped 10 mL reaction vessels with stirring. The reaction
temperature was monitored by Infrared Temperature Control
System with an infrared sensor to measure temperature. The
power required to maintain the target temperature was controlled
by the Discovery System. The reaction times under microwave
conditions reflect actual reaction times at a given temperature.
The reaction was monitored by LC/MS with UV detection (254
nm) and TLC. Melting points were measured on a Buchi M–560
Melting Point apparatus. All melting points are uncorrected.
Preparative column chromatography was carried out with silica
gel 60 (40–63 ꢁm). LC/MS spectra were recorded using a high-
perfomance liquid chromatograph (UFLC Shimadzu) and LCMS-
2020 Shimadzu mass-selective detector. Parameters for LC/MS
analyses: Nucléodur RP-C18 (10 µm), 250×4.6 mm; gradient
elution of solvent A = CH3CN/H2O:1/9 + 0.1% formic acid and
solvent B = CH3CN + 0.1% formic acid; eluent flow: 1 mL/min;
volume of injected sample: 1 µL; UV-detectors operating at 215
and 254 nm; ionization method: electro spray ionization (positive
ions ionization mode). IR spectra were recorded with a Bruker
Alpha Platinum ATR Spectrometer. FT-IR spectra (64 scans)
were obtained at 2 cm-1 resolution using a 50 µm CaF2 solution
cell and a dry air purged Bruker Tensor 27 equipped with liquid
Table 8
Comparison of Pro-containing compounds 4c, 6c with those
obtained from 2-cyanonicotinic acid8
entry Pyrazine
trans/cis Pyridine
trans/cis
ratio
77/23
derivative
ratio
derivative
1
54/46
CO2Me
CO2Me
N
N
N
N
O
N
O
N
N
87 %
4c 49 %
2
74/26
68/32
CO2Me
OH
CO2Me
OH
N
N
N
O
N
O
N
N
N
NH2
NH2
6c 93 %
a Isolated yield.
3. Conclusions
70 %
In summary, we have developed the syntheses of various
novel 2,3-substituted pyrazine non-peptidic turn structures
possessing amidoxime, esterified with amino acid amidoxime
and chiral 1,2,4-oxadiazole residues. The 1,2,4-oxadiazole ring
closure was performed under optimized microwave-assisted

7
nitrogen cooled MCT-detector. All samples were dissolved at
concentration of 10 mM in spectrophotometric grade chloroform
(≥ 99.8 %, Sigma-Aldrich) or DMSO. NMR spectra were
Hz, CHCH3), 3.72 (3H, s, OCH3), 1.49 (3H, d, J 7.2 Hz,
ACCEPTED MANUSCRIPT
CHCH3); δC (75 MHz, CDCl3) 173.0, 160.5, 147.9, 146.7, 145.6,
129.5, 115.4, 53.2, 48.8, 18.6; HRMS (ESI, m/z): MNa+, found
257.0645. C10H10N4NaO3 requires 257.0654.
1
measured with a Bruker Avance 300 (300 MHz for H and 75
MHz for 13C) with tetramethylsilane as standard. In the cases
where a 13C spectrum reflects the presence of two species arising
from trans and cis rotamers about the X-Pro amide bond, signals
are listed as X.X (X.X), where the first signal refers to the trans
species and the second, in parentheses, to the cis. Electron spray
ionization mass spectra (ESI-MS) were recorded on a Brucker
MicroTof-Q HR spectrometer, Faculté des Sciences et
Techniques, Vandoeuvre-lès-Nancy, France.
4.3.2. Methyl (2S)-2-[(5E)-5-imino-7-oxo-5,7-
dihydro-6H-pyrrolo[3,4-b]pyrazin-6-yl]propanoate
(5a)
Purification by flash column chromatography (50%
AcOEt/CH2Cl2) gave 5a as a yellow oil (0.197 g, 28%); Rf (50%
AcOEt/CH2Cl2) 0.41; νmax 3272, 3001, 2953, 1733, 1666, 1518,
1372, 1220, 1157 cm-1; δH (300 MHz, CDCl3) 9.51 (1H, s, NH),
8.91 (1H, d, J 2.6 Hz, HAr), 8.84 (1H, d, J 2.6 Hz, HAr), 5.36–5.29
(1H, m, CHCH3), 3.74 (3H, s, OCH3), 1.77 (3H, d, J 7.3 Hz,
CHCH3); δC (75 MHz, CDCl3) 170.6, 163.6, 157.1, 149.2, 148.7,
144.7, 144.7, 53.4, 48.8, 15.6; HRMS (ESI, m/z): MH+, found
235.0826. C10H11N4O3 requires 235.0838.
Compounds 3, 4, 5, 6 were synthesized in a similar
manner as described in [8].
4.1. Methyl 3-cyanopyrazine-2-carboxylate (2)10
To a stirred suspension of pyrazine-2,3-dicarboxylic acid
monoamide 1 (10.86 g, 65 mmol) in CH2Cl2 (100 mL) at 0 °С
were added TEA (19.00 mL, 136.5 mmol) and methyl
chloroformate (11.05 mL, 143 mmol) and the mixture was
allowed to reach room temperature and stirred for 2 h. Water (30
mL) and NaHCO3 (sat.) (30 mL) were added and the mixture was
extracted with CH2Cl2 (3×50 mL). The organic phase was washed
with H2O (2×30 mL), dried over MgSO4, and evaporated to
dryness to give the title compound 2 that was further used
without additional purification.
4.3.3. Methyl (2S)-2-{[(3-cyanopyrazin-2-
yl)carbonyl]amino}-3-phenylpropanoate (4b)
Purification by flash column chromatography (70%
AcOEt/petroleum ether) gave 4b as a light-yellow solid (0.047 g,
3%); mp 97-99 °C; Rf (70% AcOEt/petroleum ether) 0.63; νmax
3402, 3389, 3059, 3031, 2954, 2853, 2237 (CN), 1736, 1686,
1508, 1374, 1261cm-1; δH (300 MHz, CDCl3) 8.89 (1H, d, J 2.3
Hz, HAr), 8.74 (1H, d, J 2.3 Hz, HAr), 8.20 (1H, d, J 7.9 Hz, NH),
7.29–7.13 (5H, m, 5×HAr), 5.11 (1H, dt, J 8.0, 6.0 Hz, CHCH2),
3.77 (3H, s, OCH3), 3.53–3.10 (2H, m, CHCH2); δC (75 MHz,
CDCl3) 171.9, 160.7, 147.8, 146.9, 145.4, 136.1, 129.8, 129.4,
128.0, 115.5, 54.2, 53.2, 38.6; HRMS (ESI, m/z): MNa+, found
333.0958. C16H14N4NaO3 requires 333.0954.
Yellow solid; yield: 9.22 g (87%); mp 70-72 ˚C; δH (300
MHz, DMSO-d6) 8.89 (2H, s, 2×HAr), 4.09 (3H, s, OMe); δC (75
MHz, DMSO-d6) 162.8, 147.7, 146.8, 146.6, 131.4, 115.0, 54.5.
4.3.4. Methyl (2S)-2-[(5E)-5-imino-7-oxo-5,7-
dihydro-6H-pyrrolo[3,4-b]pyrazin-6-yl] -3-
phenylpropanoate (5b)
4.2. 3-cyanopyrazine-2-carboxylic acid (3)
A mixture of 2 (0.978 g, 6.0 mmol), MeOH (50 mL) and 1 M
NaOH (12 mL) was stirred at room temperature for 3 h and then
concentrated in vacuo. The residue was diluted with water (20
mL) and CH2Cl2 (20 mL), and then acidified with 1 М НСl under
cooling. The resulting precipitate was filtered off, washed with
H2O (5 mL) and hexane (20 mL), and dried in vacuo. Acid 3 was
obtained in satisfactory purity and was used in the next step
without additional purification.
Purification by flash column chromatography (70%
AcOEt/petroleum ether) gave 5b as a yellow oil (1.007 g, 65%);
Rf (70% AcOEt/petroleum ether) 0.54; νmax 3279, 3062, 3029,
2953, 2847, 1737, 1666, 1413, 1374, 1244, 1123 cm-1; δH (300
MHz, CDCl3) 9.46 (1H, s, NH), 8.82 (1H, d, J 2.6 Hz, HAr), 8.75
(1H, d, J 2.6 Hz, HAr), 7.19–7.07 (5H, m, 5×HAr), 5.55 (1H, dd, J
10.9, 5.6 Hz, CHCH2), 3.77 (3H, s, OCH3), 3.74–3.61 (2H, m,
CHCH2); δC (75 MHz, CDCl3) 169.7, 163.7, 157.2, 149.1, 148.7,
144.3, 144.2, 137.2, 129.4, 129.0, 127.3, 54.4, 53.4, 35.0; HRMS
(ESI, m/z): MNa+, found 333.0958. C16H14N4NaO3 requires
333.0961.
Pale-yellow solid; yield: 0.742 g (82%); mp 157-158 ˚C; δH
(300 MHz, DMSO-d6) 9.00 (1H, d, J 2.3 Hz, HAr), 8.96 (1H, d, J
2.3 Hz, HAr); δC (75 MHz, DMSO-d6) 164.5, 155.1, 146.6, 144.6,
128.1, 116.7.
4.3.5. Methyl (2S)-1-[1-(3-cyanopyrazin-2-
yl)ethenyl]pyrrolidine-2-carboxylate (4c)
4.3. Synthesis of Compounds 4, 5(a-c); General Procedure:
To a solution of 3-cyanopyrazine-2-carboxylic acid (3) (0.740
g, 5 mmol) in THF (30 mL) were added: triethylamine (1.39 mL,
10 mmol), the corresponding methyl ester of amino acid
hydrochloride (5 mmol) and HOBt (0.675 g, 5 mmol). The
mixture was stirred at 0 °С and EDCI (0.967 g, 5.05 mmol) was
added. Then, the mixture was stirred at room temperature for 18
h. The precipitate was filtered and the filtrate was evaporated in
vacuo. The residue was diluted with CH2Cl2 (50 mL), washed
with a solution of 0.1 M HCl (3×15 mL), brine (20 mL), then
dried with MgSO4 and concentrated in vacuo.
Purification by flash column chromatography (50%
AcOEt/CH2Cl2) gave 4c as a light-yellow oil (0.382 g, 49%); Rf
(50% AcOEt/CH2Cl2) 0.60; νmax 2955, 2887, 2239 (CN), 1738,
1637, 1455, 1373, 1171 cm-1; δH (300 MHz, CDCl3) 8.74 (1H, s,
HAr), 8.71 and 8.61 (1H, 2×d, J 2.3 Hz, HAr), 4.80 and 4.64 (1H,
2×dd, J 8.4, 3.2 Hz, CH-α), 3.76–3.64 (2H, m, CH2-δ), 3.68 and
3.53 (3H, 2×s, OCH3), 2.30–1.90 (4H, m, CH2CH2-β,γ); δC (75
MHz, CDCl3) 172.0 (172.6), 162.3 (162.2), 152.2 (151.7), 146.1
(145.9), 145.6 (144.6), 130.0 (130.8), 114.9 (115.2), 60.2 (61.2),
52.9 (52.8), 49.5 (48.4), 29.4 (31.8), 25.6 (22.6); HRMS (ESI,
m/z): MK+, found 299.0541. C12H12KN4O3 requires 299.0551.
4.3.1. Methyl (2S)-2-{[(3-cyanopyrazin-2-
yl)carbonyl]amino}propanoate (4a)
4.4. Synthesis of Compounds 6a-c; General Procedure:
Purification by flash column chromatography (50%
AcOEt/CH2Cl2) gave 4a as a pale-yellow solid (0.161 g, 23%);
mp 71-72 °C; Rf (50% AcOEt/CH2Cl2) 0.63; νmax 3367, 3047,
2994, 2954, 2240 (CN), 1736, 1678, 1517, 1203, 1150 cm-1; δH
(300 MHz, CDCl3) 8.89 (1H, d, J 2.3 Hz, HAr), 8.79 (1H, d, J 2.3
Hz, HAr), 8.26 (1H, d, J 7.4 Hz, NH), 4.71 (1H, pseudo-p, J 7.2
To
a solution of (2S)-N-(3-cyanopyrazin-2-yl)carbonyl-
substituted amino acids 4 and/or methyl esters of methyl (2S)-2-
[(5E)-5-imino-7-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyrazin-6-
yl]alkanoic acids 5 (2 mmol) in MeOH (15 mL) was added
triethylamine (0.56 mL, 4 mmol) followed by hydroxylamine

8
Tetrahedron
ACCEPTED MANUSCRIPT
hydrochloride (0.278 g, 4 mmol) and the reaction was stirred at
Purification by flash column chromatography (70%
room temperature for 18 h. The mixture was then evaporated to
dryness. The residue was dissolved in ethyl acetate (100 mL) and
washed with water (20 mL). The organic layer was dried with
MgSO4 and the solvent was removed in vacuo. The residue was
purified by column chromatography.
AcOEt/CH2Cl2) gave 7a as a colorless solid (0.391 g, 84%); mp
91-94 °C; Rf (70% AcOEt/CH2Cl2) 0.30; νmax 3424, 3311, 3193,
2970, 2934, 2878, 1738, 1681, 1644, 1514, 1366, 1172, 893, 670
cm-1; νmax (CHCl3) 3436, 3410, 1764, 1743, 1711, 1688, 1645 cm-
1; δH (300 MHz, CDCl3) 8.68 (1H, s, HAr), 8.61 (1H, s, HAr), 7.82
(1H, d, J 6.7 Hz, NHCHCH3), 5.78 (2H, br s, NH2), 5.17 (1H, d,
J 8.5 Hz, NHBoc), 4.83–4.61 (1H, m, CHCH3), 4.36–4.15 (1H,
m, CHNHBoc), 3.77 (3H, s, OCH3), 2.23–2.08 (1H, m,
CH(CH3)2), 1.53 (3H, d, J 6.9 Hz, CHCH3), 1.43 (9H, s, (CH3)3),
1.01 (3H, d, J 7.1 Hz, (CH3)2), 0.97 (3H, d, J 7.0 Hz, (CH3)2); δC
(75 MHz, CDCl3) 173.6, 170.2, 164.0, 156.4, 155.7, 147.0,
145.5, 144.9, 144.4, 80.6, 58.6, 53.2, 49.2, 31.9, 28.9, 19.7, 18.7,
18.5; HRMS (ESI, m/z): MH+, found 467.2249. C20H31N6O7
requires 467.2244.
4.4.1. Methyl (2S)-2-({[3-(N'-
hydroxycarbamimidoyl)pyrazin-2-
yl]carbonyl}amino)propanoate (6a)
Purification by flash column chromatography (7%
MeOH/CH2Cl2) gave 6a as a pale-yellow oil (0.449 g, 84%); Rf
(7% MeOH/CH2Cl2) 0.26; νmax 3462, 3328, 3066, 2995, 2954,
2847, 1733, 1646, 1537, 1454, 1435, 1340, 1215, 1161 cm-1; δH
(300 MHz, CDCl3) 8.57 (1H, d, J 2.3 Hz, HAr), 8.49 (1H, d, J 2.3
Hz, HAr), 7.71 (1H, d, J 7.5 Hz, NH), 5.45 (2H, br s, NH2), 4.70
(1H, pseudo-p, J 7.2 Hz, CHCH3), 3.70 (3H, s, OCH3), 1.45 (3H,
d, J 7.2 Hz, CHCH3); δC (75 MHz, CDCl3) 173.8, 165.5, 150.4,
146.7, 145.0, 145.0, 143.5, 53.2, 49.2, 18.5; HRMS (ESI, m/z):
MK+, found 306.0599. C10H13KN5O4 requires 306.0603.
4.5.2. Methyl (2S)-2-[({3-[(Z,5S)-1-amino-5-
isopropyl-9,9-dimethyl-4,7-dioxo-3,8-dioxa-2,6-
diazadec-1-en-1-yl]pyrazin-2-yl}carbonyl)amino] -
3-phenylpropanoate (7b)
Purification by flash column chromatography (70%
AcOEt/CH2Cl2) gave 7b as a colorless solid (0.363 g, 67%); mp
86-89 °C; Rf (70% AcOEt/CH2Cl2) 0.48; νmax 3326, 2969, 2877,
2856, 1738, 1688, 1681, 1643, 1514, 1367, 1171, 898, 670 cm-1;
νmax (CHCl3) 3438, 3408, 1766, 1745, 1712, 1686, 1645 cm-1; δH
(300 MHz, CDCl3) 8.70 (1H, d, J 2.0 Hz, HAr), 8.61 (1H, d, J 2.0
Hz, HAr), 7.72 (1H, d, J 7.4 Hz, NHCHCH2), 7.30–7.18 (5H, m,
5×HAr), 5.70 (2H, s, NH2), 5.15 (1H, d, J 8.7 Hz, NHBoc), 5.05
(1H, m, CHCH2), 4.38–4.25 (1H, m, CHNHBoc), 3.73 (3H, s,
OCH3), 3.26 (2H, d, J 5.6 Hz, CHCH2), 2.16 (1H, m, CH(CH3)2),
1.45 (9H, s, (CH3)3), 1.01 and 0.97 (6H, 2×d, J 6.8 Hz, (CH3)2);
δC (75 MHz, CDCl3) 172.0, 170.1, 163.9, 156.4, 155.7, 146.9,
145.6, 145.0, 144.5, 136.4, 130.1, 129.2, 127.7, 80.7, 58.6, 54.5,
53.0, 38.5, 32.0, 29.0, 19.7, 18.5; HRMS (ESI, m/z): MK+, found
581.2121. C26H34KN6O7 requires 581.2117.
4.4.2. Methyl (2S)-2-({[3-(N'-
hydroxycarbamimidoyl)pyrazin-2-
yl]carbonyl}amino)-3-phenylpropanoate (6b)
Purification by flash column chromatography (7%
MeOH/CH2Cl2) gave 6b as a light-yellow solid (0.432 g, 63%);
mp 154-157 °C; Rf (7% MeOH/CH2Cl2) 0.38; νmax 3474, 3350,
3060, 2926, 1717, 1675, 1645, 1547, 1435, 1161, 971 cm-1; δH
(300 MHz, DMSO-d6) 9.97 (1H, s, OH), 8.79 (1H, d, J 7.5 Hz,
NH), 8.71 (1H, d, J 2.3 Hz, HAr), 8.64 (1H, d, J 2.3 Hz, HAr),
7.32–7.20 (5H, m, 5×HAr), 5.82 (2H, s, NH2), 4.70 (1H, dt, J 7.2,
6.8 Hz, CHCH2), 3.57 (3H, s, OCH3), 3.19–2.99 (2H, m,
CHCH2); δC (75 MHz, DMSO-d6) 171.3, 165.5, 148.6, 147.5,
143.9, 143.5, 142.6, 136.9, 129.1, 128.2, 126.5, 53.8, 51.7, 37.1;
HRMS (ESI, m/z): MH+, found 344.1353. C16H18N5O4 requires
344.1353.
4.5.3. Methyl (2S)-1-({3-[(Z,5S)-1-amino-5-benzyl-
9,9-dimethyl-4,7-dioxo-3,8-dioxa-2,6-diazadec-1-
en-1-yl]pyrazin-2-yl}carbonyl)pyrrolidine-2-
carboxylate (7c)
4.4.3. Methyl (2S)-1-{[3-(N'-
hydroxycarbamimidoyl)pyrazin-2-
yl]carbonyl}pyrrolidine-2-carboxylate (6c)
Purification by flash column chromatography (7%
MeOH/CH2Cl2) gave 6c as a white solid (0.545 g, 93%); mp 135-
138 °C; Rf (7% MeOH/CH2Cl2) 0.36; νmax 3479, 3369, 2983,
2959, 2919, 2871, 2850, 1733, 1651, 1620, 1557, 1453, 1416,
1282, 1156, 936 cm-1; δH (300 MHz, CDCl3) 8.57 and 8.54 (1H,
2×d, J 2.3 Hz, HAr), 8.54 and 8.51 (1H, 2×d, J 2.3 Hz, HAr), 5.48
and 5.38 (2H, 2×s, NH2), 4.74 and 4.11 (1H, 2×dd, J 8.5, 3.8 Hz,
CH-α), 3.77 and 3.55 (3H, 2×s, OCH3), 3.39–3.36 and 3.29–3.23
(2H, 2×m, CH2-δ), 2.37–2.22 (1H, m, CHaHb-β), 2.05–1.87 (3H,
m, CHaHbCH2-β,γ); δC (75 MHz, CDCl3) 173.3 (172.9), 167.1,
148.9 (149.2), 148.6, 143.9, 143.4 (143.7), 142.7 (142.5), 59.3
(61.1), 53.0 (52.8), 48.3 (47.3), 30.2 (31.5), 25.2 (23.5); HRMS
(ESI, m/z): MH+, found 294.1197. C12H16N5O4 requires 294.1210.
Purification by flash column chromatography (70%
AcOEt/CH2Cl2) gave 7c as a light-yellow solid (0.540 g, 100%);
mp 96-98 °C; Rf (70% AcOEt/CH2Cl2) 0.31; νmax 3444, 3330,
2977, 2935, 2878, 1742, 1711, 1644, 1498, 1455, 1365, 1163,
701 cm-1; νmax (CHCl3) 3437, 3404, 1771, 1745, 1707, 1649 cm-1;
δH (300 MHz, CDCl3) 8.67 (1H, d, J 2.3 Hz, HAr), 8.61–8.49 (1H,
m, HAr), 7.43–7.11 (5H, m, 5×HAr), 5.58 (2H, br s, NH2), 5.15
and 5.05 (1H, 2×d, J 7.8 Hz, NHBoc), 4.84–4.72 and 4.30–4.27
(1H, 2×m, CH-α), 4.72–4.58 (1H, m, CHCH2), 3.79 and 3.62
(3H, 2×s, OCH3), 3.96–3.84 and 3.52–3.37 (2H, m, CH2-δ),
3.23–3.02 (2H, m, CHCH2), 2.49–2.30 (1H, m, CHaHb-β), 2.02–
1.52 (3H, m, CHaHbCH2-β,γ), 1.44 (9H, m, (CH3)3); δC (75 MHz,
CDCl3) 173.4, 169.2, 166.3, 155.9, 153.1, 150.6, 145.6 (145.1),
143.4 (143.5), 141.1, 136.7 (136.9), 130.0, 129.4, 127.7, 80.8,
59.4 (61.4), 54.5, 52.9 (52.8), 48.7 (47.2), 39.2, 30.2 (31.5), 28.9,
25.4 (23.5); HRMS (ESI, m/z): MK+, found 579.1964.
C26H32KN6O7 requires 579.1984.
4.5. Synthesis of Compounds 7a-c; General Procedure:
Amidoxime (6) (1 mmol) was dissolved in anhydrous
acetonitrile (20 mL). After addition of N-t-butoxycarbonyl-L-
valine(phenylalanine) (1.3 mmol), 4-DMAP (0.011 g, 0.09
mmol) and DCC (0.268 g, 1.3 mmol) the solution was stirred for
24 h at room temperature. The solvent was evaporated under
reduced pressure and the crude product was purified by column
chromatography.
4.6. Synthesis of Compounds 8a-c; General Procedure:
A 5 mL CEM microwave process vessel was charged with 7
(0.2 mmol) in CH2Cl2 (5 mL) and the vessel was capped. The
mixture was stirred and heated under microwave conditions (300
W) at 150 °C for 25 min. The solution was then concentrated
under vacuum and chromatographed on silica gel to give 1,2,4-
oxadiazoles 8.
4.5.1. Methyl (2S)-2-[({3-[(Z,5S)-1-amino-5-
isopropyl-9,9-dimethyl-4,7-dioxo-3,8-dioxa-2,6-
diazadec-1-en-1-yl]pyrazin-2-
yl}carbonyl)amino]propanoate (7a)

9
4.6.1. Methyl (2S)-2-({[3-(5-{(1S)-1-[(tert-
butoxycarbonyl)amino]-2-methylpropyl}-1,2,4-
References and notes
ACCEPTED MANUSCRIPT
oxadiazol-3-yl)pyrazin-2-
yl]carbonyl}amino)propanoate (8a)
1. (a) Peterlin-Mašič, L.; Kikelj, D. Tetrahedron 2001, 57, 7073-
7105. (b) Peterlin-Mašič, L.; Cesar, J.; Zega, A.; Curr. Pharm.
Des. 2006, 12, 73-91.
Purification by flash column chromatography (60%
AcOEt/CH2Cl2) gave 8a as a colorless oil (0.068 g, 75%); Rf
(60% AcOEt/CH2Cl2) 0.68; νmax 3424, 3311, 3193, 2970, 2934,
2878, 1738, 1681, 1644, 1514, 1366, 1172, 893, 670 cm-1; νmax
(CHCl3) 3442, 3397, 1740, 1716, 1688 cm-1; δH (300 MHz,
CDCl3) 8.79 (1H, s, HAr), 8.66 (1H, s, HAr), 8.05 (1H, d, J 7.3 Hz,
NHCHCH3), 5.20 (1H, d, J 9.0 Hz, NHBoc), 5.10–4.90 (1H, m,
CHCH3), 4.64 (1H, pseudo-p, J 7.3 Hz, CHNHBoc), 3.71 (3H, s,
OCH3), 2.32–2.15 (1H, m, CH(CH3)2), 1.44 (3H, d, J 7.1 Hz,
CHCH3), 1.38 (9H, s, (CH3)3), 0.96 (6H, d, J 6.8 Hz, (CH3)2); δC
(75 MHz, CDCl3) 179.7, 173.5, 167.1, 162.4, 155.8, 146.6,
145.8, 144.5, 143.3, 81.0, 54.2, 53.3, 48.9, 33.8, 28.9, 19.2, 19.1,
18.5; LC/MS (CI, m/z): MNa+, 471; HRMS (ESI, m/z): MK+,
found 487.1702. C20H28KN6O6 requires 487.1705.
2. Fylaktakidou, K. C.; Hadjipavlou-Litina, D. J.; Litinas, K. E.;
Varella, E. A.; Nicolaides, D. N. Curr. Pharm. Des. 2008, 14,
1001-1047.
3. Bauch, E.; Reichmann, D.; Mendel, R.-R.; Bittner, F.; Manke, A.-
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4. Clement, B.; Lopian, K. Drug Met. and Disp. 2003, 31, 645-651.
5. (a) Jakopin, Z. Tetrahedron Lett. 2015, 56, 504-506. (b) Jakopin,
Z.; Dolenc, M. S. Curr. Org. Chem. 2008, 12, 850-898.
6. Borg, S.; Vollinga, R. C.; Labarre, M.; Payza, K.; Terenius, L.;
Luthman, K. J. Med. Chem. 1999, 42, 4331-4342.
7. (a) Kotthaus, J.; Hungeling, H.; Reeh, C.; Kotthaus, J.; Schade, D.;
Wein, S.; Wolffram, S.; Clement, B. Bioorg. Med. Chem. 2011,
19, 1907-1914. (b) Kitamura, S.; Fukushi, H.; Miyawaki, T.;
Kawamura, M.; Terashita, Z.-I.; Naka, T. Chem. Pharm. Bull.
2001, 49, 268-277. (c) Kode, N.-R.; Vanden Eynde, J.-J.;
Mayence, A.; Wang, G.; Huang, T.-L. Molecules 2013, 18, 11250-
11263.
4.6.2. Methyl (2S)-2-({[3-(5-{(1S)-1-[(tert-
butoxycarbonyl)amino]-2-methylpropyl}-1,2,4-
oxadiazol-3-yl)pyrazin-2-yl]carbonyl}amino)-3-
phenylpropanoate (8b)
8. Ovdiichuk, O. V.; Hordiyenko, O. V.; Medviediev, V. V.;
Shishkin, O. V.; Arrault, A. Synthesis 2015, 47, 2285-2293.
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4806-4809.
Purification by flash column chromatography (60%
AcOEt/CH2Cl2) gave 8b as a light-yellow oil (0.100 g, 95%); Rf
(60% AcOEt/CH2Cl2) 0.78; νmax 3424, 3311, 3193, 2970, 2934,
2878, 1738, 1681, 1644, 1514, 1366, 1172, 893, 670 cm-1; νmax
(CHCl3) 3442, 3395, 1742, 1715, 1689 cm-1; δH (300 MHz,
CDCl3) 8.77 (1H, s, HAr), 8.60 (1H, s, HAr), 7.95 (1H, d, J 7.4 Hz,
NHCHCH2), 7.23–7.03 (5H, m, 5×HAr), 5.19 (1H, d, J 9.2 Hz,
NHBoc), 5.07–4.95 (1H, m, CHCH2), 4.94–4.82 (1H, m,
CHNHBoc), 3.65 (3H, s, OCH3), 3.15 (2H, d, J 5.3 Hz, CHCH2),
2.32–2.15 (m, 1H, CH(CH3)2), 1.37 (9H, s, (CH3)3), 0.95 (6H, d,
J 6.3 Hz, (CH3)2); δC (75 MHz, CDCl3) 179.7, 172.0, 167.1,
162.5, 155.8, 146.6, 145.8, 144.5, 143.3, 136.3, 130.0, 129.3,
127.8, 80.9, 54.2, 53.1, 38.5, 33.8, 28.9, 19.2, 18.4; LC/MS (CI,
m/z): MNa+, 547; HRMS (ESI, m/z): MNa+, found 547.2276.
C26H32N6NaO6 requires 547.2278.
10. Holenz, J.; Karlstroem, S.; Kihlstroem, J.; Kolmodin, K.;
Lindstroem, J.; Rakos, L.; Rotticci, D.; Swahn, B.-M.; Von Berg,
S. WO Patent 2011002409, 2011; Chem. Abstr. 2011, 154,
109655.
11. Ovdiichuk, O. V.; Hordiyenko, O.; Voitenko, Z.; Arrault, A.;
Medviediev, V. Acta Cryst. E 2013, E69, o1810.
12. (a) Oussaid, B.; Moeini, L.; Martin, B.; Villemin, D.; Garrigues,
B. Synth. Commun. 1995, 10, 1451-1459. (b) Wang, Y.; Miller,
R.-L.; Sauer, D.-R.; Djuric, S.-W. Org. Lett. 2005, 7, 925-928.
13. (a) Hamzé, A.; Hernandez, J.-F.; Fulcrand, P.; Martinez, J. J. Org.
Chem. 2003, 68, 7316-7321. (b) Braga, A. L.; Lüdtke, D. S.;
Alberto, E. E.; Dornelles, L.; Filho, W. A. S.; Corbellini, V. A.;
Rosa, D. M.; Schwab, R. S. Synthesis 2004, 10, 1589-1594. (c)
Katritzky, A. R.; Shestopalov, A. A.; Suzuki, K. ARKIVOC 2005,
7, 36-55. (d) Hebert, N.; Hannah, A. L.; Sutton, S. C. Tetrahedron
Lett. 1999, 40, 8547-8550. (e) Porcheddu, A.; Cadoni, R.; De
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2003, 5, 4413-4416.
4.6.3. Methyl (2S)-1-{[3-(5-{(1S)-1-[(tert-
butoxycarbonyl)amino]-2-phenylethyl}-1,2,4-
oxadiazol-3-yl)pyrazin-2-yl]carbonyl}pyrrolidine-
2-carboxylate (8c)
15. (a) Ackermann, J.; Bleicher, K.; Ceccarelli Grenz, S. M.;
Chomienne, O.; Mattei, P.; Schulz-Gasch, T. WO Patent
2007063012, 2007; Chem. Abstr. 2007, 147, 52814. (b) Lamb, J.
P.; Engers, D. W.; Niswender, C. M.; Rodriguez, A. L.; Venable,
D. F.; Conn, J. P.; Lindsley, C. W. Bioorg. Med. Chem. Lett. 2011,
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68, 1540-1550.
17. (a) Levy, G. C.; Nelson, G. L. J. Am. Chem. Soc. 1972, 94, 4897-
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18. (a) DeRider, M. L.; Wilkens, S. J.; Waddell, M. J.; Bretscher, L.
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8644-8654.
Purification by flash column chromatography (60%
AcOEt/CH2Cl2) gave 8c as a light-yellow oil (0.079 g, 76%); Rf
(60% AcOEt/CH2Cl2) 0.48; νmax 3424, 3311, 3193, 2970, 2934,
2878, 1738, 1681, 1644, 1514, 1366, 1172, 893, 670 cm-1; νmax
(CHCl3) 3438, 3362, 1746, 1714, 1650, 1615 cm-1; νmax (DMSO)
1750, 1737, 1708, 1667, 1649 cm-1; δH (300 MHz, CDCl3) 8.72
(1H, d, J 2.2 Hz, HAr), 8.65 and 8.56 (1H, 2×d, J 2.2, 2.3 Hz,
HAr), 7.26–6.97 (5H,m, 5×HAr), 5.63 and 5.17 (1H, 2×d, J 8.1 Hz,
NHBoc), 5.43–5.28 (1H, m, CHCH2), 4.60 and 4.26 (1H, 2×dd, J
8.5, 4.0 Hz, CH-α), 3.71 and 3.50 (3H, 2×s, OCH3), 3.83–3.74,
3.47–3.41 and 3.41–3.28 (2H, 3×m, CH2-δ), 3.28–3.12 (2H, m,
CHCH2), 2.34–1.80 (4H, m, CH2CH2-β,γ), 1.24 (9H, m, (CH3)3);
δC (75 MHz, CDCl3) 180.8 (180.6), 172.8 (172.7), 166.4, 165.4,
155.4, 150.8, 145.7, 145.3 (145.1), 139.9, 135.9 (135.5), 130.0,
129.4, 127.9, 81.0, 59.5 (61.2), 53.1 (52.9), 49.9, 48.8 (47.4),
40.9, 30.1 (31.7), 28.9, 25.5 (23.3); LC/MS (CI, m/z): MNa+,
475; HRMS (ESI, m/z): MK+, found 561.1858. C26H30KN6O6
requires 561.1864.
19. Kishore, R.; Raghothama, S.; Balaram, P. Biopolymers 1987, 26,
873-891.
Supplementary Material
Supplementary data associated with this article can be found
in the online version, at http://dx.doi.org/
Acknowledgements
The authors thank Campus France for financial support of this
work.
Legends for figures and schemes.

10
Tetrahedron
ACCEPTED MANUSCRIPT
Fig. 1. Selected examples of biologically active pyrazine and amidoxime
compounds.
Fig. 2. Trans/cis conformational states of proline-containing derivatives 4c,
6c, 7c and 8c.
Fig. 3. Van’t Hoff plots for 4c, 6c, 7c, 8c in DMSO-d6.
Fig. 4. Intramolecular interactions in oxadiazole derivatives 8a-c.
Scheme 1. Synthesis of starting 3-cyanopyrazine-2-carboxylic acid 3.
Table 1
Synthesis of Ala-, Phe-, Pro-containing pseudodipeptides 4-6a
Table 2
Synthesis of 1,2,4-oxadiazole pseudopeptides 8
Table 3
Optimization of microwave assisted condensation conditions for 7b as a
model compound
Table 4
Selected carbon chemical shifts in the pyrrolidine ring of proline derivatives
in CDCl3
Table 5
Thermodynamic parameters for compounds 4c, 6c, 7c, 8c. Additionaly the
trans/cis equilibrium constant at 300K is given
Table 6
Сhemical shifts and solvent sensitivity of the NH protons in 7a-c and 8a-c
Table 7
Comparison of compounds 4, 5 (a,b) with those obtained from 2-
cyanonicotinic acid8
Table 8
Comparison of Pro-containing compounds 4c, 6c with those obtained from 2-
cyanonicotinic acid8