Article
Synthesis and evaluation of structure-affinity relation of N-(pyrazin-2-
yl)carboxamide derivatives for potent antagonists for the Adenosine
A(2B)-receptor.
Author1
1
Division of Medicinal Chemistry, Leiden University. Post Office Box 9502, 2300 RA Leiden, The Netherlands.
KEYWORDS: Adenosine A(2B) receptor, Yeast liquid growth assay, structure affinity relationship.
ABSTRACT: The A2B-receptor is part of the Adenosine-receptor family and gets activated by its endogenous ligand adeno-
sine. However, it doesn’t get activated in normal physiological conditions due to its low-affinity for the A2B-receptor.
Pathological conditions result in higher adenosine concentrations, into the micromolar, and thus only gets activated in
pathological conditions which turns this receptor in an interesting therapeutical target for pathological indications. How-
ever, as mentioned before the A2B-receptor is a low-affinity receptor and thus there is a need for potent antagonist. The
aim of this study is to evaluate the antagonistic effects 104 synthesized ligands with a core based on the LAS101057 by a
yeast liquid growth assay. Results indicate that ligands with an 6-(3-(electron withdrawing group)-phenyl) or just a phe-
nyl, 5-(4-(hydrogen bond acceptor)phenyl) and a small group at the carboxamide group are the most potent. Ligands 10,
58 and 66 comply to these rules and showed high potency.
■ INTRODUCTION
Adenosine is a purine nucleotide that is either released by such as PSB-11158, MRS17549 and PSB-60310. Pyramidine
cells or generated by the cleavage of adenosine 5’- structure analogues also have been synthesized such as
triphosphate (ATP) by ectonucleotidases1. It binds to four LAS3809611, a potent non-xanthine based substance which
subtypes of G-protein coupled receptors (GPCR’s), name- was later optimized to the pyrazine derivative LAS10105712.
ly the adenosine A1, A2A, A3 and A2B receptors1. In this present study, ligands with a scaffold of N-
Since its cloning in 1992, A2B remains the most cryptic of (pyrazin-2-yl)carboxamide (Figure 1), based on the main
all four adenosine receptors2,3. Long was thought that the structure of LAS101057, with varying R1, R2 and R3 groups
A2B-receptor had no physiological role due to the low were used due to its high affinity, selectivity and relatively
affinity4. However, the intracellular signaling routes were simple structure and evaluated using a yeast liquid
characterized in later years and the physiological roles growth assay. The aim of this study is to evaluate the
were investigated5. Upon activation of the A2B-receptor, it antagonist effects of the synthesized ligands in order to
mediates a stimulation of adenylate cyclase through cou- find a potent antagonist for the A2B receptor.
pling with Gs protein which in turn increases the intracel-
lular cAMP. Furthermore it also activates phospholipase C
by coupling to Gq proteins which increases the intracellu-
lar calcium levels5. However, under normal physiological
circumstances the A2B receptor doesn’t get activated due
to its low-affinity for adenosine, only pathological circum-
stances where adenosine levels rise to the micromolar
concentrations activate the receptor. Thus the A2B-
receptor is involved in pathological processes and thereby
turns this receptor in a potential therapeutic target for
many pathological indications6. Recently, the relevance of
A2b in cancer progression has been elucidated as a pro-
tumorigenic factor. It promotes cancer progression in
many ways7. A2B antagonists are thus emerging as promis- Figure 1. Main structure, N-(pyrazin-2-yl)carboxamide, of the
ing anticancer agents but also as pathological therapeutic 104 synthesized compounds which was based on the structure
agents. Most antagonist are based on a xanthine structure of LAS101057.
, Journal of Medicinal Chemistry Article
■ RESULTS AND DISCUSSION
Synthesis of the ligands
Biology “Yeast liquid growth assay”
All 104 compounds were evaluated using modified
A series of 104 ligands was designed based on the core MMY24-yeast strain which contains the human adenosine
structure of LAS10105712. The main core is a N- A2B-receptor, activation of the receptor is essential for the
growth of the yeast. The optical density of the yeast was
(pyrazine-2-yl)carboxamide (Figure 1). All series have
measured at 600 nm to determine the dose-effect relation
been synthesized by varying the R-groups attached to
of these ligands (Figure 2). IC50-values were calculated
the main core. The synthesis of one of these ligands, N-
using GraphPad Prism based on these curves of all 104
(6-methoxy-5-(4-pyridinyl)pyrazin-2-
compounds (Appendix I).
yl)cyclopropanecarboxamide (which will be further ad-
dressed as ligand 73), was adapted from the synthesis of
LAS10105713. Ligand 73 was obtained by the in-house
synthesized N-(6-chloro-5-(4-pyridinyl)pyrazine-2-
yl)cyclopropanecarboxamide (which will be simply refered
to as chloropyrazine 1d) with Suzuki coupling of 4-
methoxyphenyl with a 50% yield. The synthesis of 1d
consists of a regio-selective iodination step followed by the
Stille coupling of 4-(tributylstannyl)pyridine. After the
amide formation with cyclopropanecarbonyl chloride, 1d
is formed. The same reactions have been performed using
different reagents for the varying R-groups to give rise to
the remaining 103 compounds.
Figure 2. Dose-effect curve of imaginary test-ligand A & B. IC50-
values are calculated by the interpolation of the curve with y-
axis at 0.5
Interestingly, the R3 group seems to have the most specific
needs of all varying R-groups, since only a few ligands
show IC50 below 10.000 (Appendix I). An phenyl ring struc-
ture with a hydrogen bond acceptor (HBA) on the 4’ posi-
tion such as compound 95 and 100 results in lower IC50-
Scheme 1. Synthesis of N-(6-methoxy-5-(4-pyridinyl)pyrazine-2- values. The OH-group shows higher potency compared to
yl)cyclopropanecarboxamide. Reagents used: a) N- the 4-pyridin. However, relatively bigger HBA’s such as
iodosuccinimide, DMSO/H2O 70%; b) 4- methoxy-groups in compound 98 and 104 deny this rule.
(tributylstannyl)pyridine, PdCl2(Ph3P)2, CuI, DMF, 150 C; c)
Probably due to its size and thereby the inability to form
cyclopropanecarbonyl chloride, pyridine, 70 C; d) 4-
hydrogen bonds with the receptor. (Table 1)
methoxyphenyl boronic acid, 2M Cs2CO3, PdCl2(dppf), dioxane,
90 C, 50%.
Table 1. IC50-values of ligands in nM concentrations.
Cmpd A2BR IC50
nr R1 R 2
R3
(nM)
87 Me 2-F 4-Pyridine 89±28
93 Me 2-F 2-OH >10000
94 Me 2-F 3-OH 7227±2074
95 Me 2-F 4-OH 59±14
98 Me 2-F 4-OMe >10000
100 c Pr 2-F 4-Pyridine 75±7
104 c Pr 2-F 4-Ome >10000
2
Synthesis and evaluation of structure-affinity relation of N-(pyrazin-2-
yl)carboxamide derivatives for potent antagonists for the Adenosine
A(2B)-receptor.
Author1
1
Division of Medicinal Chemistry, Leiden University. Post Office Box 9502, 2300 RA Leiden, The Netherlands.
KEYWORDS: Adenosine A(2B) receptor, Yeast liquid growth assay, structure affinity relationship.
ABSTRACT: The A2B-receptor is part of the Adenosine-receptor family and gets activated by its endogenous ligand adeno-
sine. However, it doesn’t get activated in normal physiological conditions due to its low-affinity for the A2B-receptor.
Pathological conditions result in higher adenosine concentrations, into the micromolar, and thus only gets activated in
pathological conditions which turns this receptor in an interesting therapeutical target for pathological indications. How-
ever, as mentioned before the A2B-receptor is a low-affinity receptor and thus there is a need for potent antagonist. The
aim of this study is to evaluate the antagonistic effects 104 synthesized ligands with a core based on the LAS101057 by a
yeast liquid growth assay. Results indicate that ligands with an 6-(3-(electron withdrawing group)-phenyl) or just a phe-
nyl, 5-(4-(hydrogen bond acceptor)phenyl) and a small group at the carboxamide group are the most potent. Ligands 10,
58 and 66 comply to these rules and showed high potency.
■ INTRODUCTION
Adenosine is a purine nucleotide that is either released by such as PSB-11158, MRS17549 and PSB-60310. Pyramidine
cells or generated by the cleavage of adenosine 5’- structure analogues also have been synthesized such as
triphosphate (ATP) by ectonucleotidases1. It binds to four LAS3809611, a potent non-xanthine based substance which
subtypes of G-protein coupled receptors (GPCR’s), name- was later optimized to the pyrazine derivative LAS10105712.
ly the adenosine A1, A2A, A3 and A2B receptors1. In this present study, ligands with a scaffold of N-
Since its cloning in 1992, A2B remains the most cryptic of (pyrazin-2-yl)carboxamide (Figure 1), based on the main
all four adenosine receptors2,3. Long was thought that the structure of LAS101057, with varying R1, R2 and R3 groups
A2B-receptor had no physiological role due to the low were used due to its high affinity, selectivity and relatively
affinity4. However, the intracellular signaling routes were simple structure and evaluated using a yeast liquid
characterized in later years and the physiological roles growth assay. The aim of this study is to evaluate the
were investigated5. Upon activation of the A2B-receptor, it antagonist effects of the synthesized ligands in order to
mediates a stimulation of adenylate cyclase through cou- find a potent antagonist for the A2B receptor.
pling with Gs protein which in turn increases the intracel-
lular cAMP. Furthermore it also activates phospholipase C
by coupling to Gq proteins which increases the intracellu-
lar calcium levels5. However, under normal physiological
circumstances the A2B receptor doesn’t get activated due
to its low-affinity for adenosine, only pathological circum-
stances where adenosine levels rise to the micromolar
concentrations activate the receptor. Thus the A2B-
receptor is involved in pathological processes and thereby
turns this receptor in a potential therapeutic target for
many pathological indications6. Recently, the relevance of
A2b in cancer progression has been elucidated as a pro-
tumorigenic factor. It promotes cancer progression in
many ways7. A2B antagonists are thus emerging as promis- Figure 1. Main structure, N-(pyrazin-2-yl)carboxamide, of the
ing anticancer agents but also as pathological therapeutic 104 synthesized compounds which was based on the structure
agents. Most antagonist are based on a xanthine structure of LAS101057.
, Journal of Medicinal Chemistry Article
■ RESULTS AND DISCUSSION
Synthesis of the ligands
Biology “Yeast liquid growth assay”
All 104 compounds were evaluated using modified
A series of 104 ligands was designed based on the core MMY24-yeast strain which contains the human adenosine
structure of LAS10105712. The main core is a N- A2B-receptor, activation of the receptor is essential for the
growth of the yeast. The optical density of the yeast was
(pyrazine-2-yl)carboxamide (Figure 1). All series have
measured at 600 nm to determine the dose-effect relation
been synthesized by varying the R-groups attached to
of these ligands (Figure 2). IC50-values were calculated
the main core. The synthesis of one of these ligands, N-
using GraphPad Prism based on these curves of all 104
(6-methoxy-5-(4-pyridinyl)pyrazin-2-
compounds (Appendix I).
yl)cyclopropanecarboxamide (which will be further ad-
dressed as ligand 73), was adapted from the synthesis of
LAS10105713. Ligand 73 was obtained by the in-house
synthesized N-(6-chloro-5-(4-pyridinyl)pyrazine-2-
yl)cyclopropanecarboxamide (which will be simply refered
to as chloropyrazine 1d) with Suzuki coupling of 4-
methoxyphenyl with a 50% yield. The synthesis of 1d
consists of a regio-selective iodination step followed by the
Stille coupling of 4-(tributylstannyl)pyridine. After the
amide formation with cyclopropanecarbonyl chloride, 1d
is formed. The same reactions have been performed using
different reagents for the varying R-groups to give rise to
the remaining 103 compounds.
Figure 2. Dose-effect curve of imaginary test-ligand A & B. IC50-
values are calculated by the interpolation of the curve with y-
axis at 0.5
Interestingly, the R3 group seems to have the most specific
needs of all varying R-groups, since only a few ligands
show IC50 below 10.000 (Appendix I). An phenyl ring struc-
ture with a hydrogen bond acceptor (HBA) on the 4’ posi-
tion such as compound 95 and 100 results in lower IC50-
Scheme 1. Synthesis of N-(6-methoxy-5-(4-pyridinyl)pyrazine-2- values. The OH-group shows higher potency compared to
yl)cyclopropanecarboxamide. Reagents used: a) N- the 4-pyridin. However, relatively bigger HBA’s such as
iodosuccinimide, DMSO/H2O 70%; b) 4- methoxy-groups in compound 98 and 104 deny this rule.
(tributylstannyl)pyridine, PdCl2(Ph3P)2, CuI, DMF, 150 C; c)
Probably due to its size and thereby the inability to form
cyclopropanecarbonyl chloride, pyridine, 70 C; d) 4-
hydrogen bonds with the receptor. (Table 1)
methoxyphenyl boronic acid, 2M Cs2CO3, PdCl2(dppf), dioxane,
90 C, 50%.
Table 1. IC50-values of ligands in nM concentrations.
Cmpd A2BR IC50
nr R1 R 2
R3
(nM)
87 Me 2-F 4-Pyridine 89±28
93 Me 2-F 2-OH >10000
94 Me 2-F 3-OH 7227±2074
95 Me 2-F 4-OH 59±14
98 Me 2-F 4-OMe >10000
100 c Pr 2-F 4-Pyridine 75±7
104 c Pr 2-F 4-Ome >10000
2
