Short communication
TNF-α and IL-6 inhibitors: Conjugates of N-substituted indole and aminophenylmorpholin-3-one as anti-inflammatory agents

https://doi.org/10.1016/j.ejmech.2017.09.003Get rights and content

Highlights

  • Rationally designed molecules which are very easy to synthesize.

  • Appreciable inhibition of TNF-α and IL-6 was recorded in the presence of the compounds.

  • Identification of one compound as a potential lead for further modification.

Abstract

The conjugates obtained by the combination of indole and aminophenyl morpholinone were screened for TNF-α and IL-6 inhibition in microglial cells. Compound 4 was found to be the most potent anti-inflammatory agent as it reduced LPS induced level of inflammatory cytokines TNF-α and IL-6 by 71% and 53%, respectively. A significant decrease in NO and MMPs release from BV2 cells in culture pretreated with this compound as well as inhibition of nuclear translocation of NF-κB and AP-1 was observed. 75% inhibition of acetic acid induced algesia in swiss albino mice was noticed in the presence of compound 4. Experimental data and molecular docking studies indicate that the compounds are targeting TNF-α, iNOS and IL-6.

Introduction

Inflammation is the immune response of the body [1] but the prolonged inflammation becomes the root cause of various chronic diseases such as neurodegenerative diseases, cancer, rheumatoid arthritis, atherosclerosis, hay fever and heart diseases [2]. Amongst the different mediators; the cytokine receptors, chemokines, interferons, nuclear factor kappa-B (NF-κB), tumor necrosis factor alpha (TNF-α), nitric oxide radical, prostaglandins and leukotrienes [3] are the key factors responsible for the initiation and propagation of inflammation. When stimulated with microbial infections [4] and lipopolysaccharide (LPS), the macrophages secrete TNF-α and IL-6 which in turn induce the expression of iNOS (inducible nitric oxide synthase) in the inflammatory cells [5]. Nonetheless, nitric oxide plays significant role as a cellular signaling molecule regulating different physiological and pathological processes in the human body including vascular relaxation, platelet aggregation, blood pressure regulation, host defense and transmission of information between nerve cells in the brain [6]. But, the elevated levels of NO, produced in response to inflammation and a variety of oxidative stresses, activates COX-2 [7]– ultimately resulting in chronic neurodegenerative diseases [8], arthritis [9] and inflammatory bowel disease [10]. In addition to the activation of iNOS; TNF-α also induces apoptosis, secretion of cytokines such as IL-1, IL-6 and IL-10, and activates T cells and other inflammatory cells [11]. Hence, irrespective of a number of check-points that can be targeted for making control over inflammation but keeping in mind the primary role of TNF-α and IL-6 in inducing inflammation; the current study was planned to develop those molecules that may inhibit the release of TNF-α and IL-6.

The use of TNF-α inhibitors such as etanercept, infliximab, golimumab, adalimumab and cetrolizumab-pegol as well as the IL-6 inhibitor tocilizumab are associated with adverse side effects like aplastic anemia and congestive heart failure [12], [13]. Furthermore, the protein-based drugs used to inhibit pro-inflammatory cytokines suffer from lack of oral availability and low tissue penetrating capacity [14]. These issues necessitate the search for new inhibitors of TNF-α and other inflammatory cytokines.

The use of indomethacin, tenidap [15] and compound C [16] (A – C, chart 1) as COX-1/2 inhibitor, for reducing the production of cytokines, IL-1β, IL-6 and TNF-α and for the inhibition of LPS-induced expression of TNF-α and IL-6, respectively enabled us to chose indole [17] and 4-(4-aminophenyl)morpholin-3-one [18] moieties for the design of new molecules. Both indomethacin and tenidap have acted as lead molecules for the development of a series of anti-inflammatory agents [19]. Moreover, heterocyclic-based Schiff bases (iminic linkage) are known for good cytotoxic, anti-inflammatory, anticonvulsant, antimicrobial, anticancer and antifungal activities [20]. Therefore, taking in view the biological potential of compounds A – C (Chart 1), it was planned to introduce 4-(4-aminophenyl)morpholin-3-one unit at C-3 position of N-1 substituted indole and hence compounds 17 (Chart 1) were designed. The purpose of including compound 7 in the present studies was to check if more than one 4-(4-aminophenyl)morpholin-3-one units increase the potency of the molecule for the inhibition of TNF-α and IL-6. The compounds were also evaluated for the inhibition of nitric oxide production and were subjected to animal studies.

Section snippets

Chemistry

Reaction of indole-3-carboxaldehyde with 4-(4-aminophenyl)morpholin-3-one in MeOH containing catalytic amount of 1 N HCl at 70–75 °C furnished compound 1 (Scheme 1). The N-tosyl (2), N-benzoyl (3 and 4), and N-benzyl (56) derivatives of 1 were prepared by the reaction of 1 with tosyl chloride, benzoyl chloride, 4-chlorobenzoyl chloride, benzyl bromide and 4-fluorobenzyl chloride in the presence of NaH in DMF at room temperature (Scheme 1).

For the synthesis of compound 7;

Conclusion

The screening of rationally designed molecules helped to identify compound 1, 2 and 4 as the potential anti-inflammatory agents. These compounds exhibit pronounced activity to inhibit LPS induced activation of microglial cells and rescue the normal cell morphology. They were also observed to reduce pro-inflammatory cytokines TNF-α, IL-6, NO and MMPs release from BV2 cells in culture pretreated with these compounds. The inhibition of nuclear translocation of NF-κB and AP-1 was also noticed in

General

Melting points were determined in capillaries and are uncorrected. 1H and 13C NMR spectra were recorded on Bruker 500 MHz and 125 MHz NMR spectrometer and JEOL 400 MHz and 100 MHz NMR spectrometer, respectively using CDCl3 and/or DMSO-d6 as solvent. Chemical shifts are given in ppm with TMS as an internal reference. J values are given in Hertz. Signals are abbreviated as singlet, s; doublet, d; double-doublet, dd; triplet, t; multiplet, m. Mass spectra were recorded on micrOTOF Q II Mass

Molecular docking procedure

The molecular docking was performed using Schrodinger (Schrödinger Release 2015-4: Maestro, version 10.0, Schrödinger, LLC, New York, NY, 2014). Crystal co-ordinates of TNF-α (PDB ID 2AZ5) and iNOS (PDB ID 3E7G) were downloaded from the protein data bank (www.rcsb.org). In the first step, bond orders were assigned and hydrogen's were added by preprocess option. All water molecules were deleted. The heteroatoms are ionized by epikat biological pH to consider the protein permeability and drug

Acknowledgements

The financial assistance from Council of Scientific and Industrial Research (CSIR) (02(0203)/14/EMR-II), New Delhi and SERB-DST (EMR/2015/001548) New Delhi is gratefully acknowledged. SK thanks CSIR for Senior Research Fellowship. AS is thankful to DST-INSPIRE, New Delhi for Senior Research Fellowship. UGC, CPEPA are acknowledged for creating instrumentation facility.

References (33)

  • R.N. Saha et al.

    Regulation of inducible nitric oxide synthase gene in glial cells

    Antioxid. Redox Signal

    (2006)
  • R.M.J. Palmer et al.

    Nitric oxide release accounts for the biological activity of endothelium – derived relaxing factor

    Nature

    (1987)
    D.S. Bredt et al.

    Nitric oxide: a physiologic messenger molecule

    Annu. Rev. Biochem.

    (1994)
    V. Mathur et al.

    Physiological and pathophysiological functions of nitric oxide

    Der Pharm. Lett.

    (2010)
    S. Moncada et al.

    Nitric oxide: physiology, pathophysiology and pharmacology

    Pharmacol. Rev.

    (1991)
  • D. Salvemini et al.

    Proc. Natl. Aca. Sci.

    (1993)
  • D. Jang et al.

    Nitric oxide in arthritis

    Free Rad. Biol. Med.

    (1998)
  • G. Kolios et al.

    Nitric oxide in inflammatory bowel disease: a universal messenger in an unsolved puzzle

    Immunol

    (2004)
  • L.M. Sedger et al.

    TNF and TNF-receptors: from mediators of cell death and inflammation to therapeutic giants – past, present and future

    Cytokine Growth Factor Rev.

    (2014)
    P. Rieckmann et al.

    Tumor necrosis factor–α (TNF-α) and interleukin – 6 (IL-6) in B-Lymphocyte function

    Methods A companion methods Enzym.

    (1997)
  • Cited by (0)

    View full text