Straightforward synthesis and biological evaluation as topoisomerase I inhibitors and antiproliferative agents of hybrid Chromeno[4,3-b][1,5]Naphthyridines and Chromeno[4,3-b][1,5]Naphthyridin-6-ones

Article abstract of DOI:10.1016/j.ejmech.2019.06.032

This work describes the synthesis of hybrid tetrahydro-1,5-naphthyridine and 1,5-naphthyridine derivatives fused with heterocycles such as chromenes and chromen-2-ones or coumarins, which were synthesized in good to high general yields. The synthetic rout

Full text of DOI:10.1016/j.ejmech.2019.06.032

Accepted Manuscript
Straightforward synthesis and biological evaluation as topoisomerase I inhibitors
and antiproliferative agents of hybrid Chromeno[4,3-b][1,5]Naphthyridines and
Chromeno[4,3-b][1,5]Naphthyridin-6-ones
Endika Martín-Encinas, Gloria Rubiales, Birgitta R. Knudssen, Francisco Palacios,
Concepción Alonso
PII:
S0223-5234(19)30560-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.032
EJMECH 11435
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 May 2019
Revised Date: 12 June 2019
Accepted Date: 12 June 2019
Please cite this article as: E. Martín-Encinas, G. Rubiales, B.R. Knudssen, F. Palacios, Concepció.
Alonso, Straightforward synthesis and biological evaluation as topoisomerase I inhibitors and
antiproliferative agents of hybrid Chromeno[4,3-b][1,5]Naphthyridines and Chromeno[4,3-b]
[1,5]Naphthyridin-6-ones, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.06.032.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
Straightforward Synthesis and Biological Evaluation as
Topoisomerase I Inhibitors and Antiproliferative Agents
of Hybrid Chromeno[4,3-b][1,5]Naphthyridines and
Chromeno[4,3-b][1,5]Naphthyridin-6-ones.
Endika Martín-Encinas,a Gloria Rubiales,a Birgitta R. Knudssen,b Francisco Palacios a,* and
Concepción Alonsoa,*
a
Departamento de Química Orgánica I, Facultad de Farmacia and Centro de Investigación
Lascaray (Lascaray Research Center). Universidad del País Vasco/Euskal Herriko Unibertsitatea
(UPV/EHU). Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain.
b
Department of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center
(iNANO), Aarhus University, Aarhus 8000, Denmark.

ACCEPTED MANUSCRIPT
Straightforward Synthesis and Biological Evaluation as
Topoisomerase I Inhibitors and Antiproliferative Agents
of Hybrid Chromeno[4,3-b][1,5]Naphthyridines and
Chromeno[4,3-b][1,5]Naphthyridin-6-ones.
Endika Martín-Encinas,a Gloria Rubiales,a Birgitta R. Knudssen,b Francisco Palacios a,* and
Concepción Alonsoa,*
Dedicated to Professor Pablo Espinet on the occasion of his 70th anniversary
a
Departamento de Química Orgánica I, Facultad de Farmacia and Centro de Investigación
Lascaray (Lascaray Research Center). Universidad del País Vasco/Euskal Herriko Unibertsitatea
(UPV/EHU). Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain.
b
Department of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center
(iNANO), Aarhus University, Aarhus 8000, Denmark.
Keywords: Chromeno[4,3-b][1,5]naphthyridines, chromeno[4,3-b][1,5]naphthyridin-6-ones,
Topoisomerase I, Enzyme inhibition, Antiproliferative effect
Abbreviations: CCK8, cell counting kit; CPT, camptothecin; CPTs, camptothecin derivatives;
DDQ, dichloro-5,6-dicyanobenzoquinone; HDAr, hetero-Diels-Alder reaction; SDS, sodium
dodecyl sulfate; TopI, topoisomerase I; TLC, thin layer chromatography.
1

ACCEPTED MANUSCRIPT
ABSTRACT. This work describes the synthesis of hybrid tetrahydro-1,5-naphthyridine and 1,5-
naphthyridine derivatives fused with heterocycles such as chromenes and chromen-2-ones or
coumarins, which were synthesized in good to high general yields. The synthetic route involves
an intramolecular [4+2]-cycloaddition reaction of functionalized aldimines obtained by the
condensation of 3-aminopyridine and aldehydes containing a double or triple carbon-carbon
bond in orto position and allows the selective generation of three stereogenic centers in a short,
efficient and reliable synthesis. The subsequent dehydrogenation of the fused
tetrahydrochromeno[4,3-b][1,5]naphthyridines
and/or
tetrahydrochromeno[4,3-
b][1,5]naphthyridin-6-ones leads to the formation of the corresponding tetracyclic chromeno[4,3-
b][1,5]naphthyridine derivatives and/or chromeno[4,3-b][1,5]naphthyridin-6-ones in quantitative
yields. Some of the prepared products showed activity as inhibitors of Topoisomerase I (TopI).
Additionally, the cytotoxic behavior of these compounds has been studied in cell lines derived
from human lung adenocarcinoma (A549) and human ovarian carcinoma (SKOV03), and on
non-cancerous lung fibroblasts cell line (MRC5) where, on the last ones, the absence of
cytotoxicity
was
observed.
7-Phenyl-6H-6a,7,12,12a-tetrahydrochromeno[4,3-
b][1,5]naphthyridine 5a showed excellent cytotoxic activity with a IC50 value of 1.03±0.30 µM
against the A549 cell line and a IC50 value of 1.75±0.20 µM against the SKOV03 cell line. The
obtained results point to these compounds as good antiproliferative candidates.
2

ACCEPTED MANUSCRIPT
1. Introduction
The development of antiproliferative compounds remains as one of the main attention focus in
terms of the development of research activity. In recent years, different drugs have been
developed based on the inhibition of the enzyme topoisomerase I (TopI), since it has been
observed that it is overexpressed in tumor cells. This ubiquitous nuclear enzyme reduces
superhelical stress as well as other topological consequences generated in the separation of DNA
strands in metabolic processes such as replication, transcription and recombination [1].
Among the most representative TopI inhibitor drugs there is camptothecin I (CPT, Figure 1) and
its derivatives (CPTs) which are currently used in the systemic treatment of colon, ovarian and
small-cell lung cancers [2]. However, when being used in cancer therapy, these derivatives carry
certain drawbacks, such as lactone instability in blood plasma and cancer cell resistance [3].
Figure 1. Structures of TopI inhibitors: camptothecin (CPT) I, 1,5-naphthyridines II, indeno[2,1-
c][1,5]naphthyridines III and indeno[2,1-c][1,5]naphthyridin-7-ones IV with antiproliferative
activity.
Among other features, camptothecin (CPT) and its derivatives show angularly fused
pentacyclic heterocycles in their structural analysis. Moreover, with regard to the proposed
inhibition mechanism of TopI, the presence of polycyclic heterocycles seems to be relevant in
the effectiveness of the antiproliferative activity, probably due to an improvement in π-π*
3

ACCEPTED MANUSCRIPT
stacking interactions with the DNA base pairs [1]. Regarding polycyclic heterocycles 1,5-
naphthyridine derivatives II (Figure 1) belong to heterocycles identified as suitable candidates in
medicinal chemistry [4]. Recently, our group has reported that dinitrogenated heterocycles such
as 1,5-naphthyridines II (Figure 1) showed good inhibitory activity against Leishmanial TopI
(LTopI) enzyme [5]. Moreover, 1,5-naphthyridines may be fused through the 3 and 4 position of
the heterocycle with a carbocycle structure (indene) and the corresponding angularly fused
bioactive molecules such as 7H-indeno[2,1-c][1,5]naphthyridines III (X = CH2, Figure 1)
prepared by our group showed good TopI inhibition with antileishmanial and antiproliferative
activity [6]. What is more, after observing the behavior of these 1,5-naphthyridines when
participating in the human TopI enzymatic process, 6-(naphthalen-2-yl)-7H-indeno[2,1-
c][1,5]naphthyridin-7-one IV (X = CO, Figure 1) was used as a nanosensor and showed that it
inhibited human topoisomerase I activity by blocking enzyme–DNA dissociation in a post-
ligation step of catalysis [7].
2H-Chromenes V (X = CH2, Figure 2) and coumarins or 2H-chromen-2-ones VI (X = CO,
Figure 2) are heterocyclic derivatives with a benzene fused to a pyran [8]. They are present in a
wide number of natural products and both heterocycles constitute an important moiety in
bioactive heterocycles with pharmacological properties [9], including anticancer drugs [9,10].
Therefore, these scaffolds are very interesting substrates in the search of new drug candidates.
The low toxicity of the natural products containing chromene and their broad pharmacological
properties are attractive feature for medicinal chemists and a source of inspiration for the design
of novel therapeutic agents [9]. Moreover, naphthyridines fused with chromenes or chromenones
present diverse biological activities as antifungal, antimicrobial and antibacterial [11]. Likewise,
4

ACCEPTED MANUSCRIPT
angularly fused tricyclic chromeno[4,3-b]pyridines showed TopI inhibitory activity and
cytotoxicity [12].
The hybrid anticancer drug approach is an innovative synthetic strategy for the discovery of new
biologically active hybrid molecules [13]. Recently, the molecular hybrid approach has resulted in
several novel chemical entities with improved anticancer activity and selectivity with reduced side
effects[14].
With these considerations in mind, we believed that the development of new hybrid molecules
[15] with an angularly fused polycyclic heterocyclic skeleton, containing a 1,5-naphthyridine
fragment VII (Figure 2), and incorporating either a chromene structure (V, X = CH2, Figure 2) or
a coumarin heterocycle (VI, X = CO, Figure 2) fused through the position 2 and 3 of the
naphthyridine ring (Figure 1) may be privileged scaffolds for the search of novel
pharmaceuticals [16]. The new chromeno[4,3-b][1,5]naphthyridines VIII (Figure 2) and
chromeno[4,3-b][1,5]naphthyridin-6-ones IX (Figure 2) may improve the antiproliferative
cytotoxic properties with respect to other biologically active structures, and this strategy may be
complementary to our previous studies [6,7] with angularly tetracyclic 1,5-naphthyridines III
and IV fused with indenes through the positions 3 and 4 of the naphthyridine ring (vide supra,
Figure 1).
5

ACCEPTED MANUSCRIPT
R1
R1
O
X
R1
O
X
V (X = CH2)
VI (X = CO)
O
X
(H)N
O
HYBRIDIZATION
(H)N
NH2
R2
R2
R2
N
XI (X = CH2)
XII (X = CO)
N
N
R3
VII
R3
R3
VIII (X = CH2)
IX (X = CO)
X
Figure 2. Hybridization of chromene V and/or chromen-2-one VI with 1,5-naphthyridines VII
for the design of new bioactive chromeno[4,3-b][1,5]naphthyridines VIII (X = CH2) and
chromeno[4,3-b][1,5]naphthyridin-6-ones IX (X = CO).
In this sense, for the synthesis of the goal angularly fused new chromeno[4,3-
b][1,5]naphthyridines VIII (Figure 2) and chromeno[4,3-b][1,5]naphthyridin-6-ones IX (Figure
2), the hetero-Diels-Alder reaction (HDAr) [17] is a highly atom-economic tool, which presents
interesting applications [18]. In fact, among them the Povarov reaction may represent a direct
route to naphthyridine derivatives [19]. Furthermore, in the search of a methodology for the
preparation of molecules with higher molecular diversity, intramolecular Povarov reaction could
be considered as an important strategy for the synthesis of several ring-fused derivatives by a
single operation [20].
Bearing all this in mind, in this work we report the straightforward synthesis of chromeno[4,3-
b][1,5]naphthyidirines VIII (X = CH2, Figure 2) and chromeno[4,3-b][1,5]naphthyridin-6-ones
IX (X = CO, Figure 2) by intramolecular Povarov reaction from 3-aminopyridine X (Figure 2)
and functionalized aldehydes XI (X = CH2, Figure 2) and XII (X = CO, Figure 2). The
preparation of these new families of compounds represents an interesting challenge in organic
6

ACCEPTED MANUSCRIPT
chemistry, due to the potential interest of these molecules not only in synthetic but also in
medicinal chemistry.
2. Chemistry
The hybrid chromeno[4,3-b][1,5]naphthyridines VIII (X = CH2, Figure 2) and chromeno[4,3-
b][1,5]naphthyridin-6-ones IX (X = CO, Figure 2) were synthesized by an intramolecular
Povarov [4+2]-cycloaddition reaction. First, we started with the preparation of functionalized
aldehydes 1 (Scheme 1, X = CH2) which tailored a double bound in their structure as indicated
(see Supporting Material). Then, aldimines 3 (X = CH2), prepared in situ by condensation
reaction of 3-aminopyridine 2 and previously prepared functionalized aldehydes 1, cyclized
intramolecularly in refluxing chloroform and in the presence of BF3·Et2O (Scheme 1).
Afterwards,
the
corresponding
endo-6a,7,12,12a-tetrahydro-6H-chromeno[4,3-
b][1,5]naphthyridines 5 (X = CH2) were selectively obtained with good yields (56-87%, Scheme
1, Chart 1) in a regio- and stereospecific way. The structure of endo-6a,7,12,12a-tetrahydro-6H-
chromeno[4,3-b][1,5]naphthyridines 5 (X = CH2) was assigned by means of NMR experiments
and confirmed by HRMS (see Experimental Section).
Formation of tetrahydrochromeno[4,3-b][1,5]naphthyridine derivatives 5 may be explained by
a regio- and stereospecific intramolecular [4+2]-cycloaddition reaction between aldimines 3 to
give intermediates 4 followed by prototropic tautomerization.
7

ACCEPTED MANUSCRIPT
Scheme 1. Syntheses of chromeno[4,3-b][1,5]naphthyridine derivatives 5/10 and chromeno[4,3-
b][1,5]naphthyridin-6-one derivatives 9/11.
However, when imine 3f (R1 = H, R2 = C6H5), obtained from the corresponding aldehyde 1
and 6-bromo-3-aminopyridine 2c (R3 = Br), was heated in chloroform in the presence of
BF3·Et2O the tetrahydro-1,5-naphthyridine 5 was not isolated and the chromeno[4,3-
b][1,5]naphthyridine 10f was directly obtained instead. The formation of compound 10f could be
explained by intramolecular Povarov reaction and subsequent dehydrogenation under the
reaction conditions (Chart 2, Experimental Section).
8

ACCEPTED MANUSCRIPT
Chart 1. Structures of endo-6a,7,12,12a-tetrahydro-6H-chromeno[4,3-b][1,5]naphthyridine
derivatives 5 and endo-6a,7,12,12a-tetrahydro-6H-chromeno[4,3-b][1,5]naphthyridin-6-one
derivatives 9 obtained by intramolecular Povarov reaction.
In order to increase the molecular diversity, the methodology was extended to the preparation
of angularly fused tetracyclic derivatives 9 (X = CO), in which the chromene scaffold was
substituted by a coumarin (chromen-2-one).
As before, functionalized aldehydes 6 (Scheme 1, X = CO) were prepared as indicated (see
Supporting Material) followed by the condensation reaction of 3-aminopyridine 2 to give
aldimines 7 (X = CO). These imines 7 cyclized intramolecularly in refluxing chloroform in the
presence of 2 equivalents of Lewis acid as BF3·Et2O (Scheme 1) to afford endo-6a,7,12,12a-
tetrahydro-6H-chromeno[4,3-b][1,5]naphthyridin-6-ones 9 (X = CO) with good yields (78-95%,
Scheme 1, Chart 1) in a regio- and stereospecific way. The formation of these polycyclic
compounds 9 may be explained, as before, by a regio- and stereospecific intramolecular [4+2]-
cycloaddition reaction of aldimines 7 (X = CO) to give intermediates 8 (X = CO) followed by
prototropic tautomerization.
The methodology tolerates electron-releasing and electron-withdrawing substituents in the
aromatic aldehydes, even fluorinated ones that allow the preparation of fluoro containing
compounds, interesting substrates from a biological point of view [21]. As far as we know, this
strategy represents the first example for the preparation of 6a,7,12,12a-tetrahydro-6H-
chromeno[4,3-b][1,5]naphthyridines 5 (X = CH2, Scheme 1) and 6a,7,12,12a-tetrahydro-6H-
chromeno[4,3-b][1,5]naphthyridin-6-ones 9 (X = CO, Scheme 1).
9

ACCEPTED MANUSCRIPT
Subsequent dehydrogenation of tetrahydro-6H-chromeno[4,3-b][1,5]naphthyridines 5 (X =
CH2) and tetrahydro-6H-chromeno[4,3-b][1,5]naphthyridin-6-ones 9 (X = CO) under the
reaction conditions would afford chromeno[4,3-b][1,5]naphthyridines 10 (X = CH2) and
chromeno[4,3-b][1,5]naphthyridin-6-ones 11 (X = CO), respectively, in quantitative yields
(Scheme 1, Chart 2). This dehydrogenation reaction was performed with 1 equivalent of DDQ in
toluene at 40 ºC under microwave irradiation for 2 hours as previously reported [6b].
10

ACCEPTED MANUSCRIPT
Chart 2. Structures of chromeno[4,3-b][1,5]naphthyridines 10 and chromeno[4,3-
b][1,5]naphthyridin-6-ones 11 obtained by dehydrogenation reaction with DDQa or by
intramolecular Povarov reaction of propargyl aldimines 13 in the presence of BF3·Et2Ob.
cChromeno[4,3-b][1,5]naphthyridine 10f obtained by intramolecular Povarov reaction and
subsequent dehydrogenation under the reaction conditions.
As we previously reported, if acetylenes are used as dienophiles instead of olefins when
reacting with aldimines derived from 3-aminopyridines, the corresponding 1,5-naphthyridine
compounds may be obtained [22]. For these reasons, we decided to study the synthesis of
chromeno[4,3-b][1,5]naphthyridines 10 in a direct way by means of the intramolecular Povarov
reaction by using aldehydes 12 (Scheme 2) containing an alkyne group in the orto position. It is
noteworthy, that with this strategy, from a preparative point of view, the aromatic 1,5-
naphthyridine core may be directly obtained.
11

ACCEPTED MANUSCRIPT
The formation of chromeno[4,3-b][1,5]naphthyridines 10 can be performed by initial
condensation reaction between 3-aminopyridine 2 and functionalized aldehydes 12. Subsequent
intramolecular [4+2]-cycloaddition reaction in refluxing chloroform, in the presence of 2
equivalents of BF3·Et2O, would give intermediates 14 followed by dehydrogenation under the
reaction conditions to afford chromeno[4,3-b][1,5]naphthyridines 10 with good yields (Scheme
2, Chart 2)[23].
Scheme 2. Syntheses of 6H-chromeno[4,3-b][1,5]naphthyridines 10 by intramolecular Povarov
reaction with acetylenic aldehydes.
The methodology described in this work represents an easy, efficient and straightforward
strategy for the preparation of angularly hybrid tetrahydro-1,5-naphthyridine and 1,5-
naphthyridine derivatives fused with heterocycles such as chromenes and chromenones
(coumarins) and tolerates electron-donating and electron-withdrawing of substituents either in
the starting aldehyde and in the initial amine. Next, the biological behavior of compounds
prepared as TopI inhibitors and as antiproliferative agents has been studied.
12

ACCEPTED MANUSCRIPT
3. Biological results and discussion
3.1. Inhibition of Topoisomerase I. The inhibitory effect of the synthesized derivatives on
Topoisomerase type I (TopI) was investigated. A conventional supercoiled plasmid relaxation
assay was used to determine if the newly angularly fused tetrahydro derivatives 5 and 9, as well
as more unsaturated compounds 10 and 11 inhibit human TopI activity by converting supercoiled
plasmid DNA to relaxed DNA (Table 1). In these experiments compound samples were mixed
with enzyme followed by addition of supercoiled plasmid DNA substrate and continued
incubation for increasing time periods (15 seconds, 1 minute and 3 minutes). The reaction was
terminated by the addition of SDS. DNA relaxation products were then resolved by gel
electrophoresis in 1% agarose gel and visualized by gel red staining. Camptothecin (CPT) was
used as a positive control (Figure 3).
Figure 3. Inhibition of TopI activity along the time (15”, 1’ and 3’) by compounds 5c, 5d, 10d
and 11d and camptothecin at 100 µM: lanes 1-3, DNA+TopI+DMSO; lanes 4-6,
DNA+TopI+camptothecin 100µM; lanes 7-9, DNA+TopI+5c 100µM; lanes 10-12,
DNA+TopI+5d 100µM; lanes 13-15, DNA+TopI+10d 100µM; lanes 16-18, DNA+TopI+11d
100µM; lane 19, control DNA. Reaction samples were mixed with enzyme at 37 ºC before
adding the supercoiled DNA substrate and separated by electrophoresis on a 1% agarose gel, and
then stained with gel red, and photographed under UV light as described in the TopI mediated
DNA relaxation assay.
13

ACCEPTED MANUSCRIPT
TopI inhibitory activity of new compounds was tested by detecting the conversion of
supercoiled DNA (Sc, Figure 3) to its relaxed form in the presence of the purified enzyme and
expressed in quantitative fashion relative to the TopI inhibitory activity of camptothecin (Table
1). As shown in Figure 3, TopI relaxes supercoiled DNA even in the presence of DMSO (Figure
3, lanes 1-3), and camptothecin (CPT) inhibits the relaxation, as indicated by the increased
intensity of the band corresponding to the supercoiled DNA (Figure 3, lanes 4-6).
Based on the results of the relaxation assays, the tetrahydro-6H-chromeno[4,3-
b][1,5]naphthyridines 5, and the tetrahydro-6H-chromeno[4,3-b][1,5]naphthyridin-6-ones 9
showed low-moderate inhibitory activity, and only present remarkably activity, compound 5c (R1
= Me) containing a methyl group in the chromeno moiety and 5d (R3 = OMe) containing a
methoxy group in the naphthyridine moiety (Table 1, entries 4 and 5), which inhibited the TopI
relaxation similar to camptothecin (CPT) when added at the same concentration and in the same
time intervals. With respect to 6H-chromeno[4,3-b][1,5]naphthyridines 10 and 6H-
chromeno[4,3-b][1,5]naphthyridin-6-ones 11, the inhibitory activity is low-moderate. However,
notably inhibitory activity could be observed for compounds 10d (R3 = OMe) and 10g (R2 = 3,4-
F2C6H3) (Table 1, entries 13 and 16), which at short times inhibited TopI activity almost
completely and the effect was released at longer time intervals. Also, 6H-chromeno[4,3-
b][1,5]naphthyridin-6-one 11d (R3 = OMe) exhibited inhibitory activity at short time and a
minor effect at a later time (Table 1, entry 20). In general, a greater effect is observed in those
derivatives with a methoxy group in their structure as in compounds 5d, 10d and 11d (Charts 1
and 2). In addition, the derivative with a double substitution of fluorine 10g (R2 = 3,4-F2C6H3)
showed good activity.
14

ACCEPTED MANUSCRIPT
Table 1. TopI inhibitory activity of compounds 5, 9, 10, 11 and CPT.
% Inhibitiona
1 minute
76
Entry Compound
R1
R2
R3
15 seconds
3 minutes
1
2
Camptothecin (CPT)
80
3
5a
H
F
C6H5
C6H5
H
H
5
2
4
1
1
1
5b
5c
3
4
Me
H
H
F
C6H5
H
70
81
5
69
26
7
19
12
5
5d
5
C6H5
OMe
H
9a
6
C6H5
9b
7
C6H5
H
30
4
25
4
2
9c
8
H
H
H
F
C6H5
Br
4
9d
9
C6H5
OMe
H
4
4
4
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
11d
10
11
12
13
14
15
16
17
18
19
20
C6H5
30
2
14
2
2
C6H5
H
2
Me
H
H
H
H
H
F
C6H5
H
2
2
2
C6H5
OMe
H
86
29
4
16
30
4
15
29
4
4-MeOC6H4
C6H5
Br
3,4-F2C6H3
C6H5
H
84
25
26
4
6
6
H
2
2
C6H5
H
2
2
H
H
C6H5
Br
4
4
C6H5
OMe
72
12
12
a The activity of the compounds inhibiting TopI relaxation was expressed quantitatively by comparison with
the inhibitory activity observed for camptothecin.
3.2. In Vitro Cytotoxicity. The cytotoxicity of the new angularly fused tetracyclic hybrid
molecules 5, 9, 10 and 11 was investigated in vitro by testing the antiproliferative activities
against two human cancer cell lines: A549 (carcinomic human alveolar basal epithelial cell) and
15

ACCEPTED MANUSCRIPT
SKOV3 (human ovarian carcinoma). The cell counting kit (CCK8) assay was employed to assess
growth inhibition and, the cell proliferation inhibitory activities of the compounds are listed in
Table 2 as IC50 values.
Table 2. Antiproliferative activity of compounds 5, 9, 10, 11 and CPT.
Cytotoxicity IC50 (µM)a
Entry Comp.
R1
R2
R3
lung
ovarian
lung
MRC-5
-
A549
SKOV03
(1.0±0.06)·10-3 (5.5±0.01)·10-3
1
2
Camptothecin (CPT)
5a
5b
H
F
C6H5
C6H5
H
H
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
1.03±0.30
11.303±1.23
7.25±0.27
1.75±0.20
>50
3
5c
4
Me
H
H
F
C6H5
H
12.69±0.80
>50
5d
5
C6H5
OMe
H
5.43±2.18
9a
6
C6H5
25.42±1.82
>50
9b
7
C6H5
H
>50
>50
>50
9c
8
H
H
H
F
C6H5
Br
>50
9d
9
C6H5
OMe
H
>50
>50
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
11d
10
11
12
13
14
15
16
17
18
19
20
C6H5
>50
>50
C6H5
H
>50
>50
Me
H
H
H
H
H
F
C6H5
H
>50
>50
C6H5
OMe
H
11.74±2.38
17.89±2.00
>50
>50
4-MeOC6H4
C6H5
>50
Br
>50
3,4-F2C6H3
C6H5
H
>50
>50
H
9.03±1.47
>50
>50
C6H5
H
>50
H
H
C6H5
Br
29.00±3.15
>50
>50
C6H5
OMe
>50
aThe cytotoxicity IC50 values listed are the concentrations corresponding to 50% growth inhibition.
16

ACCEPTED MANUSCRIPT
The most cytotoxic derivative in both cancerous cell lines resulted to be the tetrahydro-6H-
chromeno[4,3-b][1,5]naphthyridine 5a (R1 = H, R2 = C6H5, R3 = H) with a IC50 value of
1.03±0.30 µM against the A549 cell line and a IC50 value of 1.75±0.20 µM against the SKOV03
cell line (Table 2, entry 2). Some other tetrahydro derivatives 5, such as 5c (R1 = Me, R2 = C6H5,
R3 = H) and 5d (R1 = H, R2 = C6H5, R3 = OMe), showed IC50 values under 10 µM against the
A549 cell line, with values of IC50 = 7.25±0.27 µM and IC50 = 5.43±2.18 µM, respectively
(Table 2, entries 4 and 5). Regarding the 6H-chromeno[4,3-b][1,5]naphthyridines 10 and 6H-
chromeno[4,3-b][1,5]naphthyridin-6-ones 11, the derivative 11a presented as low cytotoxicity as
9.03±1.47 µM against the A549 cell line (Table 2, entry 17).
According to the data presented in Table 2, in general when cytotoxicity is observed this is
higher against the human lung adenocarcinoma cell line (A549) than in the human ovarian
carcinoma cell line (SKOV3). Moreover, MRC-5 non-malignant lung fibroblasts were tested for
studying selective toxicity [24] and in contrast, none of the synthesized 6H-chromeno[4,3-
b][1,5]naphthyridines 5 nor 10 as well as 6H-chromeno[4,3-b][1,5]naphthyridin-6-ones 9 nor 11
compounds exhibited any toxicity toward MRC-5 cells (Table 2). Topoisomerase inhibitors
present limited tumor selectivity [25]. However, in our case a very high selectivity has been
observed against the human lung adenocarcinoma cell line (A549).
4. Computational analysis
Taking into account that theoretical calculations allowed the estimation of Molecular
Electrostatic Potential Surface (MEPS), HOMO-LUMO energy gap and related parameters,
which depicted the potential kinetic stability and reactivity of the target compounds [26],
theoretical studies using Density Functional Theory (DFT) [27] involving the well-known Becke
17

ACCEPTED MANUSCRIPT
three-parameter Lee-Yang-Parr function (B3LYP) [28] and 6-311G (d, p) level of theory for the
synthesized compounds were carried out.
4.1. Stereoelectronic properties. The molecular DFT-based parameters such as electronic
chemical potential (µ), chemical hardness (η), global electrophilicity (ω), maximum number of
accepted electrons (∆Nmax) and Free energy in gas and in aqueous medium for compounds 5,9-
11 are reported in Table 3.
Thus, tetrahydro-6H-chromeno[4,3-b][1,5]naphthyridines 5 and tetrahydro-6H-chromeno[4,3-
b][1,5]naphthyridin-6-ones 9 are the most stable (Table 3), with the highest gap values from 4.77
to 5.09, higher hardness and higher chemical potential than the rest of compounds, and also are
the least electrophilic. In this group, compounds 5 (Chart 1), which experimentally have shown
higher cytotoxicity and better inhibition of TopI than 9 (Tables 1 and 2), present the highest
chemical potentials (which would imply greater reactivity), are the least electrophilic and have
the lowest dipole moment. Among the tetrahydro derivatives 5 the most cytotoxic ones are
compounds 5a, 5c and 5d (Table 2) and precisely these are the least electrophilic and have the
highest values of chemical potential (Table 3, entries 1, 3 and 4).
With respect to more unsaturated compounds, 10 and 11, they seem to be more reactive than
compounds 5 and 9 of the former group presenting gap values of 3.73 to 4.00 (Table 3) with
lower chemical potential and higher electrophilicity. In this case, within the group of 1,5-
naphthyridines fused with chromene 10, the compound with the highest cytotoxicity and which
best inhibits TopI is 10d, showing, among derivatives 10 and 11, the highest chemical potential
value and the lowest electrophilicity (Table 3, entry 12). Likewise, in the case of 1,5-
naphthyridines fused with coumarin 11, the compound 11d, with good inhibitory activity, has the
18

ACCEPTED MANUSCRIPT
highest values of chemical potential, dipole moment and is the least electrophilic (Table 3, entry
19).
The computational results obtained seem to indicate that there is a direct relationship between
the low electrophilicity and the high chemical potential, with the biological activity within each
type of structure.
19

ACCEPTED MANUSCRIPT
Table 3. Calculated energies and molecular proprieties computed at B3LYP/6-311G** basis set level of theory for compounds 5,9-11
and CPT.
Dipole
EHOMO
(in a.u.)
ELUMO
(in a.u.)
Gap
(-eV)
∆G (g)
(in a.u.)
∆G (aq)
(in a.u.)
∆Nmax
(in a.u.)
η
µ
ω
(eV)
Entry Compound
moment
(debye)
1.570
(in a.u.) (in a.u.)
-995.74250
-995.75452
-0.21492
-0.21899
-0.20689
-0.21347
-0.22859
-0.23261
-0.23311
-0.22049
-0.22573
-0.22698
-0.21986
-0.21730
-0.22387
-0.22978
-0.23071
-0.24152
-0.2442
-0.03051
-0.0338
5.02
5.04
5.00
4.77
5.09
5.07
4.94
4.93
3.99
3.89
3.87
3.90
4.00
3.90
3.96
3.88
3.84
3.79
3.73
3.65
0.18441
0.18519
0.18377
0.17536
0.18718
0.18638
0.18160
0.18124
0.14674
0.14302
0.14209
0.14332
0.14700
0.14325
0.14548
0.14265
0.14127
0.13925
0.13720
0.13426
-0.12272 0.040830 0.665447
-0.12640 0.043133 0.682515
-0.12159 0.040221 0.661615
-0.11921 0.040520 0.679802
-0.13500 0.048683 0.721231
-0.13942 0.052146 0.748042
-0.14231 0.055760 0.783645
-0.12987 0.046530 0.716564
-0.15236 0.079098 1.038299
-0.15547 0.084502 1.087051
-0.14882 0.077929 1.047329
-0.14564 0.073999 1.016188
-0.15037 0.076909 1.022925
-0.15816 0.087305 1.104049
-0.15797 0.085766 1.085854
-0.17020 0.101529 1.193095
-0.17357 0.106621 1.228605
-0.17569 0.110827 1.261652
-0.16335 0.097242 1.190598
-0.15998 0.095314 1.191569
1
2
3
4
5
6
7
8
5a
5b
5c
5d
9a
9b
9c
9d
-1095.01472 -1095.026986
0.727
2.598
1.940
5.002
3.499
5.981
5.735
1.010
2.346
0.890
2.593
1.276
1.724
3.142
3.918
2.967
3.822
5.792
6.419
-1035.04548
-1110.27061
-1069.81223
-1169.08411
-3643.37008
-1184.34120
-993.387886
-1092.66012
-1032.69077
-1107.91838
-1107.91245
-3566.94648
-1191.92762
-1067.46187
-1166.73367
-3641.01952
-1181.99254
-1035.05751
-1110.28600
-1069.82773
-1169.09976
-3643.38682
-1184.35985
-993.39773
-0.0297
-0.03153
-0.04141
-0.04623
-0.05151
-0.03925
-0.07899
-0.08396
-0.07777
-0.07398
-0.07687
-0.08653
-0.08523
-0.09887
-0.10293
-0.10606
-0.09475
-0.0929
9
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
11d
CPT
-1092.66998
-1032.70067
-1107.93199
-1107.92398
-3566.95652
-1191.93771
-1067.47581
-1166.74754
-3641.03346
-1182.01049
10
11
12
13
14
15
16
17
18
19
20
-0.24531
-0.23195
-0.22711
-1182.22076 -1182.237563
Abbreviations: ∆G (g): Free energy in gas phase[a]; ∆G (aq): Free energy in aqueous medium[b];Gap: EHOMO - ELUMO; η: Hardness[c]; µ:Chemical Potentials[c];
ω:Global Electrophilicities[c] and ∆Nmax: Maximun Number of Accepted Electrons[c].
[a]
[b]
Computed a B3LYP/6-311G**+∆ZPVE level; Computed a B3LYP(PCM)/6-311G**+∆ZPVE level using water as solvent; [c]Computed at the B3LYP/6-
311G** level of theory according to the approach and equations described previously [19].
20

ACCEPTED MANUSCRIPT
4.2. Molecular Electrostatic Potential Surface (MEPS) analysis. Figure 4 shows the MEPS
[26] of 1,5-naphthyridines fused with chromene 5 and 10 and Figure 5 shows the MEPS of 1,5-
naphthyridines fused with chromenone 9 and 11 which were calculated using DFT [27] with the
standard basis set B3LYP / 6-311G (d, p) level of theory. From these calculations, it can be seen
in Figure 4 that compounds 5 and 10 have a local negative electrostatic potential close to the
nitrogen at position 8 of the tetracyclic ring system. Furthermore, in the case of compounds 5d
and 10d (Chart 1, Figure 4) due to the presence of the methoxy group (OCH3), a local negative
electrostatic potential appears in the oxygen at position 9 where the methoxy group is located,
greater than in the other member compounds of this group. Noteworthy, these compounds 5d and
10d are cytotoxic and present the highest values of TopI inhibition (Table 1 and Table 2, entries
5 and 13). Considering the limits of the electrostatic potentials calculated for these two
molecules, 5d and 10d (Figure 4), the ranges are much wider than for the rest of compounds 5
and 10, which may suggest that the electrostatic interactions with the target are an important
factor. On the other hand, the presence of a fluorine substituent (F) at position 2 of compound 5b
(with the minor cytotoxicity of this group) modifies the local positive electrostatic potential that
appears in this position for 5a, 5c and 5d. A particular electrostatic potential map is also
observed for compound 10g, with two fluorine atoms in its structure (Chart 2, Figure 4). In this
case, the positioning of local positive electrostatic potential has been modified, being situated
over the hydrogens of aromatic ring at position 7 of the tetracyclic system. By chance, this
fluorinated chromeno[4,3-b][1,5]naphthyridine 10g showed a good inhibition of TopI at short
reaction time (Table 1, entry 16) which is consistent with the biological role of fluorine
derivatives [21].
21

ACCEPTED MANUSCRIPT
-0.05770 to 0.05770
-0.05530 to 0.05530
-0.05797 to 0.05797
-0.06762 to 0.06762
5a
5b
5c
5d
-0.05052 to 0.05052
-0.04820 to 0.04820
-0.05117 to 0.05117
-0.06199 to 0.06199
10a
10b
10c
10d
-0.05121 to 0.05121
-0.04906 to 0.04906
-0.04137 to 0.04137
-0.05281 to 0.05281
10e
10f
10g
CPT
Figure 4. MEP surfaces mapped from total electron density for compounds 5, 10 and CPT.
Electrostatic potentials are displayed on a 0.002 a.u. isodensity surface. The limits of
electrostatic potentials for each molecule are under surfaces. Potential increases in the following
order: red (most negative)/orange/yellow/green/blue (most positive).
As observed in Figure 5, the most negative electrostatic potentials for compounds 9 and 11
are located over the oxygen of carbonyl group and on the nitrogen at position 8 of the tetracyclic
systems. On the other hand, the tetrahydro derivatives 9a-d, which showed low inhibitory
22

ACCEPTED MANUSCRIPT
activity of TopI, presented the most positive local electrostatic potential over the hydrogen of the
NH at position 12.
-0.05569 to 0.05569
-0.05368 to 0.05368
-0.05416 to -0.05416
-0.06521 to 0.06521
9a
9b
9c
9d
-0.06059 to 0.06059
-0.05816 to 0.05816
-0.05731 to 0.05731
-0.06376 to 0.06376
11a
11b
11c
11d
Figure 5. MEP surfaces mapped from total electron density for compounds 9 and 11.
Electrostatic potentials are displayed on a 0.002 a.u. isodensity surface. The limits of
electrostatic potentials for each molecule are under surfaces. Potential increases in the following
order: red (most negative)/orange/yellow/green/blue (most positive).
A very different electrostatic potential is obtained through these theoretical calculations for
the more unsaturated compounds 11. In this case, a small negative local electrostatic potential
appears on the nitrogen at the same position 12 for compounds 11a-d. In addition, the most
biologically active compound in this group, the derivative 11d (Table 1, entry 20), shows a local
positive electrostatic potential over the hydrogens at positions 10 and 11 and over the hydrogens
23

ACCEPTED MANUSCRIPT
of the methoxy group (OCH3) sited at position 9 (Figure 5). Conversely, for compound 11c, with
the lowest value of TopI inhibiton, the presence of bromine atom at the 9 position of the
tetracyclic system makes that a small negative electrostatic potential appears at that position.
4.3. Docking studies. Among all the compounds synthesized in this work, a molecular
docking study of those who showed significant inhibition of TopI was carried out to investigate
its plausible binding pattern and its interaction with the key amino acids and DNA nucleobases
in the active site of the TopI. The model was derived by docking of compounds 5c,d, 10d,g and
11d, in the camptothecin binding site of the camptothecin−TopI−DNA ternary complex (PDB
ID: 1T8I) [29].
The most important evaluation criterion was the observation of whether the ligands were located
between C112-TGP11 and A113-T10 nucleobases, where the DNA rupture site is located,
avoiding the re-ligation of such bases, according to the concept of interfacial inhibition proposed
by Pommier [1]. The formation of hydrogen bonds with important residues [30] and the
existence of hydrophobic interactions with TopI residues and DNA were also taken into account.
Likewise, based on the above interactions, the obtained values from the gscore parameters
indicate the virtual affinity of the ligands to the complex, and gemodel, which is the theoretical
value of the interaction energy of the ligand with the TopI/DNA complex, were considered (see
Table 4).
Table 4. gscore and gemodel values for compounds 5c,d, 10d,g, 11d and CPT TopI inhibitors.
gscore
(kcal/mol)
- 5.354
- 4.934
- 5.473
gemodel
(kcal/mol)
- 50.613
- 63.067
- 67.251
Entry
Compound
1
2
3
5c
5d
10d
24

ACCEPTED MANUSCRIPT
4
5
6
- 5.125
-5.413
-8.074
- 62.562
- 69.372
- 91.172
10g
11d
CPT
The studied compounds have a weak affinity to the receptor with a low score and interaction
energy values, which is consistent with experimental results. The different orientation modes and
the interactions of each compound with nucleobases and various amino acid residues can be seen
in Figure S1 and S2 of the supporting information. And according to the observed docking
results, the structural part corresponding to chromene or coumarin (chromenone) participates in
interactions with the active site oriented towards the minor grove, establishing π-π stacking
interactions with nucleobases and suggesting that those heterocycles may play an important role
in the interaction with the target.
4.4. In silico ADME/T analysis. The synthesized compounds were submitted to in silico
pharmacokinetic properties prediction by using the graphical interface Maestro [31] using
QikProp module of Schrödinger software. The results are included in Table S1 of Supporting
Material. Thus, estimated number of hydrogen bonds that would be donated and accepted by the
solute to the water molecules in an aqueous solution are in the range of 0.0-1.0 and the 2.75-4.50
respectively. Predicted octanol/water partition coefficient value are in the range of 3.72-5.24,
indicating that the compounds present an high value of log P. Number of metabolic reactions of
compounds 5 and 9 is high (in the range of 7 to 9), while for the compounds 10 and 11 is in the
range of 1 to 3. All compounds have % Human Oral Absortion of 100%. Prediction of binding to
human serum albumin for the compounds are in the range 0.27-9.67 and predicted brain/blood
partition coefficient are in the range of -0.067 to 0.586. Number of violations of Lipinsky’s rule
25

ACCEPTED MANUSCRIPT
of five is 0-1. Thus, it was observed that most of the compounds were found to be within the
limit of approved drug parameter range.
5. Conclusions
In summary, the preparation of hybrid compounds derived from tetrahydro-1,5-naphthyridines
and heterocycles such as chromenes and chromen-2-ones (coumarins) is reported by a regio- and
stereospecific intramolecular [4+2]-cycloaddition reaction. Subsequent dehydrogenation of 1,5-
naphthyridine core allowed the isolation of corresponding 6H-chromeno[4,3-
b][1,5]naphthyridines 10 and 6H-chromeno[4,3-b][1,5] naphthyridin-6-ones 11. Some of these
compounds showed selective inhibitory effects on human TopI mediated relaxation assays
compatible to those observed for the natural inhibitor CPT. All the prepared derivatives have
been further subjected to evaluation of their cytotoxicity against carcinomic human alveolar
basal epithelial cell line (A549) and human ovarian carcinoma cell line (SKOV3). The most
cytotoxic derivative in both cancerous cell lines resulted to be the endo 7-phenyl-6a,7,12,12a-
tetrahydro-6H-chromeno[4,3-b][1,5]naphthyridine 5a. However, in general, these preliminary
studies revealed that some of newly synthesized compounds, such as 5c, 5d and 11a exhibited
significant cytotoxic effect against A549 cell line selectively. Moreover, when cytotoxicity was
tested against non-malignant pulmonary fibroblasts (MRC-5), none of the compounds presented
any activity.
The physicochemical properties of these hybrid compounds have been evaluated, and could be
considered as candidates for drugs with promising pharmacotherapeutic profiles similar to those
of currently used drugs. In addition, docking experiments inform us about the possible mode of
26

ACCEPTED MANUSCRIPT
binding of these compounds and according to these estudies the structural part corresponding to
chromene or coumarin seems to play an important role in the interaction with the site active. The
interesting biochemical and biological features found for these compounds provide a promising
basis for further development of biologically active naphthyridine derivatives.
6. Experimental protocols
6.1 Chemistry
6.1.1. General experimental information
All reagents from commercial suppliers were used without further purification. All solvents were
freshly distilled before use from appropriate drying agents. All other reagents were recrystallized
or distilled when necessary. Reactions were performed under a dry nitrogen atmosphere.
Analytical TLCs were performed with silica gel 60 F254 plates. Visualization was accomplished
by UV light. Column chromatography was carried out using silica gel 60 (230-400 mesh
ASTM). Melting points were determined with an Electrothermal IA9100 Digital Melting Point
Apparatus without correction. NMR spectra were obtained on a Bruker Avance 400 MHz and a
Varian VXR 300 MHz spectrometers and recorded at 25 ºC. Chemical shifts for 1H NMR spectra
13
are reported in ppm downfield from TMS, chemical shifts for C NMR spectra are recorded in
ppm relative to internal deuterated chloroform (δ
= 77.2 ppm for 13C), chemical shifts for 19F
NMR are reported in ppm downfield from fluorotrichloromethane (CFCl3). Coupling constants
(J) are reported in Hertz. The terms m, s, d, t, q refer to multiplet, singlet, doublet, triplet, quartet.
13C NMR, and 19F NMR were broadband decoupled from hydrogen nuclei. High resolution mass
spectra (HRMS) was measured by EI method with a Agilent LC-Q-TOF-MS 6520 spectrometer.
6.1.2. Compounds Purity Analysis
All synthesized compounds were analyzed by HPLC to determine their purity. The analyses were
performed on Agilent 1260 infinity HPLC system (C-18 column, Hypersil, BDS, 5µm, 0.4
27

ACCEPTED MANUSCRIPT
mm×25 mm). All the tested compounds were dissolved in dichloromethane, and 5µL of the
sample was loaded onto the column. Ethanol and heptane were used as mobile phase, and the
flow rate was set at 1.0 mL/min. The maximal absorbance at the range of 190−400 nm was used
as the detection wavelength. The purity of all the tested compounds (compounds 5, 9, 10 and 11)
is >95%, which meets the purity requirement by the Journal.
6.1.3. Synthesis of aldimines 3, 7 and 13
6.1.3.1 General procedure. To a solution of the corresponding aldehyde (1 mmol) in CHCl3 (20
mL) was added the corresponding 3-aminopyridine (1 mmol,) in presence of molecular sieve
1
(4Å) and two drops of BF3·Et2O. The mixture was refluxed for 16h and was checked by H
NMR and/or 19F NMR spectroscopy. Due to the instability of the aldimines, they were used
without purification for the following reactions.
6.1.3.1.1 (E)-1-(2-(Cinnamyloxy)phenyl)-N-(pyridin-3-yl)methanimine (3a) The general
procedure was followed using aldehyde 1a (1 mmol, 0.238 g) and 3-aminopyridine (1 mmol,
1
3
2
0.094 g). H RMN (300 MHz, CDCl3):
δ = 4.71 (dd, JHH = 5.8 Hz, JHH = 1.4 Hz, 2 H, CH2),
3
3
3
6.34 (dt,3JHH = 15.8 Hz, JHH = 5.8 Hz, JHH = 5.8 Hz, 1H, =CH), 6.65 (d, JHH = 15.8 Hz,
3
4
1H,=CH), 6.92-7.01 (m, 2 H, Harom), 7.17-7.46 (m, 8H, Harom), 8.11 (dd, JHH = 7.9 Hz, JHH
=
1.6 Hz, 1H, Harom), 8.38 (dd, 3JHH = 4.7 Hz, 4JHH = 1.5 Hz, 1H, Harom), 8.43 (d, 4JHH = 2 Hz, 1H,
Harom), 8.90 (s, 1H, HC=N) ppm.
6.1.3.1.2 (E)-1-(2-(Cinnamyloxy)-5-fluorophenyl)-N-(pyridin-3-yl)methanimine (3b) The
general procedure was followed using aldehyde 1b (1 mmol, 0.256 g) and 3-aminopyridine (1
1
3
mmol, 0.094 g). H RMN (300 MHz, CDCl3):
δ = 4.67 (d, Hz, JHH = 5.5 Hz, 2 H, CH2), 6.30
(dt,3JHH = 15.8 Hz, 3JHH = 5.8 Hz, 1H, =CH), 6.63 (d, 3JHH = 15.8 Hz, 1H, =CH), 6.85-7.46 (m, 9
H, Harom), 7.78 (dd, 3JHH = 8.9 Hz, 4JHH = 3.0 Hz, 1 H, Harom), 8.37 (d, 3JHH = 5.0 Hz, 1 H, Harom),
8.41 (d, 4JHH = 2.4 Hz, 1 H, Harom) 8.83 (s, 4JHH = 1.6 Hz, HC=N) ppm.
6.1.3.1.3 (7E)-N-(2-(Cinnamyloxy)-5-methylbenzylidene)pyridin-3-amineamine (3c) The
general procedure was followed using aldehyde 1c (1 mmol, 0.252 g) and 3-aminopyridine (1
1
2
mmol, 0.094 g). H RMN (300 MHz, CDCl3):
δ
= 2.28 (s, 3 H, CH3), 4.70 (dd, Hz, JHH = 6.0
28