Supplementary material [303079]

Supplementary material

Discovery of synthetic bioactive novel hydroxyanisole pharmacophore series as a promising myeloperoxidase inhibitor (MPOI) targeting atherosclerotic CVD

Premkumar Jayaraj1, Sampath Parthasarathy2, Sanjay Rajagopalan3, Chandrakala Aluganti2, *, Rajagopal Desikan 1,2, *

1[anonimizat], [anonimizat] 632014, India.

2[anonimizat], [anonimizat], FL 32832, USA.

3[anonimizat], Cleveland, Ohio, USA.

(*) Corresponding authors: [anonimizat] & [anonimizat]

Captions:

S1 – In silico ADMET profile Values

S2 – In-Silico Molecular Docking Images

S3 – Chemical synthesis and Characterization

i) Synthetic Route-I

ii) [anonimizat]

S4 – NMR Spectral Data Images

S5 – ESI & LC-HRMS Spectral Data Images

S6 – RT-PCR Primers table

S7- UPLC-DAD purity graph for Lead molecules

S1: Molecular Docking Images

S1a: Compound-1

S1b: Compound-2

S1c: Compound-3

S1d: Compound-4

S1e: Compound-5

S1f: Compound-6

S1g: Compound-7

S1h: Compound-8

S1i: Compound-9

S2: In Silico ADMET profile Values:

TABLE S2 – A: Absorption and Distribution

*HIA- Human intestinal absorption; HOB- Human oral bioavailability; PPB- Plasma protein binding; Sub- Substrate; Inh- Inhibitor; BBB- Blood brain barrier; SCl- Subcellular localization.

TABLE S2 – B: Metabolism

*Inh- Inhibitor; Sub- Substrate

TABLE S2 – C: Toxicity

*hERG- Human either-a-go-go Inhibition; AOT- Acute oral toxicity; Ames- Ames Mutagenesis; CRC- Carcinogenicity (binary); THPT- Tetrahymena pyriformis; H-Bee T- Honey bee toxicity; Bio-D- Biodegradation; [anonimizat].

S3-Chemical synthesis and Characterization

i) Synthetic Route-I

General Synthetic procedure for compounds 1 – 9:

In a 100 [anonimizat] (such as) (194 mg, 1mmole) was taken in 15ml of dichloromethane along with thionyl chloride (217 µL, 3mmole) and refluxed for 3h at inert atmosphere. After the completion of reaction excess amount of thionyl chloride was removed by condensing process. [anonimizat]2SO4 and solvent evaporated under reduced pressure. [anonimizat] (212 mg, 1mmole) was suspended into 10 ml diethyl ether. Amines and/or alcohols (115 mg, 1mmole) in 1M of NaOH (10 ml) was added to acid chloride solution and the resultant mixture was stirred overnight at room temperature. The solvent was evaporated under reduced pressure and crude product was directly purified on column chromatography.

ii) [anonimizat] (E)-3-(4-hydroxy-3-methoxyphenyl)-N-(4-((6-methoxyquinolin-8-yl) amino) pentyl) acrylamide (1):

In a 50mL round bottom flask charged with primaqiune (259 mg, 1 mmole), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlorid (310 mg, 2 mmole), and dimethylaminopyridine (142 mg, 2 mmole) in 20 [anonimizat]. After stirring for 30 min, Ferulic acid (194 mg, 1 mmole) was added over a period of 30 min and whole mixture was allowed to stir at room temperature overnight. The progress of the reaction was monitored by TLC. The reaction mixture was extracted with dichloromethane further treated with brine solution and dried with anhydrous Mg2SO4 and organic layer was concentrated under reduced vacuum pressure. The resultant crude product was purified with column chromatography. The purified compounds were characterized with 1H and 13C NMR spectra and molecular mass was identified using high-resolution ESI mass spectrometer. The compound 1 was obtained with 79% yield as a brown solid. 1H NMR 400 MHz, CDCl3: δ 8.53 (d, J = 4.16 Hz, 1H), 7.92 (d, J = 8.16 Hz, 1H), 7.71 (q, J = 3.44 Hz, 1H), 7.54 (d, J = 3.48 Hz, 1H), 7.50 (d, J = 15.28 Hz, 1H), 7.31 (q, J = 4.28 Hz, 1H), 7.01 (d, J = 8.12 Hz, 1H), 6.96 (s, 1H), 6.89 (d, J = 8.12 Hz, 1H), 6.32 (d, J = 12.60 Hz, 1H), 6.15 (d, J = 15.60 Hz, 1H), 6.01 (d, J = 8.00 Hz, 1H), 5.67 (s, 1H), 4.26-4.25 (m, 2H), 3.90 (s, 3H), 3.86 (s, 3H), 3.65 (d, J = 4.96 Hz, 1H), 3.45-3.43 (m, 2H), 1.47-1.46 (m, 2H), 1.31 (d, J = 6.48 Hz, 5H). 13C NMR 100 MHz, CDCl3: δ 167.77, 159.45, 146.65, 144.37, 140.77, 134.83, 132.47, 130.88, 128.81, 127.48, 121.98, 121.90, 118.41, 114.66, 109.70, 96.94, 91.84, 68.17, 55.93, 55.22, 38.75, 30.37, 23.76, 22.99. ESI Mass: Calculated mass: 435.22, Obtained mass: 435.16.

Synthesis of (E)-3-(3-hydroxy-4-methoxyphenyl)-N-(4-((6-methoxyquinolin-8-yl) amino) pentyl) acrylamide (2):

Compound-2 was achieved by coupling primaquine with Iso-Ferulic acid. Refer the compound 1 (synthetic route-II) procedure and same methodology was followed to prepare the compound. The final compound 2 was archived with 74% green color solid. 1H NMR 400 MHz, CDCl3: δ 7.04 (d, J = 8.53 Hz, 1H), 7.95 (d, J = 7.84 Hz, 1H), 7.53 (q, J = 3.36 Hz, 1H), 7.48 (d, J = 15.52 Hz, 1H), 7.32 (q, J = 4.32 Hz, 1H), 7.06 (s, 1H), 6.96 (d, J = 8.20 Hz, 1H), 6.81 (d, J = 8.24 Hz, 1H), 6.33 (d, J = 16.84 Hz, 2H), 6.09 (d, J = 15.68 Hz, 1H), 5.68 (s, 1H), 4.26-4.25 (m, 1H), 3.91 (s, 3H), 3.87 (s, 3H), 3.66 (s, 1H), 3.46-3.44 (m, 2H), 1.47-1.46 (m, 2H), 1.31 (d, J = 6.36 Hz, 4H), ESI Mass: Calculated mass: 435.22, measured mass: 435.16.

Synthesis of (E)-3-(4-hydroxy-3-methoxyphenyl) allyl 2-((3-(trifluoromethyl) phenyl) amino) nicotinate (3):

Compound-3 was achieved by coupling coniferyl alcohol with niflumic acid. Refer the compound 1 (synthetic route-II) procedure and same methodology was followed to prepare the compound. The final compound-3 was archived with 81% yellow color solid. 1H NMR 400 MHz, CDCl3: δ 10.24 (s, 1H), 8.53 (d, J = 7.84 Hz, 1H), 8.49 (d, J = 4.68 Hz, 1H), 8.09 (s, 1H), 7.86 (d, J = 8.16 Hz, 1H), 7.41 (t, J = 7.88 Hz, 1H), 7.27 (d, J = 9.60 Hz, 1H), 7.11 (d, J = 8.04 Hz, 1H), 7.04 (t, J = 6.44 Hz, 2H), 6.87 (dd, J = 5.04, 7.22 Hz, 1H), 6.63 (d, J = 15.88 Hz, 1H), 6.39-6.38 (m, 1H), 4.35 (d, J = 5.44 Hz, 2H), 3.85 (s, 3H). 13C NMR 100 MHz, CDCl3: δ 166.01, 156.09, 153.79, 151.22, 141.05, 140.21, 138.80, 136.40, 130.89, 130.24, 129.32, 129.22, 128.82, 123.43, 122.93, 119.26, 119.17, 119.13, 117.18, 114.27, 110.27, 106.60, 63.52, 55.93. ESI Mass: Calculated mass: 444.13, measured mass: 444.07.

Synthesis of (E)-3-(4-hydroxy-3-methoxyphenyl) allyl 2-((3-(trifluoromethyl) phenyl) amino) benzoate (4):

Compound-4 was achieved by coupling coniferyl alcohol with flufenamic acid. Refer the compound 1 (synthetic route-II) procedure and same methodology was followed to prepare the compound. The final compound-4 was archived with 78% yellow color solid. 1H NMR 400 MHz, CDCl3: δ 9.51 (s, 1H), 8.26 (d, J = 8.00 Hz, 1H), 7.49 (s, 1H), 7.46-7.44 (m, 3H), 7.33 (d, J = 8.52 Hz, 1H), 7.29 (d, J = 7.24 Hz, 1H), 7.11 (d, J = 8.04 Hz, 1H), 7.03 (t, J = 4.76 Hz, 2H), 6.89 (t, J = 7.52 Hz, 1H), 6.62 (d, J = 15.84 Hz, 1H), 6.38-6.37 (m, 1H), 4.34 (d, J = 5.56 Hz, 2H), 3.85 (s, 3H). 13C NMR 100 MHz, CDCl3: δ 166.77, 151.40, 147.35, 141.41, 139.15, 136.05, 134.98, 132.54, 130.43, 129.94, 129.04, 124.59, 123.11, 119.75, 119.71, 119.26, 118.44, 118.12, 118.08, 114.22, 111.89, 110.28, 63.58, 55.97. ESI Mass: Calculated mass: 443.13, measured mass: 443.08.

Synthesis of (E)-3-(4-hydroxy-3-methoxyphenyl) allyl 2-((3,4-dimethylphenyl) amino) benzoate (5):

Compound-5 was achieved by coupling coniferyl alcohol with mefenamic acid. Refer the compound 1 (synthetic route-II) procedure and same methodology was followed to prepare the compound. The final compound-5 was archived with 76% yellow color solid. 1H NMR 400 MHz, CDCl3: δ 9.12 (s, 1H), 8.21 (d, J = 8.08 Hz, 1H), 7.31 (t, J = 7.92 Hz, 1H), 7.18 (d, J = 7.84 Hz, 1H), 7.11 (q, J = 7.96 Hz, 2H), 7.03 (dd, J = 7.24, 8.12 Hz, 3H), 6.81 (d, J = 8.60 Hz, 1H), 6.73 (t, J = 7.48 Hz, 1H), 6.62 (d, J = 15.84 Hz, 1H), 6.38-6.36 (m, 1H), 4.34 (d, J = 5.56 Hz, 2H), 3.86 (s, 3H), 2.31 (s, 3H), 2.16 (s, 3H). 13C NMR 100 MHz, CDCl3: δ 166.92, 151.56, 150.08, 139.42, 138.53, 138.24, 135.81, 134.82, 132.48, 132.30, 130.58, 128.86, 126.90, 125.92, 123.29, 123.11, 119.28, 116.24, 113.77, 110.36, 109.81, 63.63, 55.99, 20.60, 14.00. ESI Mass: Calculated mass: 419.17, measured mass: 419.12.

Synthesis of methyl (E)-3-(4-hydroxy-3-methoxyphenyl) acrylate (6a):

To a methanolic solution of ferulic acid (194 mg, 1 mmole) was refluxed to added few drops Con. H2SO4, resulting reaction mixture was further continued 3h and completion of the reaction was monitored by TLC. The reaction crude was extracted with dichloromethane and washed with brine solution and followed by dried with Mg2SO4 and organic layer was concentrated under reduced pressure. The resultant crude product was purified with column chromatography. The purified compound-6a were characterized by 1H and 13C NMR spectra and molecular mass was identified using LC-HRMS. The compound-6a was obtained in 87% yield with white color semi-solid. 1H NMR 400 MHz, CDCl3: δ 7.58 (d, J = 16.00 Hz, 1H), 7.01 (d, J = 8.40 Hz, 1H), 6.97 (s, 1H), 6.88 (d, J = 8.40 Hz, 1H), 6.43 (s, 1H), 6.25 (d, J = 16.00 Hz, 1H), 3.85 (s, 3H), 3.80 (s, 3H). 13C NMR 100 MHz, CDCl3: δ 167.90, 148.17, 146.96, 145.11, 126.81, 122.99, 119.85, 114.92, 109.59, 55.89, 51.63. LC-HRMS: Calculated mass [M+1]: 209.0735, measured mass [M+1]: 209.0545.

Synthesis of 3-(4-hydroxy-3-methoxyphenyl) propane hydrazide (6):

Using ferulic ester (Compound-6a) to prepare ferulic acid hydrazide (Compound-6); To an ethanolic solution of compound-6a (210 mg, 1 mm) followed the slow addition of hydrazine hydrate (0.628 ml, 20 mm), after the reaction mixture was refluxed at 24 h, progress of the reaxtion was monitor by TLC. The organic compound was extracted ethyl acetate with brine solution followed to dried with Mg2SO4 and organic layer was concentrated under reduced pressure. The resultant crude product was purified with column chromatography. The purified compound-6 was characterized by 1H and 13C NMR spectra and molecular mass was identified using LC-HRMS. The final compound-6 was obtained in 69 % yield as a brown color solid. 1H NMR 400 MHz, DMSO-d6: δ 8.92 (s, 1H), 8.66 (s, 1H), 6.72 (s, 1H), 6.63 (d, J = 8.00 Hz, 1H), 6.54 (d, J = 7.60 Hz, 1H), 4.14 (s, 2H), 3.72 (s, 3H), 2.68 (t, J = 7.20 Hz, 2H), 2.25 (t, J = 8.00 Hz, 2H). 13C NMR 100 MHz, DMSO-d6: δ 171.42, 147.82, 145.08, 132.46, 120.67, 115.74, 112.87, 55.99, 35.99, 31.16. LC-HRMS: Calculated mass [M+1]: 211.1004, measured mass [M+1]: 211.0820.

Synthesis of (E)-N-hydroxy-3-(4-hydroxy-3-methoxyphenyl) acrylamide (7):

Ferulic ester (Compound-6a) was using to prepare ferulic hydroximic acid (Compound-7), To an aqueous solution of hydroxylamine hydrochloride (2.084 g, 30 mmole), addition of 25 mm NaOH solution slowly and stirrer the reaction mixture further 30 minutes, dissolve the compound-6a (210 mg, 1 mmole) in 1,4 -Dioxane added to the above mixture further to stirrered at room temperature for 24 h. After the reaction mixture was neutralize with HCl, the crude mixture was extracted with ethyl acetate followed by brine solution, and then dried with Mg2SO4 and organic layer was concentrated under reduced pressure. The resultant crude product was purified with column chromatography. The purified final compound-7 was obtained in 87% yield as a white color solid. 1H NMR 400 MHz, DMSO-d6: δ 10.66 (s, 1H), 9.50 (s, 1H), 9.00 (s, 1NH), 7.42 (d, J = 16.00 Hz, 1H), 7.18 (s, 1H), 7.05 (d, J = 7.60 Hz, 1H), 6.85 (d, J = 8.40 Hz, 1H), 6.34 (d, J = 15.60 Hz, 1H), 3.87 (s, 3H). 13C NMR 100 MHz, DMSO-d6: δ 163.84, 148.73, 148.26, 139.22, 126.77, 121.88, 116.10, 111.32, 56.01. LC-HRMS: Calculated mass [M+1]: 210.0688, measured mass [M+1]: 210.0493.

Synthesis of methyl (E)-3-(4-hydroxy-3,5-dimethoxyphenyl) acrylate (8a):

Compound-8a was achieved by sinapic acid. Refer the compound 6a (synthetic route-II) procedure and same methodology was followed to prepare the compound. The final compound-8a was archived with 81% pale green color semi-solid. 1H NMR 400 MHz, CDCl3: δ 7.59 (d, J = 15.60 Hz, 1H), 6.76 (s, 1H), 6.29 (d, J = 15.60 Hz, 1H), 5.79 (s, 1H), 3.91 (s, 6H), 3.79 (s, 3H). 13C NMR 100 MHz, CDCl3: δ 167.61, 147.21, 145.15, 137.14, 125.87, 115.54, 105.05, 56.34, 51.65. LC-HRMS: Calculated mass [M+Na]: 261.0738, measured mass [M+Na]: 261.0446.

Synthesis of (E)-N-hydroxy-3-(4-hydroxy-3,5-dimethoxyphenyl) acrylamide (8):

Compound-8 was achieved by sinapic ester (Compound-8a). Refer the compound 6a (synthetic route-II) procedure and same methodology was followed to prepare the compound. The final compound-8 was archived with 78% dark brown color solid. 1H NMR 400 MHz, DMSO-d6: δ 10.59 (s, 1H), 8.94 (s, 1H), 8.79 (s, 1H), 7.37 (d, J = 15.60 Hz, 1H), 6.86 (s, 2H), 6.32 (d, J = 15.60 Hz, 1H), 3.79 (s, 6H). LC-HRMS: Calculated mass [M+1]: 240.0794, obtained mass [M+1]: 242.2557.

Synthesis of methyl (E)-3-(3-hydroxy-4-methoxyphenyl) acrylate (9a):

Compound-9a was achieved by Iso-Ferulic acid. Refer the compound 6a (synthetic route-II) procedure and same methodology was followed to prepare the compound. The final compound-9a was archived with 84% white color semi-solid. 1H NMR 400 MHz, CDCl3: δ 7.60 (d, J = 15.60 Hz, 1H), 7.14 (d, J = 1.88 Hz, 1H), 7.03 (dd, J = 2.00, 8.40 Hz, 1H), 6.84 (d, J = 8.00 Hz, 1H), 6.29 (d, J = 16.00 Hz, 1H), 5.71 (s, 1H), 3.92 (s, 3H), 3.79 (s, 3H). 13C NMR 100 MHz, CDCl3: δ 176.75, 148.54, 145.86, 144.70, 128.03, 121.84, 115.84, 113.00, 110.53, 56.01, 51.62. LC-HRMS: Calculated mass [M+1]: 209.0735, measured mass [M+1]: 209.0547.

Synthesis of (E)-N-hydroxy-3-(3-hydroxy-4-methoxyphenyl) acrylamide (9):

Compound-9 was achieved by Iso-Ferulic ester (Compound-9a). Refer the compound-7 (synthetic route-II) procedure and same methodology was followed to prepare the compound. The final compound-9 was achieved with 76% white color solid. 1H NMR 400 MHz, DMSO-d6: δ 10.66 (s, 1H), 9.20 (s, 1H), 8.97 (s, 1H), 7.31 (d, J = 5.60 Hz, 1H), 6.98 (s, 1H), 6.94 (t, J = 6.80 Hz, 2H), 6.23 (d, J = 15.60 Hz, 1H), 3.79 (s, 3H). 13C NMR 100 MHz, DMSO-d6: δ 163.64, 149.65, 174.15, 138.95, 128.14, 120.73, 116.79, 113.65, 112.52, 56.05. LC-HRMS: Calculated mass [M+1]: 210.0688, measured mass [M+1]: 210.0498.

S4 – NMR Spectral Data Images

S4a: Compound-1 1H NMR

S4b: Compound-1 13C NMR

S4c:Compound-2 1H NMR

S4d: Compound-3 1H NMR

S4e: Compound-3 13C NMR

S4f: Compound-4 1H NMR

S4g: Compound-4 13C NMR

S4h: Compound-5 1H NMR

S4i: Compound-5 13C NMR

S4j: Compound-6a 1H NMR

S4k: Compound-6a 13C NMR

S4l: Compound-6 1H NMR

S4m: Compound-6 13C NMR

S4n: Compound-7 1H NMR

S4o: Compound-7 13C NMR

S4p: Compound-8a 1H NMR

S4q: Compound-8 13C NMR

S4r: Compound-8 1H NMR

S4s: Compound-9a 1H NMR

S4t: Compound-9a 13C NMR

S4u: Compound-9 1H NMR

S4v: Compound-9 13C NMR

S5: ESI & LC-HRMS Spectral Data Images

S5a: Compound-1 ESI Mass

S5b: Compound-2 ESI Mass

S5c: Compound-3 ESI Mass

S5d: Compound-4 ESI Mass

S5e: Compound-5 ESI Mass

S5f: Compound-6a LC-HRMS

S5g: Compound-6 LC-HRMS

S5h: Compound-7 LC-HRMS

S5i: Compound-8a LC-HRMS

S5j: Compound-8 LC-HRMS

S5k: Compound-9a LC-HRMS

S5l: Compound-9 LC-HRMS

S6 – RT-PCR Primers table

Table S6: List of oligonucleotide primers used for RT-PCR

S7- UPLC-DAD purity graph for Lead molecules

Figure-S7: UPLC-DAD purity graph for Lead molecules. S7A) Compound 7 purity graph, S7B) Compound 8 purity graph.

S7A)

S7B)