Synthesis, cytotoxic, anti-lipoxygenase and anti-acetylcholinesterase capacities of novel derivatives from harmine

Abstract

We synthesized two series of new hydrazide harmine derivatives. The reaction of harmine 1 with ethyl acetate chloride afforded the corresponding ethyl ester 2. The treatment of 2 with hydrazine hydrate gave the hydrazide 3 which further converted into hydrazones 4a–j and dihydrazides 5a–c. A series of new triazoles 7a–f has also been prepared from the suitable propargyl harmine 6. The synthesized derivatives were characterized by ¹H-NMR, ¹³C-NMR, and HRMS and evaluated for their activities against MCF7, HCT116 OVCAR-3, acetylcholinesterase and 5-lipoxygenase. The most hydrazones derivatives 4a-j have a good cytotoxic activity against all cell lines, when 4a, 4d, 4f and 4 g are more active than 1 (against OVCAR-3 IC₅₀ 16.7–2.5 μM). The compound 6 was the most active (IC₅₀=1.9 μM) against acetylcholinesterase. Some compounds exhibited significant activity against 5-lipoxygenase (IC₅₀=30.9–63.1 μM).

Keywords:

• Acetylcholinesterase
• cytotoxic
• hydrazide
• 5-lipoxygenase
• triazoles

Introduction

Heterocyclic compounds have attracted intense interest given to their varied applications in medicinal chemistry and organic chemistry as synthetic organic blocks. Among the various classes of heterocyclic compounds, we found alkaloids which are present in a broad range of plants1. These entities have been shown to serve as analgesic2, antiallergic3 and hallucinogenic4. Alkaloids are also endowed with a number of biological activities, including anticancer5, anti-acetylcholinesterase6, anti-inflammatory7 and antioxidant8 activities.

β-Carboline alkaloids, commonly known as harmala’s alkaloids, were first identified and isolated from Peganum harmala9. Harmine is among the β-carboline alkaloids, which can be found in some medicinal plants such as Banisteriopsis caapi, Eurycoma longifolia and abundantly in the seeds of P. harmala10. From ancient times, this alkaloid drew increasing interest due to its diverse pharmacological and biological activities11. It is known to be anti-acetylcholinesterase, antimicrobial, antifungal and cytotoxic12. The latest research showed that harmine can inhibit the breast cancer protein BCRP so it was used as resistance to anticancer drugs13. Harmine derivatives14 have received attention of researchers owing to their anti-alzheimer15, anticancer16 and antiproliferative17 activities.

Hydrazides have widespread interest in organic chemistry; they are generally prepared by the treatment of esters with hydrazine18. They are useful as a precursor for the construction of several organic compounds with a broad spectrum of biological activities19. For instance, hydrazides are used for the synthesis of several heterocyclic compounds such as pyrroles20, pyrazoles21, 1,3-thiazoles22, thiadiazoles23, 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines24 and hydrazones25. On the other hand, hydrazones are molecules containing azomethine group26. It has been reported that hydrazones possess many biological activities such as anticancer27, anti-inflammatory28, antibacterial29, anti-acetylcholinesterase and anti-butyrylcholinesterase30 activities. Recently, some hydrazones are synthesized as a drug in order to combat same diseases31.

1,2,3-Triazoles have attracted the interest of medicinal chemists due to their diverse biological activities such as anti-tumor, antimicrobial32 and anti-inflammatory33. The general method for the synthesis of 1,2,3-triazoles is the 1,3-dipolar cycloaddition, known as Huisgen cycloaddition, between azides and a terminal alkyne34. Sharpless invented the click chemistry approach using copper (I)-catalyzed azidealkyne cycloaddition (CuAAC), this reaction leads to the formation of 1, 4-disubstituted 1,2,3-triazoles in high yields and short times and avoid isomeric mixture35.

All these observations encouraged us to prepare new class of heterocyclic compounds incorporating harmine moiety. In this work, we reported the synthesis of 2-(7-methoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl) acetohydrazide 3 and its use as a building block in the synthesis of some new harmine derivatives 4a–j and 5a–c. On the other hand, we prepared a new triazoles harmine derivatives 7a–f using copper (I)-catalyzed cycloaddition. The cytotoxic, anti-5-lipoxygenase (5-LOX) and anti-acetylcholinesterase (AChE) activities of all synthesized derivatives were evaluated and discussed.

Materials and methods

Chemistry

Melting points were taken on a Buchi-510 capillary melting point apparatus. ¹H (300 MHz) and ¹³C (75 MHz) NMR spectra were recorded on a Bruker AM-300 spectrometer, using CDCl₃, CD₃OD and DMSO-d₆ as solvent and non-deuterated residual solvent as internal standard. Chemicals shifts (δ) are given in parts per million (ppm) and coupling constants (J) in Hertz. Commercial harmine was ordered from Sigma Aldrich French (St. Quentin Fallavier, France) (2 g).

Procedure for the preparation of compound 2

Under argon atmosphere, harmine 1 (1 g, 4.7 mmol) was dissolved in anhydrous DMF (40 mL). Acetylacetate chloride (1.5 equiv.) was added in the presence of sodium hydride (2 equiv.). The mixture was stirred for 48 h at room temperature, then poured into ice-cold water, and extracted with dichloromethane. After removal of solvent, the resulting residue was purified by silica gel column chromatography (EtOAc) to give compound 2 (850 mg, 60%)36.

Ethyl-2–(7-methoxy-1-methyl-9H-pyrido[3,4-b] indol-9-yl)acetate 2

Yellow solid, Yield 60%, m.p.: 130 °C, ES-HRMS m/z [M + H]⁺299.1396. ¹H-NMR (300 MHz, CDCl₃) δ_H: 8.29 (1H, d, J = 5.4 Hz, H-3), 7.95 (1H, d, J = 8.7 Hz, H-5), 7.72 (1H, d, J = 5.4 Hz, H-4), 6.89 (1H, dd, J = 8.7 Hz and J = 2.1 Hz, H-6), 6.74 (1H, d, J = 2.1 Hz, H-8), 5.15 (2H, s, H-14), 4.23 (2H, q, J = 7.2 Hz, CH₂), 3.90 (3H, s, CH₃-O), 2.92 (3H, s, CH₃), 1.24 (3H, t, J = 7.2 Hz, CH₃). ¹³C-NMR (75 MHz, CDCl₃) δ_C: 168.2 (C = O), 160.7 (C-7), 143.04 (C-1), 139.9 (C-13), 138.2 (C-3), 135.1 (C-10), 129.4 (C-11), 122.0 (C-5), 114.8 (C-12), 111.9 (C-4), 108.9 (C-6), 92.5 (C-8), 61.4 (CH₂), 52.2 (O-CH₃), 46.3 (C-14), 22.3 (CH₃), 13.6 (CH₃).

Procedure for the preparation of hydrazide 3

Wishing the preparation of hydrazide 3 by exploiting the new ester function, compound 2 was stirred with hydrazine hydrate (1 equiv.) in ethanol for 24 h37. Once the reaction is completed, the precipitate is filtered then recrystallized in ethanol. The hydrazide 3 was obtained in 75% yield.

2–(7-methoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl)acetohydrazide 3

White solid, Yield 75%, m.p.: 210 °C, ES-HRMS m/z 285.1354 [M + H]⁺. ¹H-NMR (300 MHz, DMSO-d₆) δ_H: 9.40 (1H, s, N-H), 8.61 (1H, d, J = 5.4 Hz, H-3), 7.95 (1H, d, J = 8.7 Hz, H-5), 7.72 (1H, d, J = 5.4 Hz, H-4), 6.89 (1H, dd, J = 8.7 Hz and J = 2.1 Hz, H-6), 6.74 (1H, d, J = 1.8 Hz, H-8), 5.15 (2H, s, H-14), 4.30 (2H, s, NH₂), 3.87 (3H, s, CH₃-O), 2.87 (3H, s, CH₃). ¹³C-NMR (75 MHz, DMSO-d₆) δ_C: 167.5 (C = O), 160.5 (C-7), 143.4 (C-1), 134.0 (C-13), 137.9 (C-3), 135.5 (C-10), 128.4 (C-11), 122.1 (C-5), 114.58 (C-12), 112.0 (C-4), 109.1 (C-6), 93.9 (C-8), 55.6 (O-CH₃), 46.0 (C-14), 22.7 (CH₃).

General procedure for the preparation of compounds 4a–j

A mixture of compound 3 (1 mmol) and the appropriate aromatic aldehydes (1 mmol) in 1,4-dioxane (20 mL) was boiled under reflux for 24 h37. After completion of the reaction, the solvent was then removed under reduced pressure. The resulting residue was purified by silica gel column chromatography (EP/EtOAc, 2:8) to give compounds 4a–j.

Compound 4a: N′-(4-(tert-butyl)benzylidene)-2–(7-methoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl)acetohydrazide

White solid, Yield 65%, m.p.: 205 °C, ES-HRMS m/z 429.2287 [M + H]⁺. ¹H-NMR (300 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_H: 10,37 (1H, s, NH), 8.26 (1H, d, J = 5.4 Hz, H-3), 7.91 (1H, d, J = 8.7 Hz, H-5), 7.68 (1H, d, J = 5.4 Hz, H-4),7.73 (1H, s, CH = N), 7.48 (2H, d, J = 8.4 Hz, H-3′ and H-5′), 7.41 (2H, d, J = 8.4 Hz, H-2′ and H-6′), 6.86 (1H, dd, J = 8.7 and J = 2.1 Hz, H-6), 6.80 (H, d, J = 2.1 Hz, H-8), 5.62 (2H, s, H-14), 3.79 (3H, s, CH₃-O), 2.90 (3H, s, CH₃), 1.32 (9H, s, 3CH₃). ¹³C-NMR (75 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_C: 169.6 (C = O), 160.8 (C-7), 153.7 (C = N), 150.0 (C-1′), 145.7 (C-1), 143.6 (C-13), 139.9 (C-3), 137.3 (C-10), 135.5 (C-4′), 129.9 (C-3′ and C-5′), 129.4 (C-11), 126.6 (C-2′ and C-6′), 125.2 (C-5), 114.6 (C-12), 112.0 (C-4), 109.1 (C-6), 92.6 (C-8), 55.1 (O-CH₃), 45.6 (C-14), .4 (C-H), 30.6 (3CH₃), 21.7 (CH₃).

Compound 4b: N'-(4-(dimethylamino)benzylidene)-2–(7-methoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl)acetohydrazide

Yellow solid, Yield 78%, m.p. 166 °C, ES-HRMS m/z 416.2080 [M + H]⁺. ¹H-NMR (300 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_H: 7.91 (1H, d, J = 5.4 Hz, H-3), 7.95– (1H, d, J = 8.1 Hz, H-5), 7.75 (1H, s, H-2′), 7.71 (1H, d, J = 5.1 Hz, H-4), 7.55 (1H, s, CH = N), 7.28 (1H, d, J = 8.4 Hz, H-6′), 6.95 (2H, d, J = 8,4 Hz, H-5′), 6.85 (2H, d, J = 7.8 Hz, H-6′), 6.65 (2H, m, H-6 and H-8), 5.62 (2H, s, H-14), 3.81 (3H, s, CH₃-O), 2.97 (6H, s, 2CH₃), 2.85 (3H, s, CH₃). ¹³C-NMR (75 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_C: 174.3 (C = O), 166.8 (C-7), 152.7 (C = N), 147.0 (C-1′), 146.7 (C-1), 143.6 (C-13), 138.9 (C-3), 137.1 (C-10), 130.2(C-4′), 129.3 (C-11), 128.1 (C-2′ and C-6′), 122.1 (C-5), 114.6 (C-12), 112.2 (C-3′ and C-5′), 111.2 (C-4), 109.5 (C-6), 92.4 (C-8), 55.1 (O-CH₃), 45.5 (C-14), 39.5 (2CH₃), 20.7 (CH₃).

Compound 4c: N′-(3,4-dimethoxybenzylidene)-2–(7-methoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl) acetohydrazide

White solid, Yield 80%, m.p.: 242 °C, ES-HRMS m/z 433.1865 [M + H]⁺. ¹H-NMR (300 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_H: 8.23 (1H, d, J = 5.1 Hz, H-3), 7.95 (1H, d, J = 8.1 Hz, H-5), 7.75 (1H, s, CH = N), 7.73 (1H, d, J = 5.4 Hz, H-4), 7.28 (1H, d, J = 8.4 Hz, H-2′), 6.95 (2H, d, J = 8.4 Hz, H-5′ and H-6′), 6.86 (2H, m, H-6 and H-8), 5.68 (2H, s, H-14), 3.94 (6H, s, CH₃-O), 3.84 (3H, s, CH₃-O), 2.95 (3H, s, CH₃). ¹³C-NMR (75 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_C: 169.6 (C = O), 162.1 (C-7), 151.3 (C = N), 150.2 (C-4′), 149.3 (C-3′), 146.2 (C-1), 144.1 (C-13), 140.2 (C-3), 137.6 (C-10), 135.9 (C-11), 130.1 (C-1′), 126.0 (C-5), 122.2 (C-6′), 115.0 (C-12), 112.6 (C-4),110.7 (C-2′), 109.6 (C-5′), 108.1 (C-6), 93.0 (C-8), 56.0 (O-CH₃), 55.9 (O-CH₃), 55.8 (O-CH₃), 46.1 (C-14), 22.3 (CH₃).

Compound 4d: 2–(7-methoxy-1-methyl-9H-pyrido [3,4-b]indol-9-yl)-N'-(4–(1,1,2,2-tetrafluoroethyl)benzylidene)acetohydrazide

Yellow solid, Yield 70%, m.p.: 252 °C, ES-HRMS m/z 473.1601 [M + H]⁺. ¹H-NMR (300 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_H: 7.53 (1H, d, J = 5.4 Hz, H-3), 7.33 (1H, d, J = 8.4 Hz, H-5), 7.15 (1H, d, J = 5.4 Hz, H-4), 6.92 (2H, m, H-2′ and H-6′), 6.68 (2H, m, H-3′ and H-5′), 6.26 (1H, dd, J = 8.4 Hz and J = 2.1 Hz, H-6), 6.17 (1H, d, J = 2.1 Hz, H-8), 5.47 (1H, tt, J = 2.7 Hz and J = 52.8 Hz, H-F₂), 5.04 (2H, s, H-14), 3.24 (3H, s), 2.70 (3H, s). ¹³C-NMR (75 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_C: 173.9 (C = O), 165.8 (C-7), 153.3 (C = N), 148.3 (C-1), 144.2 (C-13), 141.4 (C-3), 139.9 (C-10), 134.1 (C-1′), 133.8 (CF₂-H), 129.8 (C-11), 129,4 (C-2′ and C-6′), 127.4 (C-3′ and C-5′), 126.5 (C-3′ and C-5′), 124.3 (CF₂), 118.9 (C-5), 114.5 (C-12), 111.5 (C-4), 108.2 (C-6), 97.0 (C-8), 59.6 (O-CH₃), 51.3 (C-14), 25.7 (CH₃).

Compound 4e: 2–(7-methoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl)-N'-(4-(methylthio) benzylidene)acetohydrazide

Yellow solid, Yield 90%, m.p.: 246 °C, ES-HRMS m/z 419.1551 [M + H]⁺. ¹H-NMR (300 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_H: 8.06 (1H, d, J = 5.4 Hz, H-3), 7.86 (1H, d, J = 8.7 Hz, H-5), 7.80 (1H, s, CH = N), 7.65 (1H, d, J = 5.4 Hz, H-4), 7.48 (1H, d, J = 8.4 Hz, H-2′ and H-6′),7.17 (2H, d, J = 8.4 Hz, H-3′ and H-5′), 6.79 (2H, dd, J = 8.7 and J = 2.1 Hz, H-6), 6.73 (H, d, J = 2.1 Hz, H-8), 5.57 (2H, s, H-14), 3.75 (3H, s, CH₃-O), 2.80 (3H, s, CH₃), 2.40 (3H, s, CH₃-S). ¹³C-NMR (75 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_C: 173.2 (C = O), 164.8 (C-7), 149.0 (C = N), 147.6 (C-1), 145.6 (C-4′), 143.8 (C-13), 140.8 (C-3), 139.5 (C-10), 133.6 (C-1′), 131.0 (C-11), 130.9 (C-2′ and C-6′), 129.5 (C-3′ and C-5′), 125.9 (C-5), 118.4 (C-12), 115.9 (C-4), 113.1 (C-6), 96.6 (C-8), 58.9 (O-CH₃), 49.5 (C-14), 25.1 (CH₃-S), 18.4 (CH₃).

Compound 4f: 2–(7-methoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl)-N'-(4-methoxybenzylidene)acetohydrazide

White solid, Yield 60%, m.p.: 264 °C, ES-HRMS m/z 403.1768 [M + H]⁺. ¹H-NMR (300 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_H: 7.95 (1H, d, J = 5.4 Hz, H-3), 7.79 (1H, d, J = 8.4 Hz, H-5), 7.72 (1H, s, CH = N), 7.59 (1H, d, J = 5.4 Hz, H-4),7.44 (2H, d, J = 8.7 Hz, H-2′ and H-6′), 6.77 (2H, d, J = 8.7 Hz, H-3′ and H-5′),6.71 (1H, d, J = 1.8 Hz, H-8), 6.67 (2H, dd, J = 8.1 Hz and J = 1.8 Hz, H-6), 5.51 (2H, s, H-14), 3.69 (6H, s, 2(CH₃-O)), 2.72 (3H, s). ¹³C-NMR (75 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_C: 172.2 (C = O), 163.2 (C-7), 162.9 (C-4′), 151.3 (C = N), 145.8 (C-1), 141.7 (C-13), 138.7 (C-3), 137.5 (C-10),132.4 (C-2′ and C-6′), 130.7 (C-11), 127.7 (C-1′), 123.8 (C-5), 116.3 (C-12), 115.7 (C-3′ and C-5′), 113.9 (C-4), 111.6 (C-6), 94.7 (C-8), 56.8 (O-CH₃), 56.6 (O-CH₃), 48.4 (C-14), 20.4 (CH₃).

Compound 4 g: N'-(4-chloro-3-nitrobenzylidene)-2–(7-methoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl)acetohydrazide

White solid, Yield 65%, m.p.: 176 °C, ES-HRMS m/z 452.1125 [M + H]⁺. ¹H-NMR (300 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_H: 8.10 (1H, d, J = 5.4 Hz, H-3), 7.95 (1H, d, J = 6.9 Hz, H-5), 7.76 (2H, m, CH = N and H-2′), 7.61 (2H, m, H-6′ and H-5′), 7.40 (1H, d, J = 5.4 Hz, H-4), 6.70 (2H, m, H-8 and H-6), 5.51 (2H, s, H-14), 3.67 (3H, s, CH₃-O), 2.73 (3H, s, CH₃). ¹³C-NMR (75 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_C: 171.2 (C = O), 163.1 (C-7), 146.0 (C-3′), 143.6 (C-1), 141.0 (C-13), 137.4 (C-3), 135.4 (C-10), 133.7 (C-6′), 132.5 (C-1′), 129.5 (C-4′ and C-5′), 129.5 (C-11), 124.7 (C-2′), 124.0 (C-5), 116.3 (C-12), 114.1 (C-4), 111.4 (C-6), 94.6 (C-8), 56.9 (O-CH₃), 47.4 (C-14), 22.5 (CH₃).

Compound 4 h: N'-(4-formylbenzylidene)-2–(7-methoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl) acetohydrazide

White solid, Yield 50%, m.p.: 294 °C, ES-HRMS m/z 401.1601 [M + H]⁺. ¹H-NMR (300 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_H: 10.07 (1H, s, HC = O), 8.30 (1H, s, CH = N), 8.19 (1H, d, J = 5.4 Hz, H-3), 8.11 (1H, d, J = 8.4 Hz, H-5), 8.30 (1H, s, CH = N), 8.08 (4H, m, H-2′, H-3′, H-5′ and H-6′), 7.88 (1H, d, J = 5.4 Hz, H-4), 7.25 (1H, s, H8), 6.90 (1H, d, J = 8.4 Hz, H-6), 5.89 (2H, s, H-14), 3.95 (3H, s, CH₃-O), 2.89 (3H, s, CH₃). ¹³C-NMR (75 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_C: 192.5 (HC = O), 170.0 (C = O), 160.6 (C-7), 143.8 (C = N), 143.0 (C-1), 140.7 (C-13), 139.5 (C-3), 137.9 (C-1′), 136.8 (C-10), 132.4 (C-4′), 129.8 (C-11), 128.5 (C-3′ and C-5′), 127.5 (C-2′ and C-6′), 122.4 (C-5), 115.3 (C-12), 113.9 (C-4), 109.3 (C-6), 93.7 (C-8), 55.6 (O-CH₃), 45.9 (C-14), 22.4 (CH₃).

Compound 4i: 2–(7-methoxy-1-methyl-9H-pyrido [3,4-b]indol-9-yl)-N'-(quinolin-6-ylmethylene) acetohydrazide

White solid, Yield 40%, m.p.: 259 °C, ES-HRMS m/z 424.1773 [M + H]⁺. ¹H-NMR (300 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_H: 8.81 (1H, d, J = 4.8 Hz, H-3), 8.53 (1H, s, HC = N), 8.35 (1H, d, J = 8.4 Hz, H-5), 8.06 (2H, d, J = 8.7 Hz, H-2′ and H-7′), 7.86 (1H, d, J = 4.8 Hz, H-4), 7.72 (4H, m, H-3′, H-4′, H-5′ and H-8′), 6.80 (1H, d, J = 8.4 and J = 1.8 Hz, H-6), 6.75 (1H, d, J = 1.8 Hz, H-8), 5.66 (2H, s, H-14), 3.82 (3H, s, CH₃-O), 2.83 (3H, s, CH₃). ¹³C-NMR (75 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_C: 173.5 (C = O), 164.9 (C-7), 153.0 (C-6′), 151.7 (C = N), 145.4 (C-1), 143.5 (C-6′a), 141.0 (C-13), 140.7 (C-3), 133.8 (C-10), 133.4 (C-4′), 133.0 (C-8′), 131.2 (C-2′ and C-7′), 128.8 (C-11), 126.5 (C-3′ and C-3′a), 126.0 (C-5′), 122.5 (C-5), 118.4 (C-12), 116.1 (C-4), 113.1 (C-6), 96.5 (C-8), 59.0 (O-CH₃), 49.7 (C-14), 25.7 (CH₃).

Compound 4j: 2–(7-methoxy-1-methyl-9H-pyrido [3,4-b]indol-9-yl)-N'-(4-morpholinobenzylidene) acetohydrazide

White solid, Yield 45%, m.p.: 266 °C, ES-HRMS m/z 458.2222 [M + H]⁺. ¹H-NMR (300 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_H: 7.96 (1H, d, J = 5.4 Hz, H-3), 7.82 (1H, d, J = 8.7 Hz, H-5), 7.67 (1H, s, CH = N), 7.59 (1H, d, J = 5.4 Hz, H-4), 7.42 (2H, d, J = 8.7 Hz, H-2′ and H-6′), 6.74 (2H, d, J = 8.7 Hz, H-3′ and H-5′), 6.68 (1H, dd, J = 8.7 and J = 1.8 Hz, H-6), 6.66 (1H, d, J = 1.8 Hz, H-8), 5.49 (2H, s, H-14), 3.67 (3H, s, CH₃-O), 3.70 (4H, m, 2CH₂), 3.15 (4H, m, 2CH₂), 2.72 (3H, s, CH₃). ¹³C-NMR (75 MHz, CDCl₃ and CD₃OD (80 v/20 v)) δ_C: 170.4 (C = O), 162.3 (C-7), 153.6 (C = N), 147.1 (C-4′), 145.2 (C-1), 141.1 (C-13), 137.9 (C-3), 136.9 (C-10), 129.9 (C-11), 129.3 (C-2′ and C-6′), 125.4 (C-5),123 (C-1′), 115.6 (C-12), 113.4 (C-4), 111.9 (C-3′ and C-5′), 110.7 (C-6), 94.0 (C-8), 67.4 (2(CH₂-O)), 56.2 (2(CH₂-N)), 56.4 (O-CH₃), 46.9 (C-14), 22.3 (CH₃).

General procedure for the preparation of compounds 5a–c

A mixture of hydrazide 3 (50 mg, 4 mmol), cyclic anhydride (1 equiv.) and 10 mL of 1,4-dioxane was refluxed38. Once the reaction is completed the solvent was then removed under reduced pressure and the resulting residue was purified by column chromatography over silica gel using EtOAc and petroleum ether (80:20) as elution systems to obtain the desired products 5a–c.

Compound 5a: N-(1,3-dioxooctahydro-2H-isoindol-2-yl)-2–(7-methoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl)acetamide

White solid, Yield 75%, m.p.: 245 °C, ES-HRMS m/z 421.1879 [M + H]⁺. ¹H-NMR (300 MHz, DMSO-d₆) δ_H: 8.20 (1H, s, NH), 8.10 (1H, d, J = 5.1 Hz, H-3), 8.01 (1H, d, J = 8.7 Hz, H-5), 7.71 (1H, d, J = 5.1 Hz, H-4), 6.82 (1H, d, J = 2.1 Hz, H-8), 6.79 (2H, dd, J = 8.7 and J = 2.1 Hz, H-6), 5.36 (2H, s, H-14), 3.80 (3H, s, CH₃-O), 2.95 (2H, t, J = 4.5 Hz, H-C = O), 2.86 (3H, s, CH₃), 1.61 (4H, m, 2CH₂), 1.24 (4H, m, 2CH₂). ¹³C-NMR (75 MHz, DMSO-d₆) δ_C: 173.2 (C = O), 167.4 (C = O), 160.5 (C-7), 143.8 (C-1), 141.0 (C-13), 138.1 (C-3), 135.3 (C-10), 128.6 (C-11), 122.2 (C-5), 114.4 (C-12), 112.1 (C-4), 109.4 (C-6), 93.8 (C-8), 55.5 (O-CH₃), 46.0 (C-14), 37.6 (CH), 23.1 (2CH₂), 22.6 (CH₃), 21.0 (2CH₂).

Compound 5b: N-(1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-methanoisoindol-2-yl)-2–(7-methoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl)acetamide

Yellow solid, Yield 40%, m.p.: 270 °C, ES-HRMS m/z 431.1714 [M + H]⁺. ¹H-NMR (300 MHz, DMSO-d₆) δ_H: 8.28 (1H, s, NH), 8.19 (1H, d, J = 5.1 Hz, H-3), 8.06 (1H, d, J = 8.7 Hz, H-5), 7.86 (1H, d, J = 5.1 Hz, H-4), 6.15 (1H, d, J = 2.1 Hz, H-8), 6.89 (2H, dd, J = 8.7 and J = 2.1 Hz, H-8), 6.17 (2H, m, H-3′and H-4′), 5.38 (2H, s, H-14), 3.91 (3H, s, CH₃-O), 3.58 (4H, m, H-1′a, H-2′, H-5′ and H-5′a), 2.92 (3H, s, CH₃), 1.56 (2H, m, H-6′). ¹³C-NMR (75 MHz, DMSO-d₆) δ_C: 173.7 (C = O), 167.4 (C-1′ and C-7′), 160.5 (C-7), 143.4 (C-1), 141.0 (C-13), 138.1 (C-3), 135.4 (C-3′ and C-4′), 134.4 (C-10), 128.6 (C-11), 122.2 (C-5), 114.3 (C-12), 112.1 (C-4), 109.4 (C-6), 93.7 (C-8), 55.6 (C-6′), 55.5 (O-CH₃), 45 (C-1′a, C-2′, C-5′ and C-5′a), 44.2 (C-14), 22.5 (CH₃).

Compound 5c: 4–(2-(2–(7-methoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl)acetyl)hydrazinyl)-4-oxobutanoic acid

White solid, Yield 80%, m.p.: 229 °C, ES-HRMS m/z 385.1508 [M + H]⁺. ¹H-NMR (300 MHz, DMSO-d₆) δ_H: 10.2 (1H, s, NH), 9.83 (1H, s, NH), 8.16 (1H, d, J = 5.1 Hz, H-3), 8.06 (1H, d, J = 8.1 Hz, H-5), 7.83 (1H, d, J = 5.1 Hz, H-4), 7.14 (1H, d, J = 2.1 Hz, H-8), 6.89 (2H, dd, J = 8.1 and J = 2.1 Hz, H-8), 5.27 (2H, s, H-14), 3.89 (3H, s, CH₃-O), 3.58 (4H, m, 2CH), 2.91 (3H, s, CH₃), 2.43 (4H, m, 2CH₂). ¹³C-NMR (75 MHz, DMSO-d₆) δ_C: 173.4 (C = O), 170.1 (C-4′), 167.4 (C-1′), 160.5 (C-7), 143.5 (C-1), 141.0 (C-13), 137.7 (C-3), 135.3 (C-10), 128.6 (C-11), 122.2 (C-5), 114.3 (C-12), 112.1 (C-4), 109.3 (C-6), 93.1 (C-8), 55.3 (O-CH₃), 45.8 (C-14), 28.7 (C-3′), 28.1 (C-2′), 22.5 (CH₃).

Procedure for the preparation of compound 6

Compound 6 was prepared as previously reported in our recent publication39.

Compound 6: 7-methoxy-1-methyl-9-(prop-2-ynyl)-9H-pyrido[3,4-b]indole

Dark brown solid, Yield 67%, m.p.: 205 °C, ES-HRMS m/z 251.1187 [M + H]⁺. ¹H-NMR (300 MHz, CD₃OD) δ_H: 8.30 (1H, d, J = 5.4 Hz, H-3), 7.96 (1H, d, J = 6.9 Hz, H-5), 7.72 (1H, d, J = 5.4 Hz, H-4), 6.92 (2H, m, H-8 and H-6), 5.18 (2H, d, J = 2.4 Hz, H-14), 3.97 (3H, s, O-CH₃), 3.13 (3H, s, CH₃), 2.36 (1H, t, J = 2.4 Hz, H-16). ¹³C-NMR (75 MHz, CD₃OD) δ_C: 160.7 (C-7), 142.4 (C-1), 140.2 (C-13), 138.1 (C-3), 134.5 (C-10), 129.5 (C-11), 122.0 (C-5), 114.8 (C-12), 111.8 (C-4), 109.1 (C-6), 92.7 (C-8), 77.9 (C-15), 73.0 (C-16), 55.2 (O-CH₃), .2 (C-14), 22.1 (CH₃).

General procedure for the preparation of compounds 7a–f

Heating conditions

To derivative 6 (0.05 g, 0.2 mmol) in refluxing toluene, the appropriate dipole (1 equiv.), in the presence of triethylamine and a catalytic amount of copper (I) was added then the mixture was refluxed for 24–48 h40. The solvent was then removed under reduced pressure. The resulting residue was purified by silica gel column chromatography (EtOAc/CH₃OH) to give corresponding triazoles 7a–f.

Microwave irradiation

To compound 6 (50 mg, 0.2 mmol) we added the appropriate dipole (1 equiv.) in the presence of triethylamine (1 equiv.), the mixture is dissolved in 1 mL of DMF in the presence of a catalytic amount of copper (I) under microwave irradiation (200 W, 100 °C) for 5–7 min41. Then, the mixture was poured into ice-cold water, and extracted three times with chloroform. Finally, after removal of the solvent in vacuum, the residue was purified by silica gel column chromatography to give the corresponding triazoles 7a–f.

Compound 7a: 7-methoxy-1-methyl-9-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-9H-pyrido[3,4-b]indole

Beige solid, Yield 78%, m.p.: 192 °C, ES-HRMS m/z 370.1778 [M + H]⁺. ¹H-NMR (300 MHz, CDCl₃) δ_H: 8.40 (1H, d, J = 5.4 Hz, H-3), 8.30 (1H, d, J = 8.7 Hz, H-5), 7.87 (1H, J = 5.4 Hz, H-4), 7.68 (5H, m, H-1′, H-2′, H-3′, H-4′ and H-5′), 7.56 (1H, s, H-16), 7.25 (1H, s, H-8), 7.19 (1H, d, J= 8.7 Hz, H-6), 6.21 (1H, s, H-14), 4.24 (3H, s, CH₃-O), 3.19 (3H, s, CH₃). ¹³C-NMR (75 MHz, CD₃OD) δ_C: 160.8 (C-7), 145.5 (C-1), 142.8 (C-13), 137.4 (C-3), 136.3 (C-10), 133.1 (C-15), 132.5 (C-1′), 129,1 (C-11), 128.3 (C-3′, C-4′ and C-5′), 124.1 (C-2′ and C-6′), 121.8 (C-5), 120.1 (C-16), 115.6 (C-12), 114.7 (C-4), 109.2 (C-6), 93.3 (C-8), 55.2 (CH₃-O), 40.6 (C-14), 20.5 (CH₃).

Compound 7b: 7-methoxy-1-methyl-9-((1-(p-tolyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-pyrido[3,4-b]indole

Yellow solid, Yield 65%, m.p.: 198 °C, ES-HRMS m/z 384.1814 [M + H]⁺. ¹H-NMR (300 MHz, CDCl₃) δ_H: 8.59 (1H, d, J = 5.1 Hz, H-3), 8,26 (1H, d, J = 8.4 Hz, H-5), 8.02 (1H, d, J = 5.1 Hz, H-4), 7.60 (1H, s, H-16), 7.59 (2H, d, J = 8.1 Hz, H-2′ and H-6′), 7.43 (2H, d, J = 8.1 Hz, H-3′ and H-5′), 7.20 (1H, s, H-8), 7.18 (1H, d, J = 8.4 Hz, H-6), 5.68 (2H, s, H-14), 4.17 (3H, s, O-CH₃), 3.25 (3H, s, CH₃), 2.66 (3H, s, CH₃). ¹³C-NMR (75 MHz, CDCl₃) δ_C: 1620.8 (C-7), 145.0 (C-1), 142.7 (C-13), 139.1 (C-3), 137.6 (C-10), 134.6 (C-1′), 133.9 (C-15), 133.6 (C-4′), 129.2 (C-2′ and C-6′), 128.5 (C-3′ and C-5′), 128.2 (C-11), 121.8 (C-5), 120.7 (C-16), 114.8 (C-12), 111.0 (C-4), 109.2 (C-6), 93.0 (C-8), 53.6 (O-CH₃), 40.7 (C-14), 22.5 (CH₃), 20.3 (CH₃).

Compound 7c: 7-methoxy-9-((1–(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1-methyl-9H-pyrido[3,4-b]indole

Brown solid, Yield 80%, m.p.: 230 °C, ES-HRMS m/z 400.1778 [M + H]⁺. ¹H-NMR (300 MHz, CDCl₃) δ_H: 8.36 (1H, d, J = 6.6 Hz, H-3), 8.03 (1H, d, J = 8.4 Hz, H-5), 7.86 (1H, d, J = 6.6 Hz, H-4), 7.50 (2H, d, J = 9 Hz, H-2′ and H-6′), 7.31 (H-16), 6.98 (1H, d, J= 2.1 Hz, H-8), 6.96 (2H, d, J = 9 Hz, H-3′ and H-5′), 6.93 (1H, dd, J = 8.4 Hz and J= 2.1 Hz, H-6), 5.95 (1H, s, H-14), 4.03 (3H, s, O-CH₃), 3.93 (3H, s, O-CH₃), 3.08 (3H, s, CH₃). ¹³C-NMR (75 MHz, CDCl₃) δ_C: 161.4 (C-7), 160.2 (C-4′), 146.1 (C-1), 143.2 (C-13), 141.1 (C-3), 135.3 (C-10), 132.2 (C-15) 130.3 (C-1′), 129.9 (C-2′ and C-6′), 122.7 (C-11), 122.3 (C-3′ and C-5′), 119.8 (C-16), 115.8 (C-5), 114.2 (C-12), 114.8 (C-4), 112.5 (C-6), 93.6 (C-8), 55.9 (O-CH₃), 55.7 (O-CH₃), 41.4 (C-14), 23.3 (CH₃).

Compound 7d: 9-((1–(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole

Brown solid, Yield 70%, m.p.: 197 °C, ES-HRMS m/z 404.1276 [M + H]⁺. ¹H-NMR (300 MHz, CDCl₃) δ_H: 8.21 (1H, d, J = 5.4 Hz, H-3), 8.00 (1H, d, J = 8.7 Hz, H-5), 7.72 (1H, d, J = 5.4 Hz, H-4), 7.53 (2H, d, J = 8.7 Hz, H-2′ and H-6′), 7.40 (2H, d, J = 8.7 Hz, H-3′ and H-5′), 7.37 (C-16), 6.94 (1H, d, J= 8.7 Hz, H-6), 6.87 (1H, s, H-8), 5.90 (2H, s, H-14), 3.93 (3H, s, O-CH₃), 2.99 (3H, s, CH₃). ¹³C-NMR (75 MHz, CDCl₃) δ_C: 160.8 (C-7), 142.6 (C-1), 139.0 (C-13), 135.8 (C-3), 134.9 (C-1′), 134.4 (C-4′), 134.2 (C-10), 133.2 (C-15), 128.3 (C-2′ and C-6′), 127.5 (C-11), 121.9 (C3′ and C-5′), 121.1 (C-5), 118.8 (C-16), 114.9 (C-12), 111.5 (C-4), 109.1 (C-6), 93.1 (C-8), 55.2 (O-CH₃), 40.6 (C-14), 29.1 (O-CH₃).

Compound 7e: 9-((1–(4-ethylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole

Yellow solid, Yield 90%, m.p.: 238 °C, ES-HRMS m/z 398.1975 [M + H]⁺. ¹H-NMR (300 MHz, CDCl₃) δ_H: 8.35 (1H, d, J = 5.4 Hz, H-3), 7.89 (1H, d, J = 8.7 Hz, H-5), 7.64 (1H, d, J = 5.4 Hz, H-4), 7.36 (2H, d, J = 8.7 Hz, H-2′ and H-6′), 7.33 (C-16), 7.13 (2H, d, J = 8.7 Hz, H-3′ and H-5′), 6.85 (1H, s, H-8), 6.82 (1H, d, J= 8.7 Hz, H-6), 5.79 (2H, s, H-14), 3.79 (3H, s, O-CH₃), 2.91 (3H, s, CH₃), 2.56 (2H, q, J = 7.5 Hz, CH₂), 1.11 (3H, t, J = 7.5 Hz, CH₂). ¹³C-NMR (75 MHz, CDCl₃) δ_C: 160.8 (C-7), 144.8 (C-1), 142.5 (C-13), 140.2 (C-4′), 137.8 (C-3), 136.3 (C-10), 135.0 (C-2′ and C-6′), 134.1 (C-1′), 132.8 (C-15), 128.4 (C-3′ and C-5′), 121.9 (C-11), 120.0 (C-5), 118.9 (C-16), 114.9 (C-12), 111.5 (C-4), 109.2 (C-6), 92.9 (C-8), 55.2 (O-CH₃), 40.7 (C-14), 27.8 (CH₂), 23.2 (CH₃), 15.2 (CH₃).

Compound 7f: 7-methoxy-1-methyl-9-((1–(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-9H-pyrido[3,4-b]indole

Yellow solid, Yield 60%, m.p.: 259 °C, ES-HRMS m/z 415.1508 [M + H]⁺. ¹H-NMR (300 MHz, CDCl₃ + CD₃OD (80/20)) δ_H: 8.60 (1H, d, J = 5.4 Hz, H-3), 8.54 (1H, d, J = 9 Hz, H-5), 8.35 (1H, d, J = 5.4 Hz, H-4), 8.24 (2H, d, J = 8.7 Hz, H-3′ and H-5′), 8.16 (2H, d, J = 8.7 Hz, H-2′ and H-6′), 7.69 (C-16), 7.17 (1H, d, J = 9 Hz, H-6), 7.14 (1H, s, H-8), 6.16 (2H, s, H-14), 4.13 (3H, s, O-CH₃), 3.25 (3H, s, CH₃). ¹³C-NMR (75 MHz, CDCl₃ + CD₃OD (80/20)) δ_C: 165.7 (C-7), 151.3 (C-1′), 150.2 (C-4′), 147.6 (C-1), 144.8 (C-13), 136.8 (C-3), 134.3 (C-10), 129.1 (C-15), 126.5 (C-3′ and C-5′), 127.5 (C-11), 124.6 (C-2′ and C-6′), 124.2 (C-5), 119.1 (C-16), 114.0 (C-12), 112.5 (C-4), 108.1 (C-6), 97.6 (C-8), 59.4 (O-CH₃), 44.4 (C-14), 22.1 (CH₃).

Biological evaluation

Cytotoxicity/anticancer assay

Cytotoxicity of compounds was estimated on three human cancer cells: OVCAR-3 (ovarian cancer), MCF-7 (breast cancer) and HCT 116 (colon cancer) as described by Bendaoud et al.42 The HCT 116 and OVCAR-3 cells were grown in RPMI-1640 medium supplemented with 10% fetal calf serum (Gibco, USA), air and 5% CO₂. While the base medium for MCF7cell line was DMEM, with phenol red and 10% fetal bovine serum. The tested compound was added to a medium containing 1 × 10⁶ cells/mL, L-glutamine (2 mM) and gentamycin (50 μg/mL), and kept at 37 °C in a fully humidified atmosphere. After 18 h of incubation at 37 °C in 5% CO₂ incubator, the tubes were centrifuged at 8000g for 10 min. The supernatant was decanted, and the pellets were taken and washed with 20 mM of phosphate-buffered saline solution. Each pellet was dissolved in 100 μL (2 mg/mL) MTT solution in a tube, incubated at 22 °C for 4 h and centrifuged at 8000g for 10 min. All the pellets were dissolved in 500 μL DMSO and read spectrophotometrically at 500 nm. Doxorubicin (HCT116 and MCF-7) and tamoxifen (OVCAR-3) were used as positive control.

5-Lipoxygenase activity assay

The anti-soybean lipoxygenase activity of the compounds was determined as described by Khlifi et al.43 with some modifications. Various concentrations of 20 μL of each compound were mixed individually with sodium phosphate buffer (pH 7.4) containing soybean lipoxygenase and 60 μL of linoleic acid (3.5 mM). However, the blank does not contain the substrate, but will be added 30 μL 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 was flowed for 10 min at 25 °C. Nordihydroguaiaretic acid (NDGA) was used as positive control.

AChE inhibitory activity assay

The acetylcholinesterase inhibition assay was performed as previously reported by Bekir et al. with some modifications44. Twenty-five  microliter of each compound at different concentrations, 50 μL of 0.1 M sodium phosphate buffer (pH 8.0) and 25 μL of AChE solution and 125 μL of 5,5′-dithiobis-2-nitrobenzoic acid were mixed and incubated for 15 min at 25 °C using a 96-well microplate. All compounds were dissolved in the DMSO then diluted in the buffer and the DMSO does not exceed 1% in the mixture. After that, 25 μL of acetylthiocholine iodide solution were added to the initial mixture and the final mixture was incubated, for 15 min, at 25 °C, then the absorbance was determined at 412 nm. Control contained all components except the tested compounds. Galantamine was used as positive control.

Results and discussion

We started our study by the preparation of the intermediate 2 ethyl-2–(7-methoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl)acetate, which was obtained by the condensation of a mixture of harmine 1, (1 equiv.) ethyl acetate chloride and (1 equiv.) sodium hydride during 48 h in dry DMF at room temperature (Scheme 1). The mass spectrum, recorded in ES-HRMS for product 2 showed a pseudo-molecular ion peak [M + H]⁺at m/z 299.1396 which is consistent with the molecular formula of C₁₇H₁₈N₂O₃. The structure was evidenced by the appearance, in the ¹H NMR spectrum, of a triplet at δ_H 1.24 (J = 7.2 Hz) attributable to the methyl group H-17, a quadruplet at δ_H 4.23 (J = 7.2 Hz) attributable to the methylene protons H-16 and a singlet at δ_H 5.15 attributable to the methylene protons H-14. The ¹³C NMR spectrum reinforced this structure by the appearance of the ester function signal at δ_C 168.2, two methylene carbons signals at δ_C 46.3 and 61.4 corresponding to C-14 and C-16, respectively, and the appearance of a signal at δ_C 13.6 attributed to the methyl carbon C-17.

Scheme 1. Synthesis of hydrazones 4a–j.

Likewise, we have studied the behavior of derivative 2 with an excess of hydrazine under refluxing EtOH during 24 h (Scheme 1). The mass spectrum recorded in ES-HRMS of compound 3, showed a pseudo-molecular ion peak [M + H]⁺at m/z 285.1354 in concordance with its molecular formula. The ¹H NMR spectrum recorded in DMSO-d₆ at 300 MHz shows the presence of new signals at δ_H 4.38 and 9.40 attributable to the NH₂ and NH functions, respectively, and the disappearance of the ethoxy ester signals at δ_H 4.23 (CH₂) and 1.24 (CH₃).

Then the condensation of hydrazide 3 with different aromatic aldehydes in refluxing 1,4-dioxane for 24 h, yielded the corresponding hydrazones 4a–j (40–80%) (Scheme 1). The structures of the new hydrazones 4a–j were confirmed on the basis of their spectroscopic data (ES-HRMS, ¹H and ¹³C NMR). Indeed, the mass spectrum recorded in ES-HRMS of the derivative 4a, as an example, showed a pseudo-molecular ion peak [M + H]⁺at 429.2287 which is consistent with its molecular formula. The ¹H NMR spectrum of this compound revealed three methyl groups as a singlet at δ_H 1.32 corresponding to the protons of the tert-butyl group. It exhibited characteristic AA′BB′ pattern for aromatic hydrogens, with two doublets at δ_H 7.48 (2H, d, J = 8.4 Hz) and 7.41 (2H, d, J = 8.4 Hz). The presence of a significant signal at δ_H 7.73 (1H, s, CH = N) attributable to the proton of the imine function reinforced the proposed structure. The ¹³C NMR spectrum of 4a confirmed the above spectral data by the observation of new signals at δ_C 150.0, 135.5, 129.9 and 126.6 relative to carbons of the introduced para-substituted aromatic ring. Moreover, this spectrum showed a signal at δ_C 153.7 corresponding to the carbon of the imine function.

On the other hand, we studied the behavior of intermediate 3 with some cyclic anhydrides in refluxing 1,4-dioxane for 48 h (Scheme 2). The reaction led to the formation of new compounds 5a–c (40–80%). Their structures were established on the basis of their spectral data (ES-HRMS, ¹H and ¹³C NMR). In fact, the ¹H NMR spectrum of compound 5a, as an example, compared with that of the hydrazide 3 used as a starting material, allowed us to note in addition to the signals relative to protons introduced by the hydrazide 3, the presence of two multiplets at δ_H 1.61 (4H, m, 2CH₂) and 1.24 (4H, m, 2CH₂) corresponding to the four methylene groups introduced by the anhydride and the disappearance of the signal related to the NH₂ protons at δ_H 4.38 (of intermediate 3). This structure was also evidenced by the observation in the ¹³C NMR spectrum of characteristic signal at δ_C 167.4 corresponding to the two carbonyls of the amide functions. The same spectrum revealed three signals at δ_C 37.6 (2CH), 23.1 (2CH₂) and 21.0 (2CH₂) easly attributable to the methine and the two methylene groups of the anhydride moiety.

Scheme 2. Synthesis of derivatives 5a–c.

In order to prepare new triazole derivatives, we started by the N-propargylation of harmine 1 with propargyl bromide in the presence of sodium hydroxide in anhydrous DMF at room temperature to give the intermediate 6 (67%) (Scheme 3 and Table 1). The structure of the propargylated harmine 6 was assigned on the basis of its spectral data (ES-HRMS, ¹H and ¹³C NMR) as previously described in our recent work39. Then, we examined the 1,3-dipolar cycloaddition reaction of intermediate 6 with azides using copper (I)-catalyzed under microwave irradiation (Scheme 3 and Table 1). As indicated in Table 1, our findings demonstrated that the reaction when conducted under heating conditions required longer reaction times (24–48 h) and 1,2,3-triazoles were obtained in lower yields while under microwave irradiation the same reaction gives the 1,2,3-triazoles in high yields (65–90%) and shorter reaction times (5–10 min) (Table 1). The structures of derivatives 7a–f were characterized on the basis of their spectral data (ES-HRMS, ¹H and ¹³C NMR). The mass spectrum recorded in ES-HRMS of derivative 7c, as an example, showed a pseudo-molecular ion peak [M + H]⁺at m/z 400.1778 which is consistent with its molecular formula (C₂₃H₂₁N₅O₂). Its ¹H NMR spectrum showed, in addition to the signals relative to protons introduced by harmine 1, a characteristic AA′BB′ pattern for the four aromatic protons [δ_H 7.50 (2H, d, J = 9 Hz, H-2′ and H-6′) and δ_H 6.96 (2H, d, J = 9 Hz, H-3′ and H-5′)]. The same spectrum showed the presence of two singlets at δ_H 3.93 and 7.31 corresponding to the methoxy group and the triazole proton H-16, respectively. The ¹³C NMR spectrum of compound 7c was also in agreement with the proposed structure showing the presence of characteristic signals at δ_C 160.2, 130.3, 129.9 and 122.3 related to carbons of the para-methoxyphenyl ring. This spectrum also revealed two signals at δ_C 119.8 and 55.9 corresponding to C-16 and the carbon of the methoxy group, respectively.

Table 1. Effect of microwave irradiation for the synthesis of 1,2,3-triazoles 7a–f.

		Conventional heating^a		Microwave irradiation^b
Entry	R	Time (h)	Yield (%)	Time (min)	Yield (%)
7a	H	24	65	5	78
7b	CH3	48	50	5	65
7c	OCH3	48	70	7	80
7d	Cl	–	–	5	70
7e	NO2	24	80	5	90
7f	C2H5	–	–	7	60

a Reflux.

b 200 W, 100 °C.

Scheme 3. Synthesis of triazoles7a–f.

Cytotoxic assay

Harmine 138 and all the synthesized compounds were tested on what concerns their cytotoxic activities against three human cell lines: HCT-116 (colon cancer), OVCAR-3 (ovarian cancer) and MCF-7 (breast cancer). According to the results given in Table 2, harmine 1 was found to be significantly more active than its ester derivative 2 which did not exhibit any effect against MCF-7 cell line but moderately active toward the two cell lines HCT-116 and OVCAR-3 (IC₅₀=36.0 ± 2.0 and 53.0 ± 3.0 μM, respectively). On the other hand, the results showed a total inactivity of the hydrazide 3 toward the three cell lines used. Its transformation into hydrazones using a series of specific arylaldehydes considerably improved this activity.

Table 2. MCF-7, HCT-116 and OVCAR-3 inhibitions (IC₅₀ μM) of harmine 1 and its derivatives expressed as IC₅₀ (μM)^a.

Compounds	MCF-7	HCT-116	OVCAR-3
1	1.3 ± 0.2^b	0.7 ± 0.03^b	18.0 ± 2.0^b
2	>100	36.0 ± 2.0	53.0 ± 3.0
3	>100	>100	>100
4a	4.0 ± 0.0	5.0 ± 0	2.5 ± 0.1
4b	>100	>100	>100
4c	>100	25.0 ± 4.0	23.7 ± 1.4
4d	4.5 ± 0.0	25.0 ± 2.0	14.0 ± 1.0
4e	4.0 ± 0.6	2.9 ± 0.4	27.3 ± 1.0
4f	27.0 ± 4.0	18.0 ± 2.0	6.4 ± 0.8
4g	2.8 ± 0.2	3.0 ± 0.3	16.7 ± 0.7
4h	18.0 ± 2.0	>100	71.0 ± 9.0
4i	87.0 ± 11.0	3.8 ± 0.5	20.0 ± 1.7
4j	11.0 ± 1.0	7.0 ± 0.0	22.0 ± 2.0
5a	>100	>100	>100
5b	>100	>100	>100
5c	>100	>100	>100
6	63.0 ± 5.0	25.0 ± 2.0	9.0 ± 1.0
7a	6.3 ± 0.7	40.0 ± 5.0	96.0 ± 8.0
7b	32.0 ± 2.0	31.0 ± 4.0	>100
7c	43.0 ± 3.0	43.0 ± 6.0	>100
7d	29.0 ± 2.0	27.0 ± 3.0	>100
7e	54.0 ± 7.0	52.0 ± 7.0	>100
7f	41.0 ± 3.0	42.0 ± 5.0	>100
Doxorubicin	0.38 ± 0.03	0.36 ± 0.03	–
Tamoxifen	–	–	0.52 ± 0.04

a Averages ± SD were obtained from three different experiments.

b Results obtained in our previous work38.

Most hydrazone derivatives 4a–j has displayed good cytotoxic activity against the three cell lines. Harmine 1 exhibited potent cytotoxic activity against the two cell lines HCT-116 and MCF-7 (IC₅₀=0.70 ± 0.03 and 1.3 ± 0.2 μM, respectively). Among the synthesized compounds, derivatives 4a, 4d and 4e bearing a p-tert-butylphenyl, p-(1,1,2,2-tetrafluoroethylphenyl and p-methylthiophenyl, respectively, as substituents at the isoxazoline moiety, exhibited an important inhibitory activity against MCF-7 cell line (CI₅₀=4.0 ± 0.0, 4.5 ± 0.0 and 4.0 ± 0.6 μM, respectively), while compound 4 g bearing a 4-chloro-3-nitro-phenyl was found to be the most active against the same cell line (IC₅₀= 2.8 ± 0.2 μM). These results were in concordance with several studies cited in the literature showing potential cytotoxic activity of some hydrazone derivatives such as 3-cyclohexyl-N′-(4-methylbenzylidene)propanehydrazide which exhibited cytotoxic activity against BT-549, SKMEL, KB and SKOV-3 cell lines with IC₅₀ values of 6.80, 6.25, 6.61 and 4.41 μM, respectively45. It has been found that the derivatives 4b and 4c bearing a 4-methylaminophenyl and a 3,4-dimethoxyphenyl were inactive toward MCF-7 cell line (IC₅₀> 100 μM).

All hydrazones 4a–j have shown cytotoxic activities against OVCAR-3 cell line expect 4b (CI₅₀>100 μM), the para-position of a dimethylamino group may explain the loss of this activity against this cell line as observed toward MCF-7 and HCT-116 cell lines. Interestingly, the derivative 4a was seven times more potent than harmine 1 with an IC₅₀ value of 2.5 ± 0.1 μM against OVCAR-3 cell line. This finding could also be explained by the presence of the p-tert-butylphenyl group attached to the hydrazone function which could also be the responsible for the potent activity of this compound against MCF-7 and HCT-116 cell lines (CI₅₀=4.0 ± 0.0 and 5.0 ± 0.0 μM, respectively). The derivative 4f bearing a p-methoxyphenyl exhibited interesting cytotoxic activity against OVCAR-3 cell line (CI₅₀=6.4 ± 0.8 μM), this compound is three times more active than harmine 1. Moreover, in the derivatives 4d and 4 g the p-(1,1,2,2-tetrafluoroethylphenyl and the p-chloro-3-nitro-phenyl systems, respectively, did not significantly improve the cytotoxic activity of harmine 1 against OVCAR-3 (IC₅₀ of 14.0 ± 1.0 and 16.7 ± 0.7 μM, respectively).

Among the derivatives of hydrazone, only the two derivatives 4b and 4 h, respectively, were inactive against the HCT-116 cell line whereas the other derivatives have shown interesting activities against this cell line with IC₅₀ ranging from 2.9 ± 0.4 to 25.0 ± 4.0 μM. The derivatives 4e, 4 g and 4i bearing p-methylthiophenyl, p-chloro-3-nitrophenyl and 6-quinolinyl systems were the most active derivatives against HCT-116 cell line with IC₅₀ values of 2.9 ± 0.4, 3.0 ± 0.3 and 3.8 ± 0.5 μM, respectively. These findings demonstrated that the cytotoxic activity of hydrazones 4a–j depends in most cases on the nature of the aromatic ring attached to the hydrazone function. Our results were found to be consistent with several previous studies showing that hydrazone derivatives exhibited potent cytotoxic activity against HCT-116 such as N-(2-(benzyloxy)benzylidene)isonicotinohydrazide and N-(2,4-bis(trifluoromethyl)benzylidene)isonicotinohydrazide with IC₅₀ values of 3.10 and 0.29 μM, respectively46.

As indicated in Table 2, the derivatives 5a–c did not exhibit any activity toward the three cell lines used. This means that the anhydride fragment introduced did not improve the activity of the hydrazide 3.

The triazole derivatives 7a–f showed moderate cytotoxic activities against MCF-7 cell line (IC₅₀=6.3 ± 0.7–54.0 ± 7.0 μM) and HCT-116 cell line (IC₅₀=27.0 ± 3.0–52.0 ± 7.0 μM). However, the derivative 7a bearing a non-substituted phenyl moiety attached to the triazole ring was found to be the most active one against MCF-7 cell line with an IC₅₀ value of 6.3 ± 0.7 μM. This finding let us to think that the substitution in para position of the phenyl group is not in favor of this activity. The OVCAR-3 seems significantly less sensitive than the two other cell lines (MCF-7 and HCT-116) to the various prepared triazole derivatives.

5-Lipoxygenase inhibition

Harmine 139 and its derivatives were tested against 5-lipoxygenase. The literature reports that some hydrazone derivatives such as 2,6-di-tert-butyl-4-(pyridin-2-yl-hydrazono-methyl)-phenol and 2,6-di-tert-butyl-4-(pyrazin-2-yl-hydrazonomethyl)-phenol showed a potential anti-inflammatory activity against 5-LOX enzyme with IC₅₀ values of 0.14 μM and 0.10 μM, respectively47. From the data of the lipoxygenase activity indicated in Table 3, harmine 1 did not show any activity against this enzyme (IC₅₀>100 μM) while the corresponding ester 2 and hydrazide 3 showed activity against 5-LOX enzyme with IC₅₀ values of 31.6 ± 2.4 and 30.9 ± 1.9 μM, respectively. This finding showed that the incorporation of the ester and hydrazide functions increased the inhibitory potency of harmine 1. Among the hydrazones 4a–j only 4b (IC₅₀=39.8 ± 1.1 μM), 4c (IC₅₀=63.1 ± 3.1 μM) and 4 h (IC₅₀=53.7 ± 4.3 μM) were found to be relatively active. These results demonstrated that the inhibitory potency of the prepared hydrazones depends on the nature of the substituent in para position of the phenyl group attached to the hydrazone function. The derivatives 5a–c and 7a–f did not exhibit any considerable activity against 5-LOX enzyme while the alkylated derivative 6 was found to be active (IC₅₀=28.2 ± 2.1 μM), its activity by comparison to that of harmine 1, is certainly due to the propargyl moiety added.

Table 3. Acetylcholinesterase and 5-lipoxygenase inhibition capacity of harmine 1 and its derivatives expressed as IC₅₀ (μM)^a.

Compound	Acetylcholinesterase	5-Lipoxygenase
1	10.4 ± 0.4^b	>100^b
2	23.9 ± 1.2	31.6 ± 2.4
3	44.6 ± 3.2	30.9 ± 1.9
4a	>100	>100
4b	>100	39.8 ± 1.1
4c	>100	63.1 ± 3.1
4d	>100	>100
4e	>100	>100
4g	>100	>100
4h	>100	53.7 ± 4.3
4i	>100	>100
4j	>100	>100
5a	>100	>100
5b	>100	>100
5c	>100	>100
6	1.9 ± 1.5^b	28.2 ± 2.1
7a	>100	>100
7b	>100	>100
7c	>100	>100
7d	>100	>100
7e	>100	>100
7f	>100	>100
Galantamine	4.1 ± 0.2	–
NDGA	–	8.1 ± 0.1

a Averages ± SD were obtained from three different experiments.

b Results obtained in our previous work38.

AChE activity assay

Harmine 138 and all synthesis compounds were tested on what concern their anti-AChE activity. As depicted in Table 3, It has been found that the introduction of the ethyl ester and hydrazide functions at the nitrogen atom in compounds 2 (CI₅₀=23.9 ± 1.2 μM) and 3 (CI₅₀=44.6 ± 3.2 μM), respectively decreases the inhibitory potency of harmine 1 (CI₅₀=10.4 ± 0.4 μM)39. The conversion of hydrazide 3 into hydrazones 4a–j and 5a–c derivatives resulted in the total loss of this activity.

On the other hand, according to our previous results39, the highest activity against acetylcholinesterase remains always exhibited by the propargylated harmine 6 (IC₅₀=1.9 ± 1.5 μM), which is found to be significantly active compared with the positive control compound (galantamine IC₅₀=4.1 ± 0.2 μM). The conversion of the alkyne in compound 6 by the introduction of a triazole ring bearing different aryls in compounds 7a–f removes completely this activity.

Conclusion

In summary, in the present paper we reported the synthesis of new hydarzones 4a–j and dihydrazides 5a–c starting from harmine 1 by using a series of aromatic aldehydes and cyclic anhydrides, respectively, as well as the synthesis of new triazole derivatives 7a–f from the previously propargylated harmine 6 via the 1,3-dipolar cycloaddition reaction using both conventional way and copper (I)-catalyzed under microwave irradiation. All derivatives were evaluated for their cytotoxic, anti-inflammatory and anti-acetylcholinesterase activities. Most hydrazone derivatives 4a–j showed good cytotoxic activities toward MCF-7, HCT-116 and OVCAR-3 cell lines, specially derivative 4a, bearing a p-tert-butylphenyl attached to its hydrazone function, which was the most active compound against OVCAR-3 cell line (IC₅₀=2.5 ± 0.1 μM) and seven times more active than harmine 1 (IC₅₀=18.0 ± 2.0 μM). However, these derivatives were inactive against AChE enzyme. Some hydrazone derivatives showed moderate activity against 5-LOX enzyme which depends to the nature of the aryl group attached to the hydrazone function. The conversion of the hydrazide 3 into dihydrazides 5a–c did not improve its activity against AChE and 5-LOX enzymes. Among the triazole derivatives, compound 7a bearing a phenyl substituent in the triazole ring showed interesting cytotoxic activity (IC₅₀=6.3 ± 0.7 μM) against MCF-7 cell line. These triazoles were found to be without any effect toward AChE and 5-LOX enzymes compared with their precursor 6 which exhibited a surprising inhibitory potency against AChE (IC₅₀=1.9 ± 1.5 μM). Encouraged by the noted significant cytotoxic activity of derivatives 4a–j, we will attempt to adopt other heterocyclic compounds, with the hope to discover more biologically active harmine derivatives.

Declaration of interest

The authors declare no conflicts of interests. The authors alone are responsible for the content and writing of this article.