MOLPRINT 2D-based identification and synthesis of novel chromene based small molecules that target PLA2: validation through chemo- and bioinformatics approaches

Abstract

Phospholipase A2 (PLA2) is known to regulate inflammation and hence it is considered as a validated drug-target by medicinal chemists. In this report, we have identified and considered a highly ranked ligand from the ZINC-drug-like compounds database that targets PLA2 via the MOLPRINT-2D based chemoinformatics drug-design approach. The computationally predicted lead molecule was found to contain a core moiety of a chromene ring, which is well known for its varied biological properties. Here, a novel and efficient retro-synthetic protocol for the synthesis of highly substituted chromene libraries was made. A one-pot synthesis of chromene was carried out using different aromatic primary alcohols, malononitrile and 4-hydroxy coumarin in the presence of a mild oxidant mixture called T3P®–DMSO, followed by a Suzuki coupling reaction to obtain the lead molecules. All of the tested compounds of the chromene series displayed inhibition of the venom PLA2 in the range of 12 to 68 μM. Among the tested compounds, 2-amino-4-(2′-methyl-[1,1′-biphenyl]-4-yl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (7b) showed maximum inhibitory efficacy against venom PLA2 with an IC₅₀ value of 12.5 μM. Furthermore, the designed PLA2 ligands bound to the active site of venom PLA2, whose binding affinity was comparable to nimesulide, indicating that the chromene moiety containing ligands could be novel lead-structures that serve as anti-inflammatory agents.

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Introduction

Phospholipase A2 (PLA2) is the most-studied membrane-bound enzyme and has a molecular weight of 14 kDa.^1–3 It is a Ca²⁺-dependent and disulphide-rich enzyme, and is mostly present in mammalian tissues and in the venoms of insects and snakes.⁴ It hydrolyzes phospholipids from cellular membranes and lipoproteins at the sn-2 position, releasing lysophospholipids and free fatty acids.⁵ PLA2s are known to aid the production of eicosanoids, prostaglandins, leukotrienes and platelet-activating factors (PAFs), which exert a hormone-like function, orchestrating various physiological events at lower concentrations.² However, higher levels may induce serious pathological conditions such as inflammation, arthritis, atherosclerosis and sepsis. Hence, PLA2, along with cyclooxygenases and lipoxygenases, regulates inflammation and associated inflammatory diseases. Among the secretary PLA2s, group IIA is known to play a key role in both acute and chronic inflammation, which is regulated by multiple intracellular signalling cascades.⁶ Besides regulation of human diseases, snake venom pathology is largely attributed to the presence of PLA2. Snake venoms have been demonstrated to be a complex mixture of PLA2, matrix metalloproteinase, hyaluronidases and other toxic non-enzymatic peptides.⁷ The combined action of these enzymatic and non-enzymatic venom components are known to induce proteolysis, haemorrhaging, necrosis, altered haemostasis, shock and several other neurological dysfunctions.⁸ Among them, venom-induced necrosis, edema and anticoagulation are directly accredited to the myotoxic PLA2 and other myotoxins present in the venom.

In view of this, the inhibition of PLA2 has been considered as a prime target in the management of inflammatory diseases and snakebites. Thus, the aim of research in this field is to identify safe and effective PLA2 inhibitors. The high structural similarity between snake venom sPLA2 and that of humans suggests using snake venom PLA2 inhibitors to design novel drugs aimed at human inflammatory diseases and vice versa.^9,10 Also, the active site of sPLA2 is composed of the substrate binding hydrophobic region and the substrate cleaving hydrophilic region, hence it is a further requirement that a PLA2 inhibitor must bear hydrophobic and hydrophilic moieties in it.¹¹ To date, several synthetic¹² and natural¹³ inhibitors have been reported as PLA2 inhibitors. However this search had limited success in finding a novel class molecules which bear both hydrophobic and hydrophilic moieties.

In continuation of our ongoing reporting on synthetic inhibitors that target PLA2^14,15 and other drug-targets,^16–22 herein we report the design of novel drug-like small molecules via a chemoinformatics approach, followed by synthesis of the lead structure via a retro-synthetic approach as the starting point. Furthermore, we validate the efficacy of these inhibitors against snake venom VRV-PL-VIIIa PLA2 isolated from Vipera russelli venom and driven by the in silico molecular interaction studies, this enabled us to discover novel inhibitors that target PLA2.

Results and discussion

MOLPRINT 2D-based identification of drug-like compounds targeting PLA2

A MOLPRINT-2D PLA2 model was queried with the drug-like molecules of the ZINC database.²³ The top ranked compounds are summarized in Table 1 & Fig. 1. Among the ranked compounds, ZINC00625534 (DCMB) was ranked 4^th. This DCMB was considered as the lead molecule as the other top ranked compounds failed to contain both hydrophobic and hydrophilic moieties. Since DCMB contained an ester linkage, we designed and prepared DCMB analogues.

Table 1 List of ranked compounds targeting PLA2 based on the designing of MOLPRINT 2D

Rank	ZINC accession	Compound name	Structure	Probability of activity
1	ZINC00299345	2-(Phenoxycarbonyl)phenyl 2-furoate		2531.0
2	ZINC08427108	(5R,5′R)-5,5′-Oxybis(3,4-dibromofuran-2(5H)-one)		2295.8
3	ZINC08427105	(5S,5′S)-5,5′-Oxybis(3,4-dibromofuran-2(5H)-one)		2295.8
4	ZINC00625534	4-(2-Amino-3-cyano-5-oxo-4H,5H-pyrano[3,2-c]chromen-4-yl)-2-methoxyphenyl benzoate		1819.7

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Fig. 1 Structural representation of the in silico ranked structure ZINC0062553 (DCMB) and its analogues.

Synthesis of DCMB analogues

The lead PLA2 inhibitor ZINC00625534 (DCMB) contains hydrophilic amino, lactone, nitrile, methoxy and ester groups as well as hydrophobic aromatic rings. To achieve an effective interaction between the sPLA2 active site and the inhibitor, we have replaced the ester group of DCMB with a C–C bond, which enhances the hydrophobicity of the inhibitor. To start the synthesis of the DCMB analogues (2-amino chromene-3-carbonitriles), we employed a retro-synthetic approach (ESI Fig. S1†) where primary alcohol 1 and malononitrile (2) reacts in the presence of T3P®–DMSO and ethyl acetate as solvent to give the Swern oxidized product, which upon further Knoevenagel condensation gives product 3.²⁴ The intermediate (3) undergoes Michael cyclization with 4-hydroxy coumarin (4) to form compound 5. Furthermore, a Suzuki coupling reaction of 5 with aromatic or pyridine boronic acids (6), where we have used [1,10-bis(diphenylphosphino)ferrocene]dichloro palladium catalyst (Pd(dppf)Cl₂) and SCS-Bi₂O₃ as the base in tetrahydrofuran as the solvent, gave the DCMB analogue 7.²⁵ The detailed chemical synthesis and characterization of the DCMB analogs is presented in Fig. 2.

Fig. 2 Synthesis scheme for the DCMB analogues 7(a–n).

Neutralization of snake venom PLA2 using the lead molecule libraries

In order to test the efficacy of the synthesized inhibitors, they were tested against a snake venom PLA2 called VRV-PL-VIIIa, isolated from Vipera russelli venom. The inhibitory effects of the series of lead molecules against PLA2 were tabulated (Table 2). All of the tested compounds displayed inhibition of the venom PLA2 in the range of 12 to 68 μM. Among the tested compounds, 7b showed maximum inhibitory efficacy against PLA2 with an IC₅₀ value of 12.5 μM (Table 3). To date, several inhibitors from synthetic materials, and also from various organisms including marine sponges, snakes, bees, plants and mammals have been reported. However, this happens to be the first report of the novel chromene molecule, which displayed a potent inhibition of snake venom PLA2.

Table 2 Newly synthesized DCMB analogues

Entry	1	6	7(a–n)	Yield^a%
a Isolated.
1				92
2				90
3				89
4				84
5				85
6				89
7				93
8				91
9				92
10				90
11				84
12				87
13				86
14				83

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Table 3 In vitro inhibition of PLA2 by DCMB analogues

Sl. No	Ligand	IC50 (μM)
a ND, not determined.
1	7a	13.61 μM
2	7b	12.50 μM
3	7c	22.67 μM
4	7d	ND^a
5	7e	ND
6	7f	40.35 μM
7	7g	63.85 μM
8	7h	Inactive
9	7i	57.03 μM
10	7j	18.95 μM
11	7k	ND
12	7l	ND
13	7m	20.54 μM
14	7n	18.94 μM

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In silico interaction studies of novel ligands that target PLA2

In order to understand the structure-based correlation with compound affinity, we conducted molecular docking studies using the crystal structure of the PLA2 from Russell’s viper that bound to nimesulide (PDB 1ZWP).²⁶ The chromene ligands were docked into the PLA2 structure using MOE.²⁷ We found the cyano functionality of our ligand series consistently replaced the nitro group of nimesulide and formed hydrogen bonds to the backbone nitrogen of Gly-32. The exposed binding site of PLA2 allows for two binding modes of our compounds that include these interactions (see Fig. 3). Both of them form π–π interactions with the readily accessible indole moiety of Trp-31. Different orientations of the core ring system allow the amine functional group of the ligands to form hydrogen bonds to either the carboxylate of Asp-49 or to the backbone carbonyl of Gly-30. Both predicted binding modes are shown in Fig. 3 for the two compounds 7a and 7b with the highest PLA2 inhibition in vitro. Structure–activity relationships within the ligand series are not straightforward to interpret as group-wise contributions since the binding modes of the ligands might change and thus give rise to different molecular interactions between PLA2 and the respective compounds.

Fig. 3 Molecular interaction studies of novel ligands that target PLA2. Predicted molecular interactions between PLA2 and DCMB analogues: PLA2 is shown as the grey cartoon with a semi-transparent surface representation. The main interaction centres Asp-49, Gly-32, Trp-31, and Gly-30 (from left to right) are highlighted as lines in atomic colouring. (A) Binding mode of nimesulide within the co-crystal structure used for docking (PDB 1ZWP⁴). 7a and 7b are predicted to bind in two different modes to PLA2. Both of them replace the nitro group of nimesulide with a cyano group and show π–π interactions with Trp-31 but form different molecular interactions via the amine group (7a: Asp-49, 7b: Gly-30).

Materials and methods

In silico design of novel small molecules that target PLA2

The ligand similarity searching protocol, as implemented in MOLPRINT-2D, was trained using bioactivity data from the ChEMBL database for the target PLA2. Bioactivity training data was extracted from the ChEMBL16 database where activity values (IC₅₀/EC₅₀/K_i/K_d) were less than or equal to 10 μM, and a ChEMBL confidence score was 8 or greater for ‘binding’ or ‘functional’ assays, giving 2499 active compounds. 1426 compounds exceeded the 10 μM threshold, which were considered to be inactive and used as negative bioactivity training data. MOLPRINT 2D descriptors were generated for the complete data set of active and inactive compounds.^28,29 The Naïve Bayes learner was subsequently trained on the training compounds and queried with the MOLPRINT 2D fingerprints of 7228 drug-like compounds from the ZINC database. The ZINC molecules were ranked in terms of ‘probability of activity’ scores generated by models. A 10-fold cross validation with a 50/50 random split of both active and inactive structures was performed, confirming the predictive power of the models.

Chemical synthesis

All reagents were commercially available reagent grade and were used without further purification. Thin layer chromatography (TLC) was conducted on 0.25 mm silica gel plates (60F254, Merck). Column chromatography separations were performed on silica gel (200–400 mesh). IR spectra were recorded on a Bruker FTIR spectrophotometer. ¹H NMR spectra were recorded on a Bruker Avance-300 instrument in CDCl₃ solvent. ¹³C NMR spectra were obtained on a Bruker Avance-300 instrument at 75 MHz in DMSO-D₆ solvent (a few on an Agilent NMR instrument in CDCl₃ solvent). Chemical shifts are expressed in ppm downfield relative to TMS. Mass spectra were recorded on an Agilent LC-MS and the elemental analyses were carried out using an Elemental Vario Cube CHNS rapid Analyzer.

General procedure for synthesis of the DCMB analogues. To the solvent mixture of ethyl acetate and DMSO (1.5 ml : 0.75 ml = 2 : 1 ratio) 4-bromo/3-bromo-4-methoxy benzyl alcohol 1 (1.0 mmol) and malononitrile 2 (1.2 mmol) are added in the presence of T3P® (2.5 mmol, 50% solution in ethyl acetate) at room temperature, which undergoes an in situ Swern oxidation followed by a Knoevenagel condensation to yield the corresponding alkene, 3, within 10 minutes. Complete formation of alkene 3 was confirmed with TLC using a hexane : EtOAc (7 : 3) system and was observed at R_f 0.78 under ultraviolet (UV) light. Without isolating alkene 3, to this reaction mixture 4-hydroxy coumarin, 4, (1.0 mmol) was added and stirred for 2–3 hours at room temperature to form compound 5.²⁴ The reaction was monitored using TLC (hexane : EtOAc 7 : 3) and compound 5 was observed at R_f 0.42 under UV light. After completion of the reaction, the mixture was diluted with about 5 ml of distilled water. The product was extracted with 10 ml of ethyl acetate and the combined organic layers were washed with 10 ml of distilled water, followed by 5 ml of a brine solution. The organic phase was dried over anhydrous Na₂SO₄. The solvent was removed under reduced pressure to afford a pure product. Compound 5 was obtained with 98% yield.

Subsequently, compound 5 (1 mmol) was heated to 70 °C with a variety of aryl/hetero boronic acids 6 (1.2 mmol) in the presence of Pd(dppf)₂Cl₂ as catalyst (0.001 mmol), SCS-Bi₂O₃ (0.5 mmol) as base in 1 ml water and 4 ml tetrahydrofuran as solvent for 8–10 hours to obtain crude DCMB analogues (7a–n) (Table 2). The formation of the final products (7a–n) was monitored using TLC (hexane : EtOAc 8 : 2). This was further purified by column chromatography using hexane : ethyl acetate as the eluent. The final product (7a–n) was eluted with a 15% hexane : ethyl acetate system (85 ml hexane : 15 ml ethyl acetate). The obtained DCMB analogues were enantiomers and were isolated as racemic mixtures. These DCMB analogues were confirmed using spectral analysis without separation of the racemic mixtures. Spectral properties were consistent with their assigned structures.

7a 4-([1,1′-biphenyl]-4-yl)-2-amino-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile. This compound was obtained by heating 2-amino-4-(4-bromophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (1 mmol) (compound 5) and phenyl boronic acid 6 (1.2 mmol) at 70 °C in the presence of Pd(dppf)₂Cl₂ as catalyst (0.001 mmol), SCS-Bi₂O₃ (0.5 mmol) as base in 1 ml water and 4 ml tetrahydrofuran as solvent for 8 hours. 7a was observed using TLC at R_f 0.58 under UV light and was isolated as a white solid using column chromatography with a 15% hexane : ethyl acetate system (85 ml hexane : 15 ml ethyl acetate).

IR ν_max: 3323 cm⁻¹ ν_(NH2), 2194 cm⁻¹ ν_(CN), 1673 cm⁻¹ ν_(C–O), 1049 cm⁻¹ ν_(C=O); ¹H NMR (CDCl₃, 300 MHz): δ 7.912–7.206 (m, 13H, Ar-H), 4.856 (s, 1H, methine); ¹³C NMR (CDCl₃, 75 MHz) δ 161.13, 160.57, 158.53, 156.08, 148.44, 145.95, 134.46, 130.35, 129.75, 128.17, 126.57, 125.46, 124.53, 123.48, 117.66, 112.60, 103.83, 60.20, 35.13; LCMS (MM: ES + APCI) (M + H)⁺ 393; anal. calcd for C₂₅H₁₆N₂O₃: C 76.62, H 4.11, N 7.14; found: C 76.14, H 4.19, N 7.42.

7b 2-amino-4-(2′-methyl-[1,1′-biphenyl]-4-yl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile. This compound was obtained by heating 2-amino-4-(4-bromophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (1 mmol) (compound 5) and o-tolylboronic acid 6 (1.2 mmol) at 70 °C in the presence of Pd(dppf)₂Cl₂ as catalyst (0.001 mmol), SCS-Bi₂O₃ (0.5 mmol) as base in 1 ml water and 4 ml tetrahydrofuran as solvent for 7 hours. 7b was observed using TLC at R_f 0.59 under UV light and was isolated as a yellow solid using column chromatography with a 15% hexane : ethyl acetate system (85 ml hexane : 15 ml ethyl acetate).

IR ν_max: 3256 cm⁻¹ ν_(NH2), 2196 cm⁻¹ ν_(CN), 1680 cm⁻¹ ν_(C–O), 1047 cm⁻¹ ν_(C=O); ¹H NMR (CDCl₃, 300 MHz): δ 7.831–7.295 (m, 12H, Ar-H), 4.161 (s, 1H, methine), 3.295 (s, 2H, –NH₂), 2.254 (s, 3H, –CH₃); LCMS (MM: ES + APCI) (M + H)⁺ 407; anal. calcd for C₂₆H₁₈N₂O₃: C 76.83, H 4.46, N 6.89; found: C 76.91, H 4.53, N 6.81.

7c 2-amino-4-(3′-methoxy-[1,1′-biphenyl]-4-yl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile. This compound was obtained by heating 2-amino-4-(4-bromophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (1 mmol) (compound 5) and (3-methoxyphenyl)boronic acid 6 (1.2 mmol) at 70 °C in the presence of Pd(dppf)₂Cl₂ as catalyst (0.001 mmol), SCS-Bi₂O₃ (0.5 mmol) as base in 1 ml water and 4 ml tetrahydrofuran as solvent for 8 hours. 7c was observed using TLC at R_f 0.52 under UV light and was isolated as a white solid using column chromatography with a 16% hexane : ethyl acetate system (84 ml hexane : 16 ml ethyl acetate).

IR ν_max: 3293 cm⁻¹ ν_(NH2), 2193 cm⁻¹ ν_(CN), 1673 cm⁻¹ ν_(C–O), 1048 cm⁻¹ ν_(C=O); ¹H NMR (CDCl₃, 300 MHz): δ 8.000–7.978 (m, 3H, Ar-H), 7.792–7.746 (m, 2H, Ar-H), 7.552–7.521 (m, 2H, Ar-H), 7.434–7.394 (m, 4H, Ar-H), 7.257 (s, 1H, Ar-H), 3.884 (s, 1H, methine), 3.846 (s, 3H, –OCH₃); ¹³C NMR (DMSO-D₆, 75 MHz) δ 162.52, 160.19, 143.72, 134.25, 131.56, 126.11, 125.87, 125.09, 124.35, 123.46, 122.92, 121.62, 120.95, 120.12, 117.57, 114.29, 112.52, 99.35, 57.28, 52.25, 35.28; LCMS (MM: ES + APCI) (M + H)⁺ 423; anal. calcd for C₂₆H₁₈N₂O₄: C 73.92, H 4.29, N 6.63; found: C 73.88, H 4.32, N 6.59.

7d 2-amino-5-oxo-4-(2′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile. This compound was obtained by heating 2-amino-4-(4-bromophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (1 mmol) (compound 5) and (2-(trifluoromethyl)phenyl)boronic acid 6 (1.2 mmol) at 70 °C in the presence of Pd(dppf)₂Cl₂ as catalyst (0.001 mmol), SCS-Bi₂O₃ (0.5 mmol) as base in 1 ml water and 4 ml tetrahydrofuran as solvent for 9 hours. 7d was observed using TLC at R_f 0.50 under UV light and was isolated as a grey solid using column chromatography with a 16% hexane : ethyl acetate system (84 ml hexane : 16 ml ethyl acetate).

IR ν_max: 3292 cm⁻¹ ν_(NH2), 2199 cm⁻¹ ν_(CN), 1669 cm⁻¹ ν_(C–O), 1033 cm⁻¹ ν_(C=O); ¹H NMR (CDCl₃, 300 MHz): δ 7.831–7.260 (m, 12H, Ar-H), 4.714 (s, 1H, methine); ¹³C NMR (CDCl₃, 75 MHz) δ 162.57, 161.43, 159.27, 158.12, 143.29, 141.25, 135.04, 132.81, 128.77, 126.92, 126.03, 125.12, 124.69, 123.14, 121.20, 120.07, 118.77, 116.83, 110.32, 100.05, 58.23, 38.48; LCMS (MM: ES + APCI) (M + H)⁺ 461; anal. calcd for C₂₆H₁₅F₃N₂O₃: C 67.83, H 3.28, N 12.38; found: C 67.90, H 3.31, N 12.41.

7e 2-amino-4-(4′-chloro-3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile. This compound was obtained by heating 2-amino-4-(4-bromophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (1 mmol) (compound 5) and (4-chloro-3-(trifluoromethyl)phenyl)boronic acid 6 (1.2 mmol) at 70 °C in the presence of Pd(dppf)₂Cl₂ as catalyst (0.001 mmol), SCS-Bi₂O₃ (0.5 mmol) as base in 1 ml water and 4 ml tetrahydrofuran as solvent for 8.5 hours. 7e was observed using TLC at R_f 0.50 under UV light and was isolated as a white solid using column chromatography with a 16% hexane : ethyl acetate system (84 ml hexane : 16 ml ethyl acetate).

IR ν_max: 3293 cm⁻¹ ν_(NH2), 2200 cm⁻¹ ν_(CN), 1667 cm⁻¹ ν_(C–O), 1050 cm⁻¹ ν_(C=O); ¹H NMR (CDCl₃, 300 MHz): δ 7.842–7.260 (m, 11H, Ar-H), 4.851 (s, 1H, methine); ¹³C NMR (CDCl₃, 75 MHz) δ 161.08, 160.50, 158.19, 141.89, 135.08, 132.50, 130.19, 128.11, 127.47, 126.09, 124.05, 123.74, 122.29, 121.08, 119.85, 117.64, 115.24, 111.48, 101.56, 60.15, 36.22; LCMS (MM: ES + APCI) (M − H)⁻ 493; anal. calcd for C₂₆H₁₄ClF₃N₂O₃: C 63.11, H 2.85, N 5.66; found: C 63.15, H 2.89, N 5.70.

7f 2-amino-5-oxo-4-(4-(pyridin-3-yl)phenyl)-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile. This compound was obtained by heating 2-amino-4-(4-bromophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (1 mmol) (compound 5) and pyridin-3-ylboronic acid 6 (1.2 mmol) at 70 °C in the presence of Pd(dppf)₂Cl₂ as catalyst (0.001 mmol), SCS-Bi₂O₃ (0.5 mmol) as base in 1 ml water and 4 ml tetrahydrofuran as solvent for 8.5 hours. 7f was observed using TLC at R_f 0.55 under UV light and was isolated as a white solid using column chromatography with a 15% hexane : ethyl acetate system (85 ml hexane : 15 ml ethyl acetate).

IR ν_max: 3323 cm⁻¹ ν_(NH2), 2195 cm⁻¹ ν_(CN), 1668 cm⁻¹ ν_(C–O), 1042 cm⁻¹ ν_(C=O); ¹H NMR (CDCl₃, 300 MHz): δ 8.922–8.918 (s, 1H, Ar-N–CH), 8.688–8.673 (d, 1H, Ar-H), 8.568–8.548 (d, 1H, Ar-H), 7.996–7.961 (m, 2H, Ar-H), 7.869–7.846 (m, 1H, Ar-H), 7.586–7.589 (m, 3H, Ar-H), 7.428–7.370 (m, 3H, Ar-H), 4.747 (s, 1H, methine), 1.566 (s, 2H, –NH₂); LCMS (MM: ES + APCI) (M + H)⁺ 394; anal. calcd for C₂₄H₁₅N₃O₃: C 73.27, H 3.84, N 10.68; found: C 73.31; H, 3.88; N, 10.73.

7g 2-amino-4-(4-(5,6-dichloropyridin-3-yl)phenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile. This compound was obtained by heating 2-amino-4-(4-bromophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (1 mmol) (compound 5) and (4,6-dichloropyridin-3-yl)boronic acid 6 (1.2 mmol) at 70 °C in the presence of Pd(dppf)₂Cl₂ as catalyst (0.001 mmol), SCS-Bi₂O₃ (0.5 mmol) as base in 1 ml water and 4 ml tetrahydrofuran as solvent for 9.5 hours. 7g was observed using TLC at R_f 0.51 under UV light and was isolated as a white solid using column chromatography with a 16% hexane : ethyl acetate system (84 ml hexane : 16 ml ethyl acetate).

IR ν_max: 3320 cm⁻¹ ν_(NH2), 2192 cm⁻¹ ν_(CN), 1665 cm⁻¹ ν_(C–O), 1046 cm⁻¹ ν_(C=O); ¹H NMR (CDCl₃, 300 MHz): δ 8.625 (s, 1H, Ar-N–H), 8.462 (s, 1H, Ar-H), 7.772 (m, 1H, Ar-H), 7.595–7.501 (m, 1H, Ar-H), 7.386–7.324 (m, 2H, Ar-H), 7.003–6.896 (m, 3H, Ar-H), 3.725 (s, 1H, methine); LCMS (MM: ES + APCI) (M + H)⁺ 463; anal. calcd for C₂₄H₁₃Cl₂N₃O₃: C 62.35, H 2.83, N 9.09; found: C 62.31, H 2.79, N 9.12.

7h 2-amino-4-(6-methoxy-[1,1′-biphenyl]-3-yl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile. This compound was obtained by heating 2-amino-4-(3-bromo-4-methoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (1 mmol) (compound 5) and phenyl boronic acid 6 (1.2 mmol) at 70 °C in the presence of Pd(dppf)₂Cl₂ as catalyst (0.001 mmol), SCS-Bi₂O₃ (0.5 mmol) as base in 1 ml water and 4 ml tetrahydrofuran as solvent for 8 hours. 7h was observed using TLC at R_f 0.56 under UV light and was isolated as a yellow solid using column chromatography with a 15% hexane : ethyl acetate system (85 ml hexane : 15 ml ethyl acetate).

IR ν_max: 3286 cm⁻¹ ν_(NH2), 2197 cm⁻¹ ν_(CN), 1667 cm⁻¹ ν_(C–O), 1052 cm⁻¹ ν_(C=O); ¹H NMR (CDCl₃, 300 MHz): δ 7.797–7.776 (s, 1H, Ar-H), 7.772–7.7.52 (m, 1H, Ar-H), 7.491–7.467 (m, 2H, Ar-H), 7.374–7.336 (m, 6H, Ar-H), 7.192–7.186 (m, 1H, Ar-H), 6.939–0.917 (m, 1H, Ar-H), 3.867 (s, 1H, methine), 3.750–3.715 (s, 3H, –OCH₃); LCMS (MM: ES + APCI) (M − H)⁻ 421; anal. calcd for C₂₆H₁₈N₂O₄: C 73.92, H 4.29, N 6.63; found: C 73.88, H4.26, N 6.67.

7i 2-amino-4-(6-methoxy-2′-methyl-[1,1′-biphenyl]-3-yl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile. This compound was obtained by heating 2-amino-4-(3-bromo-4-methoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (1 mmol) (compound 5) and o-tolylboronic acid 6 (1.2 mmol) at 70 °C in the presence of Pd(dppf)₂Cl₂ as catalyst (0.001 mmol), SCS-Bi₂O₃ (0.5 mmol) as base in 1 ml water and 4 ml tetrahydrofuran as solvent for 8.5 hours. 7i was observed using TLC at R_f 0.57 under UV light and was isolated as a white solid using column chromatography with a 15% hexane : ethyl acetate system (85 ml hexane : 15 ml ethyl acetate).

IR ν_max: 3288 cm⁻¹ ν_(NH2), 2196 cm⁻¹ ν_(CN), 1668 cm⁻¹ ν_(C–O), 1057 cm⁻¹ ν_(C=O); ¹H NMR (CDCl₃, 300 MHz): δ 7.775–7.758 (m, 1H, Ar-H), 7.356–7.348 (m, 3H, Ar-H), 7.260 (m, 3H, Ar-H), 7.030–6.902 (m, 4H, Ar-H), 4.629 (1H, methine, s), 3.734 (3H, –OCH₃, s), 2.069 (3H, –CH₃, s); LCMS (MM: ES + APCI) (M − H)⁻ 435; anal. calcd for C₂₇H₂₀N₂O₄: C 74.30, H 4.62, N 6.42; found: C 74.28, H 4.65, N 6.46.

7j 2-amino-4-(3′,6-dimethoxy-[1,1′-biphenyl]-3-yl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile. This compound was obtained by heating 2-amino-4-(3-bromo-4-methoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (1 mmol) (compound 5) and (3-methoxyphenyl)boronic acid 6 (1.2 mmol) at 70 °C in the presence of Pd(dppf)₂Cl₂ as catalyst (0.001 mmol), SCS-Bi₂O₃ (0.5 mmol) as base in 1 ml water and 4 ml tetrahydrofuran as solvent for 9.5 hours. 7j was observed using TLC at R_f 0.50 under UV light and was isolated as a brown solid using column chromatography with a 16% hexane : ethyl acetate system (84 ml hexane : 16 ml ethyl acetate).

IR ν_max: 3287 cm⁻¹ ν_(NH2), 2197 cm⁻¹ ν_(CN), 1669 cm⁻¹ ν_(C–O), 1053 cm⁻¹ ν_(C=O); ¹H NMR (CDCl₃, 300 MHz): δ 7.791–7.773 (s, 1H, Ar-H), 7.613–7.574 (m, 2H, Ar-H), 7.354–7.335 (m, 3H, Ar-H), 7.260 (m, 1H, Ar-H), 7.065–7.037 (m, 2H, Ar-H), 6.936–6.847 (m, 2H, Ar-H), 6.847 (m, 11H, Ar-H), 3.811 (s, 1H, methine), 3.779 (s, 6H –OCH₃); ¹³C NMR (DMSO-D₆, 75 MHz) δ 161.52, 160.25, 154.12, 152.74, 132.13, 126.98, 126.17, 125.01, 124.45, 123.85, 123.11, 122.92, 121.51, 119.25, 116.52, 113.32, 110.59, 100.50, 60.15, 56.72, 55.91, 36.71; LCMS (MM: ES + APCI) (M − H)⁻ 451; anal. calcd for C₂₇H₂₀N₂O₅: C 71.67, H 4.46, N 6.19; found: C 71.71, H 4.41, N 6.21.

7k 2-amino-4-(6-methoxy-2′-(trifluoromethyl)-[1,1′-biphenyl]-3-yl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile. This compound was obtained by heating 2-amino-4-(3-bromo-4-methoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (1 mmol) (compound 5) and (2-(trifluoromethyl)phenyl)boronic acid 6 (1.2 mmol) at 70 °C in the presence of Pd(dppf)₂Cl₂ as catalyst (0.001 mmol), SCS-Bi₂O₃ (0.5 mmol) as base in 1 ml water and 4 ml tetrahydrofuran as solvent for 8.2 hours. 7k was observed using TLC at R_f 0.48 under UV light and was isolated as a brown solid using column chromatography with a 17% hexane : ethyl acetate system (83 ml hexane : 17 ml ethyl acetate).

IR ν_max: 3301 cm⁻¹ ν_(NH2), 2198 cm⁻¹ ν_(CN), 1671 cm⁻¹ ν_(C–O), 1053 cm⁻¹ ν_(C=O); ¹H NMR (CDCl₃, 300 MHz): δ 7.925–7.885 (m, 2H, Ar-H), 7.815 (s, 1H, Ar-H), 7.651–7.552 (m, 2H, Ar-H), 7.450–7.321 (m, 3H, Ar-H), 7.250 (m, 2H, Ar-H), 6.982 (s, 1H, Ar-H), 4.297 (s, 1H, methine), 3.306 (s, 3H, –OCH₃); ¹³C NMR (CDCl₃, 75 MHz) δ 162.19, 161.28, 159.14, 158.34, 133.65, 131.28, 129.52, 125.87, 125.19, 124.57, 124.01, 123.85, 123.08, 122.12, 117.12, 115.28, 103.52, 59.28, 55.71, 36.17; LCMS (MM: ES + APCI) (M + H)⁺ 491; anal. calcd for C₂₇H₁₇F₃N₂O₄: C 66.12, H 3.49, N 5.71; found: C 66.13, H 3.52, N 5.73.

7l 2-amino-4-(4′-chloro-6-methoxy-3′-(trifluoromethyl)-[1,1′-biphenyl]-3-yl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile. This compound was obtained by heating 2-amino-4-(3-bromo-4-methoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (1 mmol) (compound 5) and (4-chloro-3-(trifluoromethyl)phenyl)boronic acid 6 (1.2 mmol) at 70 °C in the presence of Pd(dppf)₂Cl₂ as catalyst (0.001 mmol), SCS-Bi₂O₃ (0.5 mmol) as base in 1 ml water and 4 ml tetrahydrofuran as solvent for 9 hours. 7l was observed using TLC at R_f 0.47 under UV light and was isolated as a white solid using column chromatography with a 17% hexane : ethyl acetate system (83 ml hexane : 17 ml ethyl acetate).

IR ν_max: 3288 cm⁻¹ ν_(NH2), 2198 cm⁻¹ ν_(CN), 1671 cm⁻¹ ν_(C–O), 1055 cm⁻¹ ν_(C=O); ¹H NMR (CDCl₃, 300 MHz): δ 7.819–7.799 (d, 1H, Ar-H), 7.638–7.616 (m, 2H, Ar-H), 7.599–7.571 (m, 2H, Ar-H), 7.504 (m, 2H, Ar-H), 7.392–7.510 (m, 2H, Ar-H), 6.869–6.848 (s, 1H, Ar-H), δ 3.870 (s, 1H, methine), δ 3.787 (s, 3H –OCH₃); ¹³C NMR (DMSO-D₆, 75 MHz) δ 161.57, 160.16, 158.91, 144.15, 135.46, 133.48, 132.19, 125.45, 124.89, 124.25, 123.10, 122.59, 121.85, 121.41, 119.32, 116.28, 114.11, 113.42, 101.41, 59.12, 55.72, 36.29; LCMS (MM: ES + APCI) (M − H)⁻ 523; anal. calcd for C₂₇H₁₆ClF₃N₂O₄: C 61.78, H 3.07, N 5.34; found: C 61.74, H 3.05, N 5.30.

7m 2-amino-4-(4-methoxy-3-(pyridin-3-yl)phenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile. This compound was obtained by heating 2-amino-4-(3-bromo-4-methoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (1 mmol) (compound 5) and pyridin-3-ylboronic acid 6 (1.2 mmol) at 70 °C in the presence of Pd(dppf)₂Cl₂ as catalyst (0.001 mmol), SCS-Bi₂O₃ (0.5 mmol) as base in 1 ml water and 4 ml tetrahydrofuran as solvent for 8 hours. 7m was observed using TLC at R_f 0.51 under UV light and was isolated as a white solid using column chromatography with a 16% hexane : ethyl acetate system (84 ml hexane : 16 ml ethyl acetate).

IR ν_max: 3292 cm⁻¹ ν_(NH2), 2199 cm⁻¹ ν_(CN), 1674 cm⁻¹ ν_(C–O), 1066 cm⁻¹ ν_(C=O); ¹H NMR (CDCl₃, 300 MHz): δ 8.718–8.672 (s, 1H, Ar-H), 8.608–8.511 (s, 1H, Ar-H), 8.112–7.941 (m, 2H, Ar-H), 7.819–7.766 (m, 1H, Ar-H), 7.606–7.588 (m, 2H, Ar-H), 7.518–7.310 (m, 4H, Ar-H), 4.627 (s, 1H, methine), 3.656 (s, 3H –OCH₃) LCMS (MM: ES + APCI) (M − H)⁻ 422; anal. calcd for C₂₅H₁₇N₃O₄: C 70.91, H 4.05, N 9.92; found: C 70.89, H 4.09, N 9.90.

7n 2-amino-4-(3-(4,6-dichloropyridin-3-yl)-4-methoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile. This compound was obtained by heating 2-amino-4-(3-bromo-4-methoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (1 mmol) (compound 5) and (4,6-dichloropyridin-3-yl)boronic acid 6 (1.2 mmol) at 70 °C in the presence of Pd(dppf)₂Cl₂ as catalyst (0.001 mmol), SCS-Bi₂O₃ (0.5 mmol) as base in 1 ml water and 4 ml tetrahydrofuran as solvent for 8.5 hours. 7n was observed using TLC at R_f 0.48 under UV light and was isolated as a brown solid using column chromatography with a 16% hexane : ethyl acetate system (84 ml hexane : 16 ml ethyl acetate).

IR ν_max: 3228 cm⁻¹ ν_(NH2), 2193 cm⁻¹ ν_(CN), 1671 cm⁻¹ ν_(C–O), 1051 cm⁻¹ ν_(C=O); ¹H NMR (CDCl₃, 300 MHz): δ 8.601 (s, 1H, Ar-N–CH), 7.716–7.182 (m, 8H, Ar-H), 4.112 (s, 1H, methine), 3.603 (s, 3H, –OCH₃); LCMS (MM: ES + APCI) (M + H)⁺ 493; anal. calcd for C₂₅H₁₅Cl₂N₃O₄: C 60.99, H 3.07, N 8.54; found: C 60.96, H 3.05, N 8.59.

Substrate preparation. Briefly, a working solution of 1 mM DCMB analogues (substrate) was prepared in methanol containing 2 mM Triton X-100 in Milli-Q water. The resulting substrate solution was spun at 1500 × g for 5 min to form uniform mixed micelles.

LPC standard curve. The LPC (1-myristoyl-2-hydroxy-sn-glycerol-3-phosphocholine) standard curve was constructed using different LPC concentrations ranging from 0 to 100 μM, as reported earlier.³⁰ Briefly, a total reaction mixture of 100 μl containing an activity buffer (50 mM Tris, pH 7.5, 10 mM CaCl₂) and 2 mM LPC and 4 mM Triton X-100 (1 : 2 ratio) was incubated for 5 min at 37 °C. A quenching solution was added and vortexed for 30 s and incubated for 5 min at RT. 2 μl of the reaction mixture was pipetted to measure the RFU, as described earlier.

Neutralization of VRV-PL-VIIIa (sPLA2) by the title compounds. sPLA2 activity was assayed according to the known method.³⁰ Briefly, a 50 μl activity buffer containing 50 mM Tris–HCl buffer at pH 7.5, 10 mM CaCl₂ and 10 μl substrate stock were added and incubated for 5 min at 37 °C. Activity was initiated by adding 10 ng of sPLA2 alone or pre-incubated with a different concentration of a DCMB analogue, ranging from 0–120 μM for 5 min at 37 °C. The reaction mixture was incubated for 45 min at 37 °C. 50 μl of the quenching solution was added at a final concentration of 2 mM NaN₃, 50 μM ANS and 50 mM EGTA, vortexed for 30 s and incubated for 5 min at RT. 2 μl of this solution was pipetted to measure the relative fluorescence unit (RFU) in a Nano drop ND3300 Ver 2.8 using excitation with a UV-LED (370 ± 10 nm) and emission was recorded at 480 nm in dark conditions. Enzyme activity was calculated using eqn (1), where ΔRFU is the change in the RFU of the test (with sPLA2) with respect to the control (without sPLA2) in the presence of an inhibitor. The resultant RFU is compared with the standard curve of LPC to determine the sPLA2 activity in the presence of the inhibitor. A 4^th parameter logical (4PL) fit module of Graphpad Prism 6.05 was used to compute the IC₅₀ values.

ΔRFU_LPC = RFU_c − RFU_t (1)

Molecular docking studies. We docked the series of fourteen synthesized compounds to the crystal structure of PLA2 from Russell’s viper in complex with nimesulide (PDB 1ZWP). We used identical settings as in an earlier study on imidazopyridine¹⁴-based PLA2inhibitors docking in MOE. The protocol included a pharmacophore filter during docking to enforce a hydrogen bond acceptor feature in the position of the nitro group of nimesulide. The predicted binding modes were visualized in Pymol.³¹

Conclusion

In conclusion, we efficiently used the MOLPRINT-2D protocol and found the compound 7b, which showed maximum inhibitory efficacy against venom PLA2 with an IC₅₀ value of 12.5 μM. Structure-based in silico studies revealed reasonable binding modes of the ligands, the most active of which showed a binding affinity comparable to nimesulide. Overall, in this work we present a combination of in silico methods, novel synthetic routes, and experimental validation of PLA2 inhibitors to help modulate inflammatory diseases, and optimization of their biological activity is now envisaged as the next step.

Acknowledgements

This research was supported by University Grants Commission (41-257-2012-SR), Vision Group Science and Technology, Department of Science and Technology (No. SR/FT/LS-142/2012). Keerthy HK and Bharathkumar H thank the University Grants Commission for Basic Scientific Research Fellowships. Andreas Bender thanks Unilever and the European Research Commission (ERC Starting Grant 2013) for funding. Lewis Mervin thanks the BBSRC and AstraZeneca for funding.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra13085a

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