Design and Synthesis of N-Arylphthalimides as Inhibitors of Glucocorticoid-Induced TNF Receptor-Related Protein, Proinflammatory Mediators, and Cytokines in Carrageenan-Induced Lung Inflammation

Article abstract of DOI:10.1021/acs.jmedchem.5b00934

N-Arylphthalimides (1-10P) derived from thalidomide by insertion of hydrophobic groups were evaluated for anti-inflammatory activity, and (4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N′-[(4-ethoxyphenyl)methylidene]benzohydrazide 6P was identified as a promising anti-inflammatory agent. Further testing confirmed that compared with the control, 6P treatment resulted in a considerable decrease in CD4⁺, NF-κB p65⁺, TNF-α⁺, IL-6⁺, GITR⁺, and IL-17⁺ cell populations and an increase in the Foxp3⁺, CD4⁺Foxp3⁺, and IκBα⁺ populations in whole blood and pleural fluid of a mouse model of lung inflammation. Moreover, treatment with compound 6P decreased the proteins associated with inflammation including TNF-α, IL-6, IL-17, GITR, NF-κB, COX-2, STAT-3, and iNOS and increased the anti-inflammatory mediators such as IL-10 and IL-4. Further, histopathological examination confirmed the potent anti-inflammatory effects of compound 6P. Thus, the N-arylphthalimide derivative 6P acts as a potent anti-inflammatory agent in the carrageenan-induced lung inflammation model, suggesting that this compound may be useful for the treatment of inflammation in a clinical setting.

Full text of DOI:10.1021/acs.jmedchem.5b00934

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Article
Design and synthesis of N-aryl-phthalimides as inhibitors of
glucocorticoid- induced TNF receptor-related protein, pro-inflammatory
mediators, and cytokines in carrageenan-induced lung inflammation
Mashooq Ahmad Bhat, Mohamed A Al-Omar, Mushtaq A Ansari, Khayry M
Zuheir, Faisal Imam, Sabry M Attia, Saleh A Al-Bakheet, Ahmed Nadeem,
Hesham Korashy, Andrew Voronkov, Vladimir Berishvili, and Sheikh Ahmad
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00934 • Publication Date (Web): 10 Oct 2015
Downloaded from http://pubs.acs.org on October 12, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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of their duties.

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Graphical Abstract
Design and synthesis of N-aryl-phthalimides as inhibitors of glucocorticoid-
induced TNF receptor-related protein, pro-inflammatory mediators and
cytokines in carrageenan-induced lung inflammation
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Mashooq A. Bhat*, Mohamed A. Al-Omar, Mushtaq A. Ansari, Khairy M. A. Zoheir, Faisal
Imam, Sabry M. Attia, Saleh A. Bakheet, Ahmed Nadeem, Hesham M. Korashy, Andrey
Voronkov, Vladimir Berishvili, Sheikh F. Ahmad*
CH3
O
O
NH N
N
O
O
6
P
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Design and synthesis of N-aryl-phthalimides as inhibitors of glucocorticoid-
induced TNF receptor-related protein, pro-inflammatory mediators and
cytokines in carrageenan-induced lung inflammation
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a,♯,
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Mashooq A. Bhat *, Mohamed A. Al-Omar , Mushtaq A. Ansari , Khairy M. A. Zoheir , Faisal
b
b
b
b
b
Imam , Sabry M. Attia , Saleh A. Bakheet , Ahmed Nadeem , Hesham M. Korashy , Andrey
Voronkovc,d,e, Vladimir Berishvili , Sheikh F. Ahmad
c
b,♯,
*
aDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh
Kingdom of Saudi Arabia
bDepartment of Pharmacology and Toxicology, College of Pharmacy, King Saud University,
Riyadh, Kingdom of Saudi Arabia
cDepartment of Chemistry, Lomonosov Moscow State University, Leninskie Gory,
1
/3, Moscow 119991, Russia
dDigital BioPharm ltd., 145-157 St. John Street, London, UKEC1V 4PW
eMoscow Institute of Physics and Technology (State University), 9 Institutskiy Lane,
Dolgoprudny City, Moscow Region 141700, Russia
Author information
*
Corresponding authors
M. A. Bhat: Phone +966-558164097; E-mail: mabhat@ksu.edu.sa
*
*S. F. Ahmad: +966-569496770; E-mail: fashaikh@ksu.edu.sa
Author contributions
♯M. A. Bhat and S. F. Ahmad contributed equally to this work.
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Abstract
N-aryl phthalimides (1-10P) derived from thalidomide by insertion of hydrophobic groups were
evaluated for anti-inflammatory activity and compound (4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-
yl)-N'-[(4-ethoxyphenyl)methylidene]benzohydrazide 6P was identified as a promising anti-
inflammatory agent. Further testing confirmed that compared with the control, 6P treatment
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resulted in a considerable decrease in CD4 , NF-κB p65 , TNF-α , IL-6 , GITR and IL-17 cell
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populations and an increase in the Foxp3 , CD4 Foxp3 and IκBα populations in whole blood
and pleural fluid of a mouse model of lung inflammation. Moreover, treatment with the compound
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P decreased the proteins associated with inflammation including TNF-α, IL-6, IL-17, GITR, NF-
κB, COX-2, STAT-3, and iNOS and increased the anti-inflammatory mediators such as IL-10 and
IL-4. Further, histopathological examination confirmed the potent anti-inflammatory effects of the
compound 6P. Thus, the N-aryl phthalimide derivative 6P acts as a potent anti-inflammatory agent
in the carrageenan-induced lung inflammation model suggesting that this compound may be useful
for the treatment of inflammation in a clinical setting.
Keywords: Phthalimide derivatives, Anti-inflammatory activity, Carrageenan induced lung
inflammation; inflammatory mediators; Pleural exudate; Lung tissue.
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Introduction
Thalidomide (α-N-phthalimido glutarimide) and its derivatives are known to have
immunomodulatory and anti-inflammatory activities and represent potential preventive therapies
for autoimmune diseases. Initially introduced in 1954 as a sedative, thalidomide was later
withdrawn from the market due to its teratogenic effects. Nevertheless, thalidomide possesses
various pharmacological properties and has since been approved and used successfully for
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treatment of inflammatory and autoimmune diseases including erythema nodosum leprosum and
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multiple myeloma. Thalidomide or its analogs have shown their effect in the treatment of
rheumatoid arthritis, Crohn’s disease, prostate cancer, Behcet’s disease, chronic host-versus-graft
disease, lupus erythematosus, and HIV-associated oral ulcers.3-5 In addition, previous studies have
confirmed that thalidomide exerts its anti-inflammatory effects by suppressing the production of
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pro-inflammatory molecules including TNF-α, IL-1β, IL-6, and NO. The benefits of thalidomide
administration in patients include relief of peripheral pain and inflammation, which have been
confirmed in several animal models as well.8-9 However, the teratogenicity, peripheral neuropathy,
and other adverse effects of thalidomide have prompted the design of novel analogs that exhibit
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low toxicity and enhanced potency in blocking cytokine production. Still, few groups have
investigated structural modification of thalidomide as a means to obtain potent inhibitors of TNF-
α and inflammation. Replacement of the glutarimide moiety of thalidomide with a methyl (3,4-
dimethoxyphenyl) propionate or 2,6-diisopropylphenyl moiety, having fluorine or amino
substitution in the homocycle of thalidomide, have produced analogs with enhanced tumor
necrosis factor inhibitor activity.11-12
This manuscript describes the design, synthesis, and evaluation of the anti-inflammatory
effects of N-arylphthalimide derivatives (1-10P). The rationale underlying the design of new
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derivatives (1-10P) was to obtain more lipophilic phthalimide derivatives with potent anti-
inflammatory activities. To this end, N-arylphthalimide derivatives, which are based on a
thalidomide structural analogue (LASSBio 595), were attained by modulating the thiomorpholine
moiety with hydrophobic groups in order to increase the lipophilicity of the targeted compounds
(Figure 1).
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Carrageenan (Cg)-induced pleurisy is a well-established model of acute inflammation
characterized by a rapid influx of polymorphonuclear leukocytes (PMLs) followed by
mononuclear cell infiltration into the lungs.13-14 The onset of Cg-induced acute inflammation has
been linked to neutrophil infiltration and the production of neutrophil-derived free radicals such
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as hydrogen peroxide, superoxide, and hydroxyl radicals. Moreover, this model is often used to
assess the anti-inflammatory effects of pharmaceutical agents and the in vivo importance of
established inflammatory mediators.16-17
In the present study, we hypothesized that N-arylphthalimide derivatives (1-10P) may
affect Cg-induced lung inflammation due to anti-inflammatory and immunosuppressive activities
of phthalimide pharmacophore. Consequently, we decided to investigate whether treatment with
N-arylphthalimide derivatives could prevent the development of Cg-induced lung inflammation in
mice. Thus, we first investigated the effects of compounds (1-10P) on the malondialdehyde
(MDA) levels, and myeloperoxidase (MPO) and glutathione (GSH) activities, as these are all
markers of the inflammatory process. Further, more detailed studies were carried out on one of the
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active compounds (6P). The presence of CD4 , NF-κB p65 , TNF-α , IL-6 , GITR , IL-17 ,
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Forkhead box P3 (Foxp3 ), CD4 Foxp3 , and IκBα expressing cells in pleural exudates and in
heparinized blood were assessed using flow cytometry. Moreover, the levels of cytokines produced
by Th1 and Th2 cells were analyzed using enzyme-linked immunosorbent assay (ELISA) of
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pleural fluid following intrapleural Cg administration. We also evaluated TNF-α, IL-17, NF-κB,
COX-2, STAT-3, iNOS, and IL-10 mRNA expression in lung tissue using RT-PCR, and GITR,
COX-2, STAT-3, NF-κB p65, and IκBα protein expression in lung tissues using western blotting.
Histopathological examination of lung tissues was also performed.
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2. Results and discussion
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.1. Chemistry. 4-Amino benzoic acid hydrazide (1) was prepared by the reaction of 4-
aminobenzoate (benzocaine) with hydrazine hydrate in absolute ethanol. 4-Amino-N'-[substituted
phenylmethylidene]benzohydrazide (2a-j) were synthesized by the condensation of 1 with
different substituted benzaldehydes in the presence of ethanol and a few drops of glacial acetic
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acid. The resulting 4-Amino-N'-[substituted phenylmethylidene]benzohydrazide (2a-j) were
chosen as starting compounds to design several N-arylphthalimide derivatives (1-10P). Target
compounds,
phenylmethylidene]benzohydrazide (1-10P), were synthesized by the reaction of 4-amino-N'-
substituted phenylmethylidene]benzohydrazide (2a-j) with phthalic anhydride in glacial acetic
4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N'-[substituted
[
1
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acid with yields between 58-80% (Scheme 1). The final compounds were characterized by FT
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IR, H NMR, C NMR, and mass spectroscopy. The IR spectrum revealed bands for CONH,
-1
1
N=CH, C=O groups at 3459-3258, 3065-2836 and 1740-1686 cm , respectively. The H NMR
spectra of (1-10P) confirms the presence of Ar-H, CONH, and N=CH protons by showing signals
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at δ: 7.0-8.5, 11.4-12.2 and 8.3-10.8 ppm, respectively. Also, the C NMR of the compounds
1-10P) were found to be in agreement with their chemical structures. Additionally, all the
(
compounds gave corresponding molecular ion peaks in mass spectra as determined by the triple
quadrupole LC/MS.
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.2. Effect on general behavior and acute toxicity in mice. Compound 6P was administered by
oral gavage in a single dose of 2000 mg/kg. The animals were observed for any toxic symptoms
and death over a period of 7 days. The animals tolerated this dose without any toxic symptoms.
The animals became sluggish after treatment but recovered within 2 h of the treatment, which
confirmed the absorption of the test compound. We did not observe any signs of toxicity or death
during this period. These results indicate that compound 6P exhibits no toxicity and is well
tolerated by experimental animals at the tested dose.
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2.3.Effect of compounds (1-10P) on biochemical analysis. Our preliminary study investigated
the effect of compounds (1-10P) on oxidative parameters during the acute phase of inflammation.
We measured the level of malondialdehyde (MDA) in lung tissue from normal control (NC) mice,
carrageenan (Cg)-injected mice, and Cg-injected mice pre-treated with compounds (1-10P) at 10,
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0 and 40 mg/kg, i.p. As shown in Table 1, injection of mice with Cg resulted in an increase in
MDA as compared to mice in the NC group. In contrast, pre-treatment of Cg-injected mice with
compounds (1, 3-7, 9, and 10P) resulted in a significant reduction in lung MDA, with the most
significant effect observed for compound 6P at 20 and 40 mg/kg P<0.01). Moreover, a minimum
significant (P<0.05) reduction in MDA was also observed at 10 mg/kg. MDA, a lipid peroxidation
biomarker, leads to activation and formation of free radicals by pulmonary endothelial cells and
neutrophils, increased production of adhesion molecules, and generation of cytokines and
chemokines that elicit the recruitment of macrophages and neutrophils within the pulmonary
microvasculature.21
Carrageenan-injected animals exhibited a substantial increase in lung tissue MPO, which
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is believed to indicate PML infiltration and lipid peroxidation. Tissue MPO activity is a sensitive
and specific marker for acute inflammation and reflects polymorphonuclear cell infiltration of the
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parenchyma. Thus, we further examined the effect of compounds (1-10P) on MPO activity in the
lung tissue. The results illustrate that the MPO activity was markedly increased in the Cg group.
Importantly, treatment of mice with compounds (1-7, 9, and 10P) prior to injection with Cg
resulted in a reduction in the MPO activity. Further, the results show that compound 6P is the most
potent at reducing the activity of MPO. Therefore, these results indicate that compound 6P exhibits
marked anti-inflammatory activity.
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In addition, Cg-injected mice showed reduced GSH activity as compared to untreated mice,
an effect that was reversed in mice treated with compounds (1, 3, 6-10P). Consistent with our
initial findings, the most significant increase in GSH activity was found in mice treated with
compound 6P. GSH has pleiotropic roles including the maintenance of cells in a reduced state,
serving as an electron donor for certain anti-oxidative enzymes and in the formation of conjugates
with some harmful endogenous and xenobiotic compounds via catalysis of glutathione s-
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transferase; thus, the ameliorating effects of compound 6P on GSH demonstrated a further
beneficial effect of its administration. Together, our preliminary studies demonstrate that
compound 6P significantly decreases the level of MDA and MPO activity and increases GSH
activity in vivo. These findings indicate that in vivo anti-inflammatory activities of compound 6P
attenuate the degree of acute inflammation in this model system.
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.4. Effect of compound 6P on CD4 , Foxp3 , and CD4 Foxp3 cells in whole blood.
T cells expressing the transcription factor Foxp3 are known to play a key role in the immune system
apparatus that controls regulatory T cell (Treg) development, function, and inflammatory
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processes. Furthermore, a recent study suggested that Foxp3 expression in lung epithelial cells
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suppresses inflammation via inhibition of chemokine secretion. Therefore, we assessed the effect
of compound 6P on the composition of responding T cell populations using flow cytometry. There
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was a substantial increase in the percentage of CD4 cells in the Cg group compared with the NC
group, an effect that was reversed by treatment with compound 6P. The results revealed that the Cg
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group exhibited a significant decrease on the percentage of Foxp3 and CD4 Foxp3 cells compared
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to the NC group. Importantly, following treatment of mice with compound 6P there was a marked
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increase in the proportion of Foxp3 and CD4 Foxp3 expressing cells as compared to mice treated
with Cg alone (Figure 2A). These results suggest that the anti-inflammatory action of compound
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P in the mouse model of pleurisy can be, in part, attributed to the reduction of CD4 cells
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concomitant with an increase in Foxp3 T cells at the site of lung inflammation.
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.5. Effect of compound 6P on NF-κB p65 and IκBα cells in whole blood. NF-κB is a
transcription factor known to be a major player in regulating the expression of pro-inflammatory
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mediators that participate in the inflammatory response. Inappropriate regulation of NF-κB is
directly involved in a wide range of human disorders including inflammatory bowel disease and
numerous other inflammatory conditions. Thus, agents that inhibit NF-κB activation have anti-
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inflammatory effects. Therefore, we studied the effect of compound 6P on the percentage of cells
expressing NF-κB p65 and IκBα. As shown in Figure 3A, the proportion of NF-κB p65 positive
cells increased in the Cg group over that seen in the NC group, an effect that was ameliorated
following treatment with compound 6P. Moreover, as expected, the inverse effect was seen in
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regard to IκBα cells. Together, these results suggest that the anti-inflammatory actions of
compound 6P in the mouse model of pleurisy can be attributed to a reduction in NF-κB activation
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and corresponding increase in IκBα cells at the site of inflammation.
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.6. Effects of compound 6P on the release of carrageenan-induced pro-inflammatory cytokines
in pleural exudates. Thalidomide has been shown to selectively inhibit TNF-α production by
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human monocytes. The ability of thalidomide to inhibit IL-6 and TNF-α production has been
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associated with its clinical benefits in the treatment of many inflammatory immune diseases.29
Moreover, TNF-α and IL-6 play key roles in the induction and perpetuation of inflammation by
activating macrophages and upregulating other pro-inflammatory cytokines and endothelial
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adhesion molecules, and suppressing these pro-inflammatory cytokines have been found to
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reduce the severity of the inflammatory reaction. Thus, we assessed the effects of compound 6P
on TNF-α and IL-6 production. Administration of Cg alone resulted in a significant increase in
TNF-α and IL-6 production in the pleural exudate as compared to the NC group. Furthermore,
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treatment of mice with compound 6P caused a significant decrease in the number of TNF-α and
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IL-6 positive cells (Figure 4A). The results of our study highlight the complex network of
cytokines that contribute to the initiation and maintenance of inflammation. Furthermore, the
ability of compound 6P to suppress TNF-α and IL-6 production may be one of the factors
underlying the anti-inflammatory effects of 6P on Cg-induced lung inflammation. Administration
of Cg alone or before 4-MeH (to induce pleurisy) resulted in a significant decrease in TGF-β1
and IL-10 levels compared to the control group
2
.7. Compound 6P reduces the proportion of cells in pleural exudates expressing GITR and IL-
17. It is well known that GITR plays a co-accessory role in effector T cell activation, which is
further potentiated by the inhibition of Treg cell function, and triggering of GITR on effector CD4
T lymphocytes plays a role in the development of chronic inflammation.32 Moreover, a recent
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study provides evidence that mice lacking GITR (GITR / ) have decreased development of lung
injury, decreased PML infiltration into the lung, lower levels of TNF-α, IL-1β, and stress
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oxidative products, and decreased activation of NF-κB. The suppression of pro-inflammatory
cytokines has been found to reduce the severity of the inflammatory reaction, and IL-17 is known
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to be a critical mediator of neutrophil recruitment and migration. Therefore, we assessed the
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effect of compound 6P on the total percentage of GITR and IL-17 cells during the Cg-induced
inflammatory process. As illustrated, the significant finding of the up regulation of GITR
expression in the Cg group was more significant as compared with the NC group. Further, we
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found that compound 6P significantly reduced the total percentage of GITR and IL-17 cells
(Figure 5A). These findings further suggest that the anti-inflammatory effects of compound 6P
are due to the downregulation of the production and/or secretion of pro-inflammatory cytokines.
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.8. Effect of compound 6P on Th1 and Th2 cytokine levels in serum. The formation of pro-
inflammatory cytokines including IL-2 and IL-6 are central to the pathophysiology of
inflammation. Overexpression of the pro-inflammatory cytokine IL-2, which is crucial for the
maintenance of immune homeostasis and exhibits proinflammatory activities, is well documented
in a number of inflammatory processes.35,36 IL-2 also stimulates the production of TNF-α, IFN-γ,
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and granulocyte macrophage colony-stimulating factor (GM-CSF). In addition, IL-6 plays a
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complex role in inflammation, as it can both promote and limit neutrophil emigration. High
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concentrations of IL-6 are found during lung inflammation, and in a Cg-induced pleurisy model
endogenous IL-6 was found to play a pro-inflammatory role, as reflected by reduced PML
infiltration and diminished lung injury in IL-6−/− mice. Thus, blocking these cytokines may
prove to be therapeutically useful. To this end, an increase in IL-2 and IL-6 was observed in the
exudates of animals following the induction of Cg-induced pleurisy compared to the NC group,
and administration of compound 6P significantly decreased these cytokines (Figure 6A and 6B).
The anti-inflammatory cytokine IL-4 plays a central role in regulating the differentiation
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of antigen-stimulated naive T cells into Th2 cells that produce anti-inflammatory cytokines. IL-
is also responsible for suppressing the synthesis of pro-inflammatory cytokines by macrophages
and monocytes. Likewise, IL-10 is an anti-inflammatory cytokine with potent immunosuppressive
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properties mediated through the downregulation of pro-inflammatory cytokines and T cell-
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mediated inflammatory responses. During early phases of pleural inflammation, the role for IL-
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intrapleural IL-10 has been shown to inhibit the early phase of Cg-induced pleural inflammation
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in a murine model. Thus, we wanted to assess the expression of anti-inflammatory cytokines in
response to compound 6P. To this end, we found that administration of Cg alone resulted in a
significant decrease in both IL-4 and IL-10 levels compared to the NC group. Compound 6P
increased IL-4 and IL-10 cytokine levels over those seen in the Cg group, to levels significantly
higher than that of even the NC group (Figure 6C and 6D).
These results demonstrate that the inflammatory process resulting from the administration
of Cg into the pleural cavity caused substantially increased levels of Th1 cytokines in the pleural
exudate, an effect that was diminished following treatment with compound 6P. In contrast,
compound 6P stimulated the secretion of anti-inflammatory Th2 cytokines into the pleural
exudates. Thus, these findings show that the anti-inflammatory actions of compound 6P in this
mouse model of pleurisy can be explained by a reduction in Th1 levels concurrent with an increase
in Th2 cytokine release at the site of inflammation.
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.9. Effects of compound 6P on TNF-α, IL-17, and IL-10 mRNA expression in lung tissue. We
studied the effect of pretreating animals with compound 6P on the levels of TNF-α in the pleural
exudates at 24 h after the induction of pleurisy by Cg and found that compound 6P significantly
inhibited the expression of TNF-α mRNA (Figure 7A). Interleukin-17 is known to play a pivotal
role in the inflammatory process via stimulation of the synthesis of other pro-inflammatory
cytokines and prostaglandins.44 Furthermore, IL-17A is a critical mediator of neutrophil
recruitment and migration. Thus, we studied the effect of pretreating animals with compound 6P
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on the expression levels of IL-17 in the lung tissue at 24 h after the induction of pleurisy by Cg
injection. The Cg group exhibited a significant increase in IL-17 mRNA expression in lung tissues
compared with the NC group and compound 6P downregulated IL-17 mRNA expression (Figure
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B).
IL-10 mainly induces immunosuppressive effects through the downregulation of
macrophage functions in addition to reducing monocyte/macrophage production of pro-
inflammatory cytokines such as TNF-α and IL-6. Thus, IL-10 is a very important regulator of
inflammation.45 To this end, Cg-injection induces a significant decrease in IL-10 mRNA
expression compared with the NC group, whereas 6P compound treatment upregulates IL-10
expression (Figure 7C). These results indicate that compound 6P not only induces its anti-
inflammatory effect by readjusting the delicate balance among pro-inflammatory and anti-
inflammatory cytokines at the level of release, but also affects this cytokine balance at the level of
gene expression.
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.10. Effects of compound 6P on NF-κB p65, STAT-3, iNOS, and COX-2 mRNA expression in
lung tissue. Transcription of most inflammatory mediators, including iNOS and COX-2, are
controlled by NF-κB, which is kept in an inactive state in resting cells through binding to IκB.46
Agents that inhibit NF-κB also cause decreased iNOS expression and therefore, may have
beneficial therapeutic effects for the treatment of inflammatory diseases. It has previously been
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shown that thalidomide is capable of suppressing NF-κB activation. Therefore, we performed
experiments to evaluate the possible effect of compound 6P on Cg-induced NF-κB activation.
There was a significant difference in NF-κB p65 levels between NC and Cg-induced groups with
the Cg-induced group exhibiting markedly increased NF-κB p65 expression. Treatment with
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compound 6P significantly inhibited NF-κB p65 activation as compared with the Cg control group
(
Figure 8A).
A recent study indicated that STAT-3 is both upregulated and activated in the lung
following acute lung injury, and this activation is dependent on the presence of macrophages,
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neutrophils, and cytokines. Thus, we wanted to assess if STAT-3 expression is affected by
treatment with 6P. While the Cg-treated group exhibited marked upregulation of STAT-3
expression compared with the NC group, pretreatment of mice with compound 6P resulted in
downregulation of STAT-3 mRNA expression (Figure 8B).
The activation of iNOS catalyzes the formation of a large amount of NO, which plays a
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key role in the pathogenesis of a variety of inflammatory diseases. A significant increase in iNOS
mRNA expression was detected by RT-PCR analysis in lungs of mice subjected to Cg-induced
pleurisy 24 h after Cg injection. Compound 6P treatment of mice significantly attenuated this
iNOS expression (Figure 8C).
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The expression of COX-2 is increased by pro-inflammatory mediators. Furthermore,
previous studies of two unique models of lung injury, one using IL-1β and the other TNF-α,
provide convincing evidence that an increase in COX-2 gene and protein expression can be
induced under inflammatory conditions.51 Therefore, we further characterized the effects of
compound 6P treatment on Cg-induced inflammation by measuring COX-2 mRNA levels in
untreated control mice, Cg-injected mice, and Cg-injected mice pre-treated with compound 6P.
These experiments showed that Cg-induced lung inflammation was associated with a significant
induction in the mRNA expression of COX-2 in lung tissue, as compared to the NC group.
Strikingly, pre-treatment with compound 6P significantly reversed the changes in expression of
COX-2 mRNA (Figure 8D). further supporting an anti-inflammatory role for this novel compound.
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.11. Effect of compound 6P on GITR, COX-2, STAT3, NF-κB p65, and IκBα protein
expression.
We further examined the anti-inflammatory effect of compound 6P in Cg-injected mice by
measuring protein expression of inflammatory mediators. In this regard, a substantial increase in
GITR protein level was observed in the lung tissue isolated from Cg-treated mice versus untreated,
control mice. Acute administration of compound 6P prior to induction of lung pleurisy by Cg
resulted in a significant reduction in GITR protein (approximately 1.7 at the 20 mg/kg dose and
4.1 fold at the 40 mg/kg dose) as compared to mice in the Cg-treated group (Figure 9A). A similar
trend was found for COX-2 protein expression with compound 6P treatment diminishing COX-2
expression by approximately 2.6 and 3.7 fold over that of the Cg group (Figure 9B). Similar results
were found regarding STAT-3 expression with approximately 2 and 2.8 fold reductions in
activation following 6P pre-treatment of Cg-treated mice (Figure 9C). In regard to NF-κB
signaling, we confirmed that nuclear translocation of the NF-κB p65 subunit is induced in mice by
Cg-injection; however, pre-treatment with compound 6P significantly inhibited this NF-κB p65
activation (approximately 2.2 and 3.8 fold) compared with the Cg group. Moreover, IκBα protein
levels in lungs were substantially reduced by Cg treatment, an effect that was countered by
compound 6P (approximately 4.2 and 7.8 fold) (Figure 9D).
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.12. Histopathological examination of the effects of compound 6P.
Histological examination of lung tissues revealed that in comparison to the healthy, patent alveoli
of untreated controls, lungs from Cg-injected mice showed dense inflammation with lobar lung
pneumonia and thickened alveolar lung septum with occasionally obliterated alveoli (Figure 10).
These sections also contain acute and inflammatory cells, interalveolar eosinophilic secretions,
fibrils, and extravagated red blood cells. Pre-treatment of Cg-injected mice with compound 6P
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induced a protective effect at both doses resulting in reduced inflammation and inflammatory cell
infiltration, an increase in patent areas of the lung alveoli, and overall healthier looking areas with
few congestive stage patches.
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.13. Docking studies of the compound (1-10P) to the transcription factor NF-κB p65.
Assuming the binding of compounds (1-10P) to the transcription factor NF-κB p65, the PDB
structures 1MY5 and 2RAM were analyzed for potential binding sites. To determine the binding
sites the software package OEDOCKING (OpenEye) was used. PDB structure 2RAM represents
a novel DNA recognition mode for the NF-κB p65 homodimer. In this case the docking of test
compounds into detected sites suggests an interaction of the compounds with the nucleotide
residues of DNA. Results of the docking are shown in (Figure 11). In addition, the potential
interaction of p65 with compounds based on the 1MY5 structures (NF-κB p65 subunit
dimerization domain homodimer) and 2RAM have been considered. For this purpose, the search
space of binding sites was restricted to p65 only. For the p65 from two different systems unique
binding sites have been identified. Docking studies were carried out for the largest sites of each
structure. Results of docking into the p65 from the 1MY5 are shown in (Figure 12), results for the
site with the highest docking score for docked compounds. Involvement of the following amino
acids of p65 were indicated in the interaction with the test compounds (1-10P): GLU225, SER238
and ARG236. Based on chemical structure, it is likely that compounds form hydrogen bonds with
ARG236 and SER238. Results of docking to p65, extracted from the complex with DNA, are
shown in Figure 13. As for 1MY5, the results for the site with the highest docking score are shown.
The following interactions are possible for this site and compounds: 6P can form hydrogen bond
with amino acids VAL121 and LYS123, and a hydrophobic interaction with ring of TYR36. Table
3
shows the values of docking scores for compounds (1-10P) that were docked into sites of the
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p65 from 2RAM and 1MY5 PDB structures, respectively. Docking studies have shown the ability
of this series of compounds (1-10P) to bind with the factor p65. Depending upon the particular
complex binding can occur at different binding sites. If indeed the intended type of interaction is
realized, then by the values of scoring functions the stage at which inhibition occurs may be
assumed. However, the form of binding sites, namely, their location on the surface of the protein,
as well as a relatively small value of the scoring function may also mean a much more complex
mechanism of action of compounds in this series, not including the bonding directly with p65.
Matching docking results against biological test findings allows assuming existence of correlation
between the docking score and GSH level. The logarithm of the difference between activity of the
particular enzyme in a disease state and its level under the test compound was chosen as a
quantitative characteristic of biological activity of the test compound. Figure 14 and figure 15
shows diagrams of the docking score dependency from such a value for 1MY5 PDB-structure.
Dependencies are essentially the same under all concentrations of test compound and reflect a
positive correlation between absolute value of docking score and logarithmic difference. In case
of 2RAM structure the correlation between these parameters is expressed to a much lesser extent.
It can be explained by lack of binding to the transcription factor in a complex with DNA. Besides,
based on SAR, it is not possible making any univocal conclusion on existence of relations between
docking score and activity of the MDA&MPO. Presumably, other mechanisms that require further
research are involved in the process. The biological activity of the compound 6P which was
selected for further study is higher than that which would be expected based on data from docking.
Nevertheless, it has a fairly high docking score and generally fits the relationship previously
discussed.
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The results presented herein indicate that the inflammation seen in the Cg-induced pleurisy
mouse model is significantly attenuated by the pre-treatment of mice with compound 6P. Our
findings suggest that this N-aryl phthalimide derivative may serve as a potent anti-inflammatory
therapy. Further, compound 6P (Clog P = 3.14) was among the most lipophilic compounds in the
series due to the the insertion of the para-ethoxy group at the terminal phenyl ring. In fact, not
only did compound 6P significantly decrease the levels of MPO and MDA, it also increased the
activity of GSH, while downregulating Th1 and upregulating Th2 cytokines in pleural exudates.
Further, treatment with compound 6P inhibits the inflammatory response, as judged by decreased
T cell subsets, expression of GITR, and production of inflammatory cytokines, pro-inflammatory
mediators, and NF-κB expression in lung tissue. Together, these effects result in a decrease in
tissue injury.
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acute inflammation. Moreover, the results of this study suggest that compound 6P possesses
considerable anti-inflammatory activity, and that N-aryl phthalimide derivatives are a important
potential source of synthetic anti-inflammatory agents.
4. Materials and methods
4
.1. Biology. Heparin and λ-carrageenan (Cg) were obtained from Sigma-Aldrich (St Louis, USA).
Fluoroisothiocyanate (FITC)-labeled IL-17, TNF-α and NF-κB; Phycoerythrin (PE)-labeled
GITR, IL-6 and IκBα anti-mouse monoclonal antibodies; FcR blocking reagent
fixation/Permeabilization and Permeabilization solutions were obtained from (Miltenyi Biotech,
Germany and BD Biosciences, USA). The primers used for gene expression were purchased from
Applied Biosystems (Paisley, UK) and Genscript (Piscataway, USA). High Capacity cDNA
Reverse Transcription kit and SYBR® Green PCR Master Mix were purchased from Applied
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Biosystems (Paisley, UK). TRIzol was purchased from Life Technologies (Grand Island, USA)
Primary and secondary antibodies used for western blotting were obtained from Santa Cruz
(Dallas, USA). Nitrocellulose membrane was purchased from Bio-Rad Laboratories (Hercules,
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USA). Chemiluminescence western blot detection kits were obtained from GE Healthcare Life
Sciences (Piscataway, USA).
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.2. Animals. Female adult Balb/c mice, 6-7 weeks old and weighing 20-22 g, were obtained
from the animal house of the College of Pharmacy of King Saud University, Riyadh, KSA. All the
animals were acclimatized to laboratory conditions for at least one week before use in experiments.
The animals were maintained under standard conditions of humidity, temperature (25 ± 2°C), and
light (12-h light/12-h dark), housed in a specific pathogen-free environment and fed standard
rodent chow and given water ad libitum. Each treatment group and vehicle control group consisted
of 6 randomly-assigned animals. Animal protocol used in this study, including the use of ether as
an anaesthesia agent, has been approved by the Research Ethics Committee of College of
Pharmacy, King Saud University (Riyadh, Saudi Arabia). Studies reported in the manuscript fully
meet the criteria for animal studies specified in the ACS ethical guidelines.
4.3. Experimental design. Induction of pleurisy by λ-carrageenan (Cg) was performed as previously
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described. Briefly, mice were injected on the right side of the chest intrapleurally (i.pl.) with 0.1
ml normal saline (NaCl 0.9%) containing (1%) Cg. After pleurisy induction (24 h), animals were
sacrificed with cervical dislocation, the chest was carefully opened, and the pleural cavity was
washed with 1.0 ml of sterile phosphate buffered saline (PBS). Compounds (1-10P) were dispersed
in normal saline with 1% of carboxymethylcellulose (CMC). For the initial screening of the novel
compounds (1-10P), mice were randomly divided into twelve groups (n = 6 per group). Treatments
were given to each group in the following format: Group I, Normal control (NC) group animals
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were injected with 0.1 ml normal saline intraperitoneally (i.p.). Group II, Cg-induced pleurisy (Cg)
group, animals were injected with 0.1 mL normal saline (NaCl 0.9%) containing Cg (1%) to induce
pleurisy and lung inflammation. Groups III to XII, mice received three doses of each compound
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1-10P) (10, 20, and 40 mg/kg, i.p.) 1 h prior to Cg-induced pleurisy. Compound 6P showed
significant anti-inflammatory response and was further investigated in detail at two doses (20 and
0 mg/kg, i.p.), in the Cg-induced pleurisy mouse model. All parameters were analysed 24 after
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4.4. General behaviour and acute toxicity studies in mice. The starting dose for acute oral
toxicity studies of the test compounds was 2000 mg/kg. Compound number 6P was formulated with
CMC and administered by gavage. In brief, the test was performed according to OECD (1996)
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guidelines, and after drug administration, animals were observed individually for any sign of
toxicity, at least once during the first 30 min after dosing, periodically during the first 24 h (with
special attention given during the first 4 h), and daily thereafter. Simultaneously, general behaviors
(
reactivity, gait, motor activity, ptosis, respiration, slaving, and writhing, etc.) were also observed
for 7 days.
4.5. Malondialdehyde (MDA) measurement. Malondialdehyde (MDA) levels in the lung tissue
were determined as an indicator of lipid peroxidation as described previously.52 Lung tissue
collected at the specified time was homogenized in 1.15% (w/v) KCl solution. A 100 µL aliquot of
the homogenate was added to a reaction mixture containing 200 µL of 8.1% (w/v) SDS, 1.5 ml of
2
0% (v/v) acetic acid (pH 3.5), 1.5 mL of 0.8% (w/v) thiobarbituric acid and 700 µL distilled water.
Samples were boiled for 1 h at 95°C using glass balls as condensers. After cooling under tap water,
samples were centrifuged at 4000 x g for 10 min. and the absorbance of the supernatant was
measured at 650 nm using a spectrophotometer .
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.6. Total Glutathione (GSH) Determination. Glutathione (GSH) content was estimated in lung
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tissue using the method of Sedlak and Lindsay . The absorbance of the reaction mixture at 412 nm
was read within 5 min of addition of dithibis-2-nitrobenzoic acid.
0
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8
9
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.7. Myeloperoxidase (MPO) activity. Myeloperoxidase activity, an indicator of PML
5
5
accumulation, was determined as previously described. Twenty four hours following injection of
carrageenan, lung tissues were obtained and weighed, and each piece was homogenized in a
solution containing 05% (w/v) hexadecyltrimethyl-ammonium bromide dissolved in 10 mM
potassium phosphate buffer (pH 7) and centrifuged for 30 min. at 20000 × g at 4 °C. An aliquot of
the supernatant was then allowed to react with a solution of tetramethylbenzidine (1.6 mM) and 0.1
mM hydrogen peroxide. The rate of change in absorbance was measured spectrophotometrically at
650 nm. MPO activity was defined as the quantity of enzyme degrading 1 µM of peroxide/min at
3
7 °C and was expressed in milliunits per gram of wet tissue.
+
+
+
+
+
+
4
.8. Flow cytometric analysis of CD4 , Foxp3 , CD4 Foxp3 , NF-κB , and IκB-α cells in
whole blood. For flow cytometric analysis of CD4, Foxp3, NF-κB, and IκB-α expression, whole
blood was collected from the retro-orbital plexus (under light ether anaesthesia). Monoclonal
antibody conjugated to a fluorochrome and directed against CD4 was added directly to 100 µL of
whole blood, which was then lysed using a whole blood lysing reagent (BD Biosciences, USA).
After centrifugation at 300 × g for 5 min., the supernatant was aspirated and 1 x
fixation/permeabiliziation solution (500 μl, BD Biosciences, USA) was added to the pellet and
incubated for 10 min at room temperature in the dark. The cells were then centrifuged at 300 x g for
5
min; the supernatant was aspirated and 1 x permeabilizing solution (500 μL) and FcR blocking
reagent (10 µL, Miltenyi Biotech), were added to the pellet and incubated for 10 min. at room
temperature in the dark. After washing with 3 mL of wash buffer, cytokine-specific antibodies (20
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μL) against Foxp3, NF-κB, and IκBα (Santa Cruz and BD Biosciences, USA) were added to the cells
and incubated for 30 min at room temperature in the dark. All cells were analysed for the expression
of phenotypic markers on a Beckman Coulter flow cytometer (Beckman Coulter, USA) using
0
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Cytomics FC 500 software. To analyse the staining of cell surface markers, the lymphocytes were
first gated by their physical properties (forward and side scatter), then a second gate was drawn based
+
on immunofluorescence characteristics of the gated cells. To determine Foxp3 the cells were gated
on FSC-SSC dot plot, and then the lymphocytes were gated for analyses the percentage of
+
+
CD4 Foxp3 cells.
.9. Flow cytometric analysis of IL-6 , TNF-α , GITR and IL-17 cells in pleural exudate.
+
+
+
+
4
Twenty four after the injection of carrageenan, the animals were sacrificed by cervical dislocation
under light ether anesthesia and the chest was carefully opened then the pleural cavity was rinsed
with 1 ml saline solution containing heparin. 200 µL of this solution were pipetted directly into a
1
2 x 75 mm fluorescence-activated cell sorting tube containing 20 µL of a monoclonal PE-labeled
IL-6 and GITR and FITC-labeled TNF-α and IL-17 antibodies (Santa Cruz and BD Biosciences,
USA). The red blood cells (RBCs) were lysed by incubation in 2 mL of 1× lysis solution (BD
Biosciences, USA) for 10 min. After centrifugation at 300 x g for 5 min., the supernatant was
discarded, fixation/Permeabilization solution (500 µL; BD Biosciences, USA) was added to the
pellet, and the samples were incubated for 10 min at room temperature in the dark. Cells were then
permeabilized (500 µL, BD Biosciences, USA) and incubated for 10 min. at room temperature
followed by adding, FcR blocking reagent (10 µL, Miltenyi Biotech). After washing with 3 mL of
washing buffer, the antibodies specific for IL-6, TNF-α, GITR or IL-17 (20 µl; BD Biosciences,
USA) were added and cells were incubated for 30 min. at room temperature in the dark. After one
final wash, the stained cells were immediately analyzed by flow cytometry.57
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.10. Quantification of Th1 and Th2 cytokines in pleural exudate. 50 µL of the pleural exudate
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samples obtained from all animals, including the normal control, were collected and immediately
prepared for the analysis of cytokine levels. Cytokine levels were measured with an ELISA kit
according to the manufacturers' instructions (Biolegend, USA). The individual recombinant
cytokines provided in the kits were used to establish standard curves.
0
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.11. RNA extraction and cDNA synthesis. All the extraction procedures were performed on ice
using ice-cold reagents. Total RNA from the lung tissue homogenate of each mouse was isolated
using the TRIzol reagent (Life Technologies, Grand Island, USA) according to the manufacturer's
instructions and quantified by measuring the absorbance at 260 nm; the RNA quality was determined
by measuring the 260/280 ratio. The cDNA synthesis was performed using the high-capacity cDNA
reverse transcription kit (Applied Bio systems, Paisley, UK) according to the manufacturer's
58
instructions and as previously described. Briefly, 1.5 µg of total RNA from each sample was added
to a mixture of 2.0 µL of 10 × reverse transcriptase buffer, 0.8 µL of 25× dNTP mix (l00 mM), 2.0
µL of l0 × reverse transcriptase random primers, 1.0 µL of multiscribe reverse transcriptase and 3.2
µL of nuclease-free water. The final reaction mixture was held at 25 °C for 10 min., then heated to
37 °C for 120 min. and 85 °C for 5 min. and, finally, cooled to 4 °C.
4
.12. Real-Time Polymerase Chain Reaction (RT-PCR) determination of mRNA expression.
Quantitative analysis of the mRNA expression of target genes was performed by RT-PCR by
subjecting the cDNA generated from the above reaction to PCR amplification in 96-well optical
reaction plates an analysis on an ABI Prism 7500 System (Applied Bio systems, Paisley, UK). The
2
5 µL reaction mixture included 0.1 µL of the 10 µM forward primer and 0.1 µL of the 10 µM reverse
primer (each primer at a final concentration of 40 µM), 12.5 µL of the SYBR Green Universal Master
mix, 11.05 µL of nuclease-free water and 1.25 µL of the cDNA sample. The primers used in the
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current study (Table 2) were chosen from the PubMed database. The assay controls, namely, the
no-template controls, were incorporated onto the same plate to monitor the contamination of assay
reagents. The RT-PCR data were analyzed using the relative gene expression (i.e., ΔΔCT) method,
as described in the Applied Bio systems User Bulletin No. 2. Briefly, the data are presented as the
fold change in gene expression normalized to the endogenous reference gene (GAPDH) and relative
to a calibrator.
0
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9
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9
0
4
.13. Protein extraction and western blot analysis of GITR, COX-2, STAT-3, NF-κB p65, and
IκB-α. The total lung proteins were extracted from lung tissue using a previously described method.60
Briefly, lungs were washed in ice cold PBS, cut into small pieces, and homogenized separately in
6
1
cold protein lysis buffer containing a protease inhibitor cocktail. Total cellular proteins were
obtained by incubating the cell lysates on ice for 1 h, with intermittent vortex mixing every 10 min.,
followed by centrifugation at 12,000 x g for 10 min. at 4 °C. Protein concentrations were measured
using the Lowry method.62 Western blot analysis was performed using a previously described
6
3
method. Briefly, 25-50 µg of protein from each group was separated by 10% SDS-polyacrylamide
gel electrophoresis (PAGE) and electrophoretically transferred to a nitrocellulose membrane (Bio-
Rad, USA). Protein blots were blocked overnight at 4 C, followed by incubations with primary
antibodies against GITR, COX-2, STAT-3, NF-κB p65, or IκBα (Santa Cruz, Dallas, USA), followed
by incubation for 2 h with peroxidase-conjugated secondary antibodies at room temperature. The
GITR, COX-2, STAT-3, NF-κB p65, and IκBα bands were visualized using the enhanced
chemiluminescence method (GE Health care, Mississauga, Canada) and quantified relative to β-actin
®
bands using ImageJ , image processing program (National Institutes of Health, Bethesda, USA).
Images were taken on C-Digit chemiluminescent western blot scanner (LI-COR, Lincoln, USA).
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.14. Histopathological examination of the lung tissue. Lung tissue samples isolated from all
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groups were fixed for 1 week in buffered formaldehyde solution (10% in PBS) at room temperature,
dehydrated using graded ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ).
Tissue sections (thickness 7 μm) were deparaffinised with xylene, stained with Hematoxylin and
eosin (H&E) and then studied using light microscopy (Olympus, USA).
0
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.15. Chemistry. All the solvents were obtained from Merck. The homogeneity of the compounds
was checked by TLC performed on Silica gel G coated plates (Merck). Iodine chamber was used for
visualization of TLC spots. The FT-IR spectra were recorded in KBr pellets on a (Spectrum BX)
Perkin Elmer FT-IR spectrophotometer. Melting points were determined on a Gallenkamp melting
point apparatus, and thermometer was uncorrected. NMR Spectra were scanned in DMSO-d on a
6
Bruker NMR spectrophotometer operating at 500 MHz for H and 125.76 MHz for 13C at the
1
Research Center, College of Pharmacy, King Saud University, Saudi Arabia. Chemical shifts δ are
expressed in parts per million (ppm) relative to TMS as an internal standard and D O was added to
2
confirm the exchangeable protons. Coupling constants (J) are in hertz. The following abbreviations
are used in the assignment of NMR signals: s (singlet), d (doublet), m (multiplet). The compounds
were purified by column chromatography on silica gel (60-120/100-200 mesh) using varied polarities
of n-hexane/ethyl acetate as the eluent. The molecular masses of compounds were determined by
Agilent Triple Quadrupole 6410 TQ LC/MS equipped with ESI (electrospray ionization) source. All
tested compounds yielded data consistent with a purity of ≥95%, as measured by HPLC (Agilent
1
260 affinity) with an ELSD (Evaporative Light Scattering Detector).
Synthesis of 4-aminobenzohydrazide (1)
A mixture of (Benzocaine), ethyl 4-aminobenzoate (0.01 mol) and hydrazine hydrate (99 %, 3 mL)
was refluxed for 1 h in the presence of absolute ethanol, then left to cool to room temperature. The
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precipitate was filtered off, washed with water, dried and crystallized from ethanol to give 1 as white
crystals.
4-amino-N'-[substituted phenylmethylidene]benzohydrazide (2 a-j)
0
1
2
3
4
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0
A solution of (0.01 mol) of 1 and equimolar amount of appropriate aldehyde in 60 mL of ethanol was
heated under reflux for 1 h in presence of few drops of glacial acetic acid. The precipitate obtained
was filtered off, washed with water and crystallized from ethanol.
4-amino-N'-[(4-ethoxyphenyl)methylidene]benzohydrazide (2f):
-1
1
Yield: 70%; M.p.: 210-212 °C; IR (KBr): ν max/cm : 3420 (Ar-NH
NMR (DMSO-d ) δ ppm: 1.3 (3H, t, J = 7Hz, CH ), 4.0 (2H, q, J = 9 Hz, -OCH
O exchg.), 6.5-7.6 (8H, m, Ar-H), 8.3 (1H, s, N=CH), 11.3 (1H, s, CONH, D O exchg.); C NMR
2
), 3200 (NH), 1739 (C=O); H
6
3
2
), 5.7 (2H, s, -NH,
1
3
D
2
2
(DMSO-d
6
) δ ppm: 15.0, 63.6, 113.0, 115.1, 120.1, 127.6, 128.8, 129.7, 146.3, 152.6, 160.2; ESI
+
mass (m/z): 283.37 [M] (calculated 283.32).
General method for the synthesis of 4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N'-[substituted
phenylmethylidene]benzohydrazide (1-10P):
7
.29 mmol of 4-amino-N'-[substituted phenylmethylidene]benzohydrazide (2a-j) was dissolved
along with (6.75 mmol) of phthalic anhydride in 10 mL of glacial acetic acid and refluxed for 1 h. The
N-substituted phthalimide (1-10P) separated out on cooling. The precipitate was filtered out through
a Buchner funnel and washed twice with 30 mL of water to give the desired product. The product was
recrystallized from the ethanol.
4
-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N'-[(4-nitrophenyl)methylidene]benzohydrazide (1P)
-1
1
Yield: 80%; M.p.: 283-285 °C; IR (KBr): ν max/cm : 3258 (CONH), 2837 (N=CH), 1686 (C=O); H
NMR (DMSO-d
6
) δ ppm: 7.5-8.5 (12 H, m, Ar-H), 10.8 (1H, s, N=CH), 12.2 (1H, s, CONH, D
2
O
13
exchg.); C NMR (DMSO-d
6
) δ ppm: 118.6, 119.21, 124.0, 124.5, 127.5, 127.7, 128.3, 129.1, 130.0,
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31.1, 131.9, 135.3, 138.6, 141.2, 143.4, 145.2, 148.2, 163.2, 168.1, 168.2; ESI mass (m/z): 413.29
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
[
M-1] (calculated 414.37).
4
-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N'-[(2-nitrophenyl)methylidene]benzohydrazide (2P)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-
1
1
Yield: 65%; M.p.: 258-260 °C; IR (KBr): ν max/cm : 3398 (CONH), 2847 (N=CH), 1713 (C=O); H
NMR (DMSO-d
6
) δ ppm: 7.6-8.1 (12 H, m, Ar-H), 8.9 (1H, s, N=CH), 12.1 (1H, s, CONH, D
2
O
13
exchg.); C NMR (DMSO-d
6
) δ ppm: 118.6, 124.0, 124.3, 125.1, 127.5, 127.7, 128.3, 128.4, 128.8,
1
29.1, 129.9, 131.0, 131.2, 131.9, 132.7, 134.1, 134.2, 135.3, 135.5, 135.9, 143.6, 148.6, 148.7, 163.2,
+
165.8, 167.2, 169.2; ESI mass (m/z): 413.86 [M] (calculated 414.37).
N'-[(3,4-dichlorophenyl)methylidene]-4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzohydrazide
(3P)
-1
Yield: 70%; M.p.: 275-277 °C; IR (KBr): ν max/cm : 3350 (CONH), 2927 (N=CH), 1709 (C=O);
1H NMR (DMSO-d
) δ ppm: 7.5-8.1 (11 H, m, Ar-H), 8.3 (1H, s, N=CH), 12.2 (1H, s, CONH, D O
6
2
1
3
exchg.); C NMR (DMSO-d
6
) δ ppm: 124.0, 124.4, 127.5, 127.7, 128.5, 128.7, 128.8, 129.8, 129.9,
1
31.1, 132.8, 134.3, 135.3, 135.5, 135.6, 143.2, 163.1, 167.2; ESI mass (m/z): 437.16 [M-1]+
(
calculated 438.26).
4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N'-[(2-methoxyphenyl)methylidene]benzohydrazide
(4P)
-1
1
Yield: 60%; M.p.: 253-255 °C; IR (KBr): ν max/cm : 3354 (CONH), 3065 (N=CH), 1736 (C=O); H
NMR (DMSO-d
s, CONH, D
6
) δ ppm: 3.8 (3H, s, -OCH ), 7.0-8.0 (12 H, m, Ar-H), 8.8 (1H, s, N=CH), 11.9 (1H,
3
13
2
O exchg.); C NMR (DMSO-d
6
) δ ppm: 56.1, 112.3, 121.2, 124.0, 126.0, 127.5, 127.7,
+
1
3
4
28.6, 128.8, 132.0, 135.3, 143.9, 158.2, 165.8, 167.2; ESI mass (m/z): 398.29 [M-1] (calculated
99.39).
-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N'-[(3-hydroxyphenyl)methylidene]benzohydrazide (5P)
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
-1
1
Yield: 58%; M.p.: 270-272 °C; IR (KBr): ν max/cm : 3352 (CONH), 2927 (N=CH), 1739 (C=O); H
NMR (DMSO-d
6
) δ ppm: 7.1-8.0 (12 H, m, Ar-H), 8.3 (1H, s, N=CH), 9.6 (1H, s, -OH, D
2
O exchg.),
1
3
1
1
1.9 (1H, s, CONH, D
2
O exchg.); C NMR (DMSO-d
6
) δ ppm: 113.1, 118.0, 118.7, 119.3, 124.0,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
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8
9
0
24.3, 127.5, 128.6, 129.3, 129.9, 130.3, 132.0, 133.2, 135.2, 135.8, 136.0, 148.5, 158.1, 163.0,
+
167.2; ESI mass (m/z): 384.14 [M-1] (calculated 385.37).
4
-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N'-[(4-ethoxyphenyl)methylidene]benzohydrazide (6P)
-1
1
Yield: 75%; M.p.: 248-250 °C; IR (KBr): ν max/cm : 3420 (CONH), 2927 (N=CH), 1739 (C=O); H
NMR (DMSO-d
6
) δ ppm: 1.3 (3H, t, CH
3
), 4.0 (2H,q, J = 5.5 Hz, -OCH
2
), 7.0-8.0 (12 H, m, Ar-H),
) δ ppm: 15.0, 63.7, 115.2,
13
8
1
.4 (1H, s, N=CH), 11.8 (1H, s, CONH, D
2
O exchg.); C NMR (DMSO-d
6
24.0, 127.1, 127.5, 127.7, 128.6, 128.8, 129.1, 129.2, 131.9, 135.1, 135.3, 148.4, 162.8, 167.2; ESI
+
mass (m/z): 412.10 [M-1] (calculated 413.42).
N'-[1,3-benzodioxol-5-ylmethylidene]-4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzohydrazide
(
7P)
Yield: 60%; M.p.: 240-242 °C; IR (KBr): ν max/cm : 3459 (CONH), 2927 (N=CH), 1739 (C=O); H
NMR (DMSO-d ) δ ppm: 6.0 (2H, s, -OCH O-), 7.0-8.0 (12 H, m, Ar-H), 8.3 (1H, s, N=CH), 11.8
(1H, s, CONH, D O exchg.); C NMR (DMSO-d ) δ ppm: 102.0, 105.6, 108.9, 118.6, 123.9, 124.0,
24.3, 124.4, 127.5, 127.7, 128.6, 128.8, 128.9, 129.1, 129.3, 129.9, 131.9, 133.3, 135.2, 135.3, 135.8,
-1
1
6
2
1
3
2
6
1
1
+
35.9, 148.4, 149.6, 162.9, 165.9, 167.1; ESI mass (m/z): 413.73 [M] (calculated 413.38).
4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N'-[furan-2-ylmethylidene]benzohydrazide (8P)
-1
1
Yield: 70%; M.p.: 283-285 °C; IR (KBr): ν max/cm : 3354 (CONH), 2926 (N=CH), 1736 (C=O); H
NMR (DMSO-d
6
) δ ppm: 7.7-8.1 (11 H, m, Ar-H), 8.3 (1H, s, N=CH), 11.4 (1H, s, CONH, D
exchg.); C NMR (DMSO-d ) δ ppm: 124.0, 124.4, 127.7, 128.8, 129.9, 132.0, 135.3, 135.9, 165.8,
67.1; ESI mass (m/z): 358.20 [M-1] (calculated 359.33).
2
O
1
3
6
+
1
2
8
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Journal of Medicinal Chemistry
1
2
3
4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N'-[(E)-(3-nitrophenyl)methylidene]benzohydrazide
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
9P)64
-
1
1
Yield: 60 %; M.p.: 210-212 °C; IR (KBr): ν max/cm : 3298 (CONH), 2854 (N=CH), 1712 (C=O); H
NMR (DMSO-d ) δ ppm: 7.5-8.5 (12 H, m, Ar-H), 10.6 (1H, s, N=CH), 12.2 (1H, s, CONH, D
exchg.); C NMR (DMSO-d ) δ ppm: 119.2, 121.3, 124.6, 127.8, 128.2, 129.1, 130.0, 130.3, 130.9,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
2
O
1
3
6
32.3, 133.8, 136.7, 139.0, 143.3, 145.3, 148.7, 163.2, 167.8, 168.3; ESI mass (m/z): 414. 30 [M]+
1
(calculated 414.37).
4
-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N'-[(E)-(3,4-
dimethoxyphenyl)methylidene]benzohydrazide (10P)64
-1
1
Yield: 68%; M.p.: 280-282 °C; IR (KBr): ν max/cm : 3447 (CONH), 2836 (N=CH), 1740 (C=O); H
NMR (DMSO-d
6
) δ ppm: 3.8 (6H, s, 2×-OCH
3
), 7.0-8.0 (11 H, m, Ar-H), 8.4 (1H, s, N=CH), 11.8
) δ ppm: 55.94, 56.0, 108.7, 111.9, 118.6, 122.4,
13
(1H, s, CONH, D
2
O exchg.); C NMR (DMSO-d
6
1
24.0, 124.4, 127.4, 127.5, 127.7, 128.6, 128.8, 129.9, 131.9, 133.4, 135.1, 135.3, 148.7, 149.5, 151.2,
+
162.9, 165.8, 167.2; ESI mass (m/z): 429.40 [M] (calculated 429.42).
4
.16. Docking studies of the compounds (1-10P). Structures of protein-ligand complexes were
downloaded from the RCSB Protein Data Bank (PDB). Toolkit OEDOCKING3.0.1 (OpenEye) was
used for docking and analysis of binding sites. Library conformer’s compounds (1-10P) was
generated by the program OMEGA2.5.1.4 (OpenEye). Detection of binding sites was performed by
the program MAKE RECEPTOR3.0.1 from the toolkit OEDOCKING with “Molecular cavity
detection” option. Docking compounds into these sites was performed by the program FRED with
standard options.
2
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