Synthesis of novel chromene scaffolds for adenosine receptors

Article abstract of DOI:10.1039/c1ob05305a

A one-pot procedure was developed for the synthesis of novel 3-[amino(methoxy)methylene]-2-oxo-3,4-dihydro-2H-chromen-4-yl) -3-cyanoacetamides and chromeno[3,4-c]pyridine-1-carbonitriles from the reaction of 2-oxo-2H-chromene-3-carbonitriles and cyanoacetamides. These chromene derivatives were identified as new scaffolds for adenosine receptors and the hits 3a, 3c, 5a, and 5b were found.

Full text of DOI:10.1039/c1ob05305a

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Organic &
Biomolecular
Chemistry
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PAPER
Synthesis of novel chromene scaffolds for adenosine receptors†
Marta Costa,a Filipe Areias,a,b Marian Castro,b Jose Brea,b Mar´ıa I. Lozab and Fernanda Proenc¸a*a
Received 24th February 2011, Accepted 25th March 2011
DOI: 10.1039/c1ob05305a
A one-pot procedure was developed for the synthesis of novel
3-[amino(methoxy)methylene]-2-oxo-3,4-dihydro-2H-chromen-4-yl)-3-cyanoacetamides and
chromeno[3,4-c]pyridine-1-carbonitriles from the reaction of 2-oxo-2H-chromene-3-carbonitriles and
cyanoacetamides. These chromene derivatives were identified as new scaffolds for adenosine receptors
and the hits 3a, 3c, 5a, and 5b were found.
Introduction
Adenosine receptors are distributed throughout the body, regu-
lating different cellular functions and can be considered attractive
targets for therapeutic agents.1,2 Different compounds proved to
be active on these receptors, displaying pharmacological activity
namely for the treatment of cardiovascular, inflammatory or
neurodegenerative diseases and cancer.1,2 The active molecules
usually belong to the purine family, but compounds with the
pyrazolo-triazolo-pyrimidine, dihydropyridine and quinazoline-
urea core unit were also identified as active.1 To our knowledge,
the interaction of chromene derivatives with adenosine receptors
was never reported.
The chromene scaffold is present in a variety of biologically
active compounds3 and their synthesis has been widely explored
in the literature.4 The 2-oxo-2H-chromene-3-carbonitriles, in par-
ticular, are usually prepared from the Knoevenagel condensation
of salicylaldehydes with malononitrile, a reaction that can be per-
formed in aqueous base followed by acidic treatment.5 Only a few
studies have been reported on the reactivity of these compounds
with nucleophiles5a and the reaction with cyanoacetamides was
never explored.
Results and discussion
Chemical synthesis
Preliminary studies on the reaction of 2-oxochromene 2a, prepared
according to a previously reported procedure5c with the commer-
cially available 2-cyanoacetamide 1a, were performed in methanol
and triethylamine (Scheme 1). After 2 days at room temperature,
the white solid that precipitated from solution was collected and
Scheme 1 Proposed mechanism for the formation of compounds 3a, 5a
and 9 in the reaction of chromene 2a with 2-cyanoacetamide 1a.
aDepartment of Chemistry, University of Minho, Campos de Gualtar, 4710-
057, Braga, Portugal. E-mail: fproenca@quimica.uminho.pt; Fax: +0-351-
253-604383; Tel: +0-351-253604379
identified by 1H NMR as a 1 : 1 : 1 mixture of compounds 3a, 5a,
and 9. The pure compound 3a was isolated from the mother liquor
in 22% yield. The solid mixture containing 3a, 5a and 9 evolved to a
1 : 1 mixture of 5a and 9 after reflux in methanol and triethylamine
bDepartment of Pharmacology, Universidade de Santiago de Compostela,
15782, Santiago de Compostela, Spain
† Electronic supplementary information (ESI) available: copies of 1H and
13C NMR spectra of all new compounds. See DOI: 10.1039/c1ob05305a
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Table 1 Synthesis of chromene derivatives 3
for 2.5 h. Compound 5a (8.5%) was separated from this mixture
upon stirring at room temperature in a combination of ethanol,
water and acetic acid. Compound 9 (5.4%) was also isolated from
the mother liquor after partial evaporation of the solvent.
Previous work on the condensation of a 3-cyanochromene with
ethylcyanoacetate in aqueous ammonia reported the synthesis of
a molecule analogous to 5a.6 The product, isolated in 25% yield,
was characterized by elemental analysis, mass spectrometry and
IR spectroscopy.
Entry
Compound 1
R
Product 3, yield
The results of our preliminary study suggest that compound
3a is the first product generated in solution and may evolve to
5a by intramolecular cyclization between the amide nitrogen and
the substituent on C-3 (Pathway A). Alternatively, product 9 can
be generated after reflux in methanol and triethylamine, probably
arising from ring-opening of the chromene moiety, induced by
the nucleophilic solvent, to generate intermediate 6. Two sets of
intramolecular cyclizations lead to compound 7 which oxidizes to
8. Depending on the substituents in the ring, 2-iminochromenes
can equilibrate with the phenolic nitrile by ring opening of the
pyrane unit, leading to the formation of 9.
Compounds 3a and 5a, isolated in modest yield in this synthetic
study, proved to be active on adenosine receptors, encouraging
the search for a selective experimental procedure to generate these
scaffolds.
To prepare the target compounds 3a–l, several amides 1b–l were
synthesized by the direct combination of methyl-2-cyanoacetate
and primary or secondary amines, using a previously described
procedure (Scheme 2).4k
1
2
1a
1b
NH2
3a, 27%
3b, 27%
3
4
5
6
7
8
9
1c
1d
1e
1f
3c, 33%
3d, 30%
3e, 63%
3f, 34%
3g, 80%
3h, 42%
3i, 60%
1g
1h
1i
10
11
1j
3j, 31%
3k, 37%
Scheme 2 Reaction of methyl cyanoacetate with primary or secondary
amines.
1k
Amides 1a–l were isolated in 49–100% yield and reacted
with 8-methoxy-2-oxo-2H-chromene-3-carbonitriles 2a, under the
reaction conditions identified in Table 1. Compounds 3a–l were
isolated in 22–80% yield. The use of ethanol as solvent failed
to lead to the analogous structures 3a–l when th◦e reaction was
performed in the presence of triethylamine at 40 C. The NMR
spectra for compound 3a showed two sets of bands assigned to
two diastereomers mainly on the basis of the vicinal coupling
constants between the protons on the side chain methyne group
(CH–CN) and the proton on the ring carbon C-4¢. The values
for J = 4.1 Hz and J = 6.3 Hz reveal a different stereochemical
disposition of these substituents with dihedral angles around
45◦/135◦ and 30◦/145◦ respectively, according to the Karplus
equation.7 The H–C correlation spectra (HMBC and HMQC)
allowed the complete identification of the proton and carbon
signals for each diastereomer. Compounds 3b–l, incorporating a
substituted amide, led to a single species in the NMR spectra. In
this case, it is possible that the volume of the amide substituent
hampers the formation of the most hindered diastereomer. For
compounds 3b and 3c, where a primary amide is present, the value
of the coupling constant between the methylene C–H and the C4–
H (J = 7.2 and 7.6 Hz) indicated a dihedral angle around 20◦/155◦.
The bulkier secondary amine in compounds 3d–l resulted in
12
1l
3l, 80%
coupling constants of around 4 Hz and dihedral angles close to
45◦/130◦.
The stereochemistry of the amino(methoxy)methylene side
chain was not assigned with certainty. The push-pull effect,
associated with the presence of two electron-donating substituents
conjugated with the carbonyl group, was considered responsible
for a low rotational barrier of the exocyclic double bond.
Compounds 5a–d were isolated in 26–36% yield when the
commercially available 2-cyanoacetamide 1a was combined with
2-oxo-2H-chromenes 2 in methanol and the mixture was refluxed
for 1–2 h (Table 2). The uncertainty in the relative position of
the amino and methoxy substituents favoured the intramolecu-
lar cyclization of intermediate 3 under these mild temperature
conditions. All compounds were fully characterized by 1H NMR,
13C NMR, correlation techniques (HMBC and HMQC), infrared
spectroscopy and elemental analysis (or high-resolution mass
spectra).
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Table 2 Synthesis of chromene derivatives 5
active compounds, showing affinities in the submicromolar range
at adenosine receptors.
Chromene 3a presented pKi values between 6.6 and 6.8 for
adenosine A1, A2A and A2B receptors. Chromene 3c showed pKi
values of 6.1 and 5.8 at A1 and A2B receptors, respectively.
Chromene 5a presented a pKi value of 6.9 for both A1 and A2A
receptors and lower affinity (5.9) for A2B receptor. Chromene 5b
presented pKi values in the range of 6.0–6.3 for A1, A2B and A3
receptors and a lower affinity (5.5) for A2A.
Entry
R1
Product 5, yield
When we compare the affinities of the chromenes 3a and 3c
for A1, A2A and A2B receptors, we observe higher affinities for
compound 3a, which leads us to suggest that the existence of a
primary amino group in the 3-[amino(methoxy)methylene] group
of these chromenes is important for their affinity for these receptor
subtypes.
In chromenes 5, the replacement of the 7-OCH3 group in
chromene 5a by a 7-OH group in chromene 5b led to a decrease
in affinity for A1 and A2A receptors and an increase for the A3
receptor.
1
2
3
4
7-OCH3
7-OH
9-Cl
5a, 26%
5b, 36%
5c, 35%
5d, 32%
9-Br
Table 3 Binding affinities of the new compounds at human A1, A2A, A2B
and A3 receptors (pKi or inhibition percentage at 10 mM)
Among these 4 hits, chromenes 3a, 5a and 5b show affinities
at submicromolar concentrations for both A1 and A2A receptors.
This pharmacological profile can be of therapeutic interest in the
light of the efficacy shown by compounds with dual affinity at A1
and A2A receptors in animal models of Parkinson’s disease.8
Conclusions
Adenosine receptors
In summary, we developed a one-pot procedure for the syn-
thesis of novel 3-[amino(methoxy)methylene]-2-oxo-3,4-dihydro-
Entry
Compd
A1
A2A
A2B
A3
1
2
3
4
5
6
7
8
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
5a
5b
5c
5d
6.8 0.1
35 1%
6.1 0.2
61 1%
14 1%
38 2%
29 2%
20 1%
31 3%
18 3%
20 1%
58 2%
6.9 0.2
6.1 0.3
51 1%
n.d.a
6.6 0.1
46 5%
49 2%
49 2%
24 5%
65 3%
37 5%
35 1%
41 4%
26 2%
30 4%
35 3%
6.9 0.3
5.5 0.2
22 1%
n.d.a
6.6 0.3
39 2%
5.8 0.2
57 5%
25 5%
29 4%
23 4%
41 1%
26 4%
14 1%
12 4%
17 5%
36 1%
21 1%
26 5%
27 5%
13 5%
13 3%
6.3 0.3
18 1%
n.d.a
2H-chromen-4-yl)-3-cyanoacetamides
3
and chromeno[3,4-
c]pyridine-1-carbonitriles 5 from readily available cyanoac-
etamides and 2-oxo-2H-chromene-3-carbonitriles in methanol
and triethylamine. The substituted 2-oxochromene 3 was isolated
1
5%
◦
when the reaction was performed at 40 C and reflux conditions
24 5%
47 5%
48 4%
40 2%
39 4%
18 5%
5.9 0.4
6.0 0.2
29 1%
n.d.a
resulted in the formation of the tricyclic product 5. These new
chromene scaffolds proved to be active at adenosine receptors
and 4 hits were identified in this study with affinities in the
submicromolar range.
9
10
11
12
13
14
15
16
Experimental
General
a n.d. – not determined
Chemicals and organic solvents were purchased from commercial
sources and used without further purification. All new compounds
were fully characterized by elemental analysis and spectroscopic
data. The NMR spectra were recorded at room temperature on
a Varian Unity Plus (1H: 300 MHz, 13C: 75 MHz) and a Bruker
Pharmacological evaluation
A complete in vitro evaluation of the affinity of the novel
compounds at human adenosine A1, A2A, A2B and A3 was carried
out by radioligand binding assays at receptors heterologously
expressed in mammalian cells lines. All the 15 chromenes tested
displayed affinity for adenosine receptors at the concentration of
10 mM, displacing the radioligand binding to a different extent as
shown in Table 3.
The pKi values were calculated for 4 hits identified as com-
pounds showing an inhibition percentage of the radioligand
binding (%inhib.) above 70% at the concentration of 10 mM
(Table 3). The data revealed 3-[amino(methoxy)methylene]-2-oxo-
3,4-dihydro-2H-chromen-4-yl)-3-cyanoacetamides 3a and 3c and
chromeno[3,4-c]pyridine-1-carbonitriles 5a and 5b as the most
1
Avance 3400 (1H: 400 MHz, 13C: 100 MHz), including the H–
13C correlation spectra (HMQC and HMBC) and deuterated
DMSO was used as solvent. Chemical shifts (d) were reported
in parts per million (ppm) and the coupling constants, J, are
reported in hertz (Hz). The melting points were determined on
a Stuart SMP3 melting point apparatus and are uncorrected.
The purities of all tested compounds are higher than 95% by
elemental analysis, which were performed on a LECO CHNS-
932 instrument and were reported to be within 0.4% of calculated
values. High resolution mass spectra (HRMS) were obtained from
the C.A.C.T.I. – Universidade de Vigo.
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Compound 1a is commercially available. The synthesis of
compounds 1b–e are reported in reference 4k and compounds
1f–l were synthesized using the method described in this reference.
Compounds 2 were prepared according to previously reported
procedures5c and the synthesis of compound 2 with R1 = 9-Cl was
previously reported.5b
Anal. Calcd for C13H14N3OF: C, 63.16; H, 5.67; N, 17.00. Found:
C, 63.21; H, 5.62; N, 17.04.
3-Oxo-3-[4-(pyridin-2-yl)piperazin-1-◦yl]propanenitrile 1k. Off-
1
white solid, 90% yield. Mp 101–103 C. H NMR (300 MHz,
DMSO-d6) d 3.40–3.60 (m, 8H, 4 CH2), 4.09 (s, 2H, CN-CH2),
6.84 (d, J = 8.4 Hz, 1H, Ar–H), 6.65 (t, J = 5.7 Hz, 1H, Ar–H), 7.54
(td, J = 7.8 Hz, 1.8 Hz, 1H, Ar–H), 8.11 (dd, J = 4.8 Hz, 1.8 Hz,
1H, Ar–H). 13C NMR (75 MHz, DMSO-d6) d 24.85 (CN-CH),
41.29 (CH), 44.15 (CH), 44.33 (CH), 44.89 (CH), 107.32 (Ar-CH),
113.37 (Ar-CH), 137.66 (Ar-CH), 147.56 (Ar-CH), 158.55 (Ar-C),
161.68 (C O). IR (Nujol mull) n 3050, 2256, 1660, 1592, 1567
cm-1. Anal. Calcd for C12H14N4O·0.2H2O: C, 61.64; H, 6.16; N,
23.97. Found: C, 61.80; H, 6.09; N, 23.94.
3-Oxo-3-(pyrro◦lidin-1-yl)propanenitrile 1f. White solid, 88%
1
yield. Mp 76–78 C. H NMR (300 MHz, DMSO-d6) d 1.76 (q,
J = 7.2 Hz, 2H, CH2), 1.85 (q, J = 6.3 Hz, 2H, CH2), 3.89 (s, 2H,
CN-CH2), 3.28 (t, J = 6.9 Hz, 2H, CH2), 4.35 (q, J = 7.2 Hz, 2H,
CH2). 13C NMR (75 MHz, DMSO-d6) d 24.0 (CH), 25.4 (CN-
CH), 25.5 (CH), 45.8 (CH), 46.1 (CH), 116.0 (CN), 160.8 (C O).
IR (Nujol mull) n 3084, 2256, 1651, 1597, 1556, 1521 cm-1. Anal.
Calcd for C7H10N2O·0.05H2O: C, 60.48; H, 7.27; N, 20.16. Found:
C, 60.38; H, 7.24; N, 20.17.
3-Oxo-3-[4-(pyrimidin-2-yl)piperazin-◦1-yl]propanenitrile
1l.
1
Yellow solid, 88% yield. Mp 143–145 C. H NMR (300 MHz,
DMSO-d6) d 3.50–3.60 (m, 2H, CH2), 3.68–3.80 (m, 4H, 2 CH2),
3.40–3.46 (m, 2H, CH2), 4.08 (s, 2H, CN-CH2), 6.66 (t, J =
4.8 Hz, 1H, Ar–H), 8.38 (d, J = 4.8 Hz, 2H, 2 Ar–H). 13C NMR
(75 MHz, DMSO-d6) d 24.86 (CN-CH), 41.40 (CH), 42.82 (CH),
43.02 (CH), 44.98 (CH), 110.52 (Ar-CH), 157.99 (2 Ar-CH),
161.02 (Ar-C), 161.73 (C O). IR (Nujol mull) n 2254, 1655,
1583, 1548 cm-1. Anal. Calcd for C11H13N5O: C, 57.14; H, 5.62;
N, 30.30. Found: C, 57.00; H, 5.88; N, 30.06.
3-(Morpholin-4-yl)-3-oxopropanenitrile 1g. Orange solid, 93%
yield. Mp 82–84 ◦C. 1H NMR (300 MHz, DMSO-d6) d 3.32 (t, J =
4.8 Hz, 2H, CH2), 3.51–3.59 (m, 4H, 2 CH2), 3.42 (t, J = 4.5 Hz,
2H, CH2), 4.02 (s, 2H, CN-CH2). 13C NMR (75 MHz, DMSO-
d6) d 24.64 (CN-CH), 42.03 (CH), 45.76 (CH), 65.76 (CH), 65.83
(CH), 116.13 (CN), 161.71 (C O). IR (Nujol mull) n 2264, 1652,
1618, 1532 cm-1. Anal. Calcd for C7H10N2O2: C, 54.54; H, 6.49; N,
18.18. Found: C, 54.53; H, 6.40; N, 18.16.
General procedure for the synthesis of compounds 3a–l
3-[4-(2-Hydroxyethyl)piperazin-1-yl]-◦3-oxopropanenitrile
1h.
1
Orange solid, 100% yield. Mp 78–80 C. H NMR (300 MHz,
DMSO-d6) d 2.31–2.42 (m, 6H, 3 CH2), 3.30 (t, J = 4.8 Hz, 2H,
CH2), 3.42 (t, J = 5.1 Hz, 2H, CH2), 3.48 (t, J = 6.6 Hz, 2H,
Compound
1 (0.48 mmol) was added to a suspension
of 8-methoxy-2-oxo-2H-chromene-3-carbonitrile 2a (0.09 g,
0.45 mmol) in methanol (3.5 mL) and triethylamine (0.7 mL)
and the reaction mixture was heated at 40 ◦C, in a water bath, for
30 min to 1 h and 45 min. For 3a–i and 3l, the reaction mixture
was concentrated in the rotary evaporator and cooled in an ice
bath for a few minutes. Diethyl ether (1–2 mL) was added and
the solid was filtered and washed with diethyl ether. For 3j and
3k the solvents were removed in the rotary evaporator and the
residue purified by flash chromatography using dichloromethane
as eluent. The solvent was removed in the rotary evaporator and a
few drops of methanol and diethyl ether (3 mL) were added to the
oil to precipitate the product. The solid was filtered and washed
with diethyl ether.
NCH2CH2), 4.01 (s, 2H, CN-CH2), 4.20–4.60 (brs, 1H, OH). 13
C
NMR (75 MHz, DMSO-d6) d 24.71 (CN-CH), 41.70 (CH), 45.37
(CH), 52.57 (CH), 52.95 (CH), 58.47 (N-CH2-CH2), 59.96 (CH2-
OH), 116.22 (CN), 161.36 (C O). IR (Nujol mull) n 3500–3050
(br), 2258, 1652, 1612 cm-1. Anal. Calcd for C9H15N3O2·0.2H2O:
C, 53.84; H, 7.68; N, 20.94. Found: C, 53.92; H, 7.59; N, 20.81.
3-[4-(3,4-Dichlorophenyl)piperazin-1-◦yl]-3-oxopropane-nitrile 1i.
1
White solid, 49% yield. Mp 131–133 C. H NMR (400 MHz,
DMSO-d6) d 3.57 (t, J = 3.9 Hz, 2H, CH2), 3.19 (t, J = 4.2 Hz,
2H, CH2), 3.24 (t, J = 4.5 Hz, 2H, CH2), 3.46 (t, J = 3.3 Hz,
2H, CH2), 4.08 (s, 2H, CN-CH2), 6.94 (dd, J = 6.8 Hz, 2.1 Hz,
1H, Ar–H), 7.15 (d, J = 2.1 Hz, 1H, Ar–H), 7.40 (d, J = 6.9 Hz,
1H, Ar–H). 13C NMR (100 MHz, DMSO-d6) d 24.75 (CN-CH),
41.13 (CH), 44.76 (CH), 47.10 (CH), 47.40 (CH), 115.66 (Ar-
CH), 116.55 (CN), 116.74 (Ar-CH), 119.97 (Ar-C-Cl), 130.50
(Ar-CH), 131.53 (Ar-C-Cl), 150.23 (Ar-C), 161.61 (C O). IR
(Nujol mull) n 3100, 2258, 1653, 1586, 1546 cm-1. Anal. Calcd
for C13H13N3OCl2·0.1H2O: C, 51.97; H, 4.41; N, 13.99. Found: C,
51.97; H, 4.51; N, 13.72.
2-(3-[Amino(methoxy)methylene]-8-methoxy-2-oxo-3,4-dihydro-
2H-chromen-4-yl)-2-cyanoacetamide 3a. Off-white solid, 22%
yield. Mp 181–183 ◦C. Two sets of bands were present in the
NMR spectra and were assigned to diastereomers A and B in a
1
1 : 1 ratio. H NMR, 3a(A) (300 MHz, DMSO-d6) d 3.64 (s, 3H,
OCH3), 3.82 (s, 6H, Ar-OCH3),* 3.92 (d, J = 4.1 Hz, 1H, C2–H),
4.50 (d, J = 4.1 Hz, 1H, C4¢–H), 6.82 (dd, J = 7.1 Hz, 2.1 Hz, 1H,
C5¢–H), 7.00–7.05 (m, 2H, C7¢–H),* 7.06 (t, J = 8.1 Hz, 1H, C6¢–H),
7.40 (s, 1H, C1-NH2), 7.52 (s, 1H, C1-NH2), 7.82 (s, 2H, C3¢-NH2).
13C NMR, 3a(A) (75 MHz, DMSO-d6) d 35.47 (C4¢), 47.49 (C2),
50.72 (OCH3), 55.66 (2 Ar–OCH3),* 71.81 (C3¢), 111.68 (C7¢),
117.45 (CN), 120.00 (C5¢), 124.00 (C4a¢), 124.34 (C6¢), 139.42 (C8a¢),
146.98 (2 C8¢),* 162.60 (C2¢), 165.39 (C1), 167.96 (C-NH2(OCH3)).
1H NMR, 3a(B) (300 MHz, DMSO-d6) d 3.57 (s, 3H, OCH3),
3.61 (d, J = 6.3 Hz, 3H, C2–H), 3.82 (s, 6H, Ar-OCH3),* 4.44 (d,
J = 6.3 Hz, 1H, C4¢–H), 6.91 (dd, J = 7.2 Hz, 1.2 Hz, 1H, C5¢–H),
7.00–7.05 (m, 2H, C7¢–H),* 7.09 (t, J = 7.5 Hz, 1H), 7.29 (s, 1H,
C6¢–H, C1-NH2), 7.54 (s, 1H, C1-NH2), 7.81 (s, 2H, C3¢-NH2).
3-[4-(4-Fluorophenyl)piperazin-1-yl]-3◦-oxopropanenitrile
1j.
1
Orange solid, 78% yield. Mp 130–132 C. H NMR (300 MHz,
DMSO-d6) d 3.58 (t, J = 4.8 Hz, 2H, CH2), 3.01–3.12 (m, 4H, 2
CH2), 3.48 (t, J = 4.8 Hz, 2H, CH2), 4.09 (s, 2H, CNCH2), 6.97
(d, J = 9.3 Hz, 2H, 2 Ar–H), 7.04 (d, J = 8.7 Hz, 2H, 2 Ar–H).
13C NMR (75 MHz, DMSO-d6) d 24.80 (CN-CH), 41.51 (CH),
45.19 (CH), 48.82 (CH), 49.16 (CH), 115.39 (2 Ar-CH), 116.19
(CN), 117.77 (2 Ar-CH), 147.54 (Ar-C), 156.33 (Ar-C-F), 161.55
(C O). IR (Nujol mull) n 3080, 2259, 1650, 1594, 1511 cm-1.
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13C NMR, 3a(B) (75 MHz, DMSO-d6) d 35.66 (C4¢), 46.94 (C2),
50.15 (OCH3), 55.66 (2 Ar–OCH3),* 71.29 (C3¢), 111.46 (C7¢),
117.30 (CN), 119.74 (C5¢), 121.99 (C4a¢), 124.07 (C6¢), 139.36 (C8a¢),
146.98 (2 C8¢),* 162.73 (C2¢), 165.34 (C1), 168.73 (C-NH2(OCH3)).
*The chemical shift for each isomer overlaps in a single signal.
IR (Nujol mull) n 3402, 3299, 2258, 1693, 1667, 1632, 1591,
1523 cm-1. HRMS (FAB) m/z: Calc. for C15H15N3O5 318.10910
[M+H]+, found: 318.10845 [M+H]+.
2-(3-[Amino(methoxy)methylene]-8-methoxy-2-oxo-3,4-dihydro-
2H-chromen-4-yl)-3-(4-methylpiperazin-1-yl)-3-oxopropanenitrile
3e. White solid, 63% yield. Mp 170–172 ◦C. 1H NMR (300 MHz,
DMSO-d6) d 2.21 (s, 3H, CH3), 2.28–2.40 (m, 2H, CH2), 2.41–2.46
(m, 2H, CH2), 3.28 (m, 1H, CH), 3.48 (m, 2H, CH2), 3.65 (s, 3H,
OCH3), 3.66 (m, 1H, CH), 3.83 (s, 3H, Ar-OCH3), 4.23 (d, J =
3.9 Hz, 1H, C4¢–H), 4.59 (d, J = 3.9 Hz, 1H, C2–H), 6.55–6.59
(m, 1H, C5¢–H), 7.00–7.05 (m, 2H, C6¢–H + C7¢–H), 7.84 (s, 2H,
NH2). 13C NMR (75 MHz, DMSO-d6) d 36.17 (C4¢), 41.76 (2
CH), 45.27 (2 CH), 45.53 (CH3), 46.02 (C2), 50.66 (OCH3),
55.65 (Ar–OCH3), 71.52 (C3¢), 111.73(C7¢), 117.45 (CN), 120.41
(C5¢), 121.08 (C4a¢), 123.88 (C6¢), 139.54(C8a¢), 146.89 (C8¢), 162.60
(C2¢),* 162.77 (C1),* 167.75 (C-NH2(OCH3)). *Assignments
interchangeable. IR (Nujol mull) n 3349, 3271, 2247, 1685, 1650,
1638, 1619, 1595 cm-1. Anal. Calcd for C20H24N4O5: C, 60.00; H,
6.00; N, 14.00. Found: C, 60.00; H, 5.94; N, 14.07.
2-(3-[Amino(methoxy)methylene]-8-methoxy-2-oxo-3,4-dihydro-
2H-chromen-4-yl)-2-cyano-N-cyclopentylacetamide 3b. White
solid, 27% yield. Mp 173–175 ◦C. 1H NMR (400 MHz, DMSO-d6)
d 1.10 (m, 2H, CH2), 1.40–1.50 (m, 4H, 2 CH2), 1.62 (m, 2H,
CH2), 3.51 (d, J = 7.2 Hz, 1H, C2–H), 3.60 (s, 3H, OCH3), 3.82 (s,
3H, Ar-OCH3), 3.83–3.90 (m, 1H, NH-CH-C4H8), 4.38 (d, J =
7.2 Hz, 1H, C4¢–H), 6.80 (dd, J = 7.4 Hz, 1.6 Hz, 1H, C5¢–H),
7.01 (dd, J = 7.6 Hz, 1.6 Hz, 1H, C7¢–H), 7.06 (t, J = 8.4 Hz,
1H, C6¢–H), 7.80 (s, 2H, NH2), 7.93 (d, J = 7.2 Hz, 1H, NH). 13
C
2-(3-[Amino(methoxy)methylene]-8-methoxy-2-oxo-3,4-dihydro-
NMR (100 MHz, DMSO-d6) d 23.20 (CH), 23.24 (CH), 31.87
(CH), 31.90 (CH), 35.75 (C4¢), 46.06 (C2), 50.36 (OCH3), 50.67
(NH-CH), 55.64 (Ar–OCH3), 71.78 (C3¢), 111.45 (C7¢), 117.34
(CN), 119.71 (C5¢), 123.76 (C4a¢), 124.14 (C6¢), 139.33 (C8a¢), 146.95
(C8¢), 162.57 (C2¢), 162.77 (C1), 168.06 (C-NH2(OCH3)). IR (Nujol
mull) n 3470, 3303, 2240, 1692, 1650, 1607, 1588, 1548, 1513 cm-1.
Anal. Calcd for C20H23N3O5: C, 62.34; H, 5.97; N, 10.91. Found:
C, 62.34; H, 5.67; N, 10.87.
2H-chromen-4-yl)-3-oxo-3-pyrrolidin-1-◦ylpropane-nitrile
3f.
1
Green solid, 35% yield. Mp 159–162 C. H NMR (300 MHz,
DMSO-d6) d 1.70–2.00 (m, 4H, 2 CH2), 3.25–3.40 (m, 2H,
CH2), 3.55–3.60 (m, 2H, CH2), 3.64 (s, 3H, OCH3), 3.83 (s,
3H, Ar-OCH3), 4.34 (d, J = 3.9 Hz, 1H, C2–H),* 4.36 (d, J =
3.9 Hz, 1H, C4¢–H),* 6.56–6.65 (m, 1H, C5¢–H), 6.90–7.10 (m, 2H,
C6¢–H + C7¢–H), 7.84 (s, 2H, NH2). *Assignments interchangeable.
13C NMR (75 MHz, DMSO-d6) d 23.73 (CH), 25.86 (CH), 35.34
(C4¢), 46.27 (2 CH), 47.06 (C2), 50.58 (OCH3), 55.65 (Ar–OCH3),
71.68 (C3¢), 111.62 (C7¢), 117.38 (CN), 120.92 (C5¢), 121.41 (C4a¢),
123.76 (C6¢), 139.57 (C8a¢), 146.80 (C8¢), 162.15 (C1), 162.86 (C2¢),
167.80 (C-NH2(OCH3)). IR (Nujol mull) n 3413, 3315, 2255,
1677, 1640, 1609, 1592, 1521 cm-1. Anal. Calcd for C19H21N3O5:
C, 61.46; H, 5.66; N, 11.32. Found: C, 61.39; H, 5.61; N, 11.29.
2-(3-[Amino(methoxy)methylene]-8-methoxy-2-oxo-3,4-dihydro-
2H-chromen-4-yl)-2-cyano-N-cyclohexylacetamide 3c. Yellow
solid, 33% yield. Mp 169–171 ◦C. 1H NMR (400 MHz, DMSO-d6)
d 0.90–1.24 (m, 5H, 2 CH2 + CH), 1.55–1.65 (m, 5H, 2 CH2 +
CH), 3.37–3.42 (m, 1H, NH-CH-C5H10), 3.54 (d, J = 7.6 Hz, 1H,
C2–H), 3.60 (s, 3H, OCH3), 3.81 (s, 3H, Ar-OCH3), 4.38 (d, J =
7.2 Hz, 1H, C4¢–H), 6.80 (dd, J = 7.4 Hz, 1.2 Hz, 1H, C5¢–H),
7.01–7.06 (m, 2H, C6¢–H + C7¢–H), 7.80 (s, 2H, NH2), 7.86 (d,
J = 8.0 Hz, 1H, NH). 13C NMR (100 MHz, DMSO-d6) d 24.17
(CH), 25.03 (CH), 31.74 (CH), 31.96 (CH), 35.72 (C4¢), 45.93
(C2), 50.34 (OCH3), 47.87 (NH-CH), 55.63 (Ar–OCH3), 71.84
(C3¢), 111.44 (C7¢), 117.39 (CN), 119.78 (C5¢), 123.76 (C4a¢), 124.12
(C6¢), 139.35 (C8a¢), 146.94 (C8¢), 162.24 (C1), 162.81 (C2¢), 168.07
(C-NH2(OCH3)). IR (Nujol mull) n 3469, 3305, 2242, 1692, 1651,
1622, 1602, 1587, 1546, 1510 cm-1. Anal. Calcd for C21H25N3O5:
C, 63.16; H, 6.27; N, 10.53. Found: C, 63.18; H, 6.35; N, 10.54.
2-(3-[Amino(methoxy)methylene]-8-methoxy-2-oxo-3,4-dihydro-
2H-chromen-4-yl)-3-morpholin-4-yl-3-oxopropane-nitrile
3g.
◦
1
Yellow solid, 80% yield. Mp 186–188 C. H NMR (300 MHz,
DMSO-d6) d 3.46–3.49 (m, 2H, CH2), 3.60–3.70 (m, 9H, 3
CH2 + OCH3), 3.83 (s, 3H, Ar-OCH3), 4.24 (d, J = 3.9 Hz, 1H,
C4¢–H), 4.59 (d, J = 3.9 Hz, 1H, C2–H), 6.57–6.62 (m, 1H, C5¢–H),
7.02–7.05 (m, 2H, C6¢–H + C7¢–H), 7.84 (s, 2H, NH2). 13C NMR
(75 MHz, DMSO-d6) d 36.11 (C4¢), 42.24 (CH), 45.83 (C2), 45.98
(CH), 50.67 (OCH3), 55.66 (Ar–OCH3), 65.98 (2 CH), 71.49 (C3¢),
111.73 (C7¢), 117.73 (CN), 120.61 (C5¢), 121.04 (C4a¢), 123.88 (C6¢),
139.54 (C8a¢), 146.87 (C8¢), 162.77 (C2¢),* 162.85 (C1),* 167.76
(C-NH2(OCH3)). *Assignments interchangeable. IR (Nujol mull)
n 3382, 3273, 2248, 1678, 1641, 1614, 1593, 1537 cm-1. Anal.
Calcd for C19H21N3O6: C, 58.92; H, 5.43; N, 10.85. Found: C,
58.89; H, 5.23; N, 10.99.
2-(3-[Amino(methoxy)methylene]-8-methoxy-2-oxo-3,4-dihydro-
2H-chromen-4-yl)-3-oxo-3-piperidin-1-y◦lpropane-nitrile
3d.
1
Yellow solid, 30% yield. Mp 161–163 C. H NMR (300 MHz,
DMSO-d6) d 1.40–1.70 (m, 6H, 3 CH2), 3.29–3.40 (m, 1H, CH),
3.45–3.60 (m, 2H, CH2), 3.65 (s, 3H, OCH3), 3.66–3.76 (m, 1H,
CH), 3.83 (s, 3H, Ar-OCH3), 4.22 (d, J = 3.9 Hz, 1H, C4¢–H),
4.57 (d, J = 3.6 Hz, 1H, C2–H), 6.50–6.69 (m, 1H, C5¢–H),
7.00–7.05 (m, 2H, C6¢–H + C7¢–H), 7.83 (s, 2H, NH2). 13C NMR
(75 MHz, DMSO-d6) d 23.79 (CH), 25.21 (CH), 26.06 (CH),
36.06 (C4¢), 42.68 (CH), 46.23 (C2 + CH), 50.62 (OCH3), 55.64
(Ar–OCH3), 71.58 (C3¢), 111.69 (C7¢), 117.56 (CN), 120.40 (C5¢),
121.10 (C4a¢), 123.84 (C6¢), 139.53 (C8a¢), 146.88 (C8¢), 162.29 (C1),
162.75 (C2¢), 167.77 (C-NH2(OCH3)). IR (Nujol mull) n 3372,
3261, 2246, 1684, 1638, 1610, 1590, 1548, 1523 cm-1. Anal. Calcd
for C20H23N3O5: C, 62.34; H, 5.97; N, 10.91. Found: C, 62.11; H,
5.88; N, 11.18.
2-(3-[Amino(methoxy)methylene]-8-methoxy-2-oxo-3,4-dihy-
dro-2H-chromen-4-yl)-3-[4-(2-hydroxyethyl)piperazin-1-yl]-3-oxo-
◦
1
propanenitrile 3h. White solid, 42% yield. Mp 129–131 C. H
NMR (300 MHz, DMSO-d6) d 2.36–2.62 (m, 6H, 3 CH2), 3.28–
3.44 (m, 2H, CH2-CH2-OH + CH2-CH2OH), 3.46–3.60 (m, 3H,
CH2 + CH2-CH2OH), 3.66–3.78 (m, 1H, CH2-CH2-OH), 3.65
(s, 3H, OCH3), 3.83 (s, 3H, Ar-OCH3), 4.23 (d, J = 3.9 Hz,
1H, C4¢–H), 4.45 (t, J = 5.1 Hz, 1H, OH), 4.58 (d, J = 3.9 Hz,
1H, C2–H), 6.58 (dd, J = 5.7 Hz, 3.3 Hz, 1H, C5¢–H), 7.02–
7.04 (m, 2H, C6¢–H + C7¢–H), 7.83 (s, 2H, NH2). 13C NMR
(75 MHz, DMSO-d6) d 36.17 (C4¢), 41.90 (CH2-CH2OH), 45.43
4246 | Org. Biomol. Chem., 2011, 9, 4242–4249
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(CH2-CH2-OH), 45.98 (C2), 50.67 (OCH3), 52.72 (CH), 53.26
(CH), 55.66 (Ar–OCH3), 58.50 (CH), 59.95 (CH), 71.53 (C3¢),
111.73 (C7¢), 117.45 (CN), 120.42 (C5¢), 121.09 (C4a¢), 123.88 (C6¢),
139.54 (C8a¢), 146.89 (C8¢), 162.54 (C2¢),* 162.76 (C1),* 167.75 (C-
NH2(OCH3)). *Assignments interchangeable. IR (Nujol mull) n
3500–3200, 2255, 1681, 1664, 1614, 1592, 1534 cm-1. Anal. Calcd
for C21H26N4O6·0.6H2O: C, 57.17; H, 6.17; N, 12.70. Found: C,
57.02; H, 5.97; N, 12.87.
CH), 117.48 (CN), 120.64 (C5¢), 121.02 (C4a¢), 123.87 (C6¢), 137.67
(Ar-CH), 139.54 (C8a¢), 146.86 (C8¢), 147.57 (Ar-CH), 158.69 (Ar-
C), 162.82 (C2¢ + C1), 167.80 (C-NH2(OCH3)). IR (Nujol mull) n
3402, 3292, 2248, 1681, 1645, 1614, 1594, 1567, 1527 cm-1. Anal.
Calcd for C24H25N5O5·0.2H2O: C, 61.72; H, 5.44; N, 15.00. Found:
C, 61.72; H, 5.43; N, 15.05.
2-(3-[Amino(methoxy)methylene]-8-methoxy-2-oxo-3,4-dihy-
dro-2H-chromen-4-yl)-3-oxo-3-(4-pyrimidin-2-ylpiperazin-1-yl)
◦
1
propanenitrile 3l. Yellow solid, 80% yield. Mp 180–182 C. H
NMR (400 MHz, DMSO-d6) d 3.57 (t, J = 5.2 Hz, 2H, CH2), 3.70–
3.81 (m, 4H, 2 CH2), 3.84–3.88 (m, 2H, CH2), 3.69 (s, 3H, OCH3),
3.83 (s, 3H, Ar-OCH3), 4.29 (d, J = 4.0 Hz, 1H, C4¢–H), 4.67 (d, J =
4.0 Hz, 1H, C2–H), 6.60–6.63 (m, 1H, C5¢–H), 6.68 (t, J = 4.8 Hz,
1H, Ar–H), 7.02–7.04 (m, 2H, C6¢–H + C7¢–H), 7.86 (s, 2H, NH2),
8.40 (d, J = 5.2 Hz, 2H, 2 Ar–H). 13C NMR (100 MHz, DMSO-
d6) d 36.14 (C4¢), 41.67 (CH), 42.87 (CH), 43.43 (CH), 44.97
(CH), 46.11 (C2), 50.67 (OCH3), 55.65 (Ar–OCH3), 71.52 (C3¢),
110.60 (Ar-CH), 111.75 (C7¢), 117.41 (CN), 120.66 (C5¢), 121.05
(C4a¢), 123.84 (C6¢), 139.54 (C8a¢), 146.84 (C8¢), 158.00 (2 Ar-CH),
161.08 (Ar-C), 162.81 (C1), 162.91 (C2¢), 167.75 (C-NH2(OCH3)).
IR (Nujol mull) n 3357, 3271, 2247, 1689, 1673, 1633, 1612, 1589,
1552, 1538, 1505 cm-1. Anal. Calcd for C23H24N6O5: C, 59.48; H,
5.17; N, 18.10. Found: C, 59.43; H, 5.07; N, 18.27.
2-(3-[Amino(methoxy)methylene]-8-methoxy-2-oxo-3,4-dihydro-
2H-chromen-4-yl)-3-[4-(3,4-dichlorophenyl)-piperazin-1-yl]-3-oxo-
◦
1
propanenitrile 3i. Yellow solid, 60% yield. Mp 164–166 C. H
NMR (400 MHz, DMSO-d6) d 3.25 (t, J = 5.2 Hz, 2H, CH2),
3.30–3.40 (m, 2H, CH2), 3.52–3.61 (m, 1H, CH2), 3.62–3.65 (m,
1H, CH2), 3.72–3.78 (m, 1H, CH2), 3.83–3.91 (m, 1H, CH2), 3.71
(s, 3H, OCH3), 3.82 (s, 3H, Ar-OCH3), 4.28 (d, J = 4.0 Hz, 1H,
C4¢–H), 4.65 (d, J = 4.0 Hz, 1H, C2–H), 6.58–6.62 (m, 1H, C5¢–H),
6.98–7.05 (m, 3H, C6¢–H + C7¢–H + Ar–H), 7.21 (d, J = 2.8 Hz,
1H, Ar–H), 7.41 (d, J = 8.8 Hz, 1H, Ar–H), 7.85 (s, 2H, NH2).
13C NMR (100 MHz, DMSO-d6) d 36.18 (C4¢), 41.13 (CH), 44.83
(CH), 46.14 (C2), 47.40 (CH), 47.78 (CH), 50.74 (OCH3), 55.64
(Ar–OCH3), 71.50 (C3¢), 111.74 (C7¢), 115.64 (Ar-CH), 116.69
(Ar-CH), 117.37 (CN), 120.04 (Ar-C-Cl), 120.61 (C5¢), 120.93
(C4a¢), 124.29 (C6¢), 130.57 (Ar-CH), 131.52 (Ar-C-Cl), 139.53
(C8a¢), 146.84 (C8¢), 150.32 (Ar-C), 162.74 (C2¢ + C1), 167.77
(C-NH2(OCH3)). IR (Nujol mull) n 3468, 3307, 2246, 1686, 1643,
1611, 1591, 1558, 1516 cm-1. Anal. Calcd for C25H24N4O5Cl2: C,
56.50; H, 4.52; N, 10.55. Found: C, 56.49; H, 4.51; N, 10.69.
General Procedure for the synthesis of compounds 5a–d
2-Cyanoacetamide 1a (0.10 g, 1.19 mmol) was added to a
suspension of the appropriate 2-oxo-2H-chromene-3-carbonitrile
2 (1.09 mmol) in methanol (4 mL) and triethylamine (0.7 mL)
and the reaction mixture was refluxed for 1–2 h. For 5c and 5d,
the suspension was cooled to room temperature and kept in an
ice bath for a few minutes. The solid was filtered and washed
with water. For 5a and 5b, the solvent was removed in the rotary
evaporator and a mixture of ethanol (2 mL), water (4 mL) and
acetic acid (1 mL) was added to the oil. The reaction mixture was
stirred at room temperature for 3–5 h, when the solid was filtered
and washed with water.
2-(3-[Amino(methoxy)methylene]-8-methoxy-2-oxo-3,4-dihy-
dro-2H-chromen-4-yl)-3-[4-(4-fluorophenyl)piperazin-1-yl]-3-oxo-
◦
1
propanenitrile 3j. White solid, 31% yield. Mp 161–163 C. H
NMR (300 MHz, DMSO-d6) d 3.08–3.20 (m, 4H, 2 CH2), 3.54–
3.61 (m, 1H, CH2), 3.60–3.70 (m, 2H, CH2), 3.85–3.92 (m, 1H,
CH2), 3.70 (s, 3H, OCH3), 3.83 (s, 3H, Ar-OCH3), 4.29 (d, J =
3.6 Hz, 1H, C4¢–H), 4.66 (d, J = 3.9 Hz, 1H, C2–H), 6.58–
6.63 (m, 1H, C5¢–H), 6.90–7.15 (m, 6H, C6¢–H + C7¢–H + 4
Ar–H), 7.85 (s, 2H, NH2). 13C NMR (75 MHz, DMSO-d6) d
36.21 (C4¢), 41.72 (CH), 45.23 (CH), 46.08 (C2), 48.92 (CH),
49.41 (CH), 50.72 (OCH3), 55.64 (Ar–OCH3), 71.52 (C3¢), 111.75
(C7¢), 115.33 (2 Ar-CH), 117.42 (CN), 117.82 (2 Ar-CH), 120.55
(C5¢), 121.02 (C4a¢), 123.86 (C6¢), 139.54 (C8a¢), 146.87 (C8¢), 147.55
(Ar-C), 156.34 (Ar-C-F), 162.68 (C2¢),* 162.77 (C1),* 167.48 (C-
NH2(OCH3)). *Assignments interchangeable. IR (Nujol mull) n
3475, 3318, 2249, 1684, 1650, 1614, 1591, 1510 cm-1. Anal. Calcd
for C25H25N4O5F: C, 62.50; H, 5.21; N, 11.67. Found: C, 62.46; H,
5.16; N, 11.65.
4-Amino-7-methoxy-2,5-dioxo-3,5-dihydro-2H-chromeno-[3,4-
c]pyridine-1-carbonitrile 5a. Green solid, 26% yield. Mp higher
than 300 ◦C. 1H NMR (400 MHz, DMSO-d6) d 3.94 (s, 3H,
OCH3), 7.42–7.50 (m, 2H, C8–H + C9–H), 8.73 (dd, J = 7.6 Hz,
2.0 Hz, 1H, C10–H), 10.26 (s, 1H, NH2), 10.37 (s, 1H, NH2),
11.61 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) d 56.31
(OCH3), 80.85 (C1),a 87.44 (C4a),a 116.19 (C8), 116.44 (C10), 116.66
(C10a), 118.66 (CN), 125.45 (C9), 139.30 (C6a), 146.10 (C10b), 147.57
(C7), 163.24 (C2),b 163.85 (C5).b a,bAssignments interchangeable. IR
(Nujol mull) n 3565, 3396, 3274, 2214, 1687, 1642, 1536, 1504 cm-1.
Anal. Calcd for C14H9N3O4·0.8H2O: C, 56.49; H, 3.56; N, 14.12.
Found: C, 56.21; H, 3.66; N, 13.91.
2-(3-[Amino(methoxy)methylene]-8-methoxy-2-oxo-3,4-dihy-
dro-2H-chromen-4-yl)-3-oxo-3-(4-pyridin-2-ylpiperazin-1-yl)pro-
panenitrile 3k. White solid, 37% yield. Mp 146–148 ◦C. 1H NMR
(300 MHz, DMSO-d6) d 3.57–3.61 (m, 4H, 2 CH2), 3.63–3.65 (m,
4H, 2 CH2), 3.70 (s, 3H, OCH3), 3.83 (s, 3H, Ar-OCH3), 4.29 (d,
J = 3.6 Hz, 1H, C4¢–H), 4.68 (d, J = 3.9 Hz, 1H, C2–H), 6.58–6.63
(m, 1H, C5¢–H), 6.68 (t, J = 6.6 Hz, 1H, Ar–H), 6.91 (d, J = 8.4 Hz,
1H, Ar–H), 6.98–7.20 (m, 2H, C6¢–H + C7¢–H), 7.57 (t, J = 7.2 Hz,
1H, Ar–H), 7.86 (s, 2H, NH2), 8.14 (d, J = 3.9 Hz, 1H, Ar–H).
13C NMR (75 MHz, DMSO-d6) d 36.17 (C4¢), 41.61 (CH), 44.21
(CH), 44.85 (CH), 44.94 (CH), 46.19 (C2), 50.75 (OCH3), 55.65
(Ar–OCH3), 71.52 (C3¢), 107.48 (Ar-CH), 111.73 (C7¢), 113.46 (Ar-
4-Amino-7-hydroxy-2,5-dioxo-3,5-dihydro-2H-chromeno-[3,4-
c]pyridine-1-carbonitrile 5b. Green solid, 36% yield. Mp higher
◦
1
than 300 C. H NMR (400 MHz, DMSO-d6) d 7.26 (dd, J =
8.0 Hz, 1.6 Hz, 1H, C8–H), 7.31 (t, J = 8.0 Hz, 1H, C9–H), 8.46
(dd, J = 8.2 Hz, 1.2 Hz, 1H, C10–H), 10.36 (brs, 3H, OH + NH2),
11.58 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) d 80.67 (C1),a
87.42 (C4a),a 115.27 (C10), 116.98 (C10a), 118.76 (C8), 119.83 (CN),
125.39 (C8), 138.67 (C6a), 145.96 (C7), 146.43 (C10b), 163.31 (C2),b
163.92 (C5).b a,bAssignments interchangeable. IR (Nujol mull) n
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3345, 3260, 3144, 2217, 1681, 1600, 1522, 1509 cm-1. Anal. Calcd
for C13H7N3O4·0.4H2O: C, 56.48; H, 2.82; N, 15.21. Found: C,
56.55; H, 2.92; N, 14.98.
Anal. Calcd for C20H24N4O4·0.3H2O: C, 61.63; H, 6.32; N, 14.38.
Found: C, 61.55; H, 6.12; N, 14.33.
Radioligand binding assays
4-Amino-9-chloro-2,5-dioxo-3,5-dihydro-2H -chromeno[3,4-
c]pyridine-◦1-carbonitrile 5c. Yellow solid, 32% yield. Mp higher
The inhibition percentage of the compounds was assayed at the
concentration of 10 mM at all adenosine receptors following
the conditions stated above. Competition binding curves at all
receptors were carried out by assaying 6 different concentrations
(range from 10 nM to 100 mM) for all the compounds showing
an inhibition percentage above 70%. The -log of the inhibition
constant (pKi) of each compound was calculated by the Cheng–
Prusoff equation, Ki = IC50/(1 + [L]/KD), where IC50 is the
concentration of compound that displaces the binding of the
radioligand by 50%, [L] is the free radioligand concentration and
KD is the dissociation constant of each radioligand. IC50 values
were obtained by non-linear regression fitting the data, using Prism
2.1 software (GraphPad, San Diego, CA).
1
than 300 C. H NMR (400 MHz, DMSO-d6) d 7.50 (d, J =
9.0 Hz, 1H, C7–H), 7.81 (dd, J = 8.8 Hz, 2.4 Hz, 1H, C8–H), 8.98
(d, J = 2.4 Hz, 1H, C10–H), 10.25 (s, 1H, NH2), 10.37 (s, 1H, NH2),
11.64 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) d 80.84 (C1),a
87.46 (C4a),a 117.40 (C10a), 118.42 (CN), 119.61 (C7), 124.92 (C10),
129.58 (C9), 133.81 (C8), 144.56 (C10b), 148.18 (C6a), 162.95 (C2),b
163.58 (C5).b a,bAssignments interchangeable. IR (Nujol mull) n
3353, 3160, 2204, 1683, 1615, 1598, 1520 cm-1. Anal. Calcd for
C13H6N3O3Cl: C, 54.26; H, 2.09; N, 14.61. Found: C, 54.52; H,
2.33; N, 14.40.
4-Amino-9-bromo-2,5-dioxo-3,5-dihydro-2H -chromeno-[3,4-
c]pyridine-◦1-carbonitrile 5d. Yellow solid, 35% yield. Mp higher
1
than 300 C. H NMR (400 MHz, DMSO-d6) d 7.45 (d, J =
Human A1 receptors
8.8 Hz, 1H, C7–H), 7.94 (dd, J = 8.8 Hz, 2.0 Hz, 1H, C8–H), 9.16
(d, J = 2.0 Hz, 1H, C10–H), 10.24 (s, 1H, NH2), 10.38 (s, 1H, NH2),
11.66 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) d 80.83 (C1),a
87.52 (C4a),a 117.45 (C10a), 117.88 (C9), 118.46 (CN), 119.85 (C7),
127.94 (C10), 136.63 (C8), 144.55 (C10b), 148.63 (C6a), 163.00 (C2),b
163.60 (C5).b a,bAssignments interchangeable. IR (Nujol mull) n
3323, 3145, 2204, 1693, 1611, 1599, 1579, 1504 cm-1. Anal. Calcd
for C13H6N3O3Br·0.2H2O: C, 46.48; H, 1.91; N, 12.52. Found: C,
46.55; H, 1.97; N, 12.77.
Adenosine A1 receptor competition binding experiments were
carried out in membranes from CHO-A1 cells (Euroscreen,
Gosselies, Belgium). On the day of the assay, membranes were
defrosted and re-suspended in incubation buffer 20 mM Hepes,
100 mM NaCl, 10 mM MgCl2, 2 UI ml-1 adenosine deaminase
(pH = 7.4). Each reaction well of a GF/C multiscreen plate
(Millipore, Madrid, Spain), prepared in duplicate, contained 15 mg
of protein, 2 nM [3H]DPCPX and the test compound. Non-specific
binding was determined in the presence of 10 mM (R)-PIA. The
◦
Synthesis of the ammonium salt of 4-(2-hydroxy-3-methoxy-
phenyl)-2,6-dioxo-1,2,3,6-tetrahydropyridine-3,5-dicarbonitrile 9
reaction mixture was incubated at 25 C for 60 min, after which
the samples were filtered and measured in a microplate beta
scintillation counter (Microbeta Trilux, Perkin Elmer, Madrid,
Spain).
2-Cyanoacetamide 1a (0.06 g, 0.68 mmol) was added to a
suspension of 2-oxo-8-methoxy-2H-chromene-3-carbonitrile 2a
(0.13 g, 0.67 mmol) in methanol (3 mL) and triethylamine (0.7 mL)
and the reaction mixture was stirred at room temperature. After 5 h
a white solid started to precipitate from solution and after 2 days
the solid was filtered and washed with methanol and identified
Human A2A receptors
Adenosine A2A receptor competition binding experiments were
carried out in membranes from HeLa-A2A cells. On the day of the
assay, membranes were defrosted and re-suspended in incubation
buffer 50 mM Tris-HCl, 1 mM EDTA, 10 mM MgCl2 and 2
UI mL-1 adenosine deaminase (pH = 7.4). Each reaction well of a
GF/C multiscreen plate (Millipore, Madrid, Spain), prepared in
duplicate, contained 10 mg of protein, 3 nM [3H]ZM241385 and
the test compound. Non-specific binding was determined in the
presence of 50 mM NECA. The reaction mixture was incubated at
25 ◦C for 30 min, after which samples were filtered and measured in
a microplate beta scintillation counter (Microbeta Trilux, Perkin
Elmer, Madrid, Spain).
1
by H NMR as a 1 : 1 : 1 mixture of compounds 3a, 5a, and 9.
The mother liquor was kept at 0◦ C for 4 days and a white solid
precipitated and was filtered and washed with methanol. The solid
was identified by 1H NMR as the pure compound 3a (22% yield).
The solid mixture containing 3a, 5a and 9 (in a 1 : 1 : 1 ratio) was
refluxed in methanol (3 mL) and triethylamine (0.4 mL) for 2.5
h. The isolated solid was identified as a 1 : 1 mixture of 5a and
9. This mixture was stirred at room temperature for 35 min in
a combination of ethanol (2 mL), water (4 mL) and acetic acid
(1 mL). Compound 5a precipitated from this reaction mixture and
was isolated in 8.5% yield. Compound 9 (5.4%) was also isolated
as a white solid from the mother liquor after partial evaporation of
the solvent. Mp higher than 300 ◦C. 1H NMR (300 MHz, DMSO-
d6) d 1.05–1.20 (m, 9H, 3 CH3), 3.00–3.20 (m, 6H, 3 CH2), 3.82 (s,
3H, OCH3), 6.63 (d, J = 7.2 Hz, 1H, C6¢–H), 6.79 (t, J = 8.1 Hz,
1H, C5¢–H), 6.95 (d, J = 7.5 Hz, 1H, C4¢–H), 8.40–9.20 (brs, 1H,
NH+-(CH2CH3)3), 8.79 (s, 1H, OH), 10.42 (s, 1H, NH). 13C NMR
(75 MHz, DMSO-d6) d 8.65 (3 CH3), 45.76 (3 CH2), 55.64 (OCH3),
82.58 (C3 + C5), 111.78 (C4¢), 118.64 (C5¢), 1118.75 (2 CN), 120.51
(C6¢), 124.24 (C1¢), 142.82 (C2¢), 147.45 (C3¢), 160.05 (C4), 163.50
(C2 + C6). IR (Nujol mull) n 3427, 3113, 2208, 1625, 1516 cm-1.
Human A2B receptors
Adenosine A2B receptor competition binding experiments were
carried out in membranes from HEK-293-A2B cells (Euroscreen,
Gosselies, Belgium) prepared following the provider’s protocol.
On the day of the assay, membranes were defrosted and re-
suspended in incubation buffer 50 mM Tris-HCl, 1 mM EDTA,
10 mM MgCl2, 0.1 mM benzamidine, 10 mg mL-1 bacitracine
and 2 UI mL-1 adenosine deaminase (pH = 6.5). Each reaction
well prepared in duplicate, contained 18 mg of protein, 35 nM
4248 | Org. Biomol. Chem., 2011, 9, 4242–4249
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[3H]DPCPX and the test compound. Non-specific binding was
determined in the presence of 400 mM NECA. The reaction mix-
ture was incubated at 25 ◦C for 30 min, after which samples were
filtered through a multiscreen GF/C microplate and measured in
a microplate beta scintillation counter (Microbeta Trilux, Perkin
Elmer, Madrid, Spain).
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Human A3 receptors
Adenosine A3 receptor competition binding experiments were
carried out in membranes from HeLa-A3 cells. On the day of the
assay, membranes were defrosted and re-suspended in incubation
buffer 50 mM Tris-HCl, 1 mM EDTA, 5 mM MgCl2 and 2
UI mL-1 adenosine deaminase (pH = 7.4). Each reaction well of
a GF/B multiscreen plate (Millipore, Madrid, Spain), prepared
in triplicate, contained 90 mg of protein, 30 nM [3H]NECA and
the test compound. Non-specific binding was determined in the
presence of 100 mM (R)-PIA. The reaction mixture was incubated
at 25 ◦C for 180 min, after which samples were filtered and
measured in a microplate beta scintillation counter (Microbeta
Trilux, Perkin Elmer, Madrid, Spain).
Acknowledgements
We gratefully acknowledge the financial support from Fundac¸a˜o
para a Cieˆncia e Tecnologia, a PhD grant awarded to Marta Costa
(SFRH/BD/31531/2006) and a post-doc grant awarded to Filipe
Areias (SFRH/BPD/26106/2005).
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This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 4242–4249 | 4249
©