Synthesis and Pharmacological Screening of Pyridopyrimidines as Effective Anti-Diarrheal Agents through the Suppression of Cyclic Nucleotide Accumulation

Article abstract of DOI:10.1002/open.201900060

The increased levels of cyclic nucleotides (cGMP and cAMP) in enterocytes trigger intracellular mechanisms of ion and fluid secretion into the lumen, causing secretory diarrhea. Twelve novel pyridopyrimidines derived from 5-(3,5-bistrifluoromethylphenyl)-

Full text of DOI:10.1002/open.201900060

DOI: 10.1002/open.201900060
Full Papers
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Synthesis and Pharmacological Screening of
Pyridopyrimidines as Effective Anti-Diarrheal Agents
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through the Suppression of Cyclic Nucleotide Accumulation
Tiago Zaminelli+,[b] Elisa Magli+,[a] Francesco Frecentese,[a] Caroline H. Lescano,[b]
Rafael Campos,[b] Irene Saccone,[a] Angela Corvino,[a] Paola Di Vaio,[a] Flavia Giordano,[a]
Paolo Luciano,[a] Ferdinando Fiorino,[a] Elisa Perissutti,[a] Vincenzo Santagada,[a]
Beatrice Severino,[a] Giuseppe Caliendo,*[a] and Gilberto De Nucci[b]
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The increased levels of cyclic nucleotides (cGMP and cAMP) in
enterocytes trigger intracellular mechanisms of ion and fluid
secretion into the lumen, causing secretory diarrhea. Twelve
novel pyridopyrimidines derived from 5-(3,5-bistrifluoromethyl-
phenyl)-1,3-dimethyl-5,11-dihydro-1H-indeno[2,1:5,6]pyrido
[2,3-d]pyrimidine-2,4,6-trione (FPIPP) were synthesized and
evaluated on intracellular cyclic nucleotide accumulation. All
compounds had no effect on either cyclic nucleotide basal
levels or on pre-contracted aortic rings. The metabolic activity
and viability in T84 cells, assessed by MTT (3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyl tetrazolium bromide) and the LDH
(lactate dehydrogenase) assays, respectively, were not affected
by incubation with the compounds (50 μM). Compound VI
almost abolished cGMP accumulation (94% inhibition) induced
by STa toxin in T834 cells and significantly reduced (69%)
forskolin-induced cAMP accumulation in Jurkat cells. Compound
VI was active in an in vivo model for diarrhea in rabbits. These
results prompted us to perform a microscopic histopathological
analysis of intestinal tissues, showing that only compound VI
preserves the intestine without significant pathological changes
and with a decreased inflammatory pattern in comparison to
FPIPP. In vitro stability test revealed that compound VI is
resistant to oxidation promoted by atmospheric oxygen.
1. Introduction
guanylate cyclase type C (GC-C) in intestinal epithelial cells,
stimulating the synthesis of 3’-5’ cyclic guanosine monophos-
phate (cGMP) from guanosine triphosphate (GTP).[6–9] STa toxin
perturbs electrolyte homeostasis in intestinal cells determining
secretory diarrhea that requires immediate rehydration. E. coli
strains are responsible of about 400 million cases of diarrhea in
5 or less years old children worldwide.[10]
Diarrhea is a major health issue, especially in developing
countries where it is estimated to be the second cause of death
in children under five years of age, killing more children than
AIDS, malaria and measles combined.[1–4] Almost 60% of deaths
associated with diarrhea are linked to enterotoxigenic strains of
bacteria, such as Escherichia coli (from here E. coli).[5] These
strains are able to interact with host epithelial cells through
specific adhesins, colonizing the small intestine. These strains
are able to synthesize numerous toxins, like enterotoxin “A”
thermostable (STa). STa binds to the membrane receptor
The
5-(3-bromophenyl)-1,3-dimethyl-5,11-dihydro-1H-
indeno-[20:10:5, 6]pyrido[2,3-d] pyrimidine-2,3,6-trione (BPIPP,
compound A, Figure 1) and some other related compounds, like
the 5-(3,5-bistrifluoromethylphenyl)-1,3-dimethyl-5,11-dihydro-
1H-indeno[2,1:5,6]pyrido[2,3-d]pyrimidine-2,4,6-trione (FPIPP,
compound B, Figure 1) suppress the synthesis of cGMP when in
presence of STa and other toxins in vitro.[11,12]
Here it is reported the design, synthesis and the pharmaco-
logical in vitro effect of twelve novel pyridopyrimidine deriva-
tives (I–XII, Table 1) on cyclic nucleotides accumulation. The
[a] Dr. E. Magli,+ Prof. F. Frecentese, Dr. I. Saccone, Dr. A. Corvino,
Dr. P. D. Vaio, Dr. F. Giordano, Dr. P. Luciano, Prof. F. Fiorino,
Prof. E. Perissutti, Prof. V. Santagada, Prof. B. Severino, Prof. G. Caliendo
Department of Pharmacy
University of Naples «Federico II»
Via D. Montesano, 49, 80131
Naples, Italy
E-mail: caliendo@unina.it
[b] Dr. T. Zaminelli,+ Dr. C. H. Lescano, Dr. R. Campos, Prof. G. De Nucci
Superior Institute of Biomedical Science
Ceará State University (UECE)
Ceará, Brazil
[+] These authors contributed equally to this work
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/open.201900060
© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This
is an open access article under the terms of the Creative Commons Attri-
bution Non-Commercial NoDerivs License, which permits use and distribu-
tion in any medium, provided the original work is properly cited, the use is
non-commercial and no modifications or adaptations are made.
Figure 1. Chemical structures of BPIPP (A) and FPIPP (B).
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Table 1. Conventional heating versus microwave irradiation in the syn-
thesis of aldehydes 3b and 3c and of pyridopyrimidines I–XII.
1
2
3
Conventional heating
(oil bath)
Microwave irradiation
Compd Time Temp Yield[a] Power Time Temp Yield[a] [%]
4
°
°
[h]
[ C]
[%]
[min] [ C]
5
6
7
8
3b
3c
I
II
III
IV
V
VI
VII
VIII
IX
X
12
12
8
8
8
8
8
8
8
8
8
8
8
70
70
88
85
200
200
400
400
400
400
400
400
400
400
400
400
400
400
120
120
40
40
40
40
40
40
40
40
40
40
40
40
70
95
94
91
75
67
64
72
85
90
78
73
80
94
96
Scheme 2. Synthetic route for intermediates 3b and 3c.
70
reflux 52
reflux 38
reflux 28
reflux 28
reflux 30
reflux 38
reflux 46
reflux 35
reflux 27
reflux 37
reflux 75
reflux 70
120
120
120
120
120
120
120
120
120
120
120
120
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
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XII. Aldehydes 2a–2c were commercially available, while 2d–
2g were synthesized as previously described.[16–19] Intermediate
3a was commercially available, while 3b and 3c were prepared
according to that reported in Scheme 2 following, with
modifications, procedures previously reported in literature.[20]
Conventional heating (oil bath) and microwave irradiation
of the reactions were compared (Table 1).
The main advantage of the microwave procedures was that
a short time of irradiation of the reaction mixtures provided the
derivatives I–XII and intermediates 3b and 3c as the major
product: when the reactions were performed under controlled
microwave irradiation conditions the reaction times were
greatly reduced from 12 h to 2 h (intermediates 3b and 3c) and
from 8 h to 40 min for final compounds I–XII.
XI
XII
8
[a] All the reactions were performed three times and the reaction time and
yields given are the average values.
most potent compound (VI) was further evaluated in an in vivo
model of diarrhea in rabbits and an histopathological analysis
of intestinal tissue treated with this compound was performed
by microscopic analysis. Moreover, bearing in mind that
dihydropyridines are reported in literature to be converted by
air oxygen into their corresponding pyridine analogues, the
chemical stability compound VI toward air oxidation was also
assessed both in solid state and in methyl alcohol solution.
Furthermore, in all the reactions we observed a significative
increase of the obtained yield that ranged from 64 to 96%
(higher in comparison to 27–70% of conventional heating).
2.2. Evaluation of Pyridopyrimidine Derivatives I–XII as cGMP
and cAMP Synthesis Inhibitors
2. Results and Discussion
The synthesized compounds were tested in vitro in order to
evaluate their effects on cyclic nucleotide accumulation.
Compound activity was determined measuring the concentra-
tion of cGMP or cAMP before and after stimulation with STa or
Forskolin, respectively, by ELISA assay. Results obtained for
cGMP and cAMP accumulation are reported in Table 2.
2.1. Synthesis of Pyridopyrimidine Derivatives
Compounds I–XII were prepared following, with modifications,
the synthetic procedures[13] depicted in Scheme 1. The synthetic
procedure is based on the Hantzsch dihydropyridine three
component cyclization.[14,15] In particular, reaction of 1,3-indan-
dione (1) with the aldehydes (2a–g) and the opportune primary
amines (3a–c) in acetic acid afforded the desired compounds I–
The basal levels of cAMP in Jurkat cells were 16�1 pmol/
10 min/mg and 140�8 pmol/10 min/mg following stimulation
by Forskolin (100 μM). The compounds had no effect on the
basal intracellular cAMP levels in T84 cells (data not shown). All
compounds significantly inhibited cAMP accumulation-induced
by forskolin (Table 2). In the absence of STa stimulation the
cGMP levels in T84 cells were 7�1 pmol/10 min/mg. When
stimulated by STa (1 μM) these levels enhanced to 471�
11 pmol/10 min/mg. None of the compounds induced changes
in intracellular cGMP levels compared to baseline group (DPBS)
in the absence of stimulation by STa (data not shown). With the
exception of compound VII, all compounds inhibited cGMP
accumulation-induced by STa (Table 1). A full concentration-
dependent curve (Figure 2) was performed for the most active
compounds (I, II, IV, VI and XI).
The results in cyclic nucleotides accumulation allowed some
preliminary considerations of structure-activity relationships
(SAR). An electronegative substituent at meta position of the
phenyl ring (carbon 5) was essential to compound activity.
Scheme 1. General procedure for the synthesis of compounds I–XII: i)
CH3COOH, sealed vessel, 120 C, MW (400 W) 40 min.
°
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Table 2. Chemical structures of pyridopyrimidine derivatives (I–XII) and their effects on cGMP accumulation induced by STa in T84 cells and cAMP
accumulation induced by Forskolin in Jurkat cells.[a]
1
2
3
Compd
Structure
cGMP
[%]
cAMP
[%]
Compd
Structure
cGMP
[%]
cAMP
[%]
4
5
6
7
8
9
A (BPIPP)
86�4
69�3
n.a.
B (FPIPP)
93�4
17�7
n.a.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
I
84�3
VII
VIII
IX
37�4
II
III
70�5
51�3
78�4
51�4
94�2
82�3
88�3
86�3
86�3
69�2
49�4
25�9
33�8
71�4
65�4
45�6
35�6
45�4
45�2
80�2
IV
V
X
XI
VI
XII
[a] T84 cells were pretreated for 10 minutes with compounds at 50 μM or vehicle (0.1% DMSO) and then treated with STa (1 μM). Jurkat cells were pretreated
for 10 minutes with compounds at 50 μM or vehicle (0.1% DMSO) and then treated with Forskolin (100 μM) and then intracellular cGMP or cAMP
accumulation were measured. Data are mean�SEM of 3–5 independent experiments assayed in duplicate
When this feature was not present, a severe decrease in the
activity was noted (compounds VII–X), both for cGMP and for
cAMP inhibition. Moreover, the presence of bicyclic aromatic
groups at position 5 (compounds VII–XII) caused loss of activity
when compared to the reference compounds. The replacement
of the methyl group with a more steric hindered ethyl group on
N1 enhanced the activity (compounds IV and VI). These
considerations are in accordance with those already published
in literature for analogue derivatives.[12]
The isosteric replacement of an oxygen with a sulfur atom
in position 2 proved to be not directly linked to the inhibitory
activity. Indeed, when this chemical modification was included
it generated compounds with an overlapping activity (i.e.
compounds I/II and XI/XII), or with better inhibitory effect for
the compound bearing the sulfur atom (i.e. VIII>VII).
the series even when compared to FPIPP, that shows a cLogP of
4.58.
Figure 2. Concentration response curves of compounds I, II, IV, VI and XI
(higher cGMP inhibition rate on T84 cells). Results are presented as
mean�SEM. n=3–5.
Finally compound VI, showing the highest effect on cGMP
accumulation, has a CLogP of 5.11. This value is the highest of
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On the basis of the obtained results, a pharmacological
mechanism of action of the synthesized compounds has been
preliminary postulated. In fact, when the STa or endogenous
mediators (guanylin and uroguanylin) interact with the receptor
GC-C inducing a conformational change in the extracellular
portion of the homotrimeric GC-C complex, two intracellular
catalytic domains dimerize and form two active catalytic clefts.
This mechanism of activation in the intestine results in
changes on cAMP or cGMP levels can promote significant
physiological effects due to the importance of these intra-
cellular signaling pathways, especially in the cardiovascular
system.[31,32] Furthermore, it should be noted here the partial
structural similarity of our compounds with dihydropyridine
type calcium blockers, such as amlodipine, widely used in clinic
as a tool to control blood pressure. These dihydropyridine
derivatives act inhibiting the calcium influx through ‘slow’
channels in vascular peripheral and coronary smooth muscle
cells, producing marked vasodilation in peripheral and coronary
vascular beds.[33] Thus, the evaluation of the here described
compounds effect on vascular tone is of fundamental impor-
tance. When incubated in T84 cells, amlodipine, nifedipine and
nimodipine induced no changes in cGMP accumulation induced
by STa (319�11, 328�29 and 273�11 pmol/10 min/mg
respectively, compared to STa 348�20 pmol/10 min/mg). For
this purpose, compounds I–XII have been evaluated in an aortic
tension model, were none of them induced changes in the
vascular tone, in contrast to the maximum vascular relaxation
induced by amlodipine (Figure 3). Interestingly, only FPIPP
1
2
3
4
5
6
7
8
9
À
stimulation of ClÀ and HCO3 secretion (by opening of the
apical CFTR ClÀ channels) and inhibition of Na+ absorption (by
blocking of the Na+/H+ exchanger).[7] Thus, the observed
decrease in the cGMP synthesis may be related to any of the
following events: (i) desensitization/allosteric modulation of the
GC-C receptor or (ii) modulation of PDE5 activity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
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Prolonged exposure of T84 cells to STa induces intracellular
mechanisms that promote the GC-C receptor desensitization,
resulting in a significant blockade in the cGMP synthesis.[21]
Several compounds have been described as inhibitors of GC-C
activity, even when exposed for shorter periods, similar and
lower than that employed in our protocols.[22–26] Thus, it would
be possible that the compounds herein described could interact
and stimulate these mechanisms, responsible for the receptor
desensitization, especially when highly stimulated by STa.
Furthermore, it is known that when STa activates GC-C, the
substrate (GMP) binds first to the regulatory site of the receptor
and while this binding is required for the subsequent binding
of substrate to the catalytic site, it does not affect the affinity of
the catalytic site.[22] Thereby, the binding of these compounds
to the regulatory site of GC-C, similarly to what can be observed
for other compounds,[25–27] could inhibit its activity.
PDE5 that is highly expressed in T84 cells[28] is characterized
by a relative specificity for cGMP hydrolysis at low substrate
levels and by the presence of high affinity-binding sites for
cGMP.[29] These binding sites are now known to be on the N-
terminal regulatory GAF domains of the enzyme. The binding of
cGMP to the PDE5 GAF-A domain stimulates the enzyme
activity 9- to 11-fold and the blockade of this binding inhibits
PDE5 activity.[29] Moreover, when cGMP levels are high (like after
stimulus by STa) and GAF-A is already occupied by cGMP, PKA
can also phosphorylate this site,[29] stabilizing the increased
catalytic activity by enhancing the affinity of cGMP binding to
the GAF-A domain.[30] However, when we incubated our
compounds in T84 cells, previously treated with 3-isobutyl-1-
methylxanthine (IBMX), a non-specific PDE inhibitor, the com-
pounds still were able to block the cGMP accumulation,
similarly to results described for BPIPP.[11]
Figure 3. Effect of compounds I–XII on pre-contracted rabbit aortic rings.
Rabbit aortic rings were pre-contracted with KCl (60 mM) and treated with
50 μM of compounds I–XII, FPIPP or Amlodipine (10 μM) for 40 minutes.
Results are presented as mean�SEM. n=3–5. One-Way ANOVA followed by
Tukey’s test; *p<0.05 compared to Amlodipine group.
(50 μM) promoted a dual effect by increasing the vascular tone
in 20% prior induce a maximal vascular relaxation similar to
amlodipine (10 μM). These observations are critically important
since, despite the structural similarity between our compounds
and dihydropyridine type calcium blockers, their effect on
vascular smooth muscle has no physiological relevance. More-
over, the inhibition of the accumulation of cyclic nucleotides by
the compounds seem to play no role in the vascular tonus,
which favors the absence of possible adverse cardiovascular
effects if systemic administrated.
2.3. Effect in Rabbit Aortic Rings
Functional studies in rabbit isolated aorta were performed to
evaluate smooth muscle relaxation induced by compounds I–
XII. Although all the compounds synthesized in this study were
able to block the accumulation of cGMP and cAMP when
stimulated by toxin or pharmacological agents, none of them
induced changes in the basal levels of these cyclic nucleotides.
This feature is extremely important, considering that small
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2.4. Cell Metabolic Activity (MTT) and Cytotoxicity (LDH)
Table 4. Effect of compounds on cell LDH release (LDH cytotoxicity assay).
1
2
3
4
5
6
7
8
9
Treatment
1 h
Mean
[%]
24 h
S.E.M Mean
[%]
48 h
S.E.M Mean
[%]
All the compounds evaluated in this study showed some effect
on stimulated synthesis of cGMP and cAMP. Therefore, in order
to investigate the possibility of our results be due not to an
intrinsic activity of each compound, but by a toxic effect on
cells, like metabolic alterations or loss of viability, the MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
and the LDH (Lactate dehydrogenase) assays were employed.
The first assay requires cells that are actively able to
metabolize MTT to an insoluble formazan precipitate.[34] To
investigate changes in the metabolic activity, all the com-
pounds were incubated for 1 hour, 24 hours and 48 hours in
protein free medium. Results evidenced that no changes in
cellular metabolism occurred at all tested times, suggesting the
complete absence of interference on the metabolic activity of
T84 cells and excluding the possibility of a metabolic interfer-
ence in cells during the assay of cGMP stimulation (Table 3).
S.E.M
Vehicle (DMSO
0.1%)
Positive control
1.9
1.0
1.6
0.5
3.9
2.2
0.4
2.7
61.4%* 1.5
65.8*
65.5*
(LDH)
STa (1 μM)
FPIPP (50 μM)
I
2.3
2.9
2.1
2.2
1.3
3.2
1.7
3.5
1.6
2.3
1.5
2.2
4.0
3.2
0.5
0.3
0.2
0.5
0.3
0.5
0.5
0.6
0.1
0.1
0.6
1.6
1.9
0.8
2.8
4.2
2.7
6.1
7.8*
5.6
8.6*
6.1
2.1
2.9
1.1
2.3
3.3
4.4
0.4
0.8
1.1
1.5
0.9
1.3
1.8
1.2
0.3
0.2
0.4
0.3
0.9
0.4
3.7
9.3
12.5*
9.2
1.1
1.5
0.7
1.1
0.8
1.4
0.4
0.3
2.4
0.2
0.9
1.5
0.3
2.4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
II
III
IV
V
VI
VII
VIII
IX
X
11.3*
11.7*
13.5*
11.8*
11.1*
13.3*
13.5*
10.8*
11.5*
11.3*
XI
XII
[a] T84 cells were treated with 50 μM of compounds I–XII or vehicle (DPBS
with 0.1% DMSO) for 1, 24 and 48 hours. Results are normalized to the
vehicle group. n=3; Two-Way ANOVA followed by Tukey’s test; *p<0.05
compared to vehicle group.
Table 3. Effect of compounds on cell metabolic activity (MTT assay).[a]
1 h
24 h
48 h
Treatment
Mean [%] S.E.M Mean [%] S.E.M Mean [%] S.E.M
Vehicle
Triton (0.1%)
STa (1 μM)
FPIPP (50 μM) 98.2
I
100.0
54.9*
96.8
–
100.0
45.2*
94.9
96.4
85.7
95.7
93.2
94.4
92.1
95.2
90.1
93.8
94.1
96.6
94.6
96.0
–
100.0
38.4*
90.5
93.3
84.5
96.0
86.2
93.4
88.8
91.9
89.4
89.6
95.2
92.8
90.6
86.5
–
1.1
1.2
1.3
1.1
2.4
1.6
2.5
3.3
2.2
1.1
1.4
1.4
3.7
1.0
1.6
1.2
1.0
1.1
2.6
2.3
3.7
1.6
4.0
1.8
2.7
2.6
2.7
3.8
3.3
1.8
1.3
1.2
1.9
5.5
3.4
3.4
5.1
3.7
3.0
3.9
1.6
4.7
3.5
4.0
2.4
line with those of nontoxic compounds and are moreover
similar to the results obtained with BPIPP.[11]
91.8
90.8
101.1
99.3
101.3
96.2
91.7
90.5
90.6
91.4
92.7
90.6
II
III
IV
V
VI
VII
VIII
IX
X
2.5. Measurement of Diarrhea In Vivo in a Model of Rabbit
Ileal Loops
In order to investigate the efficacy of compound VI in reducing
bacterial toxin-induced diarrhea (dependent on the activation
of cGMP pathways and / or cAMP) in vivo, as well as the
possibility of modulation of the inflammatory process and cell
lesions induced by this toxin, we used the technique previously
described by Alcantara et al.[36] In particular, a midline abdomi-
nal incision was made to expose the small bowel of rabbits
previously anesthetized. Then 6–8 loops of 3 cm each were
ligated and compound VI or FPIPP+STa toxin were injected.
The in vivo tests were performed with an allowance and
according to ethics committee approval.
The data reported in Figure 4 shows that both, compound
VI and FPIPP are effective in reducing the accumulation of fluid
in the intestinal tissue when stimulated by the STa toxin.
Statistically, as well as the observations in cell culture, both
compounds do not differ from each other.
However, histopathological findings point to a relevant
biological difference between the two compounds. According
to these results, the treatment with compound VI prevents
microscopic changes in the intestinal tissue, preserving tissue
architecture, highlighting mild mucosal hyperplasia, associated
with deposition of mucoid material and rare hematic focus,
decreased inflammatory pattern when compared to FPIPP
treated tissue, represented by less lymphocytes, plasmocytes
and rare eosinophils. The submucosa and lamina propria are
preserved, with mild edema and lymphatic dilatation, that can
XI
XII
[a] T84 cells were treated with 50 μM of compounds I–XII or vehicle (DPBS
with 0.1% DMSO) for 1, 24 or 48 hours. Results are normalized to the
vehicle group. n=3; One-Way ANOVA followed by Tukey’s test; *p<0.05
compared to vehicle group.
Finally, complementary assays were performed in order to
investigate cytotoxicity induced by the compounds. Intracellular
enzymes are usually released after damage to the cell
membrane, thus, the larger the rate of release of enzyme, the
greater the extent of cell death/damage which occurred in that
period.[35] In order to investigate, we measured the LDH levels in
T84 cells incubated with compounds for 1 hour, 24 hours and
48 hours. Also in this case results (Table 4) were encouraging
and evidenced complete absence of cytotoxicity for all the
compounds at 1 h; when analyzing data at 24 h and 48 h we
noticed that all compounds evidenced a very low toxic effect at
24 h with exception of compounds III and V (7.8% and 8.6%,
respectively), while a general increasing toxicity trend at 48 h
was noticed. In this latter case all the compounds, as expected,
showed effects ranging between 9.2% and 13.5%, that are in
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scope stage and illumination adjusted with the aid of the
condenser and diaphragm. Then, image was focused with a 10x
and 40x objective. Tissue architecture was evaluated through-
out the slide and histopathological findings were measured
according to the following score:
0) Preserved tissue absent from inflammatory cellularity and
necrosis;
1) Mild tissue disorganization with rare inflammatory cells;
2) Moderate tissue disorganization with sketches of necrosis
and/or discrete inflammatory infiltrate aggregate;
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3) Clear tissue disorganization with mild necrosis traces and/or
moderate inflammatory aggregates;
4) Loss of tissue architecture with features of moderate
necrosis and/or accentuated inflammatory aggregates;
5) Loss of complete tissue architecture with severe necrosis
and/or marked inflammatory aggregates.
Figure 4. Intraluminal injection of compound VI and/or FPIPP (1 mg/loop) in
Heat Stable Toxin (STa, 20 μg/mL) treated rabbit ileal loops. *P<0.05 (SHAM
vs. STa; SHAM vs. FPIPP); #P<0.05 (STa vs. Compound VI; STa vs. FPIPP).
One-way ANOVA, complemented by Tukey’s test.
The obtained images are reported in Figure 5.
In particular, the microscopic inspection allowed the
observation of the tissue modifications due to the exposure to
STa toxin alone or in combination with compound VI or FPIPP;
the experiment results for each case together with the relative
anti-inflammatory score are reported as follows:
be characterized as a hyperplasia of intestinal mucosa associ-
ated with lymphoplasmocitary aggregates.
FPIPP compound, however, does not demonstrate such
activity in the intestinal tissue. Although it is able to reduce the
accumulation of fluids induced by the toxin, there is an intense
loss of tissue architecture with features of moderate necrosis
and/or accentuated inflammatory aggregates. Also, a significant
deposition of serocellular material, with moderate epithelial
hyperplasia of the villi, focal area of mucosal dilatation, a
multifocal lymphoplasmacytic inflammatory infiltrate, evidenc-
ing a moderate arrangement in nests, mixing pronounced
suppurative/exudative traits in the middle of the mucosa
transition. In the submucosal region, there is moderate tissue
edema, with the spacing of the collagen fibers and lymphatic
dilatation, that can be characterized as a severe diffuse
lymphoplasmocitary enteritis, associated with exudative traits.
2.6.1. No Treatment with STa Toxin (Vehicle only)
Histopathological evaluation (Figure 5A) reveals preserved
tissue architecture, highlighting mild mucosal edema and
deposition of superficial secretory material. In submucosa and
muscular layer are observed discrete foci of vascular congestion
and edema of the collagen fibers.
Conclusion of analysis: preserved intestine without signifi-
cant pathological changes, with an inflammatory score of 0
(preserved tissue absent from inflammatory cellularity and
necrosis).
2.6. Histopathological Analysis of Intestinal Tissue
2.6.2. Treatment with STa Toxin and Post-Treatment with
Vehicle
The microscopic analysis of stained slides was made in a Nikon
binocular microscope (E200). First, slides were placed in micro-
Histopathological evaluation (Figure 5B) reveals
a discrete
deposition of serocellular material, with moderate epithelial
hyperplasia of the villi, mixing focal area of mucosal dilatation.
In the middle of the epithelial layer is observed a multifocal
lymphoplasmacytic inflammatory infiltrate, evidencing a moder-
ate arrangement in nests, mixing pronounced suppurative/
exudative traits in the middle of the mucosa transition. In the
submucosal region, there is moderate tissue edema, with the
spacing of the collagen fibers and lymphatic dilatation.
Conclusion of analysis: severe diffuse lymphoplasmocitary
enteritis, associated with exudative traits, with an inflammatory
score of 4 (loss of tissue architecture with features of moderate
necrosis and/or accentuated inflammatory aggregates).
Figure 5. Histopathological analysis of intestinal tissue (ileum) of rabbits
treated for 5 hours with: A) vehicle; B) STa toxin; C) Compound VI+STa
toxin; D) FPIPP+STa toxin.
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2.6.3. Pre-Treatment with Compound VI and Post-Treatment
with STa Toxin
2.7.2. Methyl Alcohol Solution
1
2
3
4
5
6
7
8
9
Two samples of the same lot of compound VI were prepared as
follows:
Sample C: 1 mg, diluted in 1 mL of MeOH, stored at 4 C
under nitrogen atmosphere.
Sample D: 1 mg, diluted in 1 mL of MeOH, room temper-
ature, open vial.
After 4 weeks the samples were compared by visual
comparison (Figure S4), analytical RP-HPLC analyses and by LC-
HRMS.
Analytical HPLC results evidenced that prolonged storage of
a methanolic solution of the compound VI, at room temper-
ature and under air exposure, allowed the formation of a small
amount of two by-products, named “a” and “b” (Figure S5).
Sample D was therefore further investigated by LC-HRMS in
order to characterize these compounds.
By-product “a” revealed a molecular weight of 538.1189
corresponding to a molecule having a general formula of
C25H17O4N3F6; we propose that this compound corresponds to
the N-hydroxy derivative of compound VI. By-product “b”,
characterized by a molecular weight of 520.1081, has been
identified as a pyridine derivative, obtained by oxidation of
compound VI. The chemical structure of compounds “a” and
“b” are showed in Figure S6.
Histopathological evaluation (Figure 5C) reveals preserved tis-
sue architecture, highlighting mild mucosal hyperplasia, asso-
ciated with deposition of mucoid material and rare hematic
focus. In the middle of the mucosa there is a discrete mixed
inflammatory pattern, represented by lymphocytes, plasmo-
cytes and rare eosinophils. The submucosa and lamina propria
are preserved, with mild edema and lymphatic dilatation.
Conclusion of analysis: hyperplasia of intestinal mucosa
associated with lymphoplasmocitary aggregates, with an in-
flammatory score of 2 (moderate tissue disorganization with
sketches of necrosis and/or discrete inflammatory infiltrate
aggregate).
°
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2.6.4. Pre-Treatment with FPIPP and Post-Treatment with STa
Toxin
Histopathological evaluation (Figure 5D) reveals a discrete
deposition of serocellular material, with moderate epithelial
hyperplasia of the villi, mixing focal area of mucosal dilatation.
In the middle of the epithelial layer is observed a multifocal
lymphoplasmacytic inflammatory infiltrate, evidencing a moder-
ate arrangement in nests, mixing pronounced suppurative/
exudative traits in the middle of the mucosa transition. In the
submucosal region, there is moderate tissue edema, with the 3. Conclusions
spacing of the collagen fibers and lymphatic dilatation.
Conclusion of analysis: severe diffuse lymphoplasmocitary
enteritis, associated with exudative traits, with an inflammatory
score of 4 (loss of tissue architecture with features of moderate
necrosis and/or accentuated inflammatory aggregates).
Research results reported in this work allowed the identification
of pyridopyrimidines acting as a promising class of anti-
diarrheal agents. The molecules were prepared by an optimized
synthetic methodology that was found as the best strategy for
the obtaining all compounds in high yields and purity. The
most notable derivatives were compounds VI and IV, with a
partial selectivity towards cGMP and cAMP, respectively. In vitro
assays demonstrated that none of the synthesized compounds
affects vascular tonus of pre-contracted aortic rings when
compared to amlodipine. Moreover, the metabolic activity and
viability in T84 cells were not affected by compounds I–XII as
MTT and LDH assays evidenced.
Compound VI was then selected for a specific in vivo insight
focused to the evaluation of its anti-diarrheal activity in rabbits;
the experiments performed on rabbit ileum ligations evidenced
a potent effect of compound VI and FPIPP. This prompted us to
perform a microscopic histopathological analysis of intestinal
tissues aimed to the evaluation of tissue architecture. The
images allowed to reveal that only compound VI preserves
intestine, without significant pathological changes and with a
decreased inflammatory pattern in comparison to FPIPP. Finally,
bearing in mind that the oxidative aromatization of 1,4-DHPs to
the corresponding pyridines has been extensively studied and
that a plethora of oxidants including air have been applied in
the aromatization reactions, compound VI was further inves-
tigated for its chemical stability and revealed to be suitable for
a prolonged storage in solid state. This derivative therefore
candidates as “lead compound” of this class of pyridopyrimi-
2.7. Air Exposure Stability Evaluation for Compound VI
In order to evaluate the chemical stability of compound VI,
identified as the most active compound towards cGMP
accumulation, we decided to perform experiments designed to
assess its resistance against air oxidation, both in solid state and
in methyl alcohol solution. In particular the procedure was as
below reported:
2.7.1. Solid State
Two samples of the same lot of compound VI were prepared as
follows:
°
Sample A: 10 mg, stored at 4 C under nitrogen atmosphere.
Sample B: 10 mg, room temperature, open vial.
After 4 weeks the samples A and B were compared by visual
inspection, analytical RP-HPLC and ESI-MS. Results, reported in
Figures S1–S3 (supplementary materials), suggest no oxidation
of the compound.
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available in Supplementary Materials (Figures 7 S, 8 S and 9 S,
respectively).
dines, an interesting scaffold for the generation of molecules
for possible use as efficient anti-diarrheal drugs.
1
2
3
4
5
6-Amino-1,3-Dimethyl-2-Thioxo-2,3-Dihydropyrimidin-4(1H))-
One (3b)
Experimental Section
6
7
8
9
To a stirred solution of 1,3-dimethylthiourea (1.83 g, 17.6 mmol) in
acetic anhydride (30 mL) was added cyanoacetic acid (1.5 g,
17.6 mmol), and the resulting mixture was transferred into a sealed
Chemistry
5-(Trifluoromethyl)thiophene-3-carbaldehyde was purchased from
Aurora Building Blocks, 1-ethyl-3-methylurea was purchased from
AKos Building Blocks and all the other commercial products have
been purchased from Sigma Aldrich. All the reactions have been
followed by TLC, carried out on Merck silica gel 60 F254 (E. Merck,
AG, Darmstadt, Germany) plates with fluorescent indicator and the
plates have been visualized with UV light (254 nm). Microwave
reactions were performed using a microwave oven (ETHOS 1600,
Milestone) especially designed for organic synthesis. Microwave
reactions were performed in sealed tubes and were performed by
microwave program which was composed by appropriate ramping
and holding steps. Identification of the optimum profile power/
time and temperature for the synthesis was preliminarily assessed.
The temperature of the stirred reaction mixture was monitored
directly by a microwave-transparent fluoroptic probe inserted into
the reaction mixture; irradiation time and power were monitored
with the “easyWAVE” software package. No further yield increasing
was evidenced when the irradiation power or time were increased
due to decomposition of the starting materials. Preparative
chromatographic purifications have been performed using silica gel
column (Kieselgel 60). Solutions have been concentrated with a
Büchi rotary evaporator at low pressure. Melting points, determined
using a Büchi Melting Point B-540 instrument, are uncorrected and
represent values obtained on recrystallized or chromatographically
purified material. Molecular weights of intermediates have been
assessed by electrospray ionization mass spectrometry (ESI/MS)
performed on an API 2000 Applied Biosystems mass spectrometer.
Analytical RP-HPLC analyses were performed using a Phenomenex
Kinetex C18 column (2.5 μm, 4.6×300 mm). The column was
connected to a Rheodyne model 7725 injector, a Shimadzu-10
ADsp HPLC system, a Shimadzu SPD-20 A/SPD-20 AV UV-VIS
detector set to 254 nm. The analytical determinations employed
solvent A: 100% acetonitrile in 0.1% TFA, and B: 100% H2O in 0.1%
TFA. The operational conditions involved a linear gradient of 0–
100% acetonitrile with 0.1% TFA, which developed in 30 minutes
at a flow rate of 1 mL/min. LC-HRMS was carried out on a Dionex
Ultimate 3000 quaternary system coupled to a hybrid linear ion
trap LTQ Orbitrap XLTM Fourier Transform MS (FTMS) equipped
with an ESI ION MAXTM source (Thermo-Fisher, San Josè, CA, USA).
The same instrument was used for HRMS (infusions) of final
compounds. Chromatographic separation was accomplished by
using a Kinetex C18 column (100×2.1 mm, 1.7 μm) (Phenomenex,
Le Pecq, France) maintained at room temperature and eluted at
0.2 mLminÀ 1 with water (eluent A) and 95% acetonitrile/water
°
vessel and heated by microwave irradiation at 70 C (200 W power)
for 2 hours. The reaction mixture was concentrated and the
resulting oily residue was diluted with H2O (40 mL) and treated
with 5 N NaOH (15 mL). The precipitate thus formed was collected
by filtration, washed with cold water and purified by recrystalliza-
tion from MeOH/H2O to give 2.65 g of 6-amino-1,3-dimethyl-2-
thioxo-2,3-dihydropyrimidin-4(1H)-one.
10
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1
°
Yield: 2.86 g, 95%. m.p. 289 C. H-NMR (400 MHz, DMSO-d6): δ 7.01
(s, 2H, NH2), 5.01 (s, 1H), 3.74 (s, 3H), 3.50 (s, 3H). ESI-MS (MW
171.05): 172.1 (M+H+). Anal. calcd for C6H9N3OS: C 42.09, H 5.30, O
9.34, found: C 42.49, H 5.00, O 9.44.
Synthesis of 6-Amino-1-Ethyl-3-Methylpyrimidine-2,4(1H,
3H)-Dione (3c)
To a stirred solution of 1-ethyl-3-methylurea (1.36 g, 13.3 mmol) in
acetic anhydride (30 mL) was added cyanoacetic acid (1.13 g,
13.3 mmol), and the resulting mixture was transferred into a sealed
°
vessel and heated by microwave irradiation at 70 C (200 W power)
for 2 hours. The reaction mixture was concentrated and the
resulting oily residue was diluted with H2O (40 mL) and treated
with 5 N NaOH (15 mL). The precipitate thus formed was collected
by filtration, washed with cold water and purified by recrystalliza-
tion from MeOH/H2O to give 1.91 g of 6-amino-1-ethyl-3-methylpyr-
imidine-2,4(1H,3H)-dione.
1
°
Yield: 2.11 g, 94%. m.p. 232 C. H-NMR (400 MHz, DMSO-d6): δ 6.76
(s, 2H, NH2), 4.64 (s, 1H), 3.80 (q, 2H), 3.29 (s, 3H), 1.08 (t, 3H). ESI-
MS (MW 169.09): 170.1 [M+H]+. Anal. calcd for C7H11N3O2: C 49.70,
H 6.55, O 18.91, found: C 49.30, H 6.15, O 18.71.
5-(4-Trifluoromethylthiophen)-1,3-Dimethyl-5,11-Dihydro-1-
H-Indeno-[2’,1’:5,6]Pyrido[2,3-d]Pyrimidine-2,4,6-Trione (I)
To acetic acid (15 mL) in a round bottom flask, 1,3-indandione (1,
0.56 g, 3.87 mmol), 5-(trifluoromethyl)thiophene-3-carbaldehyde
(2c, 0.697 g, 3.87 mmol) and 6-amino-1,3-dimethyluracil (3a, 0.5 g,
3.22 mmol) were added. T The mixture was then transferred into a
°
sealed vessel and heated by microwave irradiation at 120 C (400 W
power) for 40 minutes (2 min ramping step; 38 min holding step). It
°
was then cooled to 0 C. The resulting precipitate was filtered and
rinsed with cold water to obtain the crude product. Purification on
silica gel column (CH2Cl2/CH3OH, 9,5/0,5, v/v) afforded the desired
(eluent B), both containing 30 mM acetic acid. The H and 13C-NMR
1
spectra have been recorded on a Varian Mercury Plus 400 MHz
instrument. All spectra have been recorded in DMSO-d6. The
following abbreviations are used to describe peak patterns when
appropriate: s (singlet), bs (broad singlet), d (doublet), dd (double
doublet), t (triplet), q (quartet), m (multiplet).
°
compound I as an orange powder. Yield: 1.28 g, 91%. m.p. 265 C.
1H-NMR (400 MHz, DMSO-d6): δ 9.88 (s, 1H, NH), 7.85 (d, 1H), 7.59 (d,
1H), 7.56 (s, 1H), 7.46 (t, 1H), 7.32 (t, 1H), 5.73 (s, 1H), 4.89 (s, 1H),
3.56 (s, 3H), 3.13 (s, 3H). 13C-NMR (100 MHz, DMSO-d6): δ 190.9,
161.5, 155.5, 154.5, 151.1, 146.8, 133.6, 132.6, 130.8, 130.6, 127.0,
124.0, 122.1, 121.5, 121.4, 121.3, 108.9, 91.2, 31.3, 30.0, 29.0. HRMS
445.93459. Anal. calcd for C21H14F3N3O3S: C 56.63, H 3.17, O 10.78,
found: C 56.43, H 3.07, O 10.98.
Analytical purification of each product was obtained by silica gel
column chromatography and by crystallization from the appropri-
ate solvent. Analyses indicated by the symbols of the elements
were whitin �0.4 of the theoretical values. H NMR, 13C NMR and
1
MS data for all final compounds were consistent with the proposed
structures. Representative HRMS, 1H and 13C-NMR spectra are
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132.9, 128.9, 128.6, 127,9, 123.3, 121.6, 108.6, 93.7, 37.3, 35.0, 27.7.
HRMS 524.03679. Anal. calcd for C24H15F6N3O2S: C 55.07, H 2.89, O
6.11, found: C 55.37, H 2.99, O 6.41.
5-(4-Trifluoromethylthiophen)-1,3-Dimethyl-5,11-Dihydro-1-
H-Indeno-[2’,1’:5,6]Pyrido[2,3-d]
Pyrimidine-2-Thioxo-4,6-Dione (II)
1
2
3
4
5
6
7
8
9
The compound was obtained with the same procedure described
for compound I, by reaction of 1,3-indandione (1, 170 mg,
5-(3,5-Bistrifluoromethylphenyl)-1-ethyl3-methyl-5,11-di-
hydro-1H-indeno-[2’,1’:5,6] Pyrido[2,3-d]
pyrimidine-2,4,6-trione (VI)
1.16 mmol),
5-(trifluoromethyl)thiophene-3-carbaldehyde
(2c,
209 mg, 1.16 mmol) and 6-amino-1,3-dimethyl-2-thioxo-2,3-dihy-
dropyrimidin-4(1H)-one (3b, 166 mg, 0.97 mmol). Yield: 0.335 g,
1
The compound was obtained with the same procedure described
for compound I, by reaction of 1,3-indandione (1, 708 mg,
4.85 mmol), 3,5-bistrifluoromethylbenzaldehyde (2a, 1.17 g,
4.85 mmol) and 6-amino-1-ethyl-3-methylpyrimidine-2,4(1H,3H)-di-
°
75%. m.p. 178 C. H-NMR (400 MHz, DMSO-d6): δ 10.09 (s, 1H, NH),
7.87 (d, 1H), 7.66 (d, 1H), 7.60 ( t, 1H), 7.49 (s, 1H), 7.33 (t, 1H), 5.95
(s, 1H), 4.96 (s, 1H), 4.07 (s, 3H), 3.58 (s, 3H). 13C-NMR (100 MHz,
CD3OD): δ 193.2, 178.3, 161.5, 155.4, 152.2, 146.8, 146.8, 133.5,
131.5, 130.7, 130.6, 126.9, 126.9, 122.4, 121.1, 121.0, 107.8, 97.2,
39.0, 36.2, 31.3. HRMS 461.95812. Anal. calcd for C21H14F3N3O2S2: C
54.66, H 3.06, O 6.93, found: C 54.36, H 3.36, O 6.63.
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one (3c, 683 mg, 4.04 mmol). Yield: 2.01 g, 85%. m.p. 166 C. 1H-
°
NMR (400 MHz, DMSO-d6): δ 9.87 (s, 1H, NH), 7.94 (d, 1H), 7.91 (s,
2H), 7.88 (s, 1H), 7.50 (t, 1H), 7.37 (t, 1H), 7.30 (d, 1H), 5.03 (s, 1H),
4.32 (m, 2H), 3.09 (s, 3H), 1.24 (t, 3H). 13C-NMR (100 MHz, DMSO-d6):
191.3, 168.1, 161.4, 154.8, 150.7, 148.6, 145.3, 136.2, 132.7, 132.5,
131.0, 130.5, 128.9, 125.2, 121.5, 121.4, 120.8, 109.3, 91.2, 35.5, 28.1,
14.0. HRMS 522.12598. Anal. calcd for C25H17F6N3O3: C 57.59, H 3.29,
O 9.21, found: C 57.69, H 3.39, O 9.31.
5-(3-Fluoro-5-trifluoromethylphenyl)-1,3-dimethyl-5,11-dihyd-
ro-1H-indeno-[2’,1’:5,6] pyrido[2,3-d]
pyrimidine-2-thioxo-4,6-dione (III)
The compound was obtained with the same procedure described
for compound I, by reaction of 1,3-indandione (1, 1.11 g,
7.61 mmol), 3-fluoro-5-trifluoromethylbenzaldehyde (2b, 1.46 g,
7.61 mmol) and 6-amino-1,3-dimethyl-2-thioxo-2,3-dihydropyrimi-
din-4(1H)-one (3b, 1.85 g, 6.34 mmol). Yield: 2.01 g, 67%. m.p.
5-(Benzo[d][1,3]
dioxol-4-yl)-1,3-dimethyl-5,11-dihydro-1H-indeno-[2’,1’:5,6]
pyrido[2,3-d]pyrimidine-2,4,6-trione (VII)
313 C. 1H-NMR (400 MHz, DMSO-d6): δ 10.09 (s, 1H, NH), 8.03 (d,
The compound was obtained with the same procedure described
for compound I, by reaction of 1,3-indandione (1, 1.61 g,
11.06 mmol), 2,3-(methylenedioxy)benzaldehyde (2g, 1.66 g,
°
1H), 7,66 (s, 1H), 7,54 (s, 1H), 7,51 (d, 1H), 7.41 (t, 1H), 7,35 (t, 1H),
7,28 (s, 1H), 4,96 (s, 1H), 4.08 (s, 3H), 3.52 (s, 3H). 13C-NMR (100 MHz,
CD3OD): δ 193.2, 178.0, 167.5, 158.4, 156.8, 154.9, 153.0, 150.2,
143.9, 138.8, 136.6, 134.7, 133.9, 127.1, 124.3, 123.1, 122.6, 122.2,
120.4, 109.6, 92.6, 38.1, 36.0. HRMS 474.10597. Anal. calcd for
C23H15F4N3O2S: C 58.35, H 3.19, O 6.76, found: C 58.55, H 3.29, O
6.86.
11.06 mmol)
and
6-amino-1,3-dimethyluracil
(3a,
1.43 g,
9.22 mmol). Yield: 3.44 g, 90%. m.p. 300 C. 1H-NMR (400 MHz,
DMSO-d6): δ 9.88 (s, 1H, NH), 7.83 (d, 1H), 7.45 (t, 1H), 7.32 (t, 1H),
7.24 (d, 1H), 6.73 (t, 1H), 6.66 (d, 2H), 5.90 (d, 2H), 4.81 (s, 1H), 3.56
(s, 3H), 3.08 (s, 3H). 13C-NMR (100 MHz, DMSO-d6): 188.1, 168.2,
159.4, 155.9, 150.7, 146.8, 145.7, 144.2, 140.7, 136.4, 136.0, 133.6,
124.7, 124.0, 122.3, 120.9, 120.5, 116.4, 108.5, 107.6, 101.1, 30.8,
28.6. HRMS 416.08251. Anal. calcd for C23H17N3O5: C 66.50, H 4.12, O
19.26, found: C 66.90, H 4.52, O 19.66.
°
5-(3-Fluoro-5-trifluoromethylphenyl)-1-ethyl-3-methyl-5,11-
dihydro-1H-indeno-[2’,1’:5,6] Pyrido[2,3-d]
pyrimidine-2,4,6-trione (IV)
The compound was obtained with the same procedure described
for compound I, by reaction of 1,3-indandione (1, 970 mg,
6.64 mmol), 3-fluoro-5-trifluoromethylbenzaldehyde (2b, 1.27 g,
6.64 mmol) and 6-amino-1-ethyl-3-methylpyrimidine-2,4(1H,3H)-di-
5-(Benzo[d][1,3]
dioxol-4-yl)-1,3-dimethyl-5,11-dihydro-1H-indeno-[2’,1’:5,6]
pyrido[2,3-d]pyrimidine-2-thioxo-4,6-dione (VIII)
one (3c, 936 mg, 5.53 mmol). Yield: 1.64 g, 64%. m.p. 177 C. 1H-
The compound was obtained with the same procedure described
for compound I, by reaction of 1,3-indandione (1, 535 mg,
3.66 mmol), 2,3-(methylenedioxy)benzaldehyde (2g, 549 mg,
3.66 mmol) and 6-amino-1,3-dimethyl-2-thioxo-2,3-dihydropyrimi-
din-4(1H)-one (3b, 521 mg, 3.05 mmol). Yield: 1.02 g, 78%. m.p.
°
NMR (400 MHz, DMSO-d6): δ 9.81 (s, 1H, NH), 7.94 (d, 1H), 7.49 (m,
3H), 7.37 (d, 1H), 7.33 (t, 1H), 7.27 (t, 1H), 4.91 (s, 1H), 4.26 (m, 2H),
3.10 (s, 3H), 1.24 (t, 3H). 13C-NMR (100 MHz, CD3OD): δ 193.2, 165.1,
163.4, 162.7, 155,7, 152.0, 150.6, 150.5, 146.4, 137.5, 133.9, 133.5,
131.5, 126.1, 122.3, 121.7, 121.1, 119.7, 119.5, 111.5, 93.2, 36.1, 28.4,
13.8. HRMS 471.12632. Anal. calcd for C24H17F4N3O3: C 61.15, H 3.63,
O 10.18, found: C 61.25, H 3.43, O 10.38.
310 C. 1H-NMR (400 MHz, DMSO-d6): δ 10.11 (s, 1H, NH), 7.83 (d,
°
1H), 7.47 (t, 1H), 7.34 (t, 1H), 7.27 (d, 1H), 6.76 (t, 1H), 6.68 (d, 2H),
5.91 (d, 2H), 4.88 (s, 1H), 4.08 (s, 3H), 3.52 (s, 3H). 13C-NMR (100 MHz,
DMSO-d6): 193.5, 179.3, 161.5, 156.8, 149.8, 148.8, 148.18, 147.2,
143.1, 139.3, 135.2, 133.3, 129.2, 125.3, 124.0, 123.8, 112.0, 111.4,
110.0 103. 6, 98.5, 38.4, 33.0. HRMS 432.04328. Anal. calcd for
C23H17N3O4S: C 64.03, H 3.97, O 14.83, found: C 64.23, H 3.67, O
15.03.
5-(3,5-Bistrifluoromethylphenyl)-1,3-dimethyl-5,11-dihydro-1-
H-indeno-[2’,1’:5,6]pyrido[2,3-d]
pyrimidine-2-thioxo-4,6-dione (V)
The compound was obtained with the same procedure described
for compound I, by reaction of 1,3-indandione (1, 503 mg,
3.44 mmol), 3,5-bistrifluoromethylbenzaldehyde (2a, 833 mg,
3.44 mmol) and 6-amino-1,3-dimethyl-2-thioxo-2,3-dihydropyrimi-
din-4(1H)-one (3b, 490 mg, 2.87 mmol). Yield: 1.16 g, 72%. m.p.
5-(Benzo[d][1,3]
dioxol-5-yl)-1,3-dimethyl-5,11-dihydro-1H-indeno-[2’,1’:5,6]
pyrido[2,3-d]pyrimidine-2,4,6-trione (IX)
314 C. 1H-NMR (400 MHz, DMSO-d6): δ 10.14 (s, 1H, NH), 8.08 (d,
The compound was obtained with the same procedure described
for compound I, by reaction of 1,3-indandione (1, 203 mg,
1.39 mmol), 3,4-(methylenedioxy)benzaldehyde (2f, 209 mg,
1.39 mmol) and 6-amino-1,3-dimethyluracil (3a, 180 mg,
°
1H), 7.97 (s, 2H), 7.87 (s, 1H), 7.53 (t, 1H), 7.37 (t, 1H), 7.25 (d, 1H),
5.06 (s, 1H), 4.08 (s, 3H), 3.51 (s, 3H). 13C-NMR (100 MHz, DMSO-d6):
187.4, 177.0, 166.9, 157.6, 153.9, 146.8, 139.5, 136.7, 135.6, 135.3,
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1.16 mmol). Yield: 0,352 g; 73%. m.p. 338 C. 1H-NMR (400 MHz,
(Sigma-Aldrich, #D8900) supplemented with 10% FBS (Gibco,
#12657029) and penicillin-streptomycin (Gibco, #15140122) in a
°
1
2
3
4
5
6
7
8
DMSO-d6): δ 9.79 (s, 1H, NH), 7.98 (d, 1H), 7.86 (t, 1H), 7.77 (t, 1H),
7.62 (d, 1H), 6.92 (d, 1H), 6.81 (s, 1H), 6.72 (d, 1H), 5.89 (s, 2H), 4.69
(s, 1H), 3.57 (s, 3H), 3.10 (s, 3H). 13C-NMR (100 MHz, DMSO-d6): 189.2,
168.5, 159.9, 156.5, 151.5, 146.8, 145.7, 145.2, 140.9, 136.8, 135.9,
133.3, 124.6, 124.3, 122.9, 121.2, 120.3, 117.0, 109.1, 108.0, 101.1,
31.1, 27.9. HRMS 416.06943. Anal. calcd for C23H17N3O5: C 66.50, H
4.12, O 19.26, found: C 66.70, H 4.02, O 19.16.
°
humidified atmosphere of 95% air/5% CO2 at 37 C. Jurkat E6-1
cells were purchased from APABCAM (BCRJ, #0125) and were grown
in RPMI 1640 medium (Gibco, #61870-036) supplemented with 10%
FBS (Gibco, #12657029) and penicillin-streptomycin (Gibco,
#15140122) in a humidified atmosphere of 95% air/5% CO2 at
°
37 C.
5-(2,1,3-Benzothiadiazol-4-yl)-1,3-dimethyl-5,11-dihydro-1H-
indeno-[2’,1’:5,6]pyrido[2,3-d]pyrimidine-2,4,6-trione (X)
cGMP Accumulation in Cells
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37
T84 cells were grown to confluence in 12-well plates (Santa Cruz
Biotechnology, #sc-204444) and washed three times with DPBS
(pH 7.4). Cells were treated with vehicle (DPBS with DMSO [Sigma-
Aldrich, #D2650], 0.1%, 0.5 mL per well) or compounds (50 μM) in
DPBS for 10 minutes. Then, cGMP accumulation was stimulated
with enterotoxin of E. Coli (STa, 1 μM [Bachem, #404429]) for
10 minutes. Medium was aspirated and cGMP was extracted with
0.1 M HCl (0.3 mL per well) and measured by ELISA (acetylation
protocol; Cayman Chemical, #581021).
The compound was obtained with the same procedure described
for compound I, by reaction of 1,3-indandione (1, 420 mg,
2.87 mmol), 2,1,3-benzothiadiazol-4-carboxaldehyde (2e, 471 mg,
2.87 mmol) and 6-amino-1,3-dimethyluracil (3a, 359 mg,
2.39 mmol). Yield: 0.82 g, 80%. m.p. 331 C. 1H-NMR (400 MHz,
°
DMSO-d6): δ 10.08 (s, 1H, NH), 7.88 (d, 1H), 7.62 (d, 3H), 7.50 (t, 1H),
7.32 (t, 1H), 7.23 (d, 1H), 5.46 (s, 1H), 3.63 (s, 3H), 3.01 (s, 3H). 13C-
NMR (100 MHz, DMSO-d6): 191.3, 168.1, 161.3, 155.2, 155.0, 153.7,
151.0, 146.00, 140.7, 138.1, 132.7, 132.4, 130.6, 128.9, 121.2, 121.1,
121.0, 119.9, 109.7, 91.8, 32.9, 27.5. HRMS 430.15267. Anal. calcd for
C22H15N5O3S: C 61.53, H 3.52, O 11.18, found: C 61.63, H 3.42, O
11.48.
AMP Accumulation in Cells
Jurkat cells were grown to 5 million cells/mL in 75 cm2 culture
bottle (Santa Cruz Biotechnology, #sc-200263). The cells were
centrifuged at 3000×g for five minutes and washed three times
with DPBS (pH 7.4). Jurkat cells (0.1 mL of cell suspension) were
treated with vehicle (DPBS with DMSO 0.1% [Sigma-Aldrich,
D2650], 0.9 mL per vial) or compounds (50 μM) in DPBS for 10
minutes. Then, cAMP accumulation was stimulated with Forskolin
100 μM (Sigma-Aldrich, #F6886) for 10 minutes. cAMP was extracted
with 0.2 M HCl (0.1 mL per vial) and gentle shaking on an orbital
shaker for 20 minutes at room temperature. After, cells were
centrifuged at 3000×g for five minutes and the supernatant frozen
5-(2,1,3-Benzothiadiazol-5-yl)-1,3-dimethyl-5,11-dihydro-1H-
indeno-[2’,1’:5,6]pyrido[2,3-d]pyrimidine-2,4,6-trione (XI)
The compound was obtained with the same procedure described
for compound I, by reaction of 1,3-indandione (1, 468 mg,
3.20 mmol), 2,1,3-benzothiadiazol-5-carboxaldehyde (2d, 525 mg,
3.2 mmol) and 6-amino-1,3-dimethyluracil (3a, 415 mg, 2.67 mmol).
1
°
Yield: 1.08 g, 94%. m.p. 358 C. H-NMR (400 MHz, DMSO-d6): δ 9.92
(s, 1H, NH), 7.90 (m, 3H), 7.77 (d, 1H), 7.47 (t, 1H), 7.33 (t, 1H), 7.26
(d, 1H), 4.99 (s, 1H), 3.61 (s, 3H), 3.08 (s, 3H).13C-NMR (100 MHz,
DMSO-d6): 194.1, 163.8, 157.7, 157.2, 156.6, 153.7, 150.0, 148.4,
139.1, 138.6, 136.3, 135.0, 134.8, 134.2, 133.2, 126.5, 123.5, 121.7,
112.4, 93.7, 34.1, 30.7. HRMS 430.11667. Anal. calcd for C22H15N5O3S:
C 61.53, H 3.52, O 11.18, found: C 61.73, H 3.22, O 11.28.
°
at À 20 C. cAMP was measured by ELISA (acetylation protocol;
Cayman Chemical, #581001).
Preparation of Rabbit Isolated Aorta
Male rabbits (3 Kg) were anesthetized with ketamine (50 mg/kg),
isoflurane (2%) and exsanguinated. Thoracic aorta was isolated and
placed in cold Krebs solution (pH 7.4). The composition of the Krebs
solution was: NaCl (139.9 mM), KCl (2.7 mM), CaCl2 (1.8 mM); MgSO4
(0.6 mM); NaHCO3 (25 mM), KH2PO4 (0.5 mM) and Glucose
(11.5 mM). The tissue was dissected and cut in aortic rings (3 mm).
Each ring was stretched to 20 mN during 45 minutes for stabiliza-
tion. Following the stabilization period, the absence of endothelium
was evaluated in lack of acetylcholine (1 μM) induce relaxation in
ring pre contracted with phenylephrine (1 μM). After washing, the
rings were pre contracted with a hyperpolarization solution of KCl
(60 mM) and the effects of compounds were evaluated.
5-(2,1,3-Benzothiadiazol-5-yl)-1,3-dimethyl-5,11-dihydro-1H-
indeno-[2’,1’:5,6]pyrido[2,3-d]pyrimidine-2-thioxo-4,6-dione
38 (XII)
39
The compound was obtained with the same procedure described
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
for compound I, by reaction of 1,3-indandione (1, 456 mg,
3.12 mmol), 2,1,3-benzothiadiazol-5-carboxaldehyde (2d, 512 mg,
3.12 mmol) and 6-amino-1,3-dimethyl-2-thioxo-2,3-dihydropyrimi-
din-4(1H)-one (3b, 445 mg, 2.60 mmol). Yield: 1.11 g, 96%. m.p.
1
°
362 C. H-NMR (400 MHz, DMSO-d6): δ 10.15 (s, 1H, NH), 7.96 (m,
3H), 7.80 (d, 1H), 7.48 (t, 1H), 7.34 (t, 1H), 7.27 (d, 1H), 5.05 (s, 1H),
4.11 (s, 3H), 3.52 (s, 3H). 13C-NMR (100 MHz, DMSO-d6): 190.9, 176.4,
159.5, 155.0, 154.3, 153.7, 147.3, 146.6, 136.8, 136.6, 133.7, 132.6,
132.1, 131.0, 130.9, 121.4, 121.3, 120.9, 119.5, 96.0, 35.6, 21.3. HRMS
446.07465. Anal. calcd for C22H15N5O2S2: C 59.31, H 3.39, O 7.18,
found: C 59.21, H 3.29, O 7.28.
MTT Assay (Cell Metabolic Activity)
Using 96-well plates, T84 cells were grown for 48 h according to
the supplier guidelines in the presence of 10% FBS in a humidified
°
atmosphere of 95% air/5% CO2 at 37 C. Medium was aspirated and
replaced by medium protein free (100 μl/well). Compounds (50 μM)
and vehicle (Dulbecco’s PBS with DMSO 0.1% [Sigma-Aldrich,
#D2650]) were incubated for 1 hour. After incubation, medium was
aspirated and replaced by medium protein free (100 μl/well) and
10 μl MTT (Sigma-Aldrich, #M2128) solution (final concentration at
Biological Experiments
Cell Culture
Human colorectal carcinoma cell, or T84 cell, was from the
American Type Culture Collection (CCL248) and were grown in
Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham
°
0.45 mg/mL). Plates were incubated for 2 to 4 hours at 37 C and
medium aspirated and replaced by 100 μl of solubilization solution
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Full Papers
(acidified isopropanol), mixed by 15 minutes and absorbance read
at 570 nm.
An equal amount of molten wax was added using a hot glass pipet.
Samples were mixed and leaved at room temperature overnight.
The wax/xylenes mixture hardened. Samples were transferred to
1
2
°
3
4
5
6
7
8
9
60 C oven in order to melt the wax/xylenes mixture. Vials were
transferred to a heating block and wax/xylenes mixture was
LDH Cytotoxicity Assay
°
replaced. Vials were then incubated for 1 h at 60 C and moved
Cytotoxicity was measured using CaymanChem LHD Assay Kit
(#601170). In summary, T84 cells were grown in a 96-well plate at
the previously determined optimal density in 200 μl of culture
medium. Triton X-100 (10%; 20 μl) was added to wells containing
cells to get maximum release; Assay buffer (20 μl) was added to get
spontaneous release and LDH (20 μl) was added as positive control.
The compounds (50 μM) or its vehicle were added (20 μl) and plate
from oven to heating block again.
Embedding molds were prepared according to manufacturer’s
instructions and filled with molten paraffin wax using a hot glass
pipet. Sample were immediately transferred to the wax-filled mold
using a hot cut-off Pasteur pipet. An embedding ring was placed
on the mold and fill with paraffin wax. Cast blocks were leaved at
room temperature to harden completely and stored in a dry place
at room temperature.
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°
incubated in a CO2 incubator at 37 C. Following, plates were
centrifuged at 400×g for five minutes and 100 μl of cell super-
natant was transferred to a new 96-well assay plate where 100 μl of
reaction solution was added to each well. Plates were incubated
with gentle shaking on an orbital shaker for 30 minutes at room
temperature and the absorbance read at 490 nm.
A wax block containing samples was cut into a trapezoidal shape
using a razor blade. The extra wax was shaved off and the trapezoid
block was attached to the holding clamp of a microtome in order
to be sectioned, cutting at 8-μm sections. A drop of 0.2×gelatin
was putted subbing solution on a gelatin-subbed glass slid and the
ribbon of sections were then transferred onto the drop on the
subbed slide using fine brushes. The slides were transferred onto a
slide warmer or heating plate set between 45 and 50 C. When
stretching was complete slides were removed from the warmer and
the remaining subbing solution was carefully removed using a
Pasteur pipet. Slides were dried at room temperature and
Rabbit Ileal Loops
Six New Zealand white rabbits weighing 2 kg were fasted over-
night. The rabbits were anesthetized with ketamine (60–80 mg/kg)
and xylazine (5–10 mg/kg), administered intramuscularly, and a
midline abdominal incision was made to expose the small bowel.
After that, 6–8 loops of 3 cm each were ligated using double ties,
and a 1-cm interval was left between loops.
°
°
°
incubated for 2 days at 42 C to firmly attach sections to subbed
slides. Sections have been stored in a slide box with desiccant at
À 20 C.
°
Each of the control loops was injected intraluminal with a 1-mL
solution of PBS with either E. coli heat-stable toxin (STa; Sigma-
Aldrich). Immediately before the enterotoxin was administered,
loops were treated with Compound VI or FPIPP were diluted in PBS
to concentrations of 1 mg/Kg. The ileal loops were replaced
intraperitoneally, and the abdominal incision was sutured closed.
The rabbits were maintained under anesthesia until they were
euthanized 4 h later.
The cryosections slides have successively been immersed in H2O for
30 seconds with agitation by hand. The slides have been dipped
into a Coplin jar containing Mayer’s hematoxylin and agitated for
30 seconds. Then the slide have been stained with 1% eosin Y
solution for 10–30 seconds with agitation. The sections have been
dehydrated with two changes of 95% alcohol and two changes of
100% alcohol for 30 seconds each. The alcohol was extracted with
two changes of xylene and finally one or two drops of mounting
medium were added, before to cover with a coverslip.
Measurement of Intestinal Secretion
After 4 h of incubation, the ligated ileal loops were removed. The
length of each ligated ileal segment was measured. Intraluminal
fluid was extracted from each ileal loop and quantified. The volume:
length ratio (V:L) was calculated in milliliters per centimeter per
loop. The gross description (clear, serous, purulent, or bloody) of
the collected fluid was also noted.
Statistical Analysis
The Kolmogorov-Smirnov test was applied to determine the normal
distribution of the data (p>0.05). Data were analyzed using one-
way ANOVA complemented by the Tukey post hoc test. The results
are expressed as mean+SEM. Statistically significant differences
were set at p<0.05. All experiments were repeated at least three
times in duplicate.
Tissue Fixation and Histologic Coloration
In order to fix the samples, dissected ileum tissues have been
placed in labeled 20 mL snap-cap glass vials. Vials have been filled
with freshly prepared 4% PFA fixative, 4 C. Fixation was allowed to
°
Acknowledgment
°
proceed at 4 C for 24 h.
T. Z. is thankful to Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP) for financial support (process 2013/15525-1).
All the procedures were conducted in accordance with Institu-
tional guidelines, approved by Ethical Principles in Animal
Research by College for Animal Experimentation (COBEA) and
local Ethics Committee (State University of Campinas) for the Use
of Experimental Animals (CEUA #4667-1/2017).
After fixation was completed, fixative solution was replaced with
50% ethanol. Ethanol was substituted with fresh one. Tissues were
incubated for 20 min and then 50% ethanol was replaced again. A
total of three changes of ethanol, each followed by a 20 min
incubation at room temperature, was done in this way. Dehydration
was continued by incubating the samples three times in 70%
ethanol, 20 min each time (room temperature). Successively,
samples were incubated in 95% ethanol for 20 min at room
temperature for three times. Finally, incubation in 100% ethanol for
20 min at room temperature was performed three times. Ethanol
was replaced by xylene and incubation for 10 min was repeated for
three times (each with fresh xylene).
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Full Papers
M. Fitzgerald, M. Gopalakrishnan, R. J. Greg, R. F. Henry, M. W. Holladay,
L. L. King, M. E. Kort, P. R. Kym, I. Milicic, R. Tang, S. C. Turner, K. L.
Whiteaker, L. Yi, H. Zhang, J. P. Sullivan, J. Med. Chem., 2004, 47, 3163–
3179.
Conflict of interest
1
2
3
The authors declare no conflict of interest.
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Keywords: pyridopyrimidines · diarrhea · cyclic nucleotides ·
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Manuscript received: February 13, 2019
Revised manuscript received: March 12, 2019
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