Synthesis, Characterization, and Biological Evaluation of Gabapentinoid Hybrids with Isoindole-1,3-(2H) Dione Moiety as potential antioxidant, antimicrobial, and anticancer

doi:10.21203/rs.3.rs-3085868/v1

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Synthesis, Characterization, and Biological Evaluation of Gabapentinoid Hybrids with Isoindole-1,3-(2H) Dione Moiety as potential antioxidant, antimicrobial, and anticancer

https://doi.org/10.21203/rs.3.rs-3085868/v1

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Purpose: To synthesize new Isoindole-1,3-(2H) Dione derivatives by molecular hybridization of Gabapentin, and Pregabalin with phthalic anhydride derivatives, and to evaluate their biological activity as promising antioxidant, antimicrobial, and anticancer agents.

Method: Molecular hybridization was successfully achieved by two procedures; synthesized compounds were characterized using analytical and spectral methods. Free radical scavenging properties of synthesized compounds were evaluated using DPPH method. Antibacterial activity of synthesized compounds, and parent compounds was evaluated against two microbial gram-positive and gram-negative strains by well diffusion method. Furthermore, we have studied effect of compounds on proliferation, cell cycle and cell death of two human cancer cell lines (Caco-2 and HCT-116).

Results: Compounds 1,3,4 exhibited good free radical scavenging effect, compound 3 is the most effective with IC₅₀ value 2.525 µmol/mL.

All Compounds showed antibacterial activity against E. coli and Staphylococcus Aureus related to concentration while parent drugs did not exhibit any antibacterial effect. Compounds 1 and 2 showed good zone of inhibition against Escherichia Coli at micromolar concentrations, and they are more effective than gentamycin sulfate.

Treatment with studied compounds suppresses proliferation, arrests progress throughout the cell cycle and induces apoptosis in Caco-2 and HCT-116 cancer cells. Compound 2 is highly effective against Caco-2 cells and more effective than Thalidomide, with IC₅₀ value less than 1 µmol/L.

Conclusion: Our results showed that molecular hybridization of Gabapentin, and Pregabalin in Isoindole-1,3(2H) Dione moiety result in promising anti-cancer and antimicrobial molecules.

Results of this preliminary study show that halogenation of Isoindole-1,3-(2H) dione moiety improves antimicrobial, and anticancer activity, and that tetra-brominated derivatives are comparable or more effective than related tetra-chlorinated derivatives.

Isoindole-1

3-(2H) dione

molecular hybridization

antioxidant

antimicrobial

antiproliferation

apoptosis

cell cycle

Research for new, safe, and effective drugs is a continuous need over the years, especially that there are still challenging diseases with no sufficient treatments such as cancer, neurodegenerative diseases, autoimmune diseases and infectious diseases related to resistant microorganisms.

Therapeutic choices for complex heterogeneous diseases can be very limited, considering that one drug is not sufficient to control the illness in an effective way, the fact that demands the use of combination of pharmaceuticals with different pharmacotherapeutic profiles. In this context molecular hybridization has been emerged as a new strategy in medicinal chemistry for rational drug design based on the fusion of pharmacophoric units of active parent compounds, leading to a new hybrid architecture containing characteristics of both original active compounds.

Molecular hybridization approach increases the possibility of generating new active molecules, with optimized pharmacokinetic and pharmacodynamic properties compared with parent compounds, taking into consideration the advantage of available data about bioactive profile, targets, pharmacokinetic, and pharmacodynamic properties of parent compounds due to high percentage of structure similarity and homology (1) (2) (3).

The structural diversity and biological importance of nitrogen containing heterocycles have made

them attractive targets for synthesis over years. Among heterocyclic scaffolds, isoindole-1,3-(2H) dione is of particular interest in drug discovery and drug development.

This moiety has been described as multitarget structure with different biological effects, and many compounds of this scaffold are effective antimicrobial(4), anti-inflammatory(5) (6), anticonvulsant(7) (8), anti-viral (9), antioxidant (10),Xanthine oxidase inhibitors(11), and carbonic anhydrase inhibitors(11). Many compounds of this moiety are effective for the treatment of Leishmania (12), diabetes (13) (14), hyperlipidemia (13) (15), cancer (16) (17) (18) (10), Alzheimer (19) (20) (21). Recently many compounds of this moiety were synthesized and studied as SARS-CoV-2 virus, for the treatment of the breakthrough of COVID-19 pandemic(22) (23).

Furthermore, many iso indole-1,3-(2H) Dione drugs have been approved and marketed, like Thalidomide, Pomalidomide, Lenalidomide, and Apremilast (24).

Gabapentinoids, Gabapentin and pregabalin, are analogues of the inhibitory neurotransmitter γ-amino-butyric acid, (GABA), they block α2δ subunit of voltage-dependent calcium channels (VDCCs).

They are first line treatment for management of neuropathic pain, and they are used to prevent seizures(25).

The aim of this study is to synthesize novel promising drug candidates by molecular hybridization of Gabapetinoids and isoindole-1,3-(2H) dione scaffold (Fig. 1), and to evaluate their biological activity. In addition, we report a preliminary study of structure activity relationship, and mechanism of action of compounds 1–5 as anticancer candidates, and.

Two synthesis methods have been successfully applied, the conventional method and microwave assisted method (MAS). Microwave assisted method has many advantages as an ecofriendly method, short reaction time, better yield and purity of products.

Synthesized compounds were screened for their promising biological activity as antioxidants, antibacterial, and anticancer.

Antimicrobial effect was studied against gram negative Escherichia Coli and gram-positive Staphylococcus aureus. Hybrids of Pregabalin and Gabapentin show antimicrobial activity, in contrast to parent compounds. Hybrids of Pregabalin are highly effective against Escherichia Coli and more effective that standard drug Gentamycin sulfate.

Antiproliferative activity was studied against two cancer cell lines Caco-2, and HCT-116. Hybrid compounds showed significant antiproliferative effect, they arrest progression throughout cell cycle, and effectively induce apoptosis in cancer cell lines.

Compound 2 is the most effective against Caco-2 cells, 137.7 times more effective than Thalidomide with IC₅₀ = 0.00064 µmol/mL. Compound 2 inhibits progression of cell cycle to synthesis phase and induces apoptosis.

Compound 4 is the most effective against HCT-116 cell line with IC₅₀ 0.228 µmol/mL, it inhibits synthesis of DNA, and mitosis in HCT-116 cells, and induces cell death by apoptosis and necrosis.

Our results show that molecular hybridization of Gabapentin, and Pregabalin within Isoindole-1,3-(2H) dione moiety result in new promising drug like molecules, potent as antimicrobial and anticancer.

Herein we report that substitution of aromatic hydrogens of iso indole-1,3-(2H) dione moiety with bromine or chlorine atoms leads to more potent derivatives.

2.1. Synthesis and characterization:

Starting materials, and solvents were purchased from commercial sources and used without further purification.

Scheme 1 shows synthesis reactions, we used two procedures, procedure A(26), and procedure B(27). Progress of reactions were monitored by TLC on silica-gel plates (Merck 60F254).

Procedure A

0.010 mole of phthalic anhydride or its derivatives, and 0.011 mole of pregabalin (1–2) or Gabapentin (3–5) were dissolved in 5 mL dimethyl form amide (DMF) in a round bottom flask. Solution was refluxed in oil path at temperature 180 C^o, completion of reaction was determined by TLC. Reaction mixture was poured into ice cold water, crude product was precipitated, filtered out on a Buchner funnel, washed with water, dried, and purified by recrystallization from appropriate solvents.

Procedure B

Equimolar quantities of phthalic anhydride or its derivatives, and pregabalin (1–2) or Gabapentin (3–5), were grounded together in a mortar for 1 minute, then mixture was transferred to glass beaker containing a magnetic stirrer. Few drops of DMF were added to the beaker, and it was irradiated with microwaves (750 W) with continuous stirring for 5 minutes. completion of reaction was determined by TLC.

Mixture was cooled to room temperature, 50 mL ice-cold water was poured into the flask to precipitate the crude product, and it was filtered off, washed with water, dried, and recrystallized from appropriate solvent.

Melting points were determined using Stuart melting point apparatus SMP30 and used without calibration. High performance liquid chromatography was performed using Shimadzu UFLC apparatus A20.

IR spectra were recorded in KBr on Thermo Nicolet 6700 FT-IR spectrophotometer apparatus. UV spectra were recorded using JENWAY 6850 UV/Vis spectrophotometer. ¹H-NMR and ¹³C-NMR spectra were recorded on a Bruker Ultra Shield 400 Instrument (400 MHz for ¹H, 100 MHz for ¹³C).

ESI–MS experiments were performed using Agilent 6420 Triple Quadrupole LC/MS apparatus equipped with a standard ESI source. Instrument was operated in the positive-ion mode. Spectra were recorded for samples dissolved in DMSO: H₂O.

5-methyl-3-[(4,5,6,7-tetrachloro-1,3-dioxo-1,3-dihydro-2H-isoindole-2-yl) methyl] hexanoic acid (1):

Pale yellow crystals, yield 90% (procedure A)-92% (procedure B), MP 177.6–180 C^O, UV (methanol, nm): λ_max 288, 333.

IR (KBr, cm^− 1):3330.2 (OH), 2954.9, 2870 (CH₂ stretching), 1772.4 (CO), 1732.4 (CO), 1702.5 (CO), 1437(CH₂ bending), 1165 (C-N).

¹H NMR (400 MHz, DMSO-d₆, ppm): 12.009(s,1H, OH), 3.6–3.42 (m, 2H, N-CH₂), 2.18–2.35 (m,2H, CH₂-COOH), 2.18–2.048 (m,1H,N-CH₂-CH), 1.793–1.634 (m,1H,CH (CH₃)₂), 1.0785–1.267 (m,2H,CH₂-CH(CH₃)₂), 0.7587–1.0058 (2d, 6H, (CH₃)₂).

¹³C NMR (100 MHz, DMSO-d₆, ppm): 174.037 (COOH), 164.175 (2CO), 138.474 (2aromatic carbons), 128.887(2 aromatic carbons), 128,454 (2 aromatic carbons), 42.637, 41.572, 37.682, 32.157, 25.198, 23.207, 22.677.

DEPT 135 ¹³C NMR (100 MHz, DMSO-d₆, ppm): 42.592 (N-CH₂), 41.547 (CH₂-COOH), 37.659 (CH₂CH (CH₃)₂), 32.135(N-CH₂-CH-), 25.185 (CH (CH₃)₂), 23.223 (CH₃), 22.664 (CH₃).

ESI⁺ MS-MS (M/Z): 101.0, 240.8, 285.9, 287.9, 289.9, 291.9, 307.8, 309.9, 311.9, 362.1.

5-methyl-3-[(4,5,6,7-tetrabromo-1,3-dioxo-1,3-dihydro-2H-isoindole-2-yl) methyl] hexanoic acid (2):

White crystals and crystalline powder, yield: 83% (procedure A)-86% (procedure B), MP 212-212.6 C^O .

UV λ_max (methanol, nm): 288, 338. IR(KBr,cm^− 1):3399.5 (OH), 2954.6, 2869.6 (CH₂ stretching), 1769.9 (CO), 1732.3 (CO), 1703.4 (CO), 1610.7 (C = C arom.), 1561.7 (C = C arom.), 1435 (CH₂ bending), 1163.5 (C-N).

¹H NMR (DMSO-d6, 400 MHz, ppm): 11.995(s,1H, OH), 3.6–3.42 (m, 2H, N-CH₂), 2.18–2.35 (m,2H, CH₂-COOH), 2.18-2.0414 (m,1H, N-CH₂-CH), 1.776–1.636 (m,1H, CH(CH₃)₂), 1.059–1.216 (m,2H, CH₂-CH(CH₃)₂), 0.790–0.994 (2d, 6H, (CH₃)₂).

¹³C NMR (DMSO-d6, 100MHz, ppm): 174.074 (COOH), 164.493 (2CO), 136.463 (2aromatic carbons), 131.614(2 aromatic carbons), 120.831 (2 aromatic carbons), 42.796 (N-CH₂), 41.632(CH₂COOH), 37.762((CH₂-CH(CH₃)₂), 32.083(N-CH₂-CH), 25.215(CH(CH₃)₂), 23.250(CH₃), 22.652(CH₃).

DEPT 135 ¹³C NMR: 42.796 (N-CH₂), 41.632(CH₂COOH), 37.762((CH₂-CH(CH₃)₂), 32.083(N-CH₂-CH), 25.215(CH(CH₃)₂), 23.250(CH₃), 22.652(CH₃).

ESI⁺MS-MS (M/Z): 679.8 [M + DMSO + H]⁺, 604.7 [M]⁺, 588.7 [M-OH]⁺, 565.7,476.7, 463.7.

{1-[(1,3-dioxo-1,3-dihydro-2H-isoindole-2-yl) methyl] cyclohexyl} acetic acid (3):

White fine powder, yield:80% (procedure A)-84% (procedure B), MP 151–152 C^O, UV λ_max (methanol, nm):290, 302.

IR(KBr, cm^− 1): 3434 (OH), 3096 (Ar-H stretch.), 2929, 2869 (CH₂ stretching), 1770 (CO), 1720 (CO), 1710 (CO), 1611 (C = C arom), 1466 (CH₂ bending), 1195 (C-N).

¹H NMR (400 MHz, CD₃CN, ppm): 11.99(s,1H, OH), 7.91–7.78 (m, Ar-4H), 3.654(s,2H, N-CH₂), 2.304(s,2H, CH₂-COOH), 1.670–1.108 (m, 10H of cyclohexane).

¹³C NMR (100 MHz, CD₃CN, ppm): 173.287(COOH), 169.243 (2CO), 134.784(2 aromatic carbons), 132.131(2 aromatic carbons), 123.454 (2 aromatic carbons), 46.146, 38.098, 33.365, 25.768, 21.551.

DEPT 135 ¹³C NMR: 134.787 (2 aromatic carbons), 123.459 (2 aromatic carbons), 46.141 (CH₂-N), 38.098 (CH₂-COOH), 33.349 (2(CH₂) of cyclohexane), 25.767(CH₂ of cyclohexane), 21.546(2 (CH₂) of cyclohexane).

ESI⁺MS-MS (M/Z): 302.0 [M + H] ⁺, 323.9 [M + Na] ⁺, 339.9 [M + K] ⁺, 284.0[M-OH] ⁺, 256.1[M-COOH] ⁺, 242 [M-(CH₂-COOH)] ⁺, 160[M-(COOH-CH₂-C₆H₁₀)] ⁺.

{1-[(4,5,6,7-tetrachloro-1,3-dioxo-1,3-dihydro-2H-isoindole-2-yl) methyl] cyclohexyl} acetic acid (4):

White crystals yield:70% (procedure A)-75% (procedure B), MP 218-218.4 C^O, UV λ _max (methanol, nm):202, 212, 236, 333.

IR(KBr, cm¹): 3299 (OH), 2862, 2931 (CH₂ stretching), 1772 (CO), 1733 (CO), 1703 (CO), 1434 (CH₂ bending), 1157 (C-N), 736 (C-Cl).

¹H NMR (400 MHz, DMSO-d₆, ppm): 12.031(S,1H, OH), 3.662 (S,2H, N-CH₂), 2.323 (s,2H, CH₂-COOH), 1.618–1.139 (m, 10H of cyclohexane).

¹³C NMR (DMSO-d₆, 100 MHz, ppm): 173.281(COOH), 164.820 (2CO), 138.412(2 aromatic carbons), 128.975(2 aromatic carbons), 128.375 (2 aromatic carbons), 46.954, 38.212, 33.144, 25.668, 21.533.

DEPT135 (DMSO-d₆, 100 MHz, ppm): 46.948 (CH₂-N), 38.212 (CH₂COOH), 33.144 (2(CH₂) of cyclohexane), 25.690 (CH₂ of cyclohexane), 21.526(2(CH₂) of cyclohexane).

ESI⁺MS-MS (M/Z):407.9, 497.8, 387.8, 301.

{1-[(4,5,6,7-tetrabromo-1,3-dioxo-1,3-dihydro-2H-isoindole-2-yl)methyl]cyclohexyl}acetic acid (5):

Pale yellow crystals yield: 77% (procedure A)-80% (procedure B), MP 241.5–243 C^O, UV λ _max (methanol, nm):287, 336.

IR(KBr, cm¹): 3299 (OH), 2929, 2861 (CH₂ stretching), 1770 (CO), 1735 (CO), 1702 (CO), 1559 (C = C),1432 (CH₂ bending), 1155 (C-N), 669 (C-Br).

¹H NMR (400 MHz, DMSO-d₆, ppm): 12.004(S,1H, OH), 3.645 (S, 2H, N-CH₂), 2.308 (S,2H, CH₂-COOH), 1.72–1.07 (m, 10H of cyclohexane).

¹³C NMR (DMSO-d₆, 100 MHz, ppm): 173.308(COOH), 165.135 (2CO), 136.588(2 aromatic carbons), 131.689(2 aromatic carbons), 120.784 (2 aromatic carbons), 47.112(N-CH₂), 38.192 (CH₂-COOH), 33.171(2C of cyclohexane), 25.665 (1C of cyclohexane), 21.555 (2C of cyclohexane).

DEPT135-¹³C NMR (DMSO-d₆, 100 MHz, ppm): 47.059 (CH₂-N), 38.361 (CH₂-COOH), 33.226 (2(CH₂)), 25.778 (CH₂), 21.547 (2(CH₂)).

ESI⁺MS-MS (M/Z):588.3, 566.3, 475.2,452.6.

2.2. Evaluation of biological activity:

2.2.1. Antioxidant activity:

Antioxidant activity of prepared compounds was determined by measuring free radical scavenging activity using DPPH (2,2-diphenyl-1-picrylhydrazyl) according to standard method, and Ascorbic acid was used as positive control(28).

We have studied serial concentrations 250, 500, 750, and 1000 µg/mL of each compound, and Ascorbic acid in methanol.

1 mL of studied solution was mixed with 3 mL of DPPH solution, and allowed to stand in dark for 30, 60, 90, 120 minutes.

Methanol was used to prepare negative control. Pure methanol was used as a blank. Absorbance was measured at 517 nm after 30, 60, 90 and 120 minutes. All experiments were done in triplicates and the values are averaged.

DPPH scavenging activity was calculated though following equation:

Scavenging effect % = (A_{negative control} - A_sample/A_{negative control}) x 100

IC₅₀ was calculated from concentration-inhibition curve by plotting the sample concentration versus the corresponding DPPH scavenging activity.

2.2.2. Antimicrobial activity:

Antimicrobial activity of parent compounds (Pregabalin, and Gabapentin) and synthesized compounds was investigated against gram positive (Staphylococcus aureus) and gram negative (Escherichia Coli) bacteria by well diffusion method reported by Perez et al(29).

Stock solution of each compound was prepared, and diluted to prepare other concentrations through two-fold dilution method. At least 6 concentrations were studied for each compound (3200, 1600, 800, 400, 200, 100 µg/mL).

Microbial suspension was prepared in physiological saline serum, and adjusted to 0.5 McFarland standard.

Culture media was prepared by pour plate method, in which microbial suspension was added to Muller Hinton Agar at percentage 1%, then poured in petri dishes (depth 3–4 mm).

Poured Plates were left to cool then similar wells (8 mm in diameter) were made in the agar and loaded with 100 µl of the tested compound solution. Inculcated plates were incubated at 37°C for 24 h.

Antimicrobial activity was evaluated by measuring the zone of inhibition (IZ) in mm. Each experiment was carried out in triplicate and the average zone of inhibition was calculated.

Gentamycin sulfate was used as standard drug for antibacterial activity, pure solvent was used as negative control.

2.2.3. Anticancer activity:

Effect of compounds was studied on human cancer cell lines Caco-2 and HCT-116 (Sigma Aldrich).

Stock solution of each compound was prepared in DMSO, and diluted with culture medium to prepare serial concentrations. The final concentration of DMSO in wells is less or equal to 0.1%.

Viability was measured after 48 hours using XTT method, cell cycle changes of treated cells were studied by Flow Cytometry, Assay of apoptosis and necrosis cell death was performed by Annexin V/propidium iodide (PI) double staining assay method using Flow Cytometry.

2.2.3.1. Cell cultures:

Human cancer cell lines Caco-2 and HCT-116 were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 U/mL penicillin and 100 µg/mL streptomycin in sterile flasks. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

2.2.3.2. XTT assay:

Human Caco-2 and HCT-116 cultures were seeded in 96-well plates (1.5x10⁴ cells/well).

Plates were incubated for 24 hours at 37°C and humidified atmosphere containing 5% CO₂, then culture medium was removed and cells were treated with different concentrations of studied compounds.

At least six concentrations were studied for each compound, and each concentration was studied in three triplicates. DMSO alone was added to a set of wells as the solvent control, culture medium was added to another set of wells as negative control.

After treatment, plates were incubated for 48 hours at temperature 37 C^O and 5% CO₂ atmosphere.

Antiproliferative effect was measured after 48 hours by XTT assay reported by Skehan et al.(30) according to instructions of manufacturing company of the Kit (Roche).

Absorbance was measured using ELISA reader spectrophotometer at 450 nm. All experiments were carried out in triplicates.

Inhibition of cell viability was expressed as percentage and it was calculated as following:

Growth inhibition GI%= [1- (absorbance of test compound/absorbance of the negative control)]*100

Inhibition ratio was calculated as mean of results of all replicates. IC₅₀ was calculated from dose- response curve.

2.2.3.3. Cell Cycle analysis:

We have studied cell cycle phases in treated cells, compared with untreated cells, in order to get more information about the mechanism of anticancer effect of synthesized compounds.

Cells were cultured and treated with studied compounds for 72 hours, untreated cells were incubated with culture medium in the same conditions as negative control.

Cells were harvested by trypsin, samples were centrifuged, cellular precipitate was washed with phosphate-buffered saline (PBS), and resuspended in RPMI1640.

Samples were fixed by incubation with methanol in dark place for 30 minutes at -20 C^o.

Samples were centrifuged, and fixed cells were rinsed twice with PBS. Staining solution (DNA fluorochrome PI in a solution containing Triton X-100 and RNase) was added, and samples were kept in dark for 30 minutes at 4–8 C^O. Stained cells were analyzed by BD FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) in accordance with the manufacturer's protocol (31).

The tests were performed in duplicates and repeated at least twice.

2.2.3.4. Apoptosis assay:

Assay of apoptosis and necrosis cell death was executed by Annexin V/propidium iodide (PI) double staining assay method in order to know effect of compounds on cell death.

Cells were treated with compounds for 72 h, untreated cells were incubated with RPMI1640 as a negative control.

Cells were harvested by trypsin, and precipitated by centrifuge. cellular precipitate was washed with PBS, and resuspended in culture medium.

Cells were fixed by incubation with methanol in dark and cold place for 30 minutes at -20 C^o.

Fixed cells were separated by centrifuge, and rinsed twice by PBS.

Cells were stained with 5 µL Annexin V and 5 µL PI and 350 µL Annexin binding buffer, and analyzed by BD FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Number of cells analyzed for each sample was 10⁴ cells(31). Gating was implemented on the basis of negativecontrol staining profiles.

The tests were performed in duplicates and repeated at least twice.

3.1. Synthesis and characterization:

New Isoindoline-1,3-dione derivatives (1–5) were synthesized by molecular hybridization of Pregabalin, or Gabapentin with phthalic anhydride derivatives. One-step synthesis reaction occurs by dehydrative condensation in the presence or absence of solvent according to Scheme 1.

Synthesis was successfully executed by two methods, the conventional method, and microwave-assisted method.

Reaction is shorter, 5–10 minutes, with microwave assisted method, while it takes more than 2 hours for completion of reaction with conventional method.

Microwave-assisted method is an eco-friendly and green chemistry method. Short reaction time, good yield, and mild conditions are additional advantages of this method, over the conventional method.

Structures of compounds are confirmed by ¹H NMR, ¹³C NMR, DEPT-135 NMR, LC-MS, FT-IR and UV-visible spectra.

Analytical and spectral results of all identification tests comply with structures of compounds.

Molecular data of compounds are tabulated in Table 1.

Table 1

Molecular and physical properties of synthesized compounds
compound No.	compound name	molecular formula	MW	purity %	mp.	Yield %
Compound 1	5-methyl-3-[(4,5,6,7-tetrachloro-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) methyl] hexanoic acid	C₁₆H₁₅Cl₄NO₄	427.11	98.50%	177.6–180	90 &92
Compound 2	5-methyl-3-[(4,5,6,7-tetrabromo-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) methyl] hexanoic acid	C₁₆H₁₅Br₄NO₄	604.91	99.91%	212-212.6	83&86
Compound 3	{1-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) methyl] cyclohexyl} acetic acid	C₁₇H₁₉NO₄	301.34	99.18%	151–152	80&84
Compound 4	{1-[(4,5,6,7-tetrachloro-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) methyl] cyclohexyl} acetic acid	C₁₇H₁₅Cl₄NO₄	439.12	99.99%	218-218.4	70 &75
Compound 5	{1-[(4,5,6,7-tetrabromo-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) methyl] cyclohexyl} acetic acid	C₁₇H₁₅Br₄NO₄	616.92	95.90%	241.5–243	77 &80

3.2. Evaluation of biological activity:

3.2.1. Antimicrobial activity:

Compounds (1–5) showed moderate to good zone of inhibition against tested bacterial strains at micromolar concentrations, in a concentration depended manner, while negative control (solvent), and parent compounds did not show any effect (Tables 2 and 3).

Compounds 1–2 are more effective than standard drug gentamycin sulfate against Escherichia Coli, and they are promising as antimicrobial agents.

Table 2

In vitro antimicrobial activity of synthesized compounds against Staphylococcus Aureus.
Zone of inhibition on Staphylococcus Aureus (mm)
conc. µg/mL	3200 µg/mL	1600 µg/mL	800 µg/mL	400 µg/mL	200 µg/mL	100 µg/mL
Pregabalin	-	-	-	-	-	-
Gabapentin	-	-	-	-	-	-
Compound 1	20	18.67	18	14.34	12	9.67
Compound 2	21.67	20	19.67	19.34	18.67	16.34
Compound 3	16	12	0	0	0	0
Compound 4	19	17.33	16.5	15.67	14.67	11.67
Compound 5	20	18.34	17.67	17	16.67	14.67
Gentamycin sulphate	26	25	24	22.34	21.34	19.34

Table 3

In vitro antimicrobial activity of synthesized compounds against Escherichia coli.
Zone of inhibition on Escherichia coli (mm)
conc. µg/mL	3200 µg/mL	1600 µg/mL	800 µg/mL	400 µg/mL	200 µg/mL	100 µg/mL
Pregabalin	-	-	-	-	-	-
Gabapentin	-	-	-	-	-	-
Compound 1	23	20	18	16	14	13
Compound 2	24	23	22.67	22.34	21.67	20.34
Compound 3	14.34	13.34	12.37	11.67	11	10.34
Compound 4	15	13.67	13	11.67	11.34	10.67
Compound 5	16	15	14	13.67	13.34	12.67
Gentamycin sulphate	21	20	17	13	0	0

Though Pregabalin and Gabapentin do not have antimicrobial effect, hybridization into unsubstituted, tetra chlorinated or tetra brominated Isoindoline-1,3(2H)-Dione moiety gives new hybrids with moderate to good antimicrobial activity against gram positive and gram-negative microorganisms.

Hybrids of pregabalin are more effective against gram negative E.coli, while hybrids of Gabapentin are more effective against gram positive Staphylococcus Aureus.

Results show that halogenation of iso indole-1,3-(2H) Dione derivatives enhance antimicrobial effect of compounds, and brominated derivatives are more effective that chlorinated ones.

3.2.2. Antioxidant activity:

We have evaluated free radical scavenging properties of compounds by DPPH free radical scavenging assay in an attempt to find new sources of antioxidants.

Results of our compounds, and antioxidant Ascorbic acid are shown in Table 4.

Table 4

Free radical scavenging activity of studied compounds and Ascorbic acid.
Conc. µg/mL	Compound 1				Compound 2
	Free radical scavenging activity%				Free radical scavenging activity%
	30 min.	60 min.	90 min.	120 min	30 min.	60 min.	90 min.	120 min
250	14.82	16.64	39.88	44.02	2.04	9.03	13.71	19.95
500	18.02	20.28	40.93	44.86	6.54	14.35	18.26	23.33
750	20.56	22.98	43.05	48.16	7.59	16.26	20.2	25.76
1000	21.06	26.25	44.26	48.88	8.38	17.29	21.48	26.87
Conc. µg/mL	Compound 3				Compound 4
	Free radical scavenging activity%				Free radical scavenging activity%
	30 min.	60 min.	90 min.	120 min	30 min.	60 min.	90 min.	120 min
250	28.94	34.47	39.84	42.58	4.2	8.4	10.85	14.85
500	41.48	44.07	49.55	56.45	27.37	38.37	42.44	44.27
750	50.4	55.57	60.12	71.45	29.93	58.62	56.86	63.97
1000	55	58.39	65.34	77.74	32.2	61.3	58.6	67.95
Conc. µg/mL	Compound 5				Ascorbic acid
	Free radical scavenging activity%				Free radical scavenging activity%
	30 min.	60 min.	90 min.	120 min	30 min.	60 min.	90 min.	120 min
250	12.32	15.52	19.35	22.04	95.42	98.02	98.13	98.23
500	14.98	19.03	20.65	23.25	96.58	98.15	98.22	98.36
750	21.2	27.84	30.52	33.84	96.65	98.16	98.26	98.41
1000	25.6	28.96	32.2	40.16	96.66	98.18	98.3	98.47

Though they are less efficient compared with Ascorbic acid, compounds (1–5) showed ability to scavenge free radicals of DPPH in a time and concentration-related manner.

Compound 3 showed considerable activity with significant IC₅₀ value 2.525 µmol/mL, and can thus ensure protection against oxidative stress caused by free radicals.

Scavenging activity may be attributed to the hydrogen-donating ability of the compounds.

3.2.3. Anti-cancer activity:

We have studied effect of compounds 1–4 on two cancer cell lines, Caco-2 and HCT-116. We were not able to study effect of compound 5 on cells due to inappropriate solubility of the compound in the safety range of DMSO.

3.2.3.1. Antiproliferative assay:

Antiproliferative effect of compounds was evaluated by XTT cell viability assay after 48-hours treatment on two cell lines, Caco-2 and HCT 116. Antiproliferative percentage and IC₅₀ values are shown in Tables 5 and 6 respectively.

Results indicate significant antiproliferative effect of studied compounds on both cell lines, and they are in good correlation with concentrations.

Table 5

Antiproliferative effect of studied compounds on Caco-2 cell line.
Compound number	Antiproliferative effect % on Caco-2 cell line						IC₅₀ (µmol/mL)
Compound 1	Conc. µg/mL	500	250	125	75	25	0.2724
Compound 1	% *GI	74.58	73.08	65.37	0	0	0.2724
Compound 2	Conc. µg/mL	125	75	35	10	5	0.000639
Compound 2	% GI	72.9	72.66	69.25	58.12	52.25	0.000639
Compound 3	Conc. µg/mL	1000	500	250	125	75	2.0845
Compound 3	% GI	69.9	39	19	0	0	2.0845
Compound 4	Conc. µg/mL	100	50	25	12.5	10	0.1450
Compound 4	% GI	72.67	44.2	8.67	0	0	0.1450

*GI%: Percentage of growth inhibition.

IC₅₀ values of compounds 1,2, and 4 against Caco-2 and HCT-116 are less than 1 µmol/mL, the fact that assures good antiproliferative effect.

Results on Caco-2 show that compound 2 has the most efficient antiproliferative effect against Caco-2 with IC₅₀ value 0.00064 µmol/mL.

Compound 4 is 14 times more effective than compound 3, this result improves that chlorination of iso indole-1,3-(2H) Dione moiety increases antiproliferative effect against Caco-2 cells.

Compound 2 is 426 times more effective than compound 1, this result improves that halogenation of iso indole-1,3-(2H) Dione compounds increases the antiproliferative activity.

Antiproliferative effect of compound 2 (IC₅₀ = 0.64 µmol/L) against Caco-2 cells is 137.7 times more effective than Thalidomide (IC₅₀ = 88 µmol/L) (32).

Table 6

Antiproliferative effect of studied compounds on HCT-116 cell line.
Compound number	Antiproliferative effect % on HCT-116 cell line						IC₅₀ (µmol/mL)
Compound 1	Conc. µg/mL	500	250	125	75	25	0.412
Compound 1	% GI	55.774	52.102	49.392	48.308	30.462	0.412
Compound 2	Conc. µg/mL	1000	500	250	125	75	0.413
Compound 2	% GI	33.133*	45.384*	50.02	49.855	49.189	0.413
Compound 3	Conc. µg/mL	1000	500	250	125	75	> 3.39
Compound 3	% GI	48.322	22.858	15.685	7.255	1.683	> 3.39
Compound 4	Conc. µg/mL	100	50	25	12.5	10	0.228
Compound 4	% GI	49.558	47.406	24.692	7.236	0	0.228

Results on HCT-116 cell line show that compound 4 is the most effective with IC₅₀ 0.228 µmol/mL, then compounds 1 and 2 having the same antiproliferative effect against HCT-116 with IC₅₀ 0.412 µmol/mL.

IC₅₀ of compound 3 is more than 3.39 µmol/mL, and it is not applicable in studied concentrations.

Compound 3 is less effective than compound 4, and other compounds against Caco-2 and HCT-116 cells, this result improves that halogenation of iso indole-1,3-(2H) Dione moiety increases antiproliferative effect against HCT-116 cells.

3.2.3.2. Cell Cycle analysis:

For further understanding of the mechanism of antiproliferative effect of compounds, we have defined percentage of cells in each phase of the cell cycle by flow cytometry using PI staining method.

Results of studying cell cycle of Caco-2 and HCT-116 cells treated with compounds for 72 hours compared with untreated cells show that all compounds strongly delay progress throughout cell cycle, the fact that explains and confirms the already outlined antiproliferative effect.

Results of cell cycle study of Caco-2 cellular line after treatment with compounds show that compounds 1 and 2 arrest cell cycle of treated cells on phase G1, compound 3 arrest cell cycle on phase S, and compound 4 arrest cell cycle on phases S and G2/M (Fig. 2).

Compounds 1 and 2 arrest progression of cell cycle to synthesis phase, compound 3 inhibits synthesis of DNA, compound 4 inhibits synthesis of DNA and inhibit mitosis as well in treated cells.

Results of cell cycle study of HCTT116 cells after treatment with compounds show that compounds arrest cell cycle at phases S and G2/M, which indicates their ability to inhibit synthesis of DNA, and mitosis as well (Fig. 3).

Results explain the antiproliferative effect of compounds on Caco-2 and HCT-116 cell line, and they are in agreement with our previous results.

3.2.3.3. Apoptosis assay:

For further understanding of method of action of synthesized compounds as promising anticancer agents, and for further understanding of mechanism of cell death induced by compounds, we have treated Caco-2 and HCT 116 cells with studied compounds for 72 hours and assayed the percentage of necrotic cells, and percentage of cells in different stages of apoptosis using Annexin V Binding, Propidium Iodide Uptake method by flow cytometry.

Results show that treatment of Caco-2 and HCT-116 cell lines with studied compounds induces cell death by apoptosis, results show decrease of the percentage of live cells, and an increase in the percentage of apoptotic cells, and/or necrotic cells compared with untreated cells.

Results of cell death assay in Caco-2 cells, confirm that all compounds effectively induce apoptosis in Caco-2 cells compared with untreated cells (Fig. 4).

Results of cell death assay in HCT-116 cells, show that all studied compounds effectively induce apoptosis, and necrosis in HCT-116 cells compared with untreated cells.

Biological importance of Iso indole-1,3-(2H) Dione scaffold has promoted us for synthesis of some promising multitarget derivatives by molecular hybridization of this moiety with Gabapentin and Pregabalin.

Five derivatives of Gabapentin, and Pregabalin hybrids with iso indole-1,3-(2H) Dione were synthesized, Two Synthesis methods have been successfully carried out.

Synthesized compounds were characterized by UV spectra, I.R spectra, ¹H-NMR ¹³C-NMR, DEPT-135 NMR and ESI-MS, results showed good agreements with the proposed chemical structures of compounds.

Compounds were screened for antimicrobial activity against two bacterial species, gram positive Staphylococcus aureus, and gram-negative Escherichia Coli, and for anticancer effect against two cancer cell lines, Caco-2 and HCT-116.

In vitro studies indicated that compounds 1, and 2 are promising antimicrobial agents, and they are more effective than Gentamycin sulfate against E. coli.

Viability studies indicate that tested compounds have significant antiproliferative effect against Caco-2 and HCT-116 cancer cells. Treatment with compounds arrest progression though cell cycle and induce cell death in treated cells.

Data Availability:

All data used to support the findings and conclusion of the study are available from the corresponding author upon request.

Conflicts of Interest:

The authors declare that they have no conflict of interest.

Funding statement:

This work was funded by Damascus University.

Authors’ Contributions:

All authors have read and approved the manuscript.

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Scheme 1 is available in Supplementary Files section.

No competing interests reported.

Scheme1.png
Scheme 1 Synthesis reactions of compounds.

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Synthesis, Characterization, and Biological Evaluation of Gabapentinoid Hybrids with Isoindole-1,3-(2H) Dione Moiety as potential antioxidant, antimicrobial, and anticancer

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Abstract

Figures

1. Introduction

2. Material and method

2.1. Synthesis and characterization:

2.2. Evaluation of biological activity:

2.2.1. Antioxidant activity:

2.2.2. Antimicrobial activity:

2.2.3. Anticancer activity:

2.2.3.1. Cell cultures:

2.2.3.2. XTT assay:

2.2.3.3. Cell Cycle analysis:

2.2.3.4. Apoptosis assay:

3. Results and Discussion

3.1. Synthesis and characterization:

3.2. Evaluation of biological activity:

3.2.1. Antimicrobial activity:

3.2.2. Antioxidant activity:

3.2.3. Anti-cancer activity:

3.2.3.1. Antiproliferative assay:

3.2.3.2. Cell Cycle analysis:

3.2.3.3. Apoptosis assay:

4. Conclusion

Declarations

References

Scheme

Additional Declarations

Supplementary Files

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