| Summary, etc. |
Chronic obstructive pulmonary disease (COPD) is a progressive disease, meaning that over time it typically worsens, and it is characterized by long-term breathing problems and poor airflow, shortness of breath and a sputum-producing cough. Many inflammatory cells and mediators participate in COPD symptoms such as bronchitis and emphysema, so antagonizing their receptors become a challenge for a medicinal chemist to find new chemical entities (NCEs) that play this role. Many treatments had been developed for COPD as inhaled corticosteroids, inhibitors of inducible nitric oxide synthase(i-NOS), leukotriene inhibitors, adhesion-molecule blockers, chemokine inhibitors, TNF-α inhibitors, NFκB inhibitors, p38 MAPK inhibitors, Phosphoinositide-3-kinase (PI3K) inhibitors and Peroxisome proliferator-activated receptors (PPARs) activators. <br/><br/>The wide distribution of phosphodiestrase-4 (PDE-4) enzymes in inflammatory cells and in the lung in which PDE-4 inactivates 3',5'-cyclic adenosine monophosphate (cAMP) which is highly expressed in inflammatory cells, and so, inhibitors of PDE-4 enzyme cause a decrease in inflammatory response which is increased in COPD. Therefore, selective phosphodiestrase-4 (PDE-4) inhibitor treatment has been a very effective treatment strategy. PDE-4 enzymes are classified into 4 subtypes (4 A-D) which show a similarity of 78% in the active catalytic site. It has been revealed that selective inhibition of PDE-4B showed a potent anti-inflammatory effect not associated with emesis side effect accompanied with the inhibition of PDE-4D enzyme.<br/><br/>Roflumilast was marketed in the USA in 2012 for the treatment of COPD as a PDE-4 inhibitor. Though, its side effects, such as gastrointestinal upsets, headache, and weight loss limited its therapeutic potential. Roflumilast is rapidly metabolized to its active metabolite, roflumilast N-oxide, which has twofold to threefold specificity and potency less than roflumilast. The main cytochrome P450 enzymes that are responsible for the conversion of roflumilast to its N-oxide metabolite are CYP3A4 and 1A2. As the activity ofthese two enzymes can be affected by interindividual variables, so it is can be predicted that these variables can affect roflumilast pharmacokinetic.<br/><br/> A phosphodiesterase (PDE) is an enzyme that breaks a phosphodiester bond in cAMP and cGMP. Phosphodiesterase-4 (PDE-4) is the primary enzyme that regulates the turnover of cAMP.Phosphodiestrase-4 (PDE-4) enzyme is important target in Chronic Obstructive Pulmonary Disease (COPD).Notably, PDE-4D is expressed in the chemo-sensitive trigger zone in the brainstem, and it appears to be particularly significant in nausea and vomiting. This isoenzyme appears to be less important for anti-inflammatory effects. Therefore, a selective (PDE-4) inhibitor has been identified as a very effective treatment strategyagainst inflammation. It has been revealed that selective inhibition of PDE-4B showed a potent anti-inflammatory effect without emesis as a side effect accompanied by the inhibition of the PDE-4D enzyme. <br/><br/>In the present work, we designed and synthesized new roflumilast analogues with preferential-selective PDE-4B inhibition activity with good potency. To accomplish this target; synthesis of novel series (IVa-u), (Va-i), (VIa-f), (VII), (VIIIa-i), (IX), (X) was done, aiming at obtaining new PDE-4B inhibitors hits based on the proposed pharmacophore, 1-(cyclopropylmethoxy)-2-(difluoromethoxy) benzene moiety. Forty-nine new compounds were prepared via different synthetic routes. The purity of the synthesized compounds was monitored by TLC, and detected by UV chamber. The structures of the new compounds were elucidated via microanalysis, 1H NMR, 13C NMR, and IR spectroscopy. <br/><br/>Enzyme assay was used to measure the IC50 values for the PDE-4B inhibition of all the synthesized compounds along with roflumilast as a reference drug. The biology results demonstrated that most of the examined candidates exhibited considerable inhibitory activity against PDE-4B. The six compounds (IVg, IVl, IVm, IVs, VIIIh, VIIIi) exhibited the highest potency against PDE-4B enzyme (IC50 = 7.15, 5.50, 7.19, 7.25, 7.42, 8.29 nM, respectively) with no significant inhibition difference from roflumilast (no statistical difference at p< 0.05). Compound IVl was found to be the most potent compound against PDE-4B enzyme in all series with 3-OH and 4-OCH3 substituents (IC50 = 5.50 nM) showing no significant difference with that of roflumilast (IC50 = 2.36 nM).The most potent derivatives were further tested for PDE-4D inhibition activity to investigate their PDE-4B/PDE-4D selectivity ratio.The most selective compounds are IVg, VIIIi and VIIIh.<br/>Compound IVg showed the highest selectivity towards PDE-4B isozyme more than the reference compound roflumilast, (PDE-4D/4B IC50 Ratio for compound IVg = 3.22, and for roflumilast = 3.02).The most potent compounds (IVg, IVl, IVm, IVs, VIIIh, VIIIi) were subjected to further investigation, and their effects on cAMP level and percentage of inhibition of Tumor necrosis factor-alpha (TNF-α) were studied and compared with reference compound roflumilast. Compound IVm, was superior as it showed the highest increase in the level of intracellular cAMP (6.55±0.37 pmole/mL) but still lower than the reference compound (8.40±0.79 pmole/mL). Compound IVs showed the highest % of TNF-α inhibition (77.22%) but still lower than the reference compoundroflumilast (81.58%).<br/><br/>This work was supported by the docking studies at the active site of the PDE-4B enzyme for the most potent compounds (IVg, IVl, IVm, IVs, VIIIh, VIIIi) showing good potency against PDE-4B. Simulation docking investigations were carried out to gain insight into the potential binding mechanism of the newly synthesized compounds in the PDE-4B binding site. The docking results were in good accordance with the biological screening results. All of the most potent compounds against PDE-4B showed higher docking scores (S) than roflumilast.<br/>The thesis contains: <br/>1) Introduction: <br/>This part includes a brief literature review on the different classes of COPD drugs according to their mechanism of action, PDE-4 inhibitors, and selective PDE-4B inhibitors.<br/>2) Aim of the work (Research Objectives): <br/>This part presents the aim and the rational upon which the designed compounds were synthesized.<br/>3) Theoretical discussion: <br/>It deals with the theoretical discussion of the experimental synthetic methods adopted for the synthesis of the designed compounds as well as the different analytical methods applied for the characterization and confirmation of the new compounds. Three synthetic schemes were provided to illustrate the adopted synthetic pathways.This part also includes the screening of the newly synthesized compounds for their PDE-4B inhibitory activity using roflumilast as a reference compound. The most potent derivatives were further tested for PDE-4D inhibition activity to investigate their PDE-4B/PDE-4D selectivity ratio. <br/>The level of intracellular cAMP and the percentage of inhibition of the inflammatory mediator TNF-α were also measured for additional biological evaluation. Also, it involves the results of molecular docking of compounds showing remarkable PDE-4B inhibitory activity (IVg, IVl, IVm, IVs, VIIIh, VIIIi) to study their binding mode to the active site of the enzyme in comparison to roflumilast as reference drug. <br/>4) Experimental work: <br/> In this part, the practical procedures used for the synthesis of the known starting, new intermediate, and new final compounds were described, in addition, their physical, spectral, and microanalytical data were cited. This part also includes the methods used for biological measurements and molecular docking.<br/>The following compounds were prepared:<br/> I- Reported compounds: (1 compound)<br/>• Methyl 3-(cyclopropyl methoxy)-4-(difluoromethoxy) benzoate II.<br/> II- New compounds:<br/> IIa- New intermediate:(1 compound)<br/>• 3-(Cyclopropyl methoxy)-4-(difluoromethoxy) benzohydrazide III.<br/> IIb- New target compounds:(48 compounds)<br/>1. N'-Benzylidene-3-(cyclopropylmethoxy)-4-(difluoromethoxy)benzohydrazide IVa.<br/>2. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(4-fluorobenzylidene)benzohydrazide IVb. <br/>3. N'-(4-Chlorobenzylidene)-3-(cyclopropylmethoxy)-4-(difluoromethoxy)benzohydrazide IVc. <br/>4. N'-(3-Bromobenzylidene)-3-(cyclopropylmethoxy)-4-(difluoromethoxy)benzohydrazide IVd. <br/>5. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(4-methoxybenzylidene) benzohydrazide IVe. <br/>6. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(4-methylbenzylidene)benzohydrazide IVf.<br/>7. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(4-nitrobenzylidene)benzohydrazide IVg. <br/>8. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(3-methoxybenzylidene) benzohydrazide IVh. <br/>9. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(3,4-dimethoxybenzylidene) benzohydrazide IVi. <br/>10. 3-(Cyclopropylmethoxy)-N'-(2,6-dichlorobenzylidene)-4-(difluoromethoxy) benzohydrazide IVj. <br/>11. 3-(Cyclopropylmethoxy)-N'-(3,4-dichlorobenzylidene)-4-(difluoromethoxy) benzohydrazide IVk. <br/>12. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(3-hydroxy-4-methoxybenzylidene) benzohydrazide IVl. <br/>13. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(4-hydroxy-3-methoxybenzylidene) benzohydrazide IVm. <br/>14. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(furan-2-ylmethylene)benzohydrazide IVn. <br/>15. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(3-phenylallylidene)benzohydrazide IVo. <br/>16. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(3-nitrobenzylidene)benzohydrazide IVp. <br/>17. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(2-(trifluoromethyl)benzylidene) benzohydrazide IVq. <br/>18. N'-(2-Chlorobenzylidene)-3-(cyclopropylmethoxy)-4-(difluoromethoxy)benzohydrazide IVr.<br/>19. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(2-nitrobenzylidene)benzohydrazide IVs. <br/>20. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(4-(dimethylamino)benzylidene) benzohydrazide IVt. <br/>21. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(2-hydroxybenzylidene) benzohydrazide IVu.<br/>22. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(1-phenylethylidene)benzohydrazide Va.<br/>23. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(1-(4-fluorophenyl)ethylidene) benzohydrazide Vb.<br/>24. N'-(1-(4-Chlorophenyl)ethylidene)-3-(cyclopropylmethoxy)-4-(difluoromethoxy) benzohydrazide Vc.<br/>25. N'-(1-(4-Bromophenyl)ethylidene)-3-(cyclopropylmethoxy)-4-(difluoromethoxy) benzohydrazide Vd.<br/>26. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(1-(4-methoxyphenyl)ethylidene) benzohydrazide Ve.<br/>27. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(1-(p-tolyl)ethylidene)benzohydrazide Vf.<br/>28. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(1-(4-nitrophenyl)ethylidene) benzohydrazide Vg.<br/>29. N'-(1-(3-Aminophenyl)ethylidene)-3-(cyclopropylmethoxy)-4-(difluoromethoxy) benzohydrazide Vh. <br/>30. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(1-(3,4-dimethoxyphenyl) ethylidene)benzohydrazide Vi. <br/>31. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(2-oxoindolin-3-ylidene) benzohydrazide VIa.<br/>32. N'-(5-Bromo-2-oxoindolin-3-ylidene)-3-(cyclopropylmethoxy)-4-(difluoromethoxy) benzohydrazide VIb. <br/>33. N'-(5-Chloro-2-oxoindolin-3-ylidene)-3-(cyclopropylmethoxy)-4-(difluoromethoxy) benzohydrazide VIc.<br/>34. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(5-nitro-2-oxoindolin-3-ylidene) benzohydrazide VId.<br/>35. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(5-methoxy-2-oxoindolin-3-ylidene)benzohydrazide VIe. <br/>36. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(1-methyl-2-oxoindolin-3-ylidene) benzohydrazide VIf.<br/>37. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N-(4,5,6,7-tetrachloro-1,3-dioxo isoindolin-2-yl)benzamide VII.<br/>38. N'-Benzoyl-3-(cyclopropylmethoxy)-4-(difluoromethoxy)benzohydrazideVIIIa.<br/><br/>39. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(4-fluorobenzoyl)benzohydrazide VIIIb.<br/><br/>40. N'-(4-Chlorobenzoyl)-3-(cyclopropylmethoxy)-4-(difluoromethoxy)benzohydrazide VIIIc.<br/><br/>41. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(4-iodobenzoyl)benzohydrazide VIIId.<br/><br/>42. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(4-methylbenzoyl)benzohydrazide VIIIe.<br/><br/>43. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(4-nitrobenzoyl)benzohydrazide VIIIf. <br/><br/>44. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(4-methoxybenzoyl)benzohydrazide VIIIg.<br/><br/>45. N'-(3-(Cyclopropylmethoxy)-4-(difluoromethoxy)benzoyl)-3,4,5-trimethoxy benzohydrazide VIIIh.<br/><br/>46. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N'-(3,5-dinitrobenzoyl)benzohydrazide VIIIi.<br/><br/>47. N'-Cyclohexylidene-3-(cyclopropylmethoxy)-4-(difluoromethoxy)benzohydrazide IX.<br/><br/>48. 3-(Cyclopropylmethoxy)-4-(difluoromethoxy)-N-(2,5-dimethyl-1H-pyrrol-1-yl) benzamide X.<br/><br/>• The final target synthesized derivatives (IVa-u), (VIa-i), (VIa-f), (VII), (VIIIa-i), (IX), (X) are listed in Scheme (1), Scheme (2), Scheme (3).<br/>5) References: <br/>This part comprises two hundred ninety-six references stated in the thesis. <br/>6) Arabic summary.<br/> |
