PdCl2-catalyzed synthesis of a new class of isocoumarin derivatives containing aminosulfonyl / aminocarboxamide moiety: First identification of a isocoumarin based PDE4 inhibitor

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Highlights

  • Isocoumarins were explored for the first time as a new class of PDE4 inhibitors.

  • Synthesis involved PdCl2-catalyzed construction of the 4-allyl substituted 3-aryl isocoumarin ring.

  • Compound 3u (PDE4B IC50 ∼ 0.54 μM) reduced paw swelling, inflammation and pannus formation.

  • 3u also reduced pro-inflammatory gene expression/mRNA levels and showed good safety in Zebrafish.

  • 3u is a potential agent for inflammatory diseases.

Abstract

While anti-inflammatory properties of isocoumarins are known their PDE4 inhibitory potential was not explored previously. In our effort the non-PDE4 inhibitor isocoumarins were transformed into the promising inhibitors via introducing an aminosulfonyl/aminocarboxamide moiety to the C-3 benzene ring attached to the isocoumarin framework. This new class of isocoumarins were synthesized via a PdCl2-catalyzed construction of the 4-allyl substituted 3-aryl isocoumarin ring starting from the appropriate 2-alkynyl benzamide derivative. Several compounds showed good inhibition of PDE4B in vitro and the SAR indicated superiority of aminosulfonamide moiety over aminocarboxamide in terms of PDE4B inhibition. Two compounds 3q and 3u with PDE4B IC50 = 0.43 ± 0.11 and 0.54 ± 0.19 μM and ≥ 2-fold selectivity over PDE4D emerged as initial hits. The participation of aminosulfonamide moiety in PDE4B inhibition and the reason for selectivity though moderate shown by 3q and 3u was revealed by the in silico docking studies. In view of potential usefulness of moderately selective PDE4B inhibitors the compound 3u (that showed PDE4 selectivity over other PDEs) was further evaluated in adjuvant induced arthritic rats. At an intraperitoneal dose of 30 mg/kg the compound showed a significant reduction in paw swelling (in a dose dependent manner), inflammation and pannus formation (in the knee joints) as well as pro-inflammatory gene expression/mRNA levels and increase in body weight. Moreover, besides its TNF-α inhibition and no significant toxicity in an MTT assay the compound did not show any adverse effects in a thorough toxicity studies e.g. teratogenicity, hepatotoxicity, cardiotoxicity and apoptosis in zebrafish. Thus, the isocoumarin 3u emerged as a new, safe and moderately selective PDE4B inhibitor could be useful for inflammatory diseases possibly including COVID-19.

Introduction

Isocoumarins represent one of the important class of naturally occurring aromatic lactones possessing numerous biological properties including anti-inflammatory activities [1]. For example, a highly substituted isocoumarin derivative i.e. paepalantine (A, Fig. 1) isolated from the capitula of paepalanthus bromelioides has shown intestinal anti-inflammatory activity in the trinitrobenzenesulphonic acid model of rat colitis [2] whereas activities of fluorinated isocoumarins as well as 3,4-dihydroisocoumarins against TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation in mice have been documented [3]. Besides, a dual inhibitor of 5-LOX (5-lipoxygenase) and PGE2 (prostaglandin E2) production was identified via screening of a series of 3-aryl isocoumarin derivatives in HeLa cells [4]. Indeed, a naturally occurring isocoumarin thunberginol (B, Fig. 1) showed inhibition of PGE2 production in the same study. Recently, a natural isocoumarin GDC [3R-(4′-hydroxyl-3′-O-d-glucopyranosyl phenyl)-dihydro isocoumarin] (C, Fig. 1) has shown remarkable anti-inflammatory activities including the inhibition of production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) induced by LPS [5]. Interestingly, exploration of isocoumarin derivatives as inhibitor of PDE4 (phosphodiesterase 4) is rather uncommon though inhibition of calmodulin-sensitive cyclic guanosine 3′,5′-monophosphate phosphodiesterase by three natural isocoumarins (D, Fig. 1) was studied way back in 1989 [6]. Notably, the inhibitors of PDE4 are not only known to be useful for the treatment of inflammatory diseases [[7], [8], [9]] like COPD/asthma, psoriasis, psoriatic arthritis etc but also have been suggested to attenuate the cytokine storm in COVID-19 especially TNF-α [[10], [11], [12]]. The currently available inhibitors such as roflumilast and apremilast though found to be potent and effective but their emetic and other side effects emphasize the necessity for the discovery and development of alternative agents. In our effort [13,14] for the identification of new inhibitors of PDE4 we explored benzo[c]phenanthridine derivatives that were synthesized from the isocoumarin-based key intermediates (E, Fig. 2) [15]. Prompted by the reported usefulness of isocoumarins as anti-inflammatory agents we examined PDE4 inhibitory potential of E when none of these derivatives showed significant inhibition of PDE4 in vitro. However, introduction of an aminosulfonyl moiety to the benzene ring at C-3 position enhanced the inhibitory properties of the resultant analogue (F, Fig. 2) remarkably in some cases. Indeed, the role of aminosulfonyl moiety in PDE4B inhibition was further supported by the docking study of a representative molecule F-1 (Fig. 3, see also Fig. S-6 in suppl info) where one of the oxygen atom of the –NHSO2-group formed a H-bond with the HIS450 residue. Overall, with a high binding affinity (−11.9 comparable to rolipram's −10.4) F-1 interacted well with the PDE4B in silico (ID: 4MYQ) via (i) two H-bonds with HIS450 and TYR405, (ii) one aromatic pi interaction with PHE586 and (iii) a number of van der Waals/hydrophobic contacts with hydrophobic residues like TYR575, PHE618 etc. and hydrophobic regions of polar or charged residues like; SER454, ASN455, GLN456, THR579, HIS406, ASN567, GLN615 etc. Notably, in addition to participating in the hydrophobic interactions with PDE4B the allyl group enhances lipophilicity that may cause a rapid onset. Herein we present a detailed account of this study. We also examined the PDE4 inhibitory potential of the corresponding aminocarboxamide analogues designed via replacing the –NHSO2R2 of F by –NHCOR2. While PDE4 inhibition of compounds containing sulphonamide moiety has been studied earlier [16,17] the synthesis and PDE4 evaluation of isocoumarin derivatives containing aminosulfonyl/aminocarboxamide moiety was not explored previously.

Section snippets

Chemistry

The targeted new class of isocoumarin derivatives were synthesized via a Pd-catalyzed method similar to that developed by us recently (Scheme 1) [15]. This interesting approach involved the construction of a 4-allyl substituted 3-aryl isocoumarin ring starting from the appropriate 2-alkynyl benzamide derivative. Thus the commercially available terminal alkynes containing an aniline moiety were converted to the corresponding sulfonamides or amides (1a-k) (step-1 of Scheme 1) that on Sonogashira

Conclusion

In view of anti-inflammatory activities of isocoumarin derivatives a new class of PDE4B inhibitors was designed via introducing an aminosulfonamide/aminocarboxamide moiety into the C-3 benzene ring attached to an isocoumarin framework followed by docking of a representative compound into the PDE4B in silico. These compounds were synthesized via a PdCl2-catalyzed method involving the construction of a 4-allyl substituted 3-aryl isocoumarin ring starting from the appropriate 2-alkynyl benzamide

General procedure for the preparation of compound 1

Preparation of compound 1a-i: Appropriate Sulfonyl chloride (1.2 mmol) was added to a solution containing 3-ethynylaniline or 4-ethynylaniline (1.0 mmol), and pyridine (3.0 mmol) in DCM (10 mL) under a nitrogen atmosphere at 0 °C for 10 min. The resulting mixture was stirred at room temperature for overnight. After completion of the reaction (indicated by TLC) DCM was evaporated. The residue was treated with 2 N aqueous HCl (10 mL) and the mixture was diluted with ice-water (60 mL) and

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Authors acknowledge the financial support received from Department of Biotechnology, New Delhi, India (Grant No. BT/PR22126/BRB/10/1585/2017). Authors thank the Management of DRILS, Hyderabad, India, and Manipal University, Manipal, India for encouragement and support.

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