Synthesis and Pharmacological Screening of ...

Sep. 23, 2024

Synthesis and Pharmacological Screening of ...

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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&#;bistrifluoromethylphenyl)&#;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&#;dimethylthiazol&#;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.

Here it is reported the design, synthesis and the pharmacological in vitro effect of twelve novel pyridopyrimidine derivatives ( I &#; XII , Table ) on cyclic nucleotides accumulation. The 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.

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 ) 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 ) suppress the synthesis of cGMP when in presence of STa and other toxins in vitro. 11 , 12

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 , 2 , 3 , 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 guanylate cyclase type C (GC&#;C) in intestinal epithelial cells, stimulating the synthesis of 3&#;&#;5&#; cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). 6 , 7 , 8 , 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

2.&#;Results and Discussion

2.1. Synthesis of Pyridopyrimidine Derivatives

Compounds I&#;XII were prepared following, with modifications, the synthetic procedures13 depicted in Scheme&#; . The synthetic procedure is based on the Hantzsch dihydropyridine three component cyclization.14,15 In particular, reaction of 1,3&#;indandione (1) with the aldehydes (2&#; a&#;g) and the opportune primary amines (3&#; a&#;c) in acetic acid afforded the desired compounds I&#;XII. Aldehydes 2&#; a&#;2&#; c were commercially available, while 2&#; d&#;2&#; g were synthesized as previously described.16, 17, 18, 19 Intermediate 3&#; a was commercially available, while 3&#; b and 3&#; c were prepared according to that reported in Scheme&#; following, with modifications, procedures previously reported in literature.20

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Conventional heating (oil bath) and microwave irradiation of the reactions were compared (Table&#; ).

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 3&#; b and 3&#; c 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 3&#; b and 3&#; c) and from 8&#;h to 40&#;min for final compounds I&#;XII.

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

The synthesized compounds were tested in&#;vitro in order to evaluate their effects on cyclic nucleotide accumulation. Compound activity was determined measuring the concentration 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&#; .

Table 2

CompdStructurecGMP [%]cAMP [%]CompdStructurecGMP [%]cAMP [%]A (BPIPP) 86±4n.a.B (FPIPP) 93±4n.a.I 69±384±3VII 17±737±4II 70±582±3VIII 49±445±6III 51±388±3IX 25±935±6IV 78±486±3X 33±845±4V 51±486±3XI 71±445±2VI 94±269±2XII 65±480±2Open in a separate window

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&#; ). 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&#; ). A full concentration&#;dependent curve (Figure&#; ) was performed for the most active compounds (I, II, IV, VI and XI).

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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. 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).

Finally compound VI, showing the highest effect on cGMP accumulation, has a CLogP of 5.11. This value is the highest of the series even when compared to FPIPP, that shows a cLogP of 4.58.

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 stimulation of Cl&#; and HCO3 &#; secretion (by opening of the apical CFTR Cl&#; 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.

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, 23, 24, 25, 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, 26, 27 could inhibit its activity.

PDE5 that is highly expressed in T84 cells28 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 compounds still were able to block the cGMP accumulation, similarly to results described for BPIPP.11

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 changes on cAMP or cGMP levels can promote significant physiological effects due to the importance of these intracellular 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 importance. 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&#; ). Interestingly, only FPIPP (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. Moreover, 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.

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2.4. Cell Metabolic Activity (MTT) and Cytotoxicity (LDH)

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 compounds 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 interference in cells during the assay of cGMP stimulation (Table&#; ).

Table 3

&#;1&#;h24&#;h48&#;hTreatmentMean [%]S.E.MMean [%]S.E.MMean [%]S.E.MVehicle100.0&#;100.0&#;100.0&#;Triton (0.1&#;%)54.9*1.145.2*1.238.4*1.3STa (1&#;μM)96.81.294.91.090.51.2FPIPP (50&#;μM)98.21.396.41.193.31.9 I 91.81.185.72.684.55.5 II 90.82.495.72.396.03.4 III 101.11.693.23.786.23.4 IV 99.32.594.41.693.45.1 V 101.33.392.14.088.83.7 VI 96.22.295.21.891.93.0 VII 91.71.190.12.789.43.9 VIII 90.51.493.82.689.61.6 IX 90.61.494.12.795.24.7 X 91.43.796.63.892.83.5 XI 92.71.094.63.390.64.0 XII 90.61.696.01.886.52.4Open in a separate window

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&#; ) 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 line with those of nontoxic compounds and are moreover similar to the results obtained with BPIPP.11

Table 4

Treatment1&#;h24&#;h48&#;hMean [%]S.E.MMean [%]S.E.MMean [%]S.E.MVehicle (DMSO 0.1&#;%)1.91.01.60.52.20.4Positive control (LDH)61.4&#;%* 1.565.8* 3.965.5* 2.7STa (1&#;μM)2.30.52.80.43.71.1FPIPP (50&#;μM)2.90.34.20.89.31.5 I 2.10.22.71.112.5* 0.7 II 2.20.56.11.59.21.1 III 1.30.37.8* 0.911.3* 0.8 IV 3.20.55.61.311.7* 1.4 V 1.70.58.6* 1.813.5* 0.4 VI 3.50.66.11.211.8* 0.3 VII 1.60.12.10.311.1* 2.4 VIII 2.30.12.90.213.3* 0.2 IX 1.50.61.10.413.5* 0.9 X 2.21.62.30.310.8* 1.5 XI 4.01.93.30.911.5* 0.3 XII 3.20.84.40.411.3* 2.4Open in a separate window

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 abdominal 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&#; 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.

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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 be characterized as a hyperplasia of intestinal mucosa associated 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, evidencing 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. Histopathological Analysis of Intestinal Tissue

The microscopic analysis of stained slides was made in a Nikon binocular microscope (E200). First, slides were placed in microscope 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 throughout 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;

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.

The obtained images are reported in Figure&#; .

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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:

2.7. No Treatment with STa Toxin (Vehicle only)

Histopathological evaluation (Figure&#; A) 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 significant pathological changes, with an inflammatory score of 0 (preserved tissue absent from inflammatory cellularity and necrosis).

2.8. Treatment with STa Toxin and Post&#;Treatment with Vehicle

Histopathological evaluation (Figure&#; B) 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 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.

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).

2.9. Pre&#;Treatment with Compound VI and Post&#;Treatment with STa Toxin

Histopathological evaluation (Figure&#; C) reveals preserved tissue architecture, highlighting mild mucosal hyperplasia, associated 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, plasmocytes 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 inflammatory score of 2 (moderate tissue disorganization with sketches of necrosis and/or discrete inflammatory infiltrate aggregate).

2.10. Pre&#;Treatment with FPIPP and Post&#;Treatment with STa Toxin

Histopathological evaluation (Figure&#; D) 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 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.

2.11. 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:

2.12. 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.

2.13. Methyl Alcohol Solution

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 temperature, 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 temperature 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. 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., 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.

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