http://informahealthcare.com/enz ISSN: 1475-6366 (print), 1475-6374 (electronic) J Enzyme Inhib Med Chem, Early Online: 1–6 ! 2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.940932
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RESEARCH ARTICLE
Synthesis of new isoxazoline derivatives from harmine and evaluation of their anti-Alzheimer, anti-cancer and anti-inflammatory activities Insaf Filali1,2, Jalloul Bouajila2, Mansour Znati1,2, Fatima Bousejra-El Garah2, and Hichem Ben Jannet1 1
Laboratoire de Chimie He´te´rocyclique, De´partement de Chimie, Faculte´ des Sciences de Monastir, Produits Naturels et Re´activite´ (CHPNR), Equipe Chimie Me´dicinale et Produits Naturels, Universite´ de Monastir, Monastir Tunisie, France and 2Laboratoire des Interactions Mole´culaires et Re´activite´ Chimique et Photochimique, Faculte´ de pharmacie de Toulouse, Universite´ de Toulouse, Universite´ Paul-Sabatier, Toulouse, France Abstract
Keywords
In our study, a series of new harmine derivatives has been prepared by cycloaddition reaction using various arylnitrile oxides and evaluated in vitro against acetylcholinesterase and 5-lipoxygenase enzymes, MCF7 and HCT116 cancer cell lines. Some of these molecules have been shown to be potent inhibitors of acetylcholinesterase and MCF7 cell line. The greatest activity against acetylcholinesterase (IC50 ¼ 10.4 mM) was obtained for harmine 1 and cytotoxic activities (IC50 ¼ 0.2 mM) for compound 3a. Two derivatives 3e and 3f with the thiophene and furan systems, respectively, showed good activity against 5- lipoxygenase enzyme (IC50 ¼ 29.2 and 55.5 mM, respectively).
1,3-dipolar cycloaddition, anti-5-lipoxygenase, anti-acetylcholinesterase, anti-cancer, harmine, oxazolines, Peganum harmala
Introduction Alkaloids are a group of naturally occurring organic compounds with basic nitrogen atoms. It was reported that alkaloids are useful against human immunodeficiency virus (HIV) infection1, various studies have also reported alkaloids with anti-bacterial2, anti-inflammatory3, anti-malarial4, anti-oxidant5 and anti-cancer activities6. The b-carboline alkaloids are a group of alkaloids with a tricyclic pyrido[3,4-b]indole ring7. Plants, foodstuffs, marine creatures, insects, mammalians as well as human tissues and body fluids have been the main source of these compounds8. In addition, it was proved that b-carboline alkaloids are not limited to biological control of herbivores but also have medical pharmacological and biological importance. Alkaloids belonging to b-carboline group possess diverse activities such as antimicrobial, anti-HIV and anti-parasitic9. Moreover, it was found that the alkaloids belonging to b-carboline showed significant anti-tumor effect10 and potential anti-Alzheimer activity11.
Address for correspondence: Jalloul Bouajila, Laboratoire des Interactions Mole´culaires et Re´activite´ Chimique et Photochimique, Faculte´ de Pharmacie de Toulouse, Universite´ de Toulouse, Universite´ Paul-Sabatier, UMR CNRS 5623, 118 route de Narbonne, F-31062 Toulouse, France. Tel: +33-562256885. Fax: +33-562256885. E-mail: [email protected] Hichem Ben Jannet, Laboratoire de Chimie He´te´rocyclique, De´partement de Chimie, Faculte´ des Sciences de Monastir, Produits Naturels et Re´activite´ (CHPNR), Equipe Chimie Me´dicinale et Produits Naturels, Universite´ de Monastir, Avenue de l0 Environnement, 5019 Monastir Tunisie, France. Tel: +21673500279. Fax: +21673500278. E-mail: [email protected]
History Received 15 February 2014 Revised 3 June 2014 Accepted 6 June 2014 Published online 28 July 2014
b-Carbolines were first discovered in plants and characterized in Peganum harmala12. This plant, commonly known as Harmal or Syrian rue, is a perennial herbaceous, glabrous, wild-growing flowering plant, originally of Zygophyllaceae family13 but which has been recently placed in Nitrariaceae family14. This plant grows in the arid and semiarid areas and sandy soils, native to eastern Mediterranean region and distributed in Central Asia, India, South America, Southern USA and is mainly found in Middle East and North Africa15. Peganum harmala is an effective herbal medicine possessing hallucinogenic, hypothemic, antihelmitic, abortifient, lactogogue and emmenagogue properties16. Several biological activities of P. harmala have been described in literature including anti-bacterial, anti-fungal and anti-viral activities17. It exhibits cytotoxicity with regard to HL60 and K562 leukemia cell lines18. The seeds appear to possess medicinal properties19. The main chemical constituents in the seeds of P. harmala were a serial of b-carboline alkaloids such as harman, harmine, harmaline and harmalol20. There are several reports in the literature indicating a wide spectrum of therapeutic activities for these b-carbolines such as anti-nociceptive and analgesic effects, anti-proliferative, vasorelaxant and hypothermic properties21. Total alkaloids of P. harmala have been reported to possess significant cytotoxic activity on cancerous cell lines and an inhibiting activity of the synthesis of DNA22. Recent interests in harmine and structurally related compounds were stimulated by their pharmacological, neurophysiologic and biochemical activities23. Harmine has anti-microbial, antiplasmodial, anti-fungal, anti-oxidative and hallucinogenic properties24. Recent studies demonstrated that harmine and its derivatives possessed significant anti-tumor potential both in vitro and in vivo25–27. Indeed, harmine showed potent cytotoxic activity
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against MCF7, HL60, K562, KB, A549, CAKI1, 1A9 and HEL cells with IC50 values of 29.3, 8.0, 8.9, 10.3, 11.3, 8.9, 7.5 and 8.9 mM, respectively28–30. In addition, it has been shown that harmine can reverse resistance to anti-cancer drugs by inhibiting the breast cancer resistance protein BCRP31. It was also reported that harmine and its derivatives were a potent inhibitor of DYRK1A which has been found to be associated with neurofibrillary tangles (NFTs) in sporadic Alzheimer’s disease. In addition to DYRK1A inhibition, harmine has been reported to be a selective inhibitor of tau phosphorylation at multiple Alzheimer’s disease-related sites32. The inhibition of cholinesterase (ChE) can be also a new way to stop the disease of Alzheimer. Among the family of heterocyclic compounds, isoxazolines are an important class of five-membered ring heterocycles, displaying a wide variety of biological properties including anti-viral33, anti-depressant34, anti-bacterial35, anti-fungal36, antiinfluenza37 and anti-cancer38 as well as anti-inflammatory activity39. Furthermore, the isoxazolines possess significant synthetic applications40 and represent a unique class of pharmacophore present in many therapeutic agents41. Encouraged by the broad biological activity of harmine derivatives and isoxazolines biological interests, we chose harmine as a scaffold to generate novel derivatives, via 1,3dipolar cycloaddition reactions using various arylnitrile oxides. The objective of this study was the synthesis of new analogues (not cited in literature) from harmine and the evaluation of their anti-acethylcholinesterase, anti-5-lipoxygenase and anti-cancer properties.
Materials and methods Shimadzu QP-1000 EX spectrometer was used in the ESI+ experiment. 1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded on a Bruker AM-300 spectrometer, using CD3OD as solvent and none deuterated residual solvent as internal standard. Chemicals shifts () are given in parts per million (ppm) and coupling constants (J) in Hertz. Melting points were determined on a Bu¨chi 510 apparatus using capillary tubes. Extraction and isolation A total of 240 g of dried and powdered seeds of P. harmala fruits were macerated four times with 300 mL methanol at 50 C for 1 h. The extracts were combined and evaporated to dryness. The residue was dissolved in 300 mL HCl (2%) then filtered. The filtrate was extracted two times with 250-mL pentane. The aqueous acid layer was basified (pH 10) with NH4OH and extracted four times with 300-mL chloroform. The chloroform layer was combined and evaporated to dryness, then the residue (5.8 g) was purified by chromatography on silica gel eluting successively with ethyl acetate–methanol (90:10 to 0:100) to afford 1.2 g (20.7%) harmine and four fractions42. Harmine 1: 7-methoxy-1-methyl-9H -pyrido[3,4-b]indole m.p.: 321 C. ES-MS m/z 213 [M + H]+. 1H-NMR (300 MHz, CD3OD) d ppm: 8.11 (1H, d, J ¼ 5.4 Hz, H-3), 7.97 (1H, d, J ¼ 8.7 Hz, H-5), 7.78 (1H, d, J ¼ 5.4 Hz, H-4), 7.04 (1H, d, J ¼ 2.1 Hz, H-8), 6.85 (1H, dd, J ¼ 8.7, 2.4 Hz, H-6), 3.91 (3H, s), 2.77 (3H, s). 13C-NMR (75 MHz, CD3OD) d ppm: 162.5 (C-7), 144.1 (C-1), 142.0 (C-13), 137.9 (C-3), 136.2 (C-10), 130.1 (C-11), 123.5 (C-5), 116.3 (C-12), 113.3 (C-4), 110.9 (C-6), 95.3 (C-8), 55.9 (CH3-O),19.5 (CH3). The spectral data of compound 1 matched with the data reported for harmine 1 from the literature43.
J Enzyme Inhib Med Chem, Early Online: 1–6
General procedure for the preparation of compound 2 Under argon atmosphere, harmine 1 (0.35 g, 1.64 mmol) is dissolved in anhydrous Dimethylformamide (DMF) (20 mL). Allyl bromide (5 equiv.) was added in the presence of sodium hydride (2 equiv.). The mixture was stirred for 24 h at room temperature, then poured into ice-cold water, and extracted with ethyl acetate. The organic layer was dried over Na2SO4, after removal of solvent in vacuum, the resulting residue was purified by silica gel column chromatography (EtOAc) to give compound 2 (229 mg, 55.4%)44. Compound 2: 9-allyl-7-methoxy-1-methyl-9Hpyrido[3,4-b]indole Yield 55%, ES-MS m/z 253 [M + H]+. 1H-NMR (300 MHz, CD3OD) d ppm: 8.00 (1H, d, J ¼ 5.4 Hz, H-3), 7.91 (1H, d, J ¼ 8.4 Hz, H-5), 7.75 (1H, d, J ¼ 5.1 Hz, H-4), 6.81 (2H, m, H-8, H-6), 6.02 (1H, m, H-15), 5,03 (3H, m, H-14a, H-16a, H-16b), 4.52 (1H, brd, J ¼ 17.4 Hz, H-14b), 3.79 (3H, s, CH3–O), 2.80 (3H, s, CH3). 13C-NMR (75 MHz, CD3OD) d ppm: 163.0 (C-7), 145.2 (C-1), 141.6 (C-13), 137.9 (C-3), 136.5 (C-10), 135.4 (C-16), 131.3 (C-11), 123.4 (C-5), 115.9 (C-12), 115.7 (C-15), 113.6 (C-4), 111.2 (C-6), 94.0 (C-8), 56.1 (CH3–O), 47.6 (C-14), 21.8 (CH3). General procedure for the preparation of compounds 3a–f The nitrile oxides were prepared from aldoximes by halogenations followed by an in situ dehydrohalogenation using a base45. To compound 2 (0.05 g, 0.2 mmol) in refluxing dichloromethane, the appropriate nitrile oxide (2 equiv.) in the presence of triethylamine (2 equiv.) was added and the mixture was refluxed for 48 h. The resulting mixture was washed with water, and then extracted with CH2Cl2. The organic layer was dried over Na2SO4. The solvent was then removed under reduced pressure. The resulting residue was purified by preparative TLC (EtOAc/ CH3OH) to give corresponding heterocycles 3a–f. Compound 3a: 9-((4,5-dihydro-3-phenylisoxazol-5yl)methyl)-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole Yield 75%, m.p.: 98 C, ES-MS m/z 372 [M + H]+. 1H-NMR (300 MHz, CD3OD) d ppm: 8.16 (1H, d, J ¼ 5.4 Hz, H-3), 8.05 (1H, d, J ¼ 8.7 Hz, H-5), 7.95 (1H, d, J ¼ 5.4 Hz, H-4), 7.70 (2H, d, J ¼ 7.8 Hz, Ar-H), 7.46 (2H, d, J ¼ 7.8 Hz, Ar-H), 7.20 (1H, d, J ¼ 2.1 Hz, H-8), 6.96 (1H, dd, J ¼ 2.1, 8.7 Hz, H-6), 5.18 (1H, m, H-15), 4.88 (1H, dd, J ¼ 9.0, 15.9 Hz, H-14a), 4.77 (1H, dd, J ¼ 3.3, 15.9 Hz, H-14b), 3.95 (3H, s, CH3–O), 3.61 (1H, dd, J ¼ 9.8, 17.2 Hz, H-16a), 3.36 (1H, dd, J ¼ 6.3, 17.1 Hz, H-16b), 3.03 (3H, s, CH3). 13C-NMR (75 MHz, CD3OD) d ppm: 163.2 (C ¼ N), 159.0 (C-7), 145.5 (C-1), 141.7 (C-13), 137.1 (C-3), 136.8 (C-10), 132.6 (Ar-C), 131.5 (Ar-C), 130.4 (C-11), 129.9 (Ar-C), 127.8 (Ar-C), 123.6 (C-5), 115.9 (C-12), 113.8 (C-4), 111.8 (C-6), 94.9 (C-8), 81.3 (C-15), 56.2 (CH3-O), 38.9 (C-14), 30.8 (C-16), 22.5 (CH3). Compound 3b: 9-((4,5-dihydro-3-p-tolylisoxazol-5yl)methyl)-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole Yield 70%, m.p.: 158 C, ES-MS m/z 386 [M + H]+. 1H-NMR (300 MHz, CD3OD) d ppm: 8.2 (1H, d, J ¼ 5.6 Hz, H-3), 8.15 (1H, d, J ¼ 8.4 Hz, H-5), 7.89 (1H, d, J ¼ 5.1 Hz, H-4), 7.59 (2H, d, J ¼ 8.1 Hz, Ar-H), 7.29 (2H, d, J ¼ 8.1 Hz, Ar-H), 7.17 (1H, d, J ¼ 1.8 Hz, H-8), 6.96 (1H, dd, J ¼ 1.8, 8.7 Hz, H-6), 5.16 (1H, m, H-15), 4.91 (1H, dd, J ¼ 9.0, 16.5 Hz, H-14a), 4.74 (1H, dd, J ¼ 3.3, 16.5 Hz, H-14b), 3.95 (3H, s, CH3–O), 3.65 (1H, dd,
DOI: 10.3109/14756366.2014.940932
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J ¼ 10.2, 17.1 Hz, H-16a), 3.37 (1H, dd, J ¼ 6.5, 17.1 Hz, H-16b), 2.99 (3H, s, CH3), 2.39 (3H, s, CH3). 13C-NMR (75 MHz, CD3OD) d ppm: 162.2 (C ¼ N), 158.9 (C-7), 145.2 (C-1), 142.2 (C-13), 142.1 (Ar-C), 138.1(C-3), 137.0 (C-10), 131.5 (Ar-C), 130.5 (C-11), 127.8 (Ar-C), 127.6 (Ar-C), 123.4 (C-5), 116.1 (C-12), 113.5 (C-4), 111.3 (C-6), 95.0 (C-8), 81.2 (C-15), 56.1 (CH3–O), 39.0 (C-14), 30.7 (C-16) 23.0 (CH3), 21.4 (CH3). Compound 3c: 9-((4,5-dihydro-3-(4-methoxyphenyl) isoxazol-5-yl)methyl)-7-methoxy-1-methyl-9H-pyrido [3,4-b]indole Yield 65%, m.p.: 145 C, ES-MS m/z 402 [M + H]+. 1H-NMR (300 MHz, CD3OD) d ppm: 8.17 (1H, d, J ¼ 5.4 Hz, H-3), 8.14 (2H, m, H-5, H-4), 7.63 (2H, d, J ¼ 9.0 Hz, Ar-H), 7.28 (1H, d, J ¼ 1.8 Hz, H-8), 7.05 (1H, dd, J ¼ 1.8, 8.7 Hz, H-6), 7.00 (2H, d, J ¼ 9.0 Hz, Ar-H), 5.15 (1H, m, H-15), 4.84 (2H, m, H-14), 4.01 (3H, s, CH3–O), 3.87 (3H, s, CH3–O), 3.65 (2H, m, H-16), 3.13 (3H, s, CH3). 13C-NMR (75 MHz, CD3OD) d ppm: 164.2 (C ¼ N), 160.7 (Ar-C), 157.6 (C-7), 145.4 (C-1), 141.2 (C-13), 138.3 (C-3), 136.7 (C-10), 130.5 (C-11), 128.5 (Ar-C), 125.4 (Ar-C), 123.4 (C-5), 115.2 (C-12), 114.2 (Ar-C), 112.6 (C-4), 110.1 (C-6), 93.2 (C-8), 81.3 (C-15), 58.6 (CH3–O), 55.31 (CH3–O), 41.56 (C-14), 32.6 (C-16), 22.35 (CH3). Compound 3d: 9-((3-(4-chlorophenyl)-4,5dihydroisoxazol-5-yl)methyl)-7-methoxy-1-methyl-9Hpyrido[3,4-b]indole Yield 60%, m.p.: 200 C, ES-MS m/z 406 [M + H]+. 1H-NMR (300 MHz, CD3OD) d ppm: 8.16 (1H, d, J ¼ 5.4 Hz, H-3), 8.07 (1H, d, J ¼ 8.7 Hz, H-5) 7.94 (1H, d, J ¼ 5.4 Hz, H-4), 7.65 (2H, d, J ¼ 7.8 Hz, Ar-H), 7.42 (2H, d, J ¼ 7.8 Hz, Ar-H), 7.16 (1H, d, J ¼ 2.1 Hz, H-8), 6.94 (1H, dd, J ¼ 2.1, 8.7 Hz, H-6), 5.19 (1H, m, H-15), 4.9 (1H, dd, J ¼ 9.0, 15.9 Hz, H-14a), 4.77 (1H, dd, J ¼ 3, 15.9 Hz, H-14b), 3.95 (3H, s, CH3–O), 3.64 (1H, dd, J ¼ 10.2, 17.4 Hz, H-16a), 3.40 (1H, dd, J ¼ 6.6, 16.5 Hz, H-16b), 2.99 (3H, s, CH3). 13C-NMR (75 MHz, MeOD-d4): d ppm: 163.4 (C ¼ N), 158.1 (C-7), 145.7 (C-1), 141.4 (C-13), 137.3 (C-3), 136.6 (C-10), 132.3 (Ar-C), 132.1 (Ar-C), 130.1 (C-11), 129.3 (Ar-C), 129.1 (Ar-C), 123.7 (C-5), 115.8 (C-12), 113.9 (C-4), 111.9 (C-6), 95.0 (C-8), 81.6 (C-15), 56.2 (CH3–O), 38.8 (C-14), 30.7 (C-16), 22.2 (CH3). Compound 3e: 9-((3-(furan-2-yl)-4,5-dihydroisoxazol-5yl)methyl)-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole Yield 45%, m.p.: 170 C, ES-MS m/z 362 [M + H]+. 1H-NMR (300 MHz, CD3OD) d ppm: 8.15 (1H, d, J ¼ 5.4 Hz, H-3), 8.05 (1H, d, J ¼ 8.7 Hz, H-5), 7.90 (1H, d, J ¼ 5.4 Hz, H-4), 7.16 (1H, d, J ¼ 2.1 Hz, H-8), 6.95 (1H, dd, J ¼ 2.1, 8.7 Hz, H-6), 6.88 (2H, d, J ¼ 3.6 Hz, H-30 , H-40 ), 6.48 (1H, d, J ¼ 3.6 Hz, H-50 ), 5.17 (1H, m, H-15), 4.85 (1H, dd, J ¼ 3.3, 15.9 Hz, H-14a), 4.74 (1H, dd, J ¼ 9.0, 15.9 Hz, H-14b), 3.95 (3H, s, CH3–O), 3.56 (1H, m, H-16a), 3.32 (1H, m, H-16b), 2.99 (3H, s,CH3). 13C-NMR (75 MHz, CD3OD) d ppm: 163.0 (C ¼ N), 150.9 (C-7), 146.4 (C-1), 145.3 (C-20 ), 141.9 (C-13), 140.3 (C-50 ), 137.7 (C-3), 136.8 (C-10), 131.7 (C-11), 123.5 (C-5), 116.3 (C-40 ), 116.0 (C-30 ), 114.2 (C-12), 112.9 (C-4), 111.5 (C-6), 94.9 (C-8), 81.4 (C-15), 56.2 (CH3–O), 38.3 (C-14), 30.4 (C-16), 22.8 (CH3). Compound 3f: 9-((4,5-dihydro-3-(thiophen-2-yl)isoxazol-5yl)methyl)-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole Yield 50%, m.p.: 180 C, ES-MS m/z 378 [M + H]+. 1H-NMR (300 MHz, CD3OD) d ppm: 8.04 (1H, d, J ¼ 5.4 Hz, H-3), 8.94 (1H, d, J ¼ 8.7 Hz, H-5), 7.80 (1H, d, J ¼ 5.4 Hz, H-4), 7.46 (1H, dd, J ¼ 0.9, 5.1 Hz, H-50 ), 7.24 (1H, dd, J ¼ 0.9, 3.6 Hz, H-30 ),
New isoxazolines derivatives from harmine
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7.05 (1H, d, J ¼ 2.1 Hz, H-8), 7.01 (1H, dd, J ¼ 3.6, 5.1 Hz, H-40 ), 6.83 (1H, dd, J ¼ 2.1, 8.7 Hz, H-6), 5.08 (1H, m, H-15), 4.77 (1H, dd, J ¼ 16.2, 9.0 Hz, H-14a), 4.64 (1H, dd, J ¼ 3.3, 15.9 Hz, H-14b), 3.83 (3H, s, CH3–O), 3.65 (1H, dd, J ¼ 10.2, 16.8 Hz, H-16a), 3.28 (1H, dd, J ¼ 6.3, 16.5 Hz, H-16b), 2.99 (3H, s, CH3). 13 C-NMR (75 MHz, CD3OD) d ppm: 163.1 (C ¼ N), 154.7 (C-7), 145.3 (C-1), 141.9 (C-13), 137.6 (C-3), 136.8 (C-10), 132.4 (C-11), 131.8 (C-20 ), 130.2 (C-40 ), 129.3 (C-30 ), 128.6 (C-50 ), 123.5 (C-5), 115.9 (C-12), 113.7 (C-4), 111.6 (C-6), 94.8 (C-8), 81.5 (C-15), 56.1 (CH3–O), 39.6 (C-14), 30.5 (C-16), 22.7 (CH3). AChE inhibitory activity assay ChE inhibitory activity was measured using Ellman’s method, as previously reported with modifications46. In this study, 50 mL of 0.1 -M sodium phosphate buffer (pH 8.0), 25 mL of AChE solution, 25 mL of each compound and 125 mL of DTNB (5,50 dithiobis[2-nitrobenzoic acid]) were added in a 96-well microplate and incubated for 15 min at 25 C. All compounds were re-suspended in the DMSO followed by dilution in the buffer and the DMSO does not exceed 1% in the mixture. 25 mL of a solution of acetylthiocholine iodide was added and the final mixture incubated, for 15 min, at 25 C. The hydrolysis of acetylthiocholine iodide was monitored by the formation of the yellow 5-thio-2nitrobenzoate anion as a result of the reaction of DTNB with thiocholines, catalyzed by enzymes at a wavelength of 412 nm. The concentration of the compounds which caused 50% inhibition of the AChE activity (IC50) was calculated by non-linear regression analysis. The percentage of inhibition was calculated from (1 S/E) 100, where E and S were the respective enzyme activity without and with the test sample, respectively. Galanthamine was used as positive control. Cytotoxicity/anti-cancer assay Cytotoxicity of compounds was estimated on human breast cancer cells (MCF7) as described by Bendaoud et al47. with modifications. Cells were distributed in 96-well plates at 3 104 cells/well in 100 mL and then 100 mL of culture medium containing sample at various concentrations were added. Cell growth was estimated by MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. MTT is a yellow water-soluble tetrazolium salt. Metabolically active cells are able to convert the dye to water-insoluble dark blue formazan by reductive cleavage of the tetrazolium ring. All compounds were re-suspended in the DMSO followed by dilution in the buffer and the DMSO does not exceed 1% in the mixture. Doxorubicin was used as positive control. Lipoxygenase activity assay The anti-inflammatory activity of harmine 1 and its derivatives (2 and 3a–f) was determined on Soybean lipoxygenase (LOX) as described by Bylac and Racine with modifications48. Various concentrations of 20 mL of each compound was mixed individually with sodium phosphate buffer (pH 7.4) containing 5-LOX and 60 mL of linoleic acid (3.5 mM), yielding a final volume of 1 mL. The blank does not contain the substrate, but will be added 30 mL of buffer solution. All compounds were re-suspended in the DMSO followed by dilution in the buffer so that the DMSO does not exceed 1%. The mixture was incubated at 25 C for 10 min, and the absorbance was determined at 234 nm. The absorption change with the conversion of linoleic acid to 13-hydroperoxyoctadeca-9,11-dienoate (characterized by the appearance of the conjugated diene at 234 nm) was flowed for 10 min at 25 C. Nordihydroguaiaretic acid (NDGA) was used as positive control. The percentage of enzyme activity was plotted against the
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81.2–81.6 and 30.4–32.6 ppm, respectively, in addition to the carbon signals introduced by the harmine 1 skeleton. It is noted that no other derivatives were isolated from the reaction mixture, indicating the high regioselectivity of the reaction. The AChE inhibition was determined (Table 1) using an adaptation of the method described in the literature50. Values of IC50 ranging between 10.4 and 34.6 mM were obtained. The higher inhibitory activity was exhibited by the natural harmine 1 with an IC50 value of 10.4 ± 0.4 mM against AChE. Our results are in agreement with those of the literature. Indeed, harmine had been reported as a potent AChE inhibitory with IC50 value of 9.0 ± 1.1 mM51. Both compound 1 and its isoxazoline derivatives proved to inhibit AChE. The allylation of the nitrogen atom at position 9 led to the redaction of inhibitory potency (IC50 ¼ 13.5 ± 0.4 mM). Moreover, compound 3a with the phenyl system (IC50 ¼ 11.3 ± 0.7 mM) showed a good activity against the AChE enzyme, but is a little less active than harmine 1. It has been shown that the substitution in para position in the phenyl group decreases the activity regardless of the nature of substituent. A further drop in activity was noted for derivatives 3c and 3e (IC50 ¼ 15.1 ± 0.2 and 15.7 ± 0.9 mM, respectively), whose aromatic system of the isoxazoline moiety carries a methoxy group in
concentration of each compound. The IC50 value is the concentration of each compound that caused 50% enzyme inhibition.
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Results and discussion Harmine was isolated from the seeds of P. harmala using an adaptation of the method described in the literature. Harmine 1 was identified by its spectral data (1H, 13C and MS) compared with those of the literature49. In order to prepare new isoxazoline derivatives of harmine, we started by the allylation of the nitrogen atom in the five-membered ring of harmine with allyle bromide in the presence of sodium hydride to create a new double bond. The new double bond will serve in for eventual 1,3-dipolar cycloaddition reactions. This reaction has led to the preparation of the new 9-allyharmine 2 (Figure 1). The molecular formula of compound 2 was determined as C16H16N2O on the basis of ES-MS pseudomolecular peak [M + H]+ at m/z 253. The structure was evidenced by the appearance in the 1H-NMR spectrum of a multiplet at 6.0 ppm attributable to the ethylenic proton H-15 and a massif at 5.03 ppm attributable to the terminal methylenic protons H-16. The 13C-NMR spectrum reinforced this structure by the appearance of the two ethylenic carbon signals at 115.7 and 136.6 ppm and the appearance of a signal at 47.6 ppm attributed to the carbon C-14. The condensation of 2 with various arylnitrile oxides in refluxing dichloromethane for 48 h to generate corresponding heterocyclic compounds 3a–f. All novel compounds synthesized were not cited in literature. All synthesized compounds were obtained as yellow solids. Their ES-MS gave pseudo-molecular peaks [M + H]+, which are consistent with their molecular formula. The 1H-NMR spectra (300 MHz, CD3OD) of compounds 3a–f, in addition to the proton introduced by harmine 1, showed the observation of signals at 6.4–8.2 ppm corresponding to the protons of the aromatic rings attached to the isoxazoline moiety. The same 1H-NMR spectra revealed the presence of a characteristic signal at 5.1–5.2 ppm relative to H-15 and the doublet of doublets or multiplets consequent to the methylenic protons H-16 of the isoxazoline ring. The 13C-NMR spectra confirmed the above spectral data by the observation of signals at 145.3–116.0 ppm relative to carbons of the aromatic systems. The same spectra reinforced the proposed structures by the appearance of the signals of the iminic carbon C-17, C-15 (CH) and C-16 (CH2) at 162.2–163.4, Figure 1. Synthesis of derivatives 2 and 3a–f.
Table 1. Acetylcholinesterase inhibition harmine 1, harmine derivatives 2 and 3a–f. Compound
12
6
CH3
O
13 8
10.4 ± 0.4 13.5 ± 0.4 11.3 ± 0.7 34.6 ± 1.7 15.1 ± 0.2 25.1 ± 1.9 15.7 ± 0.9 27.8 ± 0.7 4.1 ± 0.2
*Averages ± SD were obtained from three different experiments. IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50% and are the mean of three independent measurements, each performed in triplicate.
N
9
5
Br
N2
11
3
4
3
10
7
of
Acetylcholinesterase IC50 (mM)*
1 2 3a 3b 3c 3d 3e 3f Galanthamine
4 5
capacity
12
6
1
NaH/ DMF
CH3
CH3
N
11 1
10
O
CH3
N
13
7 8
H
14
1
15
2 16
R
3a R = phenyl 3b R = 4'-methylphenyl 3c R = 4'-methoxyphenyl 3d R = 4'-chlorophneyl
N
(Et)3N/RefluxCH2C2
3'
4'
3e R=
4
2'
5'
5
S
1'
5'
2'
O
12
6
3'
4'
3f R=
OH
Cl
CH3
3
N
11
10
O
7
13 8
N 14
1'
O
15
N 17
R
16
3a-f
1
C CH3
New isoxazolines derivatives from harmine
DOI: 10.3109/14756366.2014.940932
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Table 2. HCT116 and MCF7 inhibitions of harmine 1, harmine derivatives 2 and 3a–f. Compound
HCT116 IC50 (mM)*
MCF7 IC50 (mM)*
1 2 3a 3b 3c 3d 3e 3f Doxorubicin
0.7 4400 9.7 16.9 4247 60.0 113.7 67.2 1.8 ± 0.2
1.3 0.5 0.2 6.2 34.6 6.2 3.1 19.4 0.8 ± 0.1
*Cytotoxicity as IC50 for each cell line, is the concentration of compound that inhibits of 50% the cell multiplication after 48 h of treatment.
para position and with the thiophene system, respectively. Compounds 3d and 3f exhibited moderate activity against the AChE enzyme (IC50 ¼ 25.1 ± 1.9 and 27.8 ± 0.7 mM, respectively). Compound 3b, whose aromatic system of the isoxazoline moiety carries a methyl group in para position, is the less active derivative (IC50 ¼ 34.6 ± 1.7 mM). These data suggest that the secondary amine function should be regarded as essential features able to confer high potency inhibition. Cytotoxicity was evaluated on two cell-lines: MCF7 breast cancer and HCT116 colon cancer. By comparing the IC50 of the various products tested towards both cellular lines, it is shown that the MCF7 line is the most sensitive to the various products tested (Table 2). Harmine 1 exhibited potent cytotoxic activity in this study against both cell lines (IC50 was 0.7–1.3 mM). Harmine was tested in several human tumor cell lines. The prepared derivatives 2, 3a, 3b, 3d, 3e and 3f exhibited cytotoxic activity against MCF7 breast cancer line. Compound 3a (IC50 ¼ 0.2 mM) exhibited the most interesting cytotoxic activity against this cell line. Also, compound 3e with the thiophene system showed a good cytotoxic activity against the same cell line (IC50 ¼ 3.1 mM). A further drop in activity was noted for derivatives 3b and 3d whose aromatic system of the isoxazoline moiety carries a methyl group and a Cl atom in para position, respectively (both IC50 ¼ 6.2 mM). The HCT116 line is less sensitive to the various tested products compared to MCF7 line. Harmine 1 showed the most interesting cytotoxic activity against this cell line (IC50 ¼ 1.3 mM). Compounds 3c and 3d whose aromatic system of the isoxazoline moiety carries a methoxy group and a Cl atom in para position respectively, are much less active to inactive by comparison with their analogues 3a and 3b. The two compounds 3e and 3f with the thiophene and furan systems, respectively, showed a much lower activity (IC50 ¼ 113.7 and IC50 ¼ 67.2 mM, respectively). In the present study, the 5-LOX inhibition was determined using an adaptation of the method described in the literature52. All compounds were analyzed on what concerns their 5-LOX inhibition to identify new anti-inflammatory compounds (Table 3). There is no report in the literature for the testing of harmine or derivatives against this enzyme. Harmine 1 and derivatives 2 and 3a–d have showed low activity against 5-LOX enzyme (IC504100 mM). Only the two derivatives 3e and 3f with the thiophene and furan systems, respectively, showed good activity against 5-LOX enzyme (IC50 ¼ 29.2 ± 3.1 and 55.5 ± 0.8 mM, respectively) comparable to the reference compound NDGA (IC50 ¼ 8.1 ± 0.1 mM).
Conclusion In conclusion, we have reported the synthesis of novel harmine derivatives via intermolecular 1,3-dipolar cycloaddition reactions
5
Table 3. 5-Lipoxygenase inhibition capacity (inhibition %, IC50 (mM)*) of harmine 1, harmine derivatives 2 and 3a–f. Compounds 1 2 3a 3b 3c 3d 3e 3f NDGA
5-Lipoxygenase 4100 4100 4100 4100 4100 4100 29.2 ± 3.1 55.5 ± 0.8 8.1 ± 0.1
*Averages ± SD were obtained from three different experiments. IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50% and are the mean of three independent measurements, each performed in triplicate.
involving nitrile oxides. Harmine 1 and its derivatives 2, 3a and 3e exhibited the most interesting cytotoxic activity against MCF7 cell line. The two derivatives 3e and 3f showed a good activity against 5-LOX enzyme.
Declaration of interest The authors declare no conflicts of interests. The authors alone are responsible for the content and writing of this article.
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RESEARCH ARTICLE
Synthesis of new isoxazoline derivatives from harmine and evaluation of their anti-Alzheimer, anti-cancer and anti-inflammatory activities Insaf Filali1,2, Jalloul Bouajila2, Mansour Znati1,2, Fatima Bousejra-El Garah2, and Hichem Ben Jannet1 1
Laboratoire de Chimie He´te´rocyclique, De´partement de Chimie, Faculte´ des Sciences de Monastir, Produits Naturels et Re´activite´ (CHPNR), Equipe Chimie Me´dicinale et Produits Naturels, Universite´ de Monastir, Monastir Tunisie, France and 2Laboratoire des Interactions Mole´culaires et Re´activite´ Chimique et Photochimique, Faculte´ de pharmacie de Toulouse, Universite´ de Toulouse, Universite´ Paul-Sabatier, Toulouse, France Abstract
Keywords
In our study, a series of new harmine derivatives has been prepared by cycloaddition reaction using various arylnitrile oxides and evaluated in vitro against acetylcholinesterase and 5-lipoxygenase enzymes, MCF7 and HCT116 cancer cell lines. Some of these molecules have been shown to be potent inhibitors of acetylcholinesterase and MCF7 cell line. The greatest activity against acetylcholinesterase (IC50 ¼ 10.4 mM) was obtained for harmine 1 and cytotoxic activities (IC50 ¼ 0.2 mM) for compound 3a. Two derivatives 3e and 3f with the thiophene and furan systems, respectively, showed good activity against 5- lipoxygenase enzyme (IC50 ¼ 29.2 and 55.5 mM, respectively).
1,3-dipolar cycloaddition, anti-5-lipoxygenase, anti-acetylcholinesterase, anti-cancer, harmine, oxazolines, Peganum harmala
Introduction Alkaloids are a group of naturally occurring organic compounds with basic nitrogen atoms. It was reported that alkaloids are useful against human immunodeficiency virus (HIV) infection1, various studies have also reported alkaloids with anti-bacterial2, anti-inflammatory3, anti-malarial4, anti-oxidant5 and anti-cancer activities6. The b-carboline alkaloids are a group of alkaloids with a tricyclic pyrido[3,4-b]indole ring7. Plants, foodstuffs, marine creatures, insects, mammalians as well as human tissues and body fluids have been the main source of these compounds8. In addition, it was proved that b-carboline alkaloids are not limited to biological control of herbivores but also have medical pharmacological and biological importance. Alkaloids belonging to b-carboline group possess diverse activities such as antimicrobial, anti-HIV and anti-parasitic9. Moreover, it was found that the alkaloids belonging to b-carboline showed significant anti-tumor effect10 and potential anti-Alzheimer activity11.
Address for correspondence: Jalloul Bouajila, Laboratoire des Interactions Mole´culaires et Re´activite´ Chimique et Photochimique, Faculte´ de Pharmacie de Toulouse, Universite´ de Toulouse, Universite´ Paul-Sabatier, UMR CNRS 5623, 118 route de Narbonne, F-31062 Toulouse, France. Tel: +33-562256885. Fax: +33-562256885. E-mail: [email protected] Hichem Ben Jannet, Laboratoire de Chimie He´te´rocyclique, De´partement de Chimie, Faculte´ des Sciences de Monastir, Produits Naturels et Re´activite´ (CHPNR), Equipe Chimie Me´dicinale et Produits Naturels, Universite´ de Monastir, Avenue de l0 Environnement, 5019 Monastir Tunisie, France. Tel: +21673500279. Fax: +21673500278. E-mail: [email protected]
History Received 15 February 2014 Revised 3 June 2014 Accepted 6 June 2014 Published online 28 July 2014
b-Carbolines were first discovered in plants and characterized in Peganum harmala12. This plant, commonly known as Harmal or Syrian rue, is a perennial herbaceous, glabrous, wild-growing flowering plant, originally of Zygophyllaceae family13 but which has been recently placed in Nitrariaceae family14. This plant grows in the arid and semiarid areas and sandy soils, native to eastern Mediterranean region and distributed in Central Asia, India, South America, Southern USA and is mainly found in Middle East and North Africa15. Peganum harmala is an effective herbal medicine possessing hallucinogenic, hypothemic, antihelmitic, abortifient, lactogogue and emmenagogue properties16. Several biological activities of P. harmala have been described in literature including anti-bacterial, anti-fungal and anti-viral activities17. It exhibits cytotoxicity with regard to HL60 and K562 leukemia cell lines18. The seeds appear to possess medicinal properties19. The main chemical constituents in the seeds of P. harmala were a serial of b-carboline alkaloids such as harman, harmine, harmaline and harmalol20. There are several reports in the literature indicating a wide spectrum of therapeutic activities for these b-carbolines such as anti-nociceptive and analgesic effects, anti-proliferative, vasorelaxant and hypothermic properties21. Total alkaloids of P. harmala have been reported to possess significant cytotoxic activity on cancerous cell lines and an inhibiting activity of the synthesis of DNA22. Recent interests in harmine and structurally related compounds were stimulated by their pharmacological, neurophysiologic and biochemical activities23. Harmine has anti-microbial, antiplasmodial, anti-fungal, anti-oxidative and hallucinogenic properties24. Recent studies demonstrated that harmine and its derivatives possessed significant anti-tumor potential both in vitro and in vivo25–27. Indeed, harmine showed potent cytotoxic activity
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against MCF7, HL60, K562, KB, A549, CAKI1, 1A9 and HEL cells with IC50 values of 29.3, 8.0, 8.9, 10.3, 11.3, 8.9, 7.5 and 8.9 mM, respectively28–30. In addition, it has been shown that harmine can reverse resistance to anti-cancer drugs by inhibiting the breast cancer resistance protein BCRP31. It was also reported that harmine and its derivatives were a potent inhibitor of DYRK1A which has been found to be associated with neurofibrillary tangles (NFTs) in sporadic Alzheimer’s disease. In addition to DYRK1A inhibition, harmine has been reported to be a selective inhibitor of tau phosphorylation at multiple Alzheimer’s disease-related sites32. The inhibition of cholinesterase (ChE) can be also a new way to stop the disease of Alzheimer. Among the family of heterocyclic compounds, isoxazolines are an important class of five-membered ring heterocycles, displaying a wide variety of biological properties including anti-viral33, anti-depressant34, anti-bacterial35, anti-fungal36, antiinfluenza37 and anti-cancer38 as well as anti-inflammatory activity39. Furthermore, the isoxazolines possess significant synthetic applications40 and represent a unique class of pharmacophore present in many therapeutic agents41. Encouraged by the broad biological activity of harmine derivatives and isoxazolines biological interests, we chose harmine as a scaffold to generate novel derivatives, via 1,3dipolar cycloaddition reactions using various arylnitrile oxides. The objective of this study was the synthesis of new analogues (not cited in literature) from harmine and the evaluation of their anti-acethylcholinesterase, anti-5-lipoxygenase and anti-cancer properties.
Materials and methods Shimadzu QP-1000 EX spectrometer was used in the ESI+ experiment. 1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded on a Bruker AM-300 spectrometer, using CD3OD as solvent and none deuterated residual solvent as internal standard. Chemicals shifts () are given in parts per million (ppm) and coupling constants (J) in Hertz. Melting points were determined on a Bu¨chi 510 apparatus using capillary tubes. Extraction and isolation A total of 240 g of dried and powdered seeds of P. harmala fruits were macerated four times with 300 mL methanol at 50 C for 1 h. The extracts were combined and evaporated to dryness. The residue was dissolved in 300 mL HCl (2%) then filtered. The filtrate was extracted two times with 250-mL pentane. The aqueous acid layer was basified (pH 10) with NH4OH and extracted four times with 300-mL chloroform. The chloroform layer was combined and evaporated to dryness, then the residue (5.8 g) was purified by chromatography on silica gel eluting successively with ethyl acetate–methanol (90:10 to 0:100) to afford 1.2 g (20.7%) harmine and four fractions42. Harmine 1: 7-methoxy-1-methyl-9H -pyrido[3,4-b]indole m.p.: 321 C. ES-MS m/z 213 [M + H]+. 1H-NMR (300 MHz, CD3OD) d ppm: 8.11 (1H, d, J ¼ 5.4 Hz, H-3), 7.97 (1H, d, J ¼ 8.7 Hz, H-5), 7.78 (1H, d, J ¼ 5.4 Hz, H-4), 7.04 (1H, d, J ¼ 2.1 Hz, H-8), 6.85 (1H, dd, J ¼ 8.7, 2.4 Hz, H-6), 3.91 (3H, s), 2.77 (3H, s). 13C-NMR (75 MHz, CD3OD) d ppm: 162.5 (C-7), 144.1 (C-1), 142.0 (C-13), 137.9 (C-3), 136.2 (C-10), 130.1 (C-11), 123.5 (C-5), 116.3 (C-12), 113.3 (C-4), 110.9 (C-6), 95.3 (C-8), 55.9 (CH3-O),19.5 (CH3). The spectral data of compound 1 matched with the data reported for harmine 1 from the literature43.
J Enzyme Inhib Med Chem, Early Online: 1–6
General procedure for the preparation of compound 2 Under argon atmosphere, harmine 1 (0.35 g, 1.64 mmol) is dissolved in anhydrous Dimethylformamide (DMF) (20 mL). Allyl bromide (5 equiv.) was added in the presence of sodium hydride (2 equiv.). The mixture was stirred for 24 h at room temperature, then poured into ice-cold water, and extracted with ethyl acetate. The organic layer was dried over Na2SO4, after removal of solvent in vacuum, the resulting residue was purified by silica gel column chromatography (EtOAc) to give compound 2 (229 mg, 55.4%)44. Compound 2: 9-allyl-7-methoxy-1-methyl-9Hpyrido[3,4-b]indole Yield 55%, ES-MS m/z 253 [M + H]+. 1H-NMR (300 MHz, CD3OD) d ppm: 8.00 (1H, d, J ¼ 5.4 Hz, H-3), 7.91 (1H, d, J ¼ 8.4 Hz, H-5), 7.75 (1H, d, J ¼ 5.1 Hz, H-4), 6.81 (2H, m, H-8, H-6), 6.02 (1H, m, H-15), 5,03 (3H, m, H-14a, H-16a, H-16b), 4.52 (1H, brd, J ¼ 17.4 Hz, H-14b), 3.79 (3H, s, CH3–O), 2.80 (3H, s, CH3). 13C-NMR (75 MHz, CD3OD) d ppm: 163.0 (C-7), 145.2 (C-1), 141.6 (C-13), 137.9 (C-3), 136.5 (C-10), 135.4 (C-16), 131.3 (C-11), 123.4 (C-5), 115.9 (C-12), 115.7 (C-15), 113.6 (C-4), 111.2 (C-6), 94.0 (C-8), 56.1 (CH3–O), 47.6 (C-14), 21.8 (CH3). General procedure for the preparation of compounds 3a–f The nitrile oxides were prepared from aldoximes by halogenations followed by an in situ dehydrohalogenation using a base45. To compound 2 (0.05 g, 0.2 mmol) in refluxing dichloromethane, the appropriate nitrile oxide (2 equiv.) in the presence of triethylamine (2 equiv.) was added and the mixture was refluxed for 48 h. The resulting mixture was washed with water, and then extracted with CH2Cl2. The organic layer was dried over Na2SO4. The solvent was then removed under reduced pressure. The resulting residue was purified by preparative TLC (EtOAc/ CH3OH) to give corresponding heterocycles 3a–f. Compound 3a: 9-((4,5-dihydro-3-phenylisoxazol-5yl)methyl)-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole Yield 75%, m.p.: 98 C, ES-MS m/z 372 [M + H]+. 1H-NMR (300 MHz, CD3OD) d ppm: 8.16 (1H, d, J ¼ 5.4 Hz, H-3), 8.05 (1H, d, J ¼ 8.7 Hz, H-5), 7.95 (1H, d, J ¼ 5.4 Hz, H-4), 7.70 (2H, d, J ¼ 7.8 Hz, Ar-H), 7.46 (2H, d, J ¼ 7.8 Hz, Ar-H), 7.20 (1H, d, J ¼ 2.1 Hz, H-8), 6.96 (1H, dd, J ¼ 2.1, 8.7 Hz, H-6), 5.18 (1H, m, H-15), 4.88 (1H, dd, J ¼ 9.0, 15.9 Hz, H-14a), 4.77 (1H, dd, J ¼ 3.3, 15.9 Hz, H-14b), 3.95 (3H, s, CH3–O), 3.61 (1H, dd, J ¼ 9.8, 17.2 Hz, H-16a), 3.36 (1H, dd, J ¼ 6.3, 17.1 Hz, H-16b), 3.03 (3H, s, CH3). 13C-NMR (75 MHz, CD3OD) d ppm: 163.2 (C ¼ N), 159.0 (C-7), 145.5 (C-1), 141.7 (C-13), 137.1 (C-3), 136.8 (C-10), 132.6 (Ar-C), 131.5 (Ar-C), 130.4 (C-11), 129.9 (Ar-C), 127.8 (Ar-C), 123.6 (C-5), 115.9 (C-12), 113.8 (C-4), 111.8 (C-6), 94.9 (C-8), 81.3 (C-15), 56.2 (CH3-O), 38.9 (C-14), 30.8 (C-16), 22.5 (CH3). Compound 3b: 9-((4,5-dihydro-3-p-tolylisoxazol-5yl)methyl)-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole Yield 70%, m.p.: 158 C, ES-MS m/z 386 [M + H]+. 1H-NMR (300 MHz, CD3OD) d ppm: 8.2 (1H, d, J ¼ 5.6 Hz, H-3), 8.15 (1H, d, J ¼ 8.4 Hz, H-5), 7.89 (1H, d, J ¼ 5.1 Hz, H-4), 7.59 (2H, d, J ¼ 8.1 Hz, Ar-H), 7.29 (2H, d, J ¼ 8.1 Hz, Ar-H), 7.17 (1H, d, J ¼ 1.8 Hz, H-8), 6.96 (1H, dd, J ¼ 1.8, 8.7 Hz, H-6), 5.16 (1H, m, H-15), 4.91 (1H, dd, J ¼ 9.0, 16.5 Hz, H-14a), 4.74 (1H, dd, J ¼ 3.3, 16.5 Hz, H-14b), 3.95 (3H, s, CH3–O), 3.65 (1H, dd,
DOI: 10.3109/14756366.2014.940932
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J ¼ 10.2, 17.1 Hz, H-16a), 3.37 (1H, dd, J ¼ 6.5, 17.1 Hz, H-16b), 2.99 (3H, s, CH3), 2.39 (3H, s, CH3). 13C-NMR (75 MHz, CD3OD) d ppm: 162.2 (C ¼ N), 158.9 (C-7), 145.2 (C-1), 142.2 (C-13), 142.1 (Ar-C), 138.1(C-3), 137.0 (C-10), 131.5 (Ar-C), 130.5 (C-11), 127.8 (Ar-C), 127.6 (Ar-C), 123.4 (C-5), 116.1 (C-12), 113.5 (C-4), 111.3 (C-6), 95.0 (C-8), 81.2 (C-15), 56.1 (CH3–O), 39.0 (C-14), 30.7 (C-16) 23.0 (CH3), 21.4 (CH3). Compound 3c: 9-((4,5-dihydro-3-(4-methoxyphenyl) isoxazol-5-yl)methyl)-7-methoxy-1-methyl-9H-pyrido [3,4-b]indole Yield 65%, m.p.: 145 C, ES-MS m/z 402 [M + H]+. 1H-NMR (300 MHz, CD3OD) d ppm: 8.17 (1H, d, J ¼ 5.4 Hz, H-3), 8.14 (2H, m, H-5, H-4), 7.63 (2H, d, J ¼ 9.0 Hz, Ar-H), 7.28 (1H, d, J ¼ 1.8 Hz, H-8), 7.05 (1H, dd, J ¼ 1.8, 8.7 Hz, H-6), 7.00 (2H, d, J ¼ 9.0 Hz, Ar-H), 5.15 (1H, m, H-15), 4.84 (2H, m, H-14), 4.01 (3H, s, CH3–O), 3.87 (3H, s, CH3–O), 3.65 (2H, m, H-16), 3.13 (3H, s, CH3). 13C-NMR (75 MHz, CD3OD) d ppm: 164.2 (C ¼ N), 160.7 (Ar-C), 157.6 (C-7), 145.4 (C-1), 141.2 (C-13), 138.3 (C-3), 136.7 (C-10), 130.5 (C-11), 128.5 (Ar-C), 125.4 (Ar-C), 123.4 (C-5), 115.2 (C-12), 114.2 (Ar-C), 112.6 (C-4), 110.1 (C-6), 93.2 (C-8), 81.3 (C-15), 58.6 (CH3–O), 55.31 (CH3–O), 41.56 (C-14), 32.6 (C-16), 22.35 (CH3). Compound 3d: 9-((3-(4-chlorophenyl)-4,5dihydroisoxazol-5-yl)methyl)-7-methoxy-1-methyl-9Hpyrido[3,4-b]indole Yield 60%, m.p.: 200 C, ES-MS m/z 406 [M + H]+. 1H-NMR (300 MHz, CD3OD) d ppm: 8.16 (1H, d, J ¼ 5.4 Hz, H-3), 8.07 (1H, d, J ¼ 8.7 Hz, H-5) 7.94 (1H, d, J ¼ 5.4 Hz, H-4), 7.65 (2H, d, J ¼ 7.8 Hz, Ar-H), 7.42 (2H, d, J ¼ 7.8 Hz, Ar-H), 7.16 (1H, d, J ¼ 2.1 Hz, H-8), 6.94 (1H, dd, J ¼ 2.1, 8.7 Hz, H-6), 5.19 (1H, m, H-15), 4.9 (1H, dd, J ¼ 9.0, 15.9 Hz, H-14a), 4.77 (1H, dd, J ¼ 3, 15.9 Hz, H-14b), 3.95 (3H, s, CH3–O), 3.64 (1H, dd, J ¼ 10.2, 17.4 Hz, H-16a), 3.40 (1H, dd, J ¼ 6.6, 16.5 Hz, H-16b), 2.99 (3H, s, CH3). 13C-NMR (75 MHz, MeOD-d4): d ppm: 163.4 (C ¼ N), 158.1 (C-7), 145.7 (C-1), 141.4 (C-13), 137.3 (C-3), 136.6 (C-10), 132.3 (Ar-C), 132.1 (Ar-C), 130.1 (C-11), 129.3 (Ar-C), 129.1 (Ar-C), 123.7 (C-5), 115.8 (C-12), 113.9 (C-4), 111.9 (C-6), 95.0 (C-8), 81.6 (C-15), 56.2 (CH3–O), 38.8 (C-14), 30.7 (C-16), 22.2 (CH3). Compound 3e: 9-((3-(furan-2-yl)-4,5-dihydroisoxazol-5yl)methyl)-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole Yield 45%, m.p.: 170 C, ES-MS m/z 362 [M + H]+. 1H-NMR (300 MHz, CD3OD) d ppm: 8.15 (1H, d, J ¼ 5.4 Hz, H-3), 8.05 (1H, d, J ¼ 8.7 Hz, H-5), 7.90 (1H, d, J ¼ 5.4 Hz, H-4), 7.16 (1H, d, J ¼ 2.1 Hz, H-8), 6.95 (1H, dd, J ¼ 2.1, 8.7 Hz, H-6), 6.88 (2H, d, J ¼ 3.6 Hz, H-30 , H-40 ), 6.48 (1H, d, J ¼ 3.6 Hz, H-50 ), 5.17 (1H, m, H-15), 4.85 (1H, dd, J ¼ 3.3, 15.9 Hz, H-14a), 4.74 (1H, dd, J ¼ 9.0, 15.9 Hz, H-14b), 3.95 (3H, s, CH3–O), 3.56 (1H, m, H-16a), 3.32 (1H, m, H-16b), 2.99 (3H, s,CH3). 13C-NMR (75 MHz, CD3OD) d ppm: 163.0 (C ¼ N), 150.9 (C-7), 146.4 (C-1), 145.3 (C-20 ), 141.9 (C-13), 140.3 (C-50 ), 137.7 (C-3), 136.8 (C-10), 131.7 (C-11), 123.5 (C-5), 116.3 (C-40 ), 116.0 (C-30 ), 114.2 (C-12), 112.9 (C-4), 111.5 (C-6), 94.9 (C-8), 81.4 (C-15), 56.2 (CH3–O), 38.3 (C-14), 30.4 (C-16), 22.8 (CH3). Compound 3f: 9-((4,5-dihydro-3-(thiophen-2-yl)isoxazol-5yl)methyl)-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole Yield 50%, m.p.: 180 C, ES-MS m/z 378 [M + H]+. 1H-NMR (300 MHz, CD3OD) d ppm: 8.04 (1H, d, J ¼ 5.4 Hz, H-3), 8.94 (1H, d, J ¼ 8.7 Hz, H-5), 7.80 (1H, d, J ¼ 5.4 Hz, H-4), 7.46 (1H, dd, J ¼ 0.9, 5.1 Hz, H-50 ), 7.24 (1H, dd, J ¼ 0.9, 3.6 Hz, H-30 ),
New isoxazolines derivatives from harmine
3
7.05 (1H, d, J ¼ 2.1 Hz, H-8), 7.01 (1H, dd, J ¼ 3.6, 5.1 Hz, H-40 ), 6.83 (1H, dd, J ¼ 2.1, 8.7 Hz, H-6), 5.08 (1H, m, H-15), 4.77 (1H, dd, J ¼ 16.2, 9.0 Hz, H-14a), 4.64 (1H, dd, J ¼ 3.3, 15.9 Hz, H-14b), 3.83 (3H, s, CH3–O), 3.65 (1H, dd, J ¼ 10.2, 16.8 Hz, H-16a), 3.28 (1H, dd, J ¼ 6.3, 16.5 Hz, H-16b), 2.99 (3H, s, CH3). 13 C-NMR (75 MHz, CD3OD) d ppm: 163.1 (C ¼ N), 154.7 (C-7), 145.3 (C-1), 141.9 (C-13), 137.6 (C-3), 136.8 (C-10), 132.4 (C-11), 131.8 (C-20 ), 130.2 (C-40 ), 129.3 (C-30 ), 128.6 (C-50 ), 123.5 (C-5), 115.9 (C-12), 113.7 (C-4), 111.6 (C-6), 94.8 (C-8), 81.5 (C-15), 56.1 (CH3–O), 39.6 (C-14), 30.5 (C-16), 22.7 (CH3). AChE inhibitory activity assay ChE inhibitory activity was measured using Ellman’s method, as previously reported with modifications46. In this study, 50 mL of 0.1 -M sodium phosphate buffer (pH 8.0), 25 mL of AChE solution, 25 mL of each compound and 125 mL of DTNB (5,50 dithiobis[2-nitrobenzoic acid]) were added in a 96-well microplate and incubated for 15 min at 25 C. All compounds were re-suspended in the DMSO followed by dilution in the buffer and the DMSO does not exceed 1% in the mixture. 25 mL of a solution of acetylthiocholine iodide was added and the final mixture incubated, for 15 min, at 25 C. The hydrolysis of acetylthiocholine iodide was monitored by the formation of the yellow 5-thio-2nitrobenzoate anion as a result of the reaction of DTNB with thiocholines, catalyzed by enzymes at a wavelength of 412 nm. The concentration of the compounds which caused 50% inhibition of the AChE activity (IC50) was calculated by non-linear regression analysis. The percentage of inhibition was calculated from (1 S/E) 100, where E and S were the respective enzyme activity without and with the test sample, respectively. Galanthamine was used as positive control. Cytotoxicity/anti-cancer assay Cytotoxicity of compounds was estimated on human breast cancer cells (MCF7) as described by Bendaoud et al47. with modifications. Cells were distributed in 96-well plates at 3 104 cells/well in 100 mL and then 100 mL of culture medium containing sample at various concentrations were added. Cell growth was estimated by MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. MTT is a yellow water-soluble tetrazolium salt. Metabolically active cells are able to convert the dye to water-insoluble dark blue formazan by reductive cleavage of the tetrazolium ring. All compounds were re-suspended in the DMSO followed by dilution in the buffer and the DMSO does not exceed 1% in the mixture. Doxorubicin was used as positive control. Lipoxygenase activity assay The anti-inflammatory activity of harmine 1 and its derivatives (2 and 3a–f) was determined on Soybean lipoxygenase (LOX) as described by Bylac and Racine with modifications48. Various concentrations of 20 mL of each compound was mixed individually with sodium phosphate buffer (pH 7.4) containing 5-LOX and 60 mL of linoleic acid (3.5 mM), yielding a final volume of 1 mL. The blank does not contain the substrate, but will be added 30 mL of buffer solution. All compounds were re-suspended in the DMSO followed by dilution in the buffer so that the DMSO does not exceed 1%. The mixture was incubated at 25 C for 10 min, and the absorbance was determined at 234 nm. The absorption change with the conversion of linoleic acid to 13-hydroperoxyoctadeca-9,11-dienoate (characterized by the appearance of the conjugated diene at 234 nm) was flowed for 10 min at 25 C. Nordihydroguaiaretic acid (NDGA) was used as positive control. The percentage of enzyme activity was plotted against the
4
I. Filali et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
81.2–81.6 and 30.4–32.6 ppm, respectively, in addition to the carbon signals introduced by the harmine 1 skeleton. It is noted that no other derivatives were isolated from the reaction mixture, indicating the high regioselectivity of the reaction. The AChE inhibition was determined (Table 1) using an adaptation of the method described in the literature50. Values of IC50 ranging between 10.4 and 34.6 mM were obtained. The higher inhibitory activity was exhibited by the natural harmine 1 with an IC50 value of 10.4 ± 0.4 mM against AChE. Our results are in agreement with those of the literature. Indeed, harmine had been reported as a potent AChE inhibitory with IC50 value of 9.0 ± 1.1 mM51. Both compound 1 and its isoxazoline derivatives proved to inhibit AChE. The allylation of the nitrogen atom at position 9 led to the redaction of inhibitory potency (IC50 ¼ 13.5 ± 0.4 mM). Moreover, compound 3a with the phenyl system (IC50 ¼ 11.3 ± 0.7 mM) showed a good activity against the AChE enzyme, but is a little less active than harmine 1. It has been shown that the substitution in para position in the phenyl group decreases the activity regardless of the nature of substituent. A further drop in activity was noted for derivatives 3c and 3e (IC50 ¼ 15.1 ± 0.2 and 15.7 ± 0.9 mM, respectively), whose aromatic system of the isoxazoline moiety carries a methoxy group in
concentration of each compound. The IC50 value is the concentration of each compound that caused 50% enzyme inhibition.
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Results and discussion Harmine was isolated from the seeds of P. harmala using an adaptation of the method described in the literature. Harmine 1 was identified by its spectral data (1H, 13C and MS) compared with those of the literature49. In order to prepare new isoxazoline derivatives of harmine, we started by the allylation of the nitrogen atom in the five-membered ring of harmine with allyle bromide in the presence of sodium hydride to create a new double bond. The new double bond will serve in for eventual 1,3-dipolar cycloaddition reactions. This reaction has led to the preparation of the new 9-allyharmine 2 (Figure 1). The molecular formula of compound 2 was determined as C16H16N2O on the basis of ES-MS pseudomolecular peak [M + H]+ at m/z 253. The structure was evidenced by the appearance in the 1H-NMR spectrum of a multiplet at 6.0 ppm attributable to the ethylenic proton H-15 and a massif at 5.03 ppm attributable to the terminal methylenic protons H-16. The 13C-NMR spectrum reinforced this structure by the appearance of the two ethylenic carbon signals at 115.7 and 136.6 ppm and the appearance of a signal at 47.6 ppm attributed to the carbon C-14. The condensation of 2 with various arylnitrile oxides in refluxing dichloromethane for 48 h to generate corresponding heterocyclic compounds 3a–f. All novel compounds synthesized were not cited in literature. All synthesized compounds were obtained as yellow solids. Their ES-MS gave pseudo-molecular peaks [M + H]+, which are consistent with their molecular formula. The 1H-NMR spectra (300 MHz, CD3OD) of compounds 3a–f, in addition to the proton introduced by harmine 1, showed the observation of signals at 6.4–8.2 ppm corresponding to the protons of the aromatic rings attached to the isoxazoline moiety. The same 1H-NMR spectra revealed the presence of a characteristic signal at 5.1–5.2 ppm relative to H-15 and the doublet of doublets or multiplets consequent to the methylenic protons H-16 of the isoxazoline ring. The 13C-NMR spectra confirmed the above spectral data by the observation of signals at 145.3–116.0 ppm relative to carbons of the aromatic systems. The same spectra reinforced the proposed structures by the appearance of the signals of the iminic carbon C-17, C-15 (CH) and C-16 (CH2) at 162.2–163.4, Figure 1. Synthesis of derivatives 2 and 3a–f.
Table 1. Acetylcholinesterase inhibition harmine 1, harmine derivatives 2 and 3a–f. Compound
12
6
CH3
O
13 8
10.4 ± 0.4 13.5 ± 0.4 11.3 ± 0.7 34.6 ± 1.7 15.1 ± 0.2 25.1 ± 1.9 15.7 ± 0.9 27.8 ± 0.7 4.1 ± 0.2
*Averages ± SD were obtained from three different experiments. IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50% and are the mean of three independent measurements, each performed in triplicate.
N
9
5
Br
N2
11
3
4
3
10
7
of
Acetylcholinesterase IC50 (mM)*
1 2 3a 3b 3c 3d 3e 3f Galanthamine
4 5
capacity
12
6
1
NaH/ DMF
CH3
CH3
N
11 1
10
O
CH3
N
13
7 8
H
14
1
15
2 16
R
3a R = phenyl 3b R = 4'-methylphenyl 3c R = 4'-methoxyphenyl 3d R = 4'-chlorophneyl
N
(Et)3N/RefluxCH2C2
3'
4'
3e R=
4
2'
5'
5
S
1'
5'
2'
O
12
6
3'
4'
3f R=
OH
Cl
CH3
3
N
11
10
O
7
13 8
N 14
1'
O
15
N 17
R
16
3a-f
1
C CH3
New isoxazolines derivatives from harmine
DOI: 10.3109/14756366.2014.940932
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Table 2. HCT116 and MCF7 inhibitions of harmine 1, harmine derivatives 2 and 3a–f. Compound
HCT116 IC50 (mM)*
MCF7 IC50 (mM)*
1 2 3a 3b 3c 3d 3e 3f Doxorubicin
0.7 4400 9.7 16.9 4247 60.0 113.7 67.2 1.8 ± 0.2
1.3 0.5 0.2 6.2 34.6 6.2 3.1 19.4 0.8 ± 0.1
*Cytotoxicity as IC50 for each cell line, is the concentration of compound that inhibits of 50% the cell multiplication after 48 h of treatment.
para position and with the thiophene system, respectively. Compounds 3d and 3f exhibited moderate activity against the AChE enzyme (IC50 ¼ 25.1 ± 1.9 and 27.8 ± 0.7 mM, respectively). Compound 3b, whose aromatic system of the isoxazoline moiety carries a methyl group in para position, is the less active derivative (IC50 ¼ 34.6 ± 1.7 mM). These data suggest that the secondary amine function should be regarded as essential features able to confer high potency inhibition. Cytotoxicity was evaluated on two cell-lines: MCF7 breast cancer and HCT116 colon cancer. By comparing the IC50 of the various products tested towards both cellular lines, it is shown that the MCF7 line is the most sensitive to the various products tested (Table 2). Harmine 1 exhibited potent cytotoxic activity in this study against both cell lines (IC50 was 0.7–1.3 mM). Harmine was tested in several human tumor cell lines. The prepared derivatives 2, 3a, 3b, 3d, 3e and 3f exhibited cytotoxic activity against MCF7 breast cancer line. Compound 3a (IC50 ¼ 0.2 mM) exhibited the most interesting cytotoxic activity against this cell line. Also, compound 3e with the thiophene system showed a good cytotoxic activity against the same cell line (IC50 ¼ 3.1 mM). A further drop in activity was noted for derivatives 3b and 3d whose aromatic system of the isoxazoline moiety carries a methyl group and a Cl atom in para position, respectively (both IC50 ¼ 6.2 mM). The HCT116 line is less sensitive to the various tested products compared to MCF7 line. Harmine 1 showed the most interesting cytotoxic activity against this cell line (IC50 ¼ 1.3 mM). Compounds 3c and 3d whose aromatic system of the isoxazoline moiety carries a methoxy group and a Cl atom in para position respectively, are much less active to inactive by comparison with their analogues 3a and 3b. The two compounds 3e and 3f with the thiophene and furan systems, respectively, showed a much lower activity (IC50 ¼ 113.7 and IC50 ¼ 67.2 mM, respectively). In the present study, the 5-LOX inhibition was determined using an adaptation of the method described in the literature52. All compounds were analyzed on what concerns their 5-LOX inhibition to identify new anti-inflammatory compounds (Table 3). There is no report in the literature for the testing of harmine or derivatives against this enzyme. Harmine 1 and derivatives 2 and 3a–d have showed low activity against 5-LOX enzyme (IC504100 mM). Only the two derivatives 3e and 3f with the thiophene and furan systems, respectively, showed good activity against 5-LOX enzyme (IC50 ¼ 29.2 ± 3.1 and 55.5 ± 0.8 mM, respectively) comparable to the reference compound NDGA (IC50 ¼ 8.1 ± 0.1 mM).
Conclusion In conclusion, we have reported the synthesis of novel harmine derivatives via intermolecular 1,3-dipolar cycloaddition reactions
5
Table 3. 5-Lipoxygenase inhibition capacity (inhibition %, IC50 (mM)*) of harmine 1, harmine derivatives 2 and 3a–f. Compounds 1 2 3a 3b 3c 3d 3e 3f NDGA
5-Lipoxygenase 4100 4100 4100 4100 4100 4100 29.2 ± 3.1 55.5 ± 0.8 8.1 ± 0.1
*Averages ± SD were obtained from three different experiments. IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50% and are the mean of three independent measurements, each performed in triplicate.
involving nitrile oxides. Harmine 1 and its derivatives 2, 3a and 3e exhibited the most interesting cytotoxic activity against MCF7 cell line. The two derivatives 3e and 3f showed a good activity against 5-LOX enzyme.
Declaration of interest The authors declare no conflicts of interests. The authors alone are responsible for the content and writing of this article.
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