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A Journal of
Accepted Article
Title: Domino Reaction of Chromone-3-carboxylic acids with
Aminoheterocycles: Synthesis of Heteroannulated Pyrido[2,3c]coumarins and their Optical and Biological Activity
Authors: Maria Miliutina, Julia Janke, Elena Chirkina, Sidra Hassan,
Abida Ejaz, Shafi Ullah Khan, Jamshed Iqbal, Aleksej
Friedrich, Stefan Lochbrunner, Anton Ivanov, Alexander
Villinger, Joanna Lecka, Jean Sevigny, and Peter Langer
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201701276
Link to VoR: http://dx.doi.org/10.1002/ejoc.201701276
Supported by
10.1002/ejoc.201701276
European Journal of Organic Chemistry
1
Domino Reaction of Chromone-3-carboxylic acids with Aminoheterocycles:
Synthesis of Heteroannulated Pyrido[2,3-c]coumarins and their Optical and
Biological Activity
Mariia Miliutina,a Julia Janke,a Elena Chirkina,a Sidra Hassan,b Syeda Abida Ejaz,b Shafi Ullah
Khan,b Jamshed Iqbal,b Aleksej Friedrich,c Stefan Lochbrunner,c Anton Ivanov,a Alexander
Villinger,a Joanna Lecka,d,e Jean Sévigny,d,e Peter Langera,f*
Institut für Chemie, Universität Rostock, Albert Einstein Str. 3a, 18059 Rostock, Germany;
peter.langer@uni-rostock.de
b
Centre for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabad, Pakistan
c
Institut für Physik, Universität Rostock, Albert-Einstein-Str. 23, 18059 Rostock, Germany
d
Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université
Laval, Québec, QC, G1V 0A6, Canada
e
f
Centre de Recherche du CHU de Québec – Université Laval, Québec, QC, G1V 4G2, Canada
Leibniz Institut für Katalyse an der Universität Rostock e.V., Albert Einstein Str. 29a, 18059
Rostock, Germany
Abstract: A series of new heteroannulated pyrido[2,3-c]coumarins were prepared by domino
reactions of chromone-3-carboxylic acid derivatives with electron-rich binucleophilic
aminoheterocycles. The products contain the core structures of coumarin, pyridine and of an
annulated five-membered heterocyclic system, such as pyrazole, pyrrole or isoazole. The
fluorescence of the products was investigated. The products inhibit ecto-5′-nucleotidase
(e5'NT) enzymatic activity.
Keywords: domino reactions; chromones; coumarins; ecto 5′-nucleotidases (e5'NT).
Introduction
Domino reactions consist of a range of consecutive and complementary bond-forming
transformations which result in products with high structural complexity. These processes are
highly effective and ecologically-friendly because of resource economy, diminished waste and
1
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Accepted Manuscript
a
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2
cost factors.1-3 Such reactions are widely spread in nature and become more and more important
in the field of natural product chemistry.4, 5 For example, the toxic vasoconstrictor palytoxin,6 the
triterpene lanosterol,7 the steroid progesterone,8 some alkaloids,9-14 fatty acids,15 flavanoids16 and
other natural products17-20 have been successfully synthesized on the basis of domino processes.
Pyrido[3,2-c]coumarins are important because of their photophysical and biological effects and
potential applications such as bright fluorescence,21 chemosensory,22 fluorescence imaging in
this type are usually obtained from 3-substituted coumarins,23,26-29 4-substituted coumarins,30-35
salicylic aldehydes,36 2-halobiarylcarboxylates,37 or from halogenated arylcarboxylates38 by
means of catalytic processes. Several authors communicated the synthesis of 5-hydroxy-5Hbenzopyrano[4,3-b]pyridine, a reduced precursor of the pyrido[3,2-c]coumarin core structure,
starting from 3-formylchromone.39-43 Pyrido[3,2-c]coumarins were obtained by reaction of
chromone-3-carboxylic acid with 3-aminocrotononitrile, a process developed by Dieter Heber
who named this transformation as an ANRORC process (Addition of nucleophile, Ring Opening
and Ring Closure). Heber et al. also studied the influence of pyrido[3,2-c]coumarins on the
regulation of the blood sugar level.24 The ring opening ring closure reactions are characteristic
for chromone chemistry and in particular for the chemistry of 3-substituted derivatives as
chromone-3-carboxylic acids.44 The 1,4-nucleophilic addition at C-2 position of a 3-substituted
chromone with successive γ-pyrone ring opening proceeds to further transformations to afford a
variety of products depending on reaction conditions, the nature of substrate substitution and the
type of nucleophile. In the present manuscript, we report what is, to the best of our knowledge, a
new and convenient synthesis of heteroannulated pyrido[3,2-c]coumarins by domino reaction of
derivatives of chromone-3-carboxylic acid44b with amino-substituted heterocycles, such as
aminopyrazoles, aminopyrroles and aminooxazoles.
The impact of the synthesized compounds was then tested on ecto-5′-nucleotidase (e5'NT) a
membrane bound protein regarded as the lymphocyte differentiation antigen CD73.45 The major
physiological role of e5'NT is to facilitate the final step of the ATP hydrolytic cascade, i.e.
adenosine production. Adenosine is considered as a master switch molecule between
extracellular pathways, involving consumption and production of ATP molecules.46 Ecto-5′-NT
has also been described as multifunctional molecule with properties that are not related to its
catalytic function.47 As an adhesive molecule, it facilitates the tumor invasiveness in human
glioblastoma.48 Along this, e5'NT is overexpressed in various human solid tumors, and correlated
with tumor invasiveness, neovascularization and metastasis, resulting in shorter life
expectancy.49 It has been observed that e5'NT was negatively regulated by estrogen receptor and
2
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Accepted Manuscript
living cells,23 decrease of the blood sugar level,24 positive inotropic effectets.25 Compounds of
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European Journal of Organic Chemistry
3
the progression of a breast cancer disease is directly related to the expression of e5'NT as well as
production of adenosine molecule. Hence, overexpression of e5'NT is used as a novel marker for
aggressive breast cancer in estrogen receptor (ER) negative cells.50 Therefore, either genetic
ablation or utilization of antibodies against e5'NT has been shown to alleviate the cancer growth
as well as their metastasis. Similarly, specific inhibitors of tumor derived e5'NT result in
alternation of adaptive immune response that ultimately results in alteration of adenosinedependent signaling pathways and their related effects. With these effects, specific and potent
with other therapeutic strategies to fight against cancer progression.51 Importantly, ADP and
ATP are physiological inhibitors of the enzyme via binding in a substrate-analogous manner,
with their inhibitory values in the low micromolar range. But these natural compounds are easily
hydrolyzed in the body. It has been found from detailed studies that the ADP analogue, α,βmethylene-ADP (AMPCP or AOPCP) can be regarded as potential inhibitor of e5'NT. Along
with adenosine diphosphate and triphosphate analogues, some other derivatives are also known
to possess significant inhibitory potential.52 Previously reported inhibitors are anthraquinone,53
sulphonamide,54 polyphenol,55 and polyoxometalates.56 But still there is a need for highly
selective and potent inhibitors of e5'NT that can be used for therapeutic intervention.56b In view
of this, we decided to evaluate our newly synthesized heteroannulated pyrido[2,3-c]coumarins
for their e5'NT inhibitory potential. Because of their potential application in life cell imaging we
also investigated their absorption and fluorescence properties.
Results and discussion
Our starting point was the development of suitable conditions for the synthesis of pyrido[3,2c]coumarin 3a by reaction of chromone-3-carboxylic acid 1a with aminopyrazole 2a (Scheme 1).
The aminopyrazole 2a and related aminoheterocycles used in this study were prepared as
previously reported in our work related to the synthesis of heteroannulated pyridines,57-64
spirocyclic 1,4-dihydropyridine compounds65 and 3,4-fused coumarins.66 The best yield of 3a (up
to 90%) was obtained under the conditions reported in entry 5 (Table 1) using acetic acid
(AcOH) as solvent (14 h, 60 °C). The use of Me3SiCl or phosphoric acid proved to be less
efficient in terms of yield (Table 1). A minor quantity of by-product 5a could also be isolated
(7%; Scheme 1). When the reaction time was shortened (only 1 h at 60 °C), we were able to
isolate a trace amount of intermediate 4a. It was possible to determine the structure of crystals
3
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inhibitors are regarded as important therapeutic agents that can be used alone or in combination
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4a, but, due to the small amounts of obtained product, we were unable to complete its full
spectroscopic characterization (vide infra).
The formation of product 3a can be explained, as outlined in Scheme 1 (path A), by conjugate
addition of the enamine moiety of 2a to the double bond of 1a, cleavage of the chromone system,
lactonization of the phenol group with the acid residue and subsequent addition of the amino
group to the carbonyl. The formation of 5a (Scheme 1; path B) can be explained by conjugate
ring. Occurring decarboxylation is a key step on the way of formation of diverse chalcones from
chromone precursors as described in the literature on the example of reaction of chromone-3carboxylic acids with indoles.44c
Scheme 1. Possible mechanism of the formation of pyrido[3,2-c]coumarin 3a and of byproducts
4a and 5a. Reagents and conditions: see Table 1, entry 5.
4
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addition, decarboxylation (to give intermediate 4a) and cyclodehydration yielding the pyridine
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5
Entry
1
2
3
4
5
6
Proportion
1a : 2a
1 : 1.5
1 : 1.5
1 : 1.5
1 : 1.05
1 : 1.05
1 : 1.05
Reaction conditions i
Solvent
Acid
Temperature
DMF
DMF
AcOH
AcOH
AcOH
AcOH
TMSCl
H3PO4
AcOH
AcOH
AcOH
AcOH
130 ˚C
135 ˚C
100 ˚C
20 ˚C
60 ˚C
120 ˚C
Yield 3a,
%
37
36
55
87
90
80
Duration
14 h
14 h
14 h
14 h
14 h
14 h
By the optimized method described above a variety of pyrido[3,2-c]coumarins 3a-v were
synthesized in good to excellent yield (Table 2). The yield of the reaction considerably depends
on the type of heterocyclic dinucleophile 2. The yields decrease from products 3a-k over
products 3l-p to products 3q-v in accordance with the decrease of the electron density of the
starting aminoheterocycles from pyrazoles 2a-c over cyanopyrroles 2d-g to isoxazoles 2h-l
(Table 2). Byproducts 5 have been isolated as minor products in many cases during the synthesis
of 3a-v. The spectra of byproducts 5 match those of previously reported clean products prepared
by reactions of 2,3-unsubstituted chromones with aminoheterocycles 2.62
Table 2. Synthesis of products 3a-v.
Precursore 1
Aminoheterocycle 2
Product 3
Isolated
yield (%)
3a,
1a
2a
90
3b,
1a
2b
96
5
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Table 1. Optimization of the synthesis of the compound 3a.
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3c,
1a
2c
87
3d,
2a
86
Accepted Manuscript
1b
3e,
1b
2b
81
3f,
1b
2c
83
3g,
1c
2a
80
3h,
1d
1e
2a
84
3i,
2a
81
6
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3j,
1f
2a
82
2b
1a
2d
Accepted Manuscript
3k,
1f
84
3l,
70
3m,
1a
31
2e
3n,
1a
48
2f
3o,
1a
51
2g
7
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3p,
1b
65
2g
2h
Accepted Manuscript
3q,
1a
33
3r,
1a
2i
42
3s,
1a
2j
19
3t,
1b
2j
1b
2k
36
3u,
33
3v,
1b
2l
45
The molecular structures of compounds 3 were determined by standard spectroscopic methods.
Moreover, structures of compounds 3c, 3d, 3j and 3m as well as 4a were unambiguously
confirmed by crystallographic analysis (Figures 1-5).
8
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Accepted Manuscript
Figure 1. X-Ray structure of the byproduct 4a.
Figure 2. X-Ray structure of 3c.
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10
Figure 3. X-Ray structure of 3d.
Figure 4. X-Ray structure of 3j.
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European Journal of Organic Chemistry
Figure 5. X-Ray structure of 3m.
UV/Vis-Spectroscopy
Fused aromatic heterocycles with intramolecular charge transfer (ICT) character are important
objectives of current research in view of their potential applications as fluorescent dyes in
organic light emitting diodes, optoelectronic devices and sensing materials.67-70 Due to their large
conjugated backbone pyrido[3,2-c]coumarins show a bright fluorescence and, therefore, these
molecules were thoroughly studied by UV/Vis-spectroscopy.
To investigate the optical properties of the synthesized pyrido[3,2-c]coumarins the absorption
and fluorescence spectra of seven selected compounds were recorded using methylcyclohexane
as solvent. The spectra are shown in Figure 6 and characteristic spectral parameters are listed in
Table 3.
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Accepted Manuscript
a)
b)
Figure 6. Absorption (solid lines) and fluorescence spectra (broken lines) of selected
compounds. For better visibility the spectra are shifted vertically with respect to each other. For
comparison, the spectra of 3d are shown in a) and b).
Table 3. Spectral parameters for selected pyrido[3,2-c]coumarins.
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λmax
(nm)
282
281
284
266
280
277.5
276
ε(λmax)
(l·mol-1·cm-1)
27380
31753
22923
31389
33635
13459
2868
λshoulder
(nm)
340
341
346
349
343
350
343
ε(λshoulder)
(l·mol-1·cm-1)
5475
5984
5167
7615
6432
2157
2010
λfl-max
(nm)
422
436
419
378
424
372
347
Φfl
(%)
9.2
12.0
7.1
9.8
8.8
14.9
6.9
Compounds 3a, 3b, and 3h have very similar absorption and fluorescence spectra. The lowest
absorption band appears at 280 nm and exhibits a structured wing extending to 410 nm. The
fluorescence spectra show vibronic structure and have their maxima between 420 nm and
440 nm. Adding a fluorine atom to the coumarin moiety has obviously no impact on the spectra,
while replacing the phenyl substituent by methylbenzene does also not change the absorption,
but shifts the fluorescence by about 10 nm to the red. Adding a methyl group to the coumarin
moiety does neither change the fluorescence nor the dominant absorption band as one can see
from the spectra of 3d. However, the long wavelength wing of the absorption becomes more
prominent and transforms almost to a band by its own. This indicates that the wing and the
shoulder, respectively, result from the transition to the lowest electronically excited singlet state
while the dominant absorption band is due to a higher lying electronically excited state. The
fluorescence quantum yields of the four compounds are similar and in the range of 10%.
Change of the substituents located at the pyrazole ring cause pronounced changes in the
absorption and fluorescence spectra (see Figure 6b). If the phenyl substituent of 3d is replaced
by a methyl group, resulting in 3f, the shoulder at the long wavelength edge of the absorption
spectrum shifts slightly to the red and becomes a separate band. The fluorescence, however,
shifts to the blue and the Stokes shift of 3f is quite small. At the same time the vibronic structure
is sharper than in 3d. Both effects indicate that the structural relaxation after optical excitation is
in 3f smaller than in 3d. This effect becomes even stronger for 3l and 3q. The width of the
lowest absorption band and of the fluorescence decreases, the fluorescence shifts to the blue and
the Stokes shift reduces the vibronic lines becomes more narrow and the 00-transition gains in
strength. All this is in line with the observation that, by going from 3d via 3f and 3l to 3q, the
geometrical changes between the electronically excited and ground state are drastically reduced.
The Strickler-Berg symmetry between the lowest absorption band and the fluorescence is,
therefore, particularly distinct in the case of 3q. At the same time decreases the overall
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Compound
3a
3b
3d
3f
3h
3l
3q
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14
absorption strength from 3d to 3q. The quantum yields are again in the order of 10% and vary by
maximal a factor of two between the compounds.
Biological structure-activity relationship (SAR)
(h-) and rat (r-) e5'NT (Table 4). Representatives of synthesized pyrrole or pyrazole derivatives
possess inhibitory potential against h-e5'NT while few pyrazole derivatives (3k, 3a, 3h, 3j and
5a) are active against r-e5'NT. In contrast, isoxazole products inhibit neither of both types of the
enzyme.
IC50 concentration range of the derivatives inhibiting the h-e5'NT lies between 0.16 to 41.2 μM.
From the evaluation of their structures, it is concluded that an aryl substituent at position 1 of the
pyrrole ring as well as the presence of an electron donating group (-CH3) at the coumarin ring,
results in the increased activity of a compound towards the target. Compound 3p, which contains
a pyrrole and pyridine ring, inhibited h-e5'NT most effectively with an IC50±SEM value of
0.16±0.02 μM. It can be suggested that the presence of an electron donating group, i.e. toluene at
the pyrrole ring, was essential for the stronger activity of the derivative 3p. Other synthesized
compounds possessing a nitrile group, e.g. 3l, 3m, 3n and 3o, show remarkably less inhibitory
potential, due to a lack of substitution at the coumarin ring. For example, compound 3o,
possessing the same substitution at the pyrrole ring as 3p but lacking any substitution at the
coumarin ring, exhibits less inhibitory activity. Additionally, pyrazolopyridine derivatives
exhibit a somewhat higher reactivity towards their active site. Among them, derivatives 3f, 3d
and 3e, containing an electron donating group (-CH3) at the coumarin ring, possess significant
inhibitory potential in the range of 0.19 to 0.63 μM. Compound 3f displays significant inhibition
with an IC50 ± SEM value of 0.19±0.01 μM, due to the less bulky substitution of the pyrazole
ring by a methyl group. However, when the methyl group was replaced by a bulky substituent,
i.e. phenyl or methylphenyl as in compounds 3d and 3e, respectively, this ultimately resulted in a
loss of activity.
Within the list of coumarine derivatives 3a, 3h, 3j and 3k inhibiting r-e5'NT, compound 3k is
the most potent inhibitor with a moderate IC50 ± SEM value of 2.55±0.09 μM. From the
structural analysis, it can be suggested that its inhibitory activity might be due to the presence of
an electron donating group (-OCH3) at the coumarin ring as well as a bulky group
14
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The synthesized derivatives were further evaluated for their inhibitory potential against human
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15
(methylphenyl) at the pyrazole ring. We assume that the presence of the methoxy group is
essential for a stronger inhibition of e5'NT enzymatic activity. The replacement of the
methylphenyl ring by a phenyl group resulted in a loss of inhibitory potential. Surprisingly,
among tested compounds the greatest inhibition of r-e5'NT shows pyrazolopyridine 5a which is
therefore selected for molecular docking analysis. This fact gives rise to an assumption that a
pyrazolopyridine bicycle is quite essential part of the molecule in terms of e5'NT inhibition.
derivatives possessing h-e5'NT inhibiting activity, whereas derivatives having a pyrazole ring
showed the maximal r-e5'NT inhibition.
Table 4. Ecto-5'-nucleotidase (h-e5'NT & r-e5'NT) inhibition data for the synthesized
compounds.
Sr. No.
Codes
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
4a
5a
Sulfamic acid
h-e5'NT
r-e5'NT
IC50±SEM (μM)
3.95±0.12
2.67±0.03
24.8±0.47
>100
0.61±0.03
>100
0.32±0.04
>100
0.63±0.09
>100
0.19±0.01
>100
>100
>100
25.1±1.21
9.21±0.67
2.1±0.05
--7.52±0.11
14.4±0.03
18.1±0.89
2.55±0.09
>100
>100
>100
>100
0.59±0.03
>100
41.2±0.21
>100
0.16±0.02
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
25.8±0.58
0.64±0.001
42.1±5.8
77.3±7.0
15
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From the structure activity relationship we conclude that the presence of pyrrole is important for
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Values are expressed as mean ± SEM of n=3. The IC50 is the concentration at which 50% of the
enzyme activity is inhibited.
Docking Studies
binding interactions with h-e5'NT and r-e5'NT, respectively. Figure 7 illustrates the putative 3D
binding interactions of compound 3p within the active site of h-e5'NT. A detailed analysis of the
binding interactions revealed that compound 3p forms a protein-ligand complex with h-e5'NT by
establishing several strong binding interaction such as π–cationic and π-π stacked interactions
with various amino acid residues of h-e5'NT. Among these bonding interactions the pyridine and
the pyrrole ring of compound 3p form π-π stacking interactions with the amino acid residues of
His243 and Arg354, respectively. The benzene ring adjacent to the pyrrole ring is involved in a
π-π stacking and an amide-π interaction with the amino acid residues of Phe500 and Gly393,
respectively. In addition exist two π-cationic interactions between the pyridine and the benzene
rings of compound 3p and the amino acid residues of Arg354 and Arg395, respectively as shown
in Fig. 7.
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Docking studies of the potent compounds 3p and 5a were carried out to identify the putative
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Figure 7. Putative binding interaction of compound 3p (olive colored) with amino acid residue
of h-e5'NT (cyan colored).
A putative bonding mode of compound 5a within the active pocket of a model structure of re5'NT is shown in Figure 8. The analysis of the binding interactions revealed that one hydrogen
bond, five π-π stacked, one π-anionic and one π-cationic interactions are formed between
compound 5a and different amino acid residues of r-e5'NT. One hydrogen bond is formed
between the hydroxyl group of compound 5a and the amino acid residue Asn392 while three π-π
stacking interactions were found between the amino acid residue Arg397 and the pyrazole, the
pyridine and the benzene ring of compound 5a. In addition, two π-π stacking interactions are also
formed by amino acid Tyr502 and the pyrazole as well as the pyridine ring of compound 5a. The
benzene ring attached to the pyrazole ring is involved in a π-anionic interaction with the amino
acid residue of Arg397 and a π-cationic interaction with the amino acid residue of Asp508.
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European Journal of Organic Chemistry
Figure 8. Putative binding interaction of potent compound 5a (sky colored) with amino acid
residue of r-e5'NT (golden colored).
In conclusion, the strong inhibitory potential of compound 3p against h-e5'NT and of compound
5a against r-e5'NT is due to strong binding interactions between these compounds and several
amino acid residues of the target enzymes.
Conclusions
In summary, we have found that a variety of pyrido[3,2-c]coumarin derivatives are readily
accessible starting with chromone-3-carboxylic acid derivatives and heterocyclic amines. The
investigated products exhibit moderately strong fluorescence. By varying the pyrazole ring and
its substituents the width of the vibronic lines and the geometry change between the ground and
the electronically excited state can be tuned. Derivatives 3p and 5a show a strong ecto 5′nucleotidases (e5'NT) inhibitory activity. The structure-activity relationship was explained
based on docking studies.
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Experimental section
General information for physicochemical analysis.
NMR spectra were recorded by a Bruker AVANCE 250 (250 MHz), a Brucker AVANCE 300
(300 MHz) and a Brucker AVANCE 500 (500 MHz) NMR spectrometer. Chemical shifts (ppm)
were given relative to the solvent; references for CDCl3 were 7.26 ppm (1H-NMR) and 77.16
NMR). Multiplets were assigned as s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), m
(multiplet), br s (broad singlet). All measurements were carried out at room temperature unless
otherwise stated. IR spectra were recorded on a Nicolet 6700 FT-IR spectrometer (ATR). The
wavelength was given in cm-1. Abbreviations: s = strong; m = middle; w = weak. Melting points
were measured on a Stanford Research Systems or Micro-Hot-Stage GalenTM III Cambridge
Instruments. Abbreviation: Mp. The melting points were not corrected. Mass spectra were
obtained on a Hewlett-Packard HP GC / MS 5890 / 5972 instrument (EI, 70 eV) by GC inlet, on
a MX-1321 and Finnigan MAT 95 XP instruments (EI, 70 eV) by direct inlet. The data were
given as mass units per charge (m/z). Column chromatography was performed on silica gel (63 –
200 mesh, Merck). Chemical yields refer to pure isolated substances. The CDP-Star
chemiluminescent substrate was obtained from Sigma Aldrich while other chemicals used in the
biochemical assay were of analytical grade.
UV/VIS-Spectroscopy
Steady-state UV/Vis absorption and fluorescence spectra were measured with a Specord 50
UV/Vis spectrophotometer (Analytic Jena) and a FluoroMax4 spectrofluorometer (Horiba
Scientific). Compounds were dissolved in methylcyclohexane and excited at 350 nm or 325 nm
for the emission experiments. Fluorescence spectra were corrected for the wavelength
dependence of the detection sensitivity. Quantum yields were determined using quinine bisulfate
dissolved in 0.05 M sulfuric acid as standard.70
General procedure for the synthesis of 3.
In a pressure tube under inert atmosphere, starting 4-oxo-4H-chromene-3-carboxylic acid 1 (1
equiv., 200 mg) and an appropriate amine 2a-l (1.05 equiv.) were dissolved in 4 mL of glacial
19
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Accepted Manuscript
ppm (13C-NMR); references for DMSO-d6 were 2.54 ppm (1H-NMR) and 39.50 ppm (13C-
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acetic acid and were stirred at 60 °C during one night (14 h). The crude product was purified by
preparative column chromatography to give 3a-v (heptane/ethylacetate-20/1, Rf ≈ 0.9, blue
fluorescence under UV light, not visible under the sun light). To isolate intermediate 4a the
reaction was maintained for 1 h under the same conditions. After that the reaction mixture was
separated by column chromatography (heptane/ethylacetate-20/1, Rf ≈ 0.6, no fluorescence under
8-Methyl-10-phenylchromeno[4,3-b]pyrazolo[4,3-e]pyridin-6(10H)-one (3a).
White crystals, yield 90%. Mp 235-236 °C. 1H NMR (250.13 MHz, CDCl3): δ= 2.67 (s, 3H,
CH3), 7.29-7.45 (m, 3H, Ar), 7.50-7.64 (m, 3H, Ar), 8.35 (d, 2H, 3J = 7.9 Hz, Ar), 8.56 (dd, 1H,
3
J = 7.9 Hz, 4J = 1.3 Hz, Ar), 8.96 (s, 1H, Py). 13C NMR (62.90 MHz, CDCl3): δ = 12.63 (CH3),
111.87 (C), 117.42 (CH), 118.09, 119.61 (2 C), 120.87 (2 CH), 124.94, 125.32, 126.32 (3 CH),
129.25 (2 CH), 132.65, 134.11 (2 CH), 139.11, 145.15, 151.05, 151.96, 152.90, 161.65 (6 C). IR
(ATR, cm-1): ~ = 1720 (s, C=O); 1608, 1591 (m, C=C, C=N); 1516 (s, C-H); 1419, 1385 (m,
CH2-H), 1252 (s, C-O-C); 1187, 1116, 1089 (m, C-O-C, C-N); 752 (C-H).MS (GC, 70eV): m/z
(%) = 327 (M+, 100), 312 (12), 77 (13), 51 (14). HRMS (ESI): calcd for C20H13N3O2 327.1006,
found 327.1008.
8-Methyl-10-p-tolylchromeno[4,3-b]pyrazolo[4,3-e]pyridin-6(10H)-one (3b).
White crystals, yield 96%. Mp 246-247 °C. 1H NMR (500.13 MHz, CDCl3): δ= 2.46 (s, 3H,
CH3, Tol), 2.71 (s, 3H, CH3), 7.35-7.43 (m, 2H, Tol + 2H, Ar), 7.59 (ddd, 1H, 3J = 8.3 Hz, 3J =
7.3 Hz, 4J = 1.7 Hz, Ar), 8.20-8.24 (m, 2H, Tol), 8.62 (d, 1H, 3J = 7.9 Hz, Ar), 9.03 (s, 1H, Py).
13
C (75.47 MHz, CDCl3): δ = 12.68, 21.24 (2 CH3), 111.80 (C), 117.46 (CH), 117.99, 119.76 (2
C), 121.08 (2 CH), 124.93, 125.40 (2 CH), 129.82 (2 CH), 132.62, 134.21 (2 CH), 136.23,
136.69, 144.93, 151.08, 151.87, 152.94, 161.83 (7 C). IR (ATR, cm-1): ~ = 1724 (s, C=O); 1607,
1505 (m, C=C, C=N); 1381 (m, CH2-H); 1253 (s, C-O-C, C-N); 818, 755 (s, C-H). MS (GC,
70eV): m/z (%) = 342 (M+1, 18), 341 (M+, 100), 340 (M-1, 27), 326 (11), 44 (12). HRMS (ESI):
calcd for C21H15O2N3 341.1163, found 341.1164.
8,10-Dimethylchromeno[4,3-b]pyrazolo[4,3-e]pyridin-6(10H)-one (3c).
20
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Accepted Manuscript
UV light, yellow spot under the sun light).
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White crystals, yield 87%. Mp 257-258 °C. 1H NMR (250.13 MHz, CDCl3): δ= 2.61 (s, 3H,
CH3), 4.15 (s, 3H, NCH3), 7.29-7.44 (m, 2H, Ar), 7.55 (dd, 1H, 3J = 7.8 Hz, 4J = 1.5 Hz, Ar),
8.62 (dd, 1H, 3J = 7.8 Hz, 4J = 1.5 Hz, Ar), 8.94 (s, 1H, Py). 13C (62.90 MHz, CDCl3): δ = 12.45
(CH3), 33.67 (NCH3), 110.92, 115.94 (2 C), 117.21 (CH), 119.55 (C), 124.61, 124.94, 132.23,
134.06 (4 CH), 143.35, 150.45, 152.38, 152.60, 161.86 (5 C). IR (ATR, cm-1): ~ = 1725 (s,
C=O); 1610, 1492 (m, C=C, C=N); 1249 (m, CH2-H); 1192, 1088 (m, C-O-C, C-N); 760 (s, CH).MS (GC, 70eV): m/z (%) = 266 (M+1, 17), 265 (M+, 100), 264 (M-1, 74). HRMS (ESI): calcd
2,8-Dimethyl-10-phenylchromeno[4,3-b]pyrazolo[4,3-e]pyridin-6(10H)-one (3d).
White crystals, yield 86%. Mp 226-227 °C. 1H NMR (300.13 MHz, CDCl3): δ= 2.52 (s, 3H,
CH3), 2.72 (s, 3H, CH3), 7.29 (s, 1H, Ar), 3.61-4.15 (m, 2H, Ar), 4.83-5.50 (m, 2H, Ar), 9.449.93 (m, 3H, Ar), 9.02 (s, 1H, Py).
13
C NMR (62.90 MHz, CDCl3): δ = 12.64, 21.26 (2 CH3),
111.93 (C), 117.19 (CH), 117.98, 119.18 (2 C), 121.01 (2 CH), 124.96, 126.32 (2 CH), 129.28 (2
CH), 133.65, 134.15 (2 CH), 134.65, 139.12, 145.15, 151.02, 151.18, 151.98, 161.84 (7 C). IR
(ATR, cm-1): ~ = 1731 (s, C=O); 1594 (m, C=C, C=N); 1500, 1418 (s, CH ); 1247, 1193 (s, C3
O-C, C-N); 796, 749 (s, C-H).MS (GC, 70eV): m/z (%) = 342 (M+1, 22), 341 (M+, 100), 340
(M+, 21), 77 (18), 51 (13). HRMS (ESI): calcd for C21H15O2N3 341.1159, found 341.1157.
2,8-Dimethyl-10-p-tolylchromeno[4,3-b]pyrazolo[4,3-e]pyridin-6(10H)-one (3e).
White crystals, yield 81%. Mp 216-217 °C. 1H NMR (250.13 MHz, CDCl3): δ = 2.45 (s, 3H,
CH3), 2.69 (s, 3H, CH3), 3.92 (s, 3H, CH3), 6.85 (d, 1H, 4J = 2.4 Hz, Ar), 6.96 (dd, 1H, 4J = 2.4
Hz, 3J = 8.7 Hz, Ar), 7.37 (d, 2H, 3J = 8.4 Hz, Tol), 8.21 (d, 2H, 3J = 8.4 Hz, Tol), 8.49 (d, 1H, 3J
= 8.7 Hz, Ar), 8.97 (s, 1H, Py). 13C NMR (62.90 MHz, CDCl3): δ = 12.67, 21.23, 55.94 (3 CH3),
101.23 (CH), 110.73, 112.87 (2 C), 112.96 (CH), 117.37 (C), 121.06 (2 CH), 126.59 (CH),
129.79 (2 CH), 134.25 (CH), 136.10, 136.76, 144.95, 151.39, 152.06, 154.39, 162.13, 163.44 (8
C). IR (ATR, cm-1): ~ = 1724 (s, C=O); 1608 (m, C=C, C=N), 1504 (m, CH ), 1383, 1253 (s,
3
+1
C-O-C, C-N), 810 (s), 755 (s, C-H). MS (GC, 70 eV): m/z (%) = 356 (M , 14), 355 (M+, 100),
354 (M-1, 16), 340 (9), 65 (7). HRMS (ESI): calcd for C22H17O2N3 355.1315, found 355.1313.
2,8,10-Trimethylchromeno[4,3-b]pyrazolo[4,3-e]pyridin-6(10H)-one (3f).
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Accepted Manuscript
for C15H11O2N3 265.0850, found 265.0851.
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White crystals, yield 83%. Mp 259-260 °C. 1H NMR (250.13 MHz, CDCl3): δ= 2.53 (s, 3H,
CH3), 2.66 (s, 3H, CH3), 4.21 (s, 3H, NCH3), 7.30 (s, 1H, Ar), 7.40 (dd, 1H, 3J = 8.4 Hz, 4J = 2.1
Hz, Ar), 8.45 (d, 1H, 3J = 1.4 Hz, Ar), 9.01 (s, 1H, Py).
13
C (62.90 MHz, CDCl3): δ = 12.47,
20.98 (2 CH3), 33.71 (NCH3), 111.02, 115.90 (2 C), 117.00 (CH), 119.15 (C), 124.64, 133.22,
134.13 (3 CH), 134.37, 143.35, 150.61, 150.76, 152.44, 162.08 (6 C). IR (ATR, cm-1): ~ = 1715
(s, C=O); 1592, 1561, 1466 (m, C=C, C=N); 1254 (m, CH2-H); 1192, 1067 (m, C-O-C, C-N);
795 (s, C-H). MS (GC, 70eV): m/z (%) = 280 (M+1, 17), 279 (M+, 100), 278 (M-1, 43), 251 (12).
2-Bromo-8-methyl-10-phenylchromeno[4,3-b]pyrazolo[4,3-e]pyridin-6(10H)-one (3g).
White crystals, yield 80%. Mp 264-265 °C. 1H NMR (300.13 MHz, CDCl3): δ= 2.74 (s, 3H,
CH3), 7.28 (d, 1H, 3J = 8.8 Hz, Ar), 7.35-7.43 (m, 1H, Ph), 7.56-7.64 (m, 2H, Ph), 7.68 (dd, 1H,
3
J = 8.8 Hz, 4J = 2.4 Hz, Ar), 8.28-8.36 (m, 2H, Ph), 8.71 (d, 1H, 3J = 2.4 Hz, Ar), 9.06 (s, 1H,
Py).
13
C (75.47 MHz, CDCl3): δ = 12.56 (CH3), 111.74, 117.86, 118.37 (3 C), 119.19 (CH),
121.17 (2 CH), 121.24 (C), 126.56, 127.80 (2 CH), 129.28 (2 CH), 134.27, 135.32 (2 CH),
138.75, 145.16, 149.77, 151.69, 151.75, 161.03 (6 C). IR (ATR, cm-1): ~ = 1735 (s, C=O); 1595,
1556, 1496 (m, C=C, C=N); 1434 (m, CH2-H); 1251, 1186 (s, C-O-C, C-N); 793, 744 (s, C-H);
683, 533 (C-Br). MS (GC, 70eV): m/z (%) = 408 (81M+1, 22), 407 (81M+, 98), 406 (80M+1, 33),
405 (80M+, 100), 404 (11), 325 (13), 77 (35), 51 (12). HRMS (ESI): calcd for
C20H12O281BrN3 407.0090, found 407.0092.
3-Fluoro-8-methyl-10-phenylchromeno[4,3-b]pyrazolo[4,3-e]pyridin-6(10H)-one (3h).
White crystals, yield 84%. Mp 254-255 °C. 1H NMR (250.13 MHz, CDCl3): δ= 2.65 (s, 3H,
CH3), 6.96-7.13 (m, 2H, Ar), 7.30 (t, 1H, 3J = 7.4 Hz, Ph), 7.52 (t, 2H, 3J = 7.8 Hz, Ph), 8.28 (d,
2H, 3J =7.8 Hz, Ph), 8.6 (dd, 1H, 3J = 8.4 Hz, 4J = 6.51 Hz, Ar), 8.96 (s, 1H, Py).
19
F NMR
(282.40 MHz, CDCl3): δ= -104.93. Dept (62.90 MHz, CDCl3): δ = 12.54 (CH3), 104.76 (CH, J =
25.7 Hz, Ar), 112.88 (CH, J = 22.5 Hz, Ar), 120.98 (2 CH, Ph), 126.37 (CH, Ph), 127.21 (CH, J
= 10.0, Ar), 129.16 (2 CH, Ph), 134.29 (CH, Py). IR (ATR, cm-1): ~ = 1738 (s, C=O); 1612,
1592 (m, C=C, C=N); 1497 (m, C-H); 1433, 1385 (m, CH2-H, C-H); 1268, 1146 (s, C-O-C, CN); 1112, 867 (m, C-F); 788, 750 (s, C-H). MS (GC, 70eV): m/z (%) = 346 (M+1, 24), 345 (M+,
100), 344 (M-1, 35), 330 (14), 77 (16). HRMS (ESI): calcd for C20H12O2FN3 345.0912, found
345.0914.
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Accepted Manuscript
HRMS (ESI): calcd for C16H13O2N3 279.1007, found 279.1008.
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2-Methoxy-8-methyl-10-phenylchromeno[4,3-b]pyrazolo[4,3-e]pyridin-6(10H)-one (3i).
White crystals, yield 81%. Mp 240-241 °C. 1H NMR (250.13 MHz, CDCl3): δ= 2.70 (s, 3H,
CH3), 3.93 (s, 3H, OCH3), 7.14 (dd, 1H, 3J = 9.0 Hz, 4J = 3.0 Hz, Ar), 7.26-7.40 (m, 1H, Ar +
1H, Ph), 7.56 (t, 2H, 3J = 7.8 Hz, Ph), 8.02 (d, 1H, 4J = 3.0 Hz, Ar), 8.35 (d, 2H, 3J = 7.8 Hz,
13
C (62.90 MHz, CDCl3): δ = 12.67, 55.91 (2 CH3), 107.73 (CH), 11.95,
118.14 (2 C), 118.54, 119.95 (2 CH), 120.16 (C), 121.01 (2 CH), 126.37 (CH), 129.24 (2 CH),
134.24 (CH), 139.10, 145.21, 147.29, 150.95, 151.92, 156.62, 161.80 (7 C). IR (ATR, cm-1): ~
= 1750 (s, C=O); 1609, 1590 (s, C=C, C=N); 1268 (s, O-CH3); 1250, 1184, 1165, 1120, 1015
(m, C-O-C, C-N); 752 (s, C-H). MS (GC, 70eV): m/z (%) = 358 (M+1, 21), 357 (M+, 100), 356
(M-1, 36), 286 (19), 77 (17). HRMS (ESI): calcd for C21H15O3N3 357.3610, found 357.3611.
3-Methoxy-8-methyl-10-phenylchromeno[4,3-b]pyrazolo[4,3-e]pyridin-6(10H)-one (3j).
White crystals, yield 82%. Mp 228-229 °C. 1H NMR (300.13 MHz, CDCl3): δ= 2.64 (s, 3H,
CH3), 3.90 (s, 3H, OCH3), 6.78 (d, 1H, 4J = 2.4 Hz, Ar), 6.93 (dd, 1H, 3J = 8.8 Hz, 4J = 2.4 Hz,
Ar), 7.34 (t, 1H, 3J = 7.4 Hz, Ph), 7.55 (t, 2H, 3J = 7.9 Hz, Ph), 8.34 (dd, 2H, 3J = 7.7 Hz, 4J = 1.0
Hz, Ph), 8.41 (d, 1H, 3J = 8.8 Hz, Ar), 8.88 (s, 1H, Py).
13
C NMR (75.47 MHz, CDCl3): δ =
12.61 (CH3), 55.91 (OCH3), 101.19 (CH), 110.76, 112.70 (2 C), 112.91 (CH), 117.45 (C),
120.80 (2 CH), 126.18, 126.48 (2 CH), 129.21 (2 CH), 134.12 (CH), 139.18, 145.16, 151.31,
152.12, 154.32, 161.92, 163.42 (7 C). IR (ATR, cm-1): ~ = 1725 (s, C=O); 1610, 1590 (s, C=C,
C=N); 1479, 1418, 1380, 1348 (m, C-H, CH2-H); 1270 (s, O-CH3); 1250, 1184, 1152, 1114,
1025 (m, C-O-C, C-N); 752 (s, C-H). MS (GC, 70eV): m/z (%) = 358 (M+1, 24), 357 (M+, 100),
356 (M-1, 13), 314 (12), 77 (13). HRMS (ESI): calcd for C19H10O5 357.1112, found 357.1113.
3-Methoxy-8-methyl-10-p-tolylchromeno[4,3-b]pyrazolo[4,3-e]pyridin-6(10H)-one (3k).
White crystals, yield 84%. Mp 268-269 °C. 1H NMR (500.13 MHz, DMSO-d6): δ= 2.45 (s, 3H,
CH3, Tol), 2.68 (s, 3H, CH3), 3.91 (s, 3H, OCH3), 6.83 (d, 1H, 3J = 2.4 Hz, Ar), 6.95 (dd, 1H, 3J
= 8.8 Hz, 4J = 2.4 Hz, Ar), 7.36 (d, 2H, 3J = 8.6 Hz, Tol), 8.20 (d, 2H, 3J = 8.6 Hz, Tol), 8.47 (d,
1H, 3J = 8.8 Hz, Ar), 8.95 (s, 1H, Py). 13C (75.47 MHz, CDCl3): δ = 12.65, 21.23 (2 CH3), 55.93
(OCH3), 101.21 (CH), 110.71, 112.85 (2 C), 112.94 (CH), 117.36 (C), 121.01 (2 CH), 126.57
(CH), 129.78 (2 CH), 134.21 (CH), 136.06, 136.76, 144.93, 151.35, 152.03, 154.37, 162.10,
163.42 (8 C). IR (ATR, cm-1): ~ = 1740 (s, C=O); 1608, 1511 (m, C=C, C=N); 1380 (m, CH 2
23
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Accepted Manuscript
Ph), 9.00 (s, 1H, Py).
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H); 1272 (s, O-CH3); 1250, 1111, 1022 (m, C-O-C, C-N); 818, 786 (s, C-H). MS (GC, 70eV):
m/z (%) = 372 (M+1, 24), 371 (M+, 100), 370 (M-1, 18), 328 (11). HRMS (ESI): calcd for
C22H17O3N3 371.1260, found 371.1259.
10-tert-Butyl-6-oxo-6,10-dihydrochromeno[4,3-b]pyrrolo[3,2-e]pyridine-8-carbonitrile (3l).
White crystals, yield 70%. Mp 342-343°C. 1H NMR (250.13 MHz, DMSO-d6): δ= 1.93 (s, 9H,
8.53 (d, 1H, 3J = 7.5 Hz, Ar), 8.68 (s, 1H, Pyr), 8.76 (s, 1H, Py).
13
C (62.90 MHz, CDCl3): δ =
29.35 (3 CH3), 59.72, 85.78, 112.69, 114.19 (4 C), 117.50 (CH), 119.89, 121.86 (2 C), 124.60,
124.92, 131.53, 131.97, 136.70 (5 CH), 147.20, 149.74, 152.47, 161.65 (4 C). IR (ATR, cm-1): ~
= 2228 (m, C≡N), 1717 (s, C=O), 1616 (m, C=N), 1403 (s, C-H/CH3), 1188 (s, C-O-C), 1087
(m, C-N), 759 (C-H). MS (GC, 70eV): m/z (%) = 317 (M+, 20), 262 (14), 261 (100), 260 (18),
205 (10), 204 (11), 178 (12), 177 (10), 57 (24), 41 (36), 39 (15), 29 (16). HRMS (ESI): calcd for
C19H15O2N3 317.1163, found 317.1164.
6-Oxo-10-phenethyl-6,10-dihydrochromeno[4,3-b]pyrrolo[3,2-e]pyridine-8-carbonitrile
(3m).
Grey crystals, yield 31%. Mp 251-252 °C. 1H NMR (MHz, ): δ= 3.27 (t, 2H, 3J = 6.9 Hz, CH2),
4.73 (t, 2H, 3J = 6.9 Hz, CH2), 7.07 (dd, 2H, 3J = 7.70 Hz, 4J = 1.6 Hz, Ar), 7.19-7.32 (m, 3H, Ar
+ CHCl3), 7.37-7.48 (m, 2H, Ar), 7.55 (s, 1H, Pyr), 7.59 (ddd, 1H, 3J = 8.2 Hz, 3J` = 7.4 Hz, 4J =
1.7 Hz, Ar), 8.64 (dd, 1H, 3J = 7.9 Hz, 3J = 1.5 Hz, Ar), 9.03 (s, 1H, Py).
13
C (62.90 MHz,
CDCl3): δ = 36.29, 47.76 (2 CH2), 86.42, 113.28, 113.84 (3 C), 117.48 (CH), 119.59, 120.41 (2
C), 124.70, 124.88, 127.48 (3 CH), 128.79, 129.10 (4 CH), 131.92, 132.17 (2 CH), 137.18 (C),
138.61 (CH), 148.27, 149.26, 152.48, 161.58 (4 C). IR (ATR, cm-1): ~ = 3110 (m, =C-H), 2224
(m, C≡N), 1728 (s, C=O), 1608 (s, C=N), 1421 (m, C-H/CH3), 1244 (s, C-O-C), 1183 (m, C-N),
752 (s), 705 (s, C-H). MS (GC, 70eV): m/z (%) = 365 (M+, 16), 274 (15), 262 (18), 261 (100), 91
(12). HRMS (ESI): calcd for С23H15O2N3 365.1159, found 365.1157.
10-(3-Chlorophenyl)-6-oxo-6,10-dihydrochromeno[4,3-b]pyrrolo[3,2-e]pyridine-8carbonitrile (3n).
24
This article is protected by copyright. All rights reserved.
Accepted Manuscript
3CH3), 7.42 (d, 1H, 3J = 7.9 Hz, Ar), 7.49 (d, 1H, 3J = 7.5 Hz, Ar), 7.64 (t, 1H, 3J = 7.6 Hz, Ar),
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Grey crystals, yield 48%. Mp 316-317 °C. 1H NMR (250.13 MHz, DMSO-d6): δ= 7.34-7.53 (m,
2H, Ar), 7.55-7.58 (m, 3H, Ar), 8.01 (dd, 1H, 3J = 8.0 Hz, Ar), 8.13 (d, 1H, 3J = 1.9, Ar), 8.40
(dd, 1H, 3J = 8.0 Hz, 4J = 1.7, Ar), 8.90 (d, 1H, 4J = 1.7, Pyr), 9.06 (s, 1H, Py). 13C (62.90 MHz,
DMSO-d6): δ = 87.08, 112.92 (2 C), 116.47 (CH), 118.55, 120.08 (2 C), 122.45, 123.51, 123.83,
124.32, 127.53, 130.14, 130.43, 131.70 (8 CH), 133.36, 136.76 (2 C), 139.82 (CH), 147.33,
151.55, 159.63, 167.03, 173.34 (5 C). IR (ATR, cm-1): ~ = 3113 (m, =C-H), 2232 (m, C≡N),
1712 (s, C=O), 1594 (s, C=N), 1409 (s, C-H), 1230 (s, C-O-C), 1103 (C-N), 871 (C-H), 754 (C(ESI): calcd for C21H1035ClO2N3 371.0456, found 371.0454; C21H1037ClO2N3 373.0427, found
373.0428.
6-Oxo-10-p-tolyl-6,10-dihydrochromeno[4,3-b]pyrrolo[3,2-e]pyridine-8-carbonitrile (3o).
White crystals, yield 51%. Mp higher 375 °C. 1H NMR (250.13 MHz, CDCl3): δ= 2.51 (s, 3H,
CH3), 7.33-7.47 (m, 2H, Tol + 2H, Ar), 7.53-7.61 (m, 1H, Ar), 7.68 (d, 2H, 3J = 8.4 Hz, Tol),
8.17 (s, 1H, Pyr), 8.51 (dd, 1H, 3J = 7.9 Hz, 4J = 1.4 Hz, Ar), 9.16 (s, 1H, Py). Due to the poor
solubility of compound 3o, no satisfactory 13C NMR could be obtained. IR (ATR, cm-1): ~ =
2224 (s, CN); 1734 (s, C=O); 1608, 1508 (m, C=C, C=N); 1421 (s, CH2-H); 1236, 1184 (s, C-OC, C-N); 750 (s, C-H). MS (GC, 70eV): m/z (%) = 352 (M+1, 25), 351 (M+, 100), 147 (10), 91
(10). HRMS (ESI): calcd for C16H13O2N3 351.1007, found 351.1008.
2-Methyl-6-oxo-10-p-tolyl-6,10-dihydrochromeno[4,3-b]pyrrolo[3,2-e]pyridine-8carbonitrile (3p).
White crystals, yield 65%. Mp 369-370 °C. 1H NMR (250.13 MHz, CDCl3): δ= 2.46 (s, 3H,
CH3), 2.52 (s, 3H, CH3), 7.26-7.39 (m, 2H, Ar), 7.46 (d, 2H, 3J = 7.9 Hz, Tol), 7.68 (d, 2H, 3J =
7.9 Hz, Tol), 8.15 (s, 1H, Pyr), 8.26 (s, 1H, Ar), 9.14 (s, 1H, Py).
13
C (62.90 MHz,): δ = 21.24,
29.38 (2 CH3), 91.85, 102.78, 112.96, 113.91 (4 C), 117.25, 118.66 (2 CH), 120.77 (C),124.53 (2
CH), 124.60 (C), 125.61 (CH), 130.53 (2 CH), 132.27, 137.43 (2 CH), 137.91, 138.05, 142.74,
149.41, 155.15, 163.78 (6 C). IR (ATR, cm-1): ~ = 2226 (s, CN); 1746 (s, C=O); 1605, 1509 (m,
C=C, C=N); 1423 (s, CH2-H); 1247, 1184 (s, C-O-C, C-N); 808 (s, C-H). MS (GC, 70eV): m/z
(%) = 366 (M+1, 25), 365 (M+, 100). HRMS (ESI): calcd for C16H13O2N3 365.1163, found
365.1164.
25
This article is protected by copyright. All rights reserved.
Accepted Manuscript
Cl). MS (GC, 70eV): m/z (%) = 373 (37M+, 35), 372 (25), 371 (35M+, 100), 158 (15). HRMS
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8-Phenyl-6H-chromeno[4,3-b]isoxazolo[4,5-e]pyridin-6-one (3q).
White crystals, yield 33%. Mp 296-297 °C. 1H NMR (300.13 MHz, CDCl3): δ= 7.42-7.53 (m,
2H, Ar), 7.60-7.66 (m, 3H, Ph), 7.69 (ddd, 1H, 3J = 8.2 Hz, 3J = 7.4 Hz, 4J = 1.6 Hz, Ar), 8.018.08 (m, 2H, Ph), 8.73 (dd, 1H, 3J = 7.9 Hz, 4J = 1.6 Hz, Ar), 9.34 (s, 1H, Py). 13C (62.90 MHz,
CDCl3): δ = 113.53, 115.10 (2 C), 117.59 (CH), 118.83 (C), 125.51, 125.97 (2 CH), 127.51 (C),
1268 (m, CH2-H); 1192 (m, C-O-C, C-N); 880, 763, 670 (s, C-H). MS (GC, 70eV): m/z (%) =
315 (M+1, 13), 314 (M+, 60), 287 (20), 286 (100), 127 (18), 77 (48), 51 (18). HRMS (ESI): calcd
for C19H10O3N2 314.0690, found 314.0691.
8-p-Tolyl-6H-chromeno[4,3-b]isoxazolo[4,5-e]pyridin-6-one (3r).
White crystals, yield 42%. 1H NMR (250.13 MHz, CDCl3): δ= 2.49 (s, 3H, CH3), 7.43 (d, 2H, 3J
= 8.0 Hz, Tol), 7.47-7.52 (m, 1H, Ar), 7.65-7.72 (m, 1H, Ar), 7.85 (d, 1H, 3J = 8.0 Hz, Ar), 7.94
(d, 1H, 3J = 8.0 Hz, Tol), 8.73 (dd, 1H, 3J = 8.0 Hz, 4J = 1.7 Hz, Ar), 9.33 (s, 1H, Py).
13
C
(300.13 MHz, CDCl3): δ = 21.71 (CH3), 111.73, 112.44 (2C), 116.04 (CH), 117.57, 119.31 (2C),
119.65 (CH), 127.74 (2CH), 127.86 (CH), 129.59 (C), 130.24 (2CH), 130.42 (C), 133.08, 133.69
(2CH), 139.79, 141.52, 150.95, 160.03 (4C). IR (ATR, cm-1): ~ = 1731 (s, C=O); 1596, 1565,
1470, 1373 (m, C=C, C=N); 1260 (s, CH2-H); 1176, 1041 (s, C-O-C, C-N); 884, 798 (s, C-H).
MS (GC, 70eV): m/z (%) = 328 (M+, 5), 303 (21), 302 (100), 301 (44), 279 (24), 274 (17), 246
(32), 91 (17), 65 (14), 57 (15), 43 (10), 41 (10). HRMS (ESI): calcd for C20H12O3N2 328.0842,
found 328.0843.
8-Methyl-6H-chromeno[4,3-b]isoxazolo[4,5-e]pyridin-6-one (3s).
White crystals, yield 36%. Mp 296-297 °C. 1H NMR (300.13 MHz, CDCl3): δ= 2.69 (s, 3H,
CH3), 7.36-7.50 (m, 2H, Ar), 7.65 (ddd, 1H, 3J = 8.2 Hz, 3J = 7.4 Hz, 4J = 1.7 Hz, Ar), 8.66 (ddd,
1H, 3J = 7.9 Hz, 4J = 1.7 Hz, 5J = 0.32 Hz, Ar), 9.03 (s, 1H, Py). 13C (62.90 MHz, CDCl3): δ =
11.03 (CH3), 114.57, 115.12 (2 C), 117.51 (CH), 118.87 (C), 125.40, 125.88, 133.73, 136.05 (4
CH), 153.25, 153.55, 156.57, 160.77, 171.56 (5 C). IR (ATR, cm-1): ~ = 1716 (s, C=O); 1596,
1574, 1382 (m, C=C, C=N); 1267 (m, CH2-H); 1189 (m, C-O-C, C-N); 753 (s, C-H). MS (GC,
70eV): m/z (%) = 253 (M+1, 15), 252 (M+, 100), 237 (29), 224 (29), 209 (15), 181 (13), 127 (21).
HRMS (ESI): calcd for C14H8O3N2 252.0529, found 252.0530.
26
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128.00, 129.74 (4 CH), 131.73, 133.90 (2 CH), 135.45 (C), 137.32 (CH), 140.22, 153.36,
160.83, 168.19 (4 C). IR (ATR, cm-1): ~ = 1723 (s, C=O); 1600, 1462, 1383 (m, C=C, C=N);
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2,8-Dimethyl-6H-chromeno[4,3-b]isoxazolo[4,5-e]pyridin-6-one (3t).
White crystals, yield 36%. Mp 292-293 °C. 1H NMR (300.13 MHz, CDCl3): δ= 2.51 (s, 3H,
CH3), 2.69 (s, 3H, CH3), 7.32 (d, 1H, 3J = 8.4 Hz, Ar), 7.46 (dd, 1H, 3J = 8.4 Hz, 4J = 1.9 Hz,
Ar), 8.47 (d, 1H, 4J = 1.3 Hz, Ar), 9.03 (s, 1H, Py). 13C (62.90 MHz, CDCl3): δ = 11.03, 21.09(2
1579, 1373 (m, C=C, C=N); 1265 (m, CH2-H); 1186, 1094 (m, C-O-C, C-N); 799 (s, C-H). MS
(GC, 70eV): m/z (%) = 267 (M+1, 20), 266 (M+, 100), 251 (22), 238 (13), 208 (17), 147 (31), 84
(13), 78 (25), 69 (16), 66 (11), 63 (26), 57 (13), 55 (11), 44 (48), 43 (19), 41 (12). HRMS (ESI):
calcd for C15H10O3N2 266.0690, found 266.0691.
2-Methyl-8-phenyl-6H-chromeno[4,3-b]isoxazolo[4,5-e]pyridin-6-one (3u).
White crystals, yield 33%. Mp 291-292 °C. 1H NMR (300.13 MHz, CDCl3): δ= 2.53 (s, 3H,
CH3), 7.34 (d, 1H, 3J = 8.4 Hz, Ar), 7.48 (dd, 1H, 3J = 8.4 Hz, 4J = 1.9 Hz, Ar), 7.59-7.66 (m,
3H, Ph), 8.00-8.08 (m, 2H, Ph), 8.51 (d, 1H, 4J = 1.4 Hz, Ar), 9.33 (s, 1H, Py). 13C (75.47 MHz,
CDCl3): δ = 21.11 (CH3), 113.37, 115.13 (2 C), 117.34 (CH), 118.37 (C), 125.62 (CH), 127.55
(C), 128.00, 129.73 (4 CH), 131.71, 134.92 (2 CH), 135.42 (C), 137.35 (CH), 151.50, 161.00,
166.73, 176.29, 185.38 (5 C). IR (ATR, cm-1): ~ = 1733 (s, C=O); 1601, 1568, 1470, 1366 (m,
C=C, C=N); 1270 (m, CH2-H); 1191 (m, C-O-C, C-N); 828, 795 (s, C-H). MS (GC, 70eV): m/z
(%) = 329 (M+1, 34), 328 (M+, 94), 301 (27), 300 (100), 149 (16), 84 (11), 77 (14), 57 (13), 44
(12), 43 (13). HRMS (ESI): calcd for C20H12O3N2 328.0847, found 328.0848.
8-(4-Methoxyphenyl)-2-methyl-6H-chromeno[4,3-b]isoxazolo[4,5-e]pyridin-6-one (3v).
White crystals, yield 45%. Mp 281-282 °C. 1H NMR (300.13 MHz, CDCl3): δ= 2.52 (s, 3H,
CH3), 3.93 (s, 3H, OCH3), 7.13 (d, 2H, 3J = 8.9 Hz, Ar`), 7.33 (d, 1H, 3J = 8.4 Hz, Ar), 7.48
(ddd, 1H, 3J = 8.4 Hz, 4J = 2.2 Hz, 5J = 0.5 Hz, Ar), 8.00 (d, 2H, 3J = 8.9 Hz, Ar`), 8.51 (d, 1H,
4
J = 1.1 Hz, Ar), 9.31 (s, 1H, Py).
13
C (62.90 MHz, CDCl3): δ = 22.58 (CH3), 55.69 (OCH3),
103.41 (C), 109.56 (CH), 113.53 (C), 115.18 (2 CH), 117.31 (CH), 119.87 (C), 125.60 (CH),
128.98 (C), 129.46 (2 CH), 134.83, 135.38 (2 C), 137.32 (CH), 143.91, 151.48, 162.39, 175.63,
184.06 (5 C). IR (ATR, cm-1): ~ = 1730 (s, C=O); 1595, 1566, 1373 (m, C=C, C=N); 1259 (m,
CH2-H); 1176, 1041 (m, C-O-C, C-N); 883, 798 (s, C-H). MS (GC, 70eV): m/z (%) = 360 (M+2,
27
This article is protected by copyright. All rights reserved.
Accepted Manuscript
CH3), 114.63, 114.98 (2 C), 117.29 (CH), 118.45 (C), 125.57, 134.75 (2 CH), 135.32 (C), 136.10
(CH), 149.03, 151.43, 153.69, 156.58, 160.98 (5 C). IR (ATR, cm-1): ~ = 1723 (s, C=O); 1602,
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13), 359 (M+1, 42), 358 (M+, 89), 331 (38), 330 (95), 329 (12), 97 (10), 78 (42), 69 (19), 63 (46),
57 (17), 55 (14), 45 (16), 44 (100), 43 (20), 41 (14), 36 (18). HRMS (ESI): calcd for
C21H14O4N2 358.0952, found 358.0954.
Biochemical Assays
The cell transfection of e5'NT was carried out according to the previously reported method.71 For
this, COS-7 were transfected with the plasmids expressing e5′NT (human and rat)72 in 10 cm
plates using Lipofectamine. The confluent cells (80-90%) were incubated at 37 °C for 5 h, in
Dulbecco’s modified Eagle’s medium (DMEM) (without fetal bovine serum (FBS)) with 6 µg of
plasmid DNA and 24 µL of Lipofectamine reagent. The transfection was stopped by adding the
same volume of DMEM/F-12 containing 20% FBS and the cells were harvested after 48-72 h.
Preparation of membrane fractions
The transfected cells (obtained in the previous step) were washed gently three times with Tris–
saline buffer at 4 °C and then collected by scraping in the harvesting buffer (95 mM NaCl, 0.1
mM phenylmethylsulfonyl fluoride (PMSF) and 45 mM Tris buffer, pH 7.5). The obtained cells
were washed again two times by following centrifugation at 300×g for 5 min. at 4 °C.73 The cells
were resuspended in the harvesting buffer containing 10 µg/mL aprotinin and then sonicated.
Nuclei and cellular debris (were discarded after centrifugation (300×g at 4 °C; 10 min). Glycerol
was added to the resulting supernatant at a final concentration of 7.5% which was kept at -80 °C
until used. The total protein was estimated by Bradford microplate assay by using Bovine serum
albumin as a reference standard.74
Ecto-5ʹ-Nucleotidase Inhibition Assay
The enzymatic assay of both human and rat e5′NTs was performed using capillary
electrophoresis (P/ACE MDQ CE system (Beckman Instruments, Fullerton, CA) as described in
previously reported data.75 According to this method, the stock solution of compounds (10 mM)
were prepared in 10% DMSO and their working solutions were prepared in assay buffer (10 mM
Tris HCl of pH 7.4 containing 1 mM CaCl2 and 2 mM MgCl2). The total assay volume of 100
µL contained 70 µL of assay buffer, 10 µL of compound followed by the addition of 10 µL of he5′NT (6.94 ng) or r-e5′NT (7.17 ng). The reaction mixture was allowed to preincubate for 10
28
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Accepted Manuscript
Cell Transfection with e5′NT
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29
min at 37 oC and then 10 µL of adenosine mono phosphosphate (AMP) substrate (0.5 mM) was
added to initiate the reaction. The reaction was again incubated for 20 min. at the same
temperature (37 oC). At the end, the reaction was stopped by keeping vials in boiling water bath
for 20 min. Aliquots of 50 µL of each reaction mixture were transferred into mini CE vial and
hydrodynamically injected into the capillary by using a pressure of 0.5 psi for 5s. The
electroosmatic separation of substrate and product peaks occurred by applying a voltage of 15
kV. The concentration of the product i.e. adenosine was estimated by calculating the area under
or the r-e5′NT enzyme were further evaluated for IC50 values. The experiments were carried out
in triplicates by following the above mentioned experimental procedure. The IC50 values were
calculated by using the non-linear regression analysis of the program PRISM 5.0 (GraphPad, San
Diego, California, USA).
Molecular Docking Studies
Molecular docking studies of potent compounds were carried out to investigate the putative
binding mode within the active site of the target enzymes. X-ray crystallographic structures of he5'NT (PDB ID: 4H2I)76 were downloaded from RCSB Protein Data Bank while a previously
established homology model of r-e5'NT was used for the docking studies. 2D structures of the
potent compounds (3p and 5a) were generated via Marvin Sketch of ChemAxon suit academic
licensed and then converted into 3D structures by mean of Molecular Builder program
implemented in Molecular Operating Environment (MOE 2014.0901).77 Water and other cocrystalized ligands in the active sites of the target enzymes were removed from the original
Protein Data Bank file. Before the docking studies both targets as well as the potent compounds
were protonated and the energy was minimized up to 0.05 gradient using the MMFF94x force
field and the Protonate 3D tool of MOE 2014.0901. Molecular docking calculations of a potent
compound with the target enzymes were performed via MOE 2014.0901.77 Before performing
molecular docking studies, the active site of h-e5'NT was selected around the co-crystallized
ligands. The ligands were docked into the active site of the protein using the Triangular
Matching docking method and 30 conformations of each ligand-protein complex were generated
based on binding free energies. Poses having lowest free binding energy values were considered
as the most stable ones with the highest affinity for interaction with the receptor. Each ligandprotein complex having the lowest binding free energy was analyzed for interactions and 3D
putative binding interactions were visualized using Discovery studio visualizer v4.78,79
29
This article is protected by copyright. All rights reserved.
Accepted Manuscript
its absorbance peak at 260 nm. The compounds exhibiting 50% inhibition of either the h-e5′NT
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30
Acknowledgment
We are grateful to the DAAD (Programm Deutsch-Pakistanische Hochschulzusammenarbeit) for
financial support. J. Iqbal is thankful to the Organization for the Prohibition of Chemical
Weapons (OPCW), The Hague, The Netherlands and Higher Education Commission of Pakistan
for the financial support through Project No. 20-3733/NRPU/R&D/14/520 for the financial
also a recipient of a “Chercheur National” Scholarship award from the “Fonds de recherche du
Québec-Santé” (FRQS).
Supporting information
Crystallographic data (excluding structure factors) for the structure 3b, 5d and 5f reported in this
paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary
publication no. 1575117-1575121 and can be obtained free of charge on application to CCDC,
12
Union
Road,
Cambridge
CB2
1EZ,
UK;
fax:
þ
44(1223)336033;
e-mail:
deposit@ccdc.cam.ac.uk, or via www.ccdc.cam.ac.uk/data_request/cif.
Supporting information consist 1H,
13
C and
19
F NMR spectra of all new compounds. This
material is available free of charge via the Internet at:
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