Anti-Cancer Activity of Gabapentin and Chiral Amino Acids-Based Hybrid-Peptides against MCF-7 Breast Cancer Cell-Line

Aims: Herein, we report the cytotoxicity of gabapentin-based peptides (11a-11j) using L-alanine and L-phenyl alanine chiral amino acids for peptide bond formation in ten efficient and straightforward steps. The in vitro MTT assays of derived molecules on the MCF-7 cell line (a human breast adenocarcinoma cell line) exhibited enhanced antitumor activity compared to the control (100% cell proliferation). Methods: The ten steps synthetic methods were adapted for the synthesis of the Gabapentinbased peptide derivatives through BOCdeBOC methods and using EDC-HCl, DMAP and commercially available solvents. All the synthesized peptides were unambiguously characterized Original Research Article Pansuriya et al.; JPRI, 33(46A): 431-446, 2021; Article no.JPRI.75639 432 with the help of spectroscopic (IR, 1 H NMR, 13 C-NMR, mass spectra, and elemental) data analysis. Results: The Compounds 11a, 11b, 11h,11i, and 11j showed a remarkable antiproliferative (cell death) activity, with % cell proliferation values ranging from 25-38 %. Conclusion: The study showed that the compounds with some specific functionalities like, benzylic and trifluoromethyl functionality enhanced the potency with comparable %cell proliferation and cell death. Based on the findings in this work and their easily accessible molecular structures, compounds 11a and 11j are worthy of further biological investigations.


INTRODUCTION
Cancer is now reached at the top, threatening human health due to the increasing incidence and mortality. Among various types of cancers, breast cancer is one of the most common types among other malignancies in women and a significant cause of mortality worldwide [1,2]. New cases of breast cancer diagnosed in 2015 accounted for approximately 12% of all new malignancy cases, and mortality accounted for 25% of all cancer cases in women. The worldwide new cases of female breast cancer are estimated to reach nearly 3.2 million per year by 2050 [3][4][5]. Despite the intensive investigation of breast cancer cell lines, the cellular and molecular mechanisms between the MCF-7 cell line and the drug polyoxometalate (POM) are still limited [6]. Anticancer peptides (ACPs) have been proved to be effective small molecules (nearly 50 amino acids) that can act specifically against cancerous cells by either a membranolytic mechanism or disruption of mitochondria [7].
Peptides and proteins are involved in various biochemical processes and physiological functions [8][9][10][11]. Approximately 34% of peptide drugs were approved by the US Food and Drug Administrations and were expected to reach USD 23 billion in 2021 [12]. It is currently act as an active hormone [13,14], neurotransmitters [15,16], growth factors [17], signaling molecules and antibiotics [18][19][20]. Peptide drugs are applicable as analgesic [21], antimicrobial [22], cardiovascular [23], CNS drugs [24,25], gastrointestinal disorder [26], hematological disorder [27], respiratory disorder [28]and anticancer agents [29][30][31]. Gabapentin (Gbp) was originally produced in a search for analogues of the neurotransmitter γ-aminobutyric acid (GABA) [32]and later on approved for various therapy by the drug agencies of some countries as therapy for partial seizures, for treatment of post-herpetic neuralgia, for adjunctive therapy for cancer pain despite the known fact of high doses of Gabapentin were associated with pancreatic acinar cell tumour in rats [33,34]. The conformational properties of Gbp in the peptide sequences are the current attention of the researcher [35][36][37][38]. In peptide design, Gabapentin may be used as a stereochemically constrained analogue of the parent chiral amino acid to enhance its potency in various cancer treatments and recently introduced as an adjunct in the multimodal management of postoperative pain after breast cancer surgery [39]. The development of gabapentin peptides could transform in vivo to release the active drug. Thus, it could be a feasible strategy to improve the physicochemical, biopharmaceutical or pharmacokinetics, and pharmacological properties of Gbp, thereby increasing its usefulness [40,41]. In November 2019, more than 20 ACPs were approved by the FDA, like Kyprolis, Lutathera, and Gallium Dotatoc Ga68 ( Fig. 1) [42,43].
Collectively, ACPs represent a promising alternative to conventional chemotherapy due to their high specificity, reduced side effects, and desired tumour penetration. However, many ACPs also have some disadvantages, including substantial toxicity and poor targeting, which seriously impair their potency. Therefore, mechanisms of effective reconstruction or modification of ACPs to improve their therapeutic properties and reduce their toxicity have become a significant research focus [44][45][46][47].
To extend our efforts towards promising anticancer agents using various natural/nonnatural precursors [48][49][50], herein, we have reported a new class of Gabapentin base hybridpeptide derivatives (11a-11j) and were evaluated for anticancer screening against MCF-7 breast cancer cells line. The structure of newly synthesized peptides was assigned based on IR, 1 H NMR, 13 C NMR, and mass spectrometry analysis.

Materials
The required chemicals and solvents for the synthesis were purchased from JSK Fine, Avra Synthesis, Finar, and Spectrochem. All the chemicals were used without further purification. Precoated plates of silica gel G60 F254 (0.2 mm, Mfg. by Merck) were used for thin-layer chromatography. Visualization was made under UV light (254 and 365nm) or with iodine vapour. Melting points were measured with the help of Optimelt MPA100, an automated apparatus. Spectral analysis of the synthesized compound was done with the help of FTIR-8400 (Shimadzu) using the ATR technique. The 1 H NMR (400 MHz) and 13 C NMR (101 MHz) spectra were recorded on the "Bruker AVANCE II Spectrometer" using DMSO-d 6 as solvent and TMS as the internal reference. Mass spectra were recorded on a Jeol-JMSD 300 mass spectrometer at 70ev. Elemental analysis was carried out by a Perkin-Elmer 2400 CHN analyzer.

Synthesis of 2-(1-(((tertbutoxycarbonyl)amino)methyl)cyclohex yl)acetic acid (KSM-1)
In 250 ml RBF, Gabapentin (10 gm, 0.58 mol) and sodium hydroxide (2.6 gm, 0.65 mol) solution dissolved in water (50 ml) was added along with slowly and dropwise addition of Boc anhydride (15.2 gm, 0.69 mol) at 0-5 o C temperature. It was stirred at RT overnight (12 hours). The progress of the reaction was monitored by TLC using Dichloromethane: methanol (9:1) as a mobile phase (Rf: 0.2). After completing the reaction, the 3-4 pH was maintained using diluted hydrochloric acid [6:4, HCl: Water] at below 5°C temperature. The reaction mixture was filtered under vacuum and dried in a vacuum dryer. It was found as a white crystalline powder with a 92% of yield.

Synthesis of 2-((tertbutoxycarbonyl)amino)propanoic acid (KSM-2)
In 250 ml RBF, L-alanine (10 gm, 1.12 mol) and sodium hydroxide pellets (5.4 gm, 1.35 mol) dissolve in water (50 ml) was added along with slowly and dropwise addition of Boc anhydride (29.3 gm, 1.35 mol) at 0-5 o C temperature. It was stirred at RT overnight. The progress of the reaction was monitored by TLC using Dichloromethane: methanol (9:1) as a mobile phase (Rf: 0.3). After completing the reaction, the pH (3-4) was maintained by dropwise addition of diluted hydrochloric acid [6:4, HCl: Water] at below 5°C temperature. The separated solid was filtered off under vacuum and was dried in a vacuum dryer at 55 o C temperature for further reaction. It was found as a white crystalline powder with a 90% of yield.

Synthesis of methyl-2-amino-3phenylpropanoate hydrochloride (4)
To a solution of L-phenylalanine 3 (10 gm, 0.60 mol) in methanol (50 ml), SOCl 2 (7.2 gm, 0.90 mol) was added in a dropwise manner by maintaining the temperature below 0°C. After addition, it was stirred at RT for 12 hours and was monitored by TLC with mobile phase Dichloromethane: methanol (8:2 ml). It was distilled off under reduced pressure and washed hexane with vigorous stirring to afford the product (4). It was used instantly in the next step without further purification.

Synthesis of methyl-2-(2-(1-(((tertbutoxycarbonyl)amino)methyl)cyclohex yl) acetamido)-3-phenylpropanoate (5)
The KSM-1 (3.0 gm, 0.11 mol) was dissolved in DMF (30 ml) and carried out at a temperature below 5°C. To this mixture, EDC . HCl (2.53 gm, 0.13 mol) and 4-Dimethylaminopyridine (4.5 gm, 0.36 mol) were added by maintaining the temperature below 5°C and stirred well for 10 minutes. It was fed by previously prepared intermediate 4 (2.39 gm, 0.11 mol) and was stirred overnight at RT. After completion of the reaction, monitored on TLC using Dichloromethane: methanol (7:3) as mobile phase, the product was poured into cold water and stirred vigorously for 1 h. Extract the product with MDC (50 ml 3). The organic layer was washed with brine followed by dried using sodium sulfate and distilled off to get the oily mass of product 5 with 88% yield.

Synthesis of methyl-2-(2-(1-(aminomethyl) cyclohexyl)acetamido)-3phenylpropanoate hydrochloride salt (6)
In a 250 ml RBF, a solution of compound 5 (3.8 gm, 0.87 mol) was dissolved in dioxane (30 ml) and cooled to 0°C temperature. After sometimes the reaction mass appeared cleared and was purged by HCl gas (1.0 hour). The reaction mixture was stirred for 3-4 hours at RT. The reaction was monitored by TLC using mobile phase Dichloromethane and methanol (9:1). It was distilled off under reduced pressure and washed twice with hexane with vigorous stirring to afford the product (6) and was found white hygroscopic solid material.

Synthesis of methyl-2-(2-(1-(2-((tertbutoxycarbonyl)amino)propanamido)met hyl)cyclohexyl) acetamido)-3phenylpropanoate (7)
The KSM-1 (1.48 gm, 0.78 mol) was dissolved in DMF (15 ml) and carried out at a temperature below 5°C. To this reaction mixture, EDC . HCl (1.80 gm, 0.94 mol) and 4-Dimethylaminopyridine (2.87 gm, 2.35 mol) were added by maintaining the temperature below 5°C and stirred well for 10 minutes. It was fed by previously prepared intermediate compound 6 (2. 9 gm, 0.78 mol) and was stirred overnight at RT. After completion of reaction monitored on TLC using Dichloromethane: methanol (7:3) as mobile phase, the product was poured into cold water and stirred vigorously for 1 hour. Extract the product with MDC (35 ml 3). The organic layer was washed with brine followed by dried using sodium sulfate and distilled off to get the yellowish oily mass of product 7 with 86% yield.

Synthesis of methyl-2-(2-(1-((2aminopropanamido) methyl) cyclohexyl) acetamido) -3-phenyl propanoate hydrochloride salt (8)
To a solution of compound 7 (3.4 gm, 0.67 mol) was dissolved in dioxane (30 ml) and cooled to 0°C temperature. After sometimes the reaction mass appeared cleared and was purged by HCl gas (1.0 hour). The reaction mixture was stirred for 3-4 hours at RT. The reaction was monitored by TLC using mobile phase Dichloromethane and methanol (9:1). It was distilled off under reduced pressure and washed twice with hexane with vigorous stirring to afford the product (8) and was found while solid material.

General procedure for the synthesis methyl-(2-(1-(2-(substituted benzamido) propanamido) methyl) cyclohexyl) acetyl)-L-phenylalaninate (9a-9c)
In round bottom flask (moisture-free), aromatic acid (0.23 mol) was dissolved in DMF (10 ml) and was charged with EDC . HCl (0.35 mmol) and 4-Dimethylaminopyridine (0.73 mol) at below 5°C temperature. It was stirred for 10 minutes at RT and added compound 8 (0.23 mol) portion-wise. The reaction mass was stirred at RT overnight and was monitored by TLC using Dichloromethane and methanol (8:2) mobile phase system. and after completing the reaction. The reaction mass was poured into crushed ice and stirred vigorously for 1 hour. Extract the product with MDC (50 ml 3). The organic layer was washed with brine followed by dried using sodium sulfate and concentrated under reduced pressure to get the oily mass of product 9a-9c with 72% yield.

General procedure for the synthesis of (2-(1-((2-(substitutedbenzamido) propanamido) methyl) cyclohexyl) acetyl)-L-phenylalanine (10a-10c)
For the hydrolysis of ester, 9a-9c (0.21 mol) was dissolved in the binary solvent system [water: THF (1:2)] (12 ml). To this, lithium hydroxide (0.32 mol) was added and the mass was stirred at RT overnight. The progress of the reaction was carried out by TLC. After completing the reaction, it was extracted with ethyl acetate (50 ml 3) to remove unreacted material and impurity. An aqueous layer was treated with 2N HCl (20 ml) to adjust pH 2. It was further extracted with MDC (50 ml 3). The MDC layer was washed with brine followed by dried using sodium sulfate and concentrated under reduced pressure to get the product 10a-10c with 79% yield.

HCl
(0.15 mmol) and 4-Dimethylaminopyridine (0.48 mol) at below 5°C temperature. It was stirred for 10 minutes at RT and added various aromatic amine (0.11 mol) portion-wise. The reaction mass was stirred at RT overnight and was monitored by TLC using Dichloromethane and methanol (8:2 ml) mobile phase system. The reaction mass was poured into crushed ice and stirred vigorously for 1 h. Extract the product with MDC (50 ml 3). The organic layer was washed with brine followed by dried using sodium sulfate and concentrated under reduced pressure to final product 11a-11j. The crude material was purified by column purification using MDC and methanol mobile phase using silica 60-120 as stationary phase.

Cell lines and cell culture
A human breast adenocarcinoma (MCF-7) cell line was obtained from National Center for Cell Science (NCCS), Pune, Maharashtra, India. MCF-7 cells were cultured in DMEM medium containing L-glutamine (2mmol/l), supplemented with 10% FBS and 1% PSN, an antibiotic mixture (Life technologies, USA). The cells were cultured in a humidified condition of 5% CO2 at 37°C. The exponentially growing cells were used in the entire study.

Treatments of compounds
Compounds were freshly prepared in cell culture grade DMSO at the stock concentration of 100mM. Exponentially growing MCF-7 cells were treated with different Compounds (100µM) for 24 h. Cells treated with DMSO (0.1%) were considered as vehicle control.

Cell proliferation assay
The cell proliferation was examined by MTT assay. Briefly, 2 X 104 MCF-7 cells were treated with series of synthesized compounds for 24 h. Thereafter, the cells were washed with DPBS and incubated with MTT (0.5mg/ml) for 4 h in dark at 37˚C. After the incubation period, the MTT was removed and DMSO was added to each well. The absorbance was recorded at 570 nm with the reference wavelength of 650 nm by using a Multimode microplate reader (SpectraMax M2e, Molecular Devices, USA). The results were represented as a percentage of cell proliferation.

Statistical analysis
The data represented were analyzed by student t-test using Sigma Stat 2.0 statistical analysis software. The normality of data was tested by the Shapiro-Wilk test before the student t-test. p values ***p≤0.001 were considered statistically significant.

Chemistry
The synthetic methods adapted for the synthesis of the Gabapentin-based peptide derivatives are represented in Scheme-1. Two parallel reactions were undertaken simultaneously to generate our Key Starting Material, i.e., KSM-1 & 2 (Boc protected KSM) formed by Gabapentin and Lalanine respectively by reaction with NaOH and Boc at 0-5 o C followed by 12 hours room temperature stirring. (Herein, we used the term overnight stirring for 12 hours RT reaction conditions). Furthermore, compound 4 was synthesized in methanol using L-phenyl alanine (3) under dropwise addition of SOCl 2 in the temperature range between 0 o C to RT, and the obtained yield was more than 60%. The Cterminal peptide bond in KSM-1 was furnished by coupling with4in the presence of EDC-HCl and DMAP in the temperature range0 o C to RT using DMF as a solvent to form a dipeptide scaffold (5). The deprotected hydrochloride salt of compound 5 was accomplished by dissolved it (5) in DCM followed by acidification with cold HCl and then stirring for 3-4 hours at room temperature. The diverse targeted motif, i.e., Tripeptide (7), was formed by coupling KSM-2 and6 (HCl salt) in the presence of EDC-HCl and DMAP at RT.
The deprotection with HCl salt formation of compound 7 followed by N-terminal amide bond formation of compound 8 in the presence of EDC-HCl and DMAP at below 5 o C temperature to afford intermediate compound 9a-9c. Basic hydrolysis of compounds 9a-9c was carried out by dissolved in THF, the addition of LiOH, and stirred at RT for overnight to form compounds 10a-10c. For the synthesis of final adducts (11a-11j), the amidic bond was generated by DMAP/EDC . HCl at RT stirring for 12 hours.
The structure of synthesized compounds 11a-11j was confirmed through various spectral data. The IR spectrum of compound 11a-11jshoweda strong absorption band at ~3635 cm -1 due to N-H stretching, secondary amine. The absorption band appeared at ~2980 cm -1 due to stretching vibrations of aromatic hydrogen. Sharp absorption peak observed at ~2927 cm -1 for -C-H stretching of a methylene group. The intense absorption peak at ~1656 cm -1 was obtained due to >C=O stretching of amide carbonyl. Moreover, absorption bands at ~1537, ~1325 cm -1 corresponding to C=C, C-N stretching, respectively. Compound 11a-11j showed a characteristic peak at ~1047 cm −1 assignable to the C-F bond. In 1 H NMR spectra, the appearance of multiplet at δ = ~1.95 ppm value in compounds 11a-11j is confirmed to the presence of methylene proton in the cyclohexane ring. The presence of -CO-CH 2 -NH-linkage was showed a singlet peak at ~2.78 ppm. Aromatic protons appeared as multiplet in the region δ = ~7.14 to ~8.45 ppm. The absorption peak at δ = ~8.66 ppm executed a proton of secondary amine in the structure. The remaining substituent's protons were in good agreement with theoretical values. 13 C NMR spectra helped us to identify the formation of the final adducts. The characteristic value δ= ~22 to ~34 ppm showed the presence of methyl group in cyclohexane ring. The signal obtained at δ= ~44 ppm can be assigned to the presence of methylene carbon between cyclohexane and amide linkage. The aromatic ring carbons were in decent covenants with the theoretical values (δ= ~124 ppm). The characteristic value at δ= ~175 ppm showed carbonyl carbon (>C=O) in compounds 11a-11j. The mass spectrum revealed a molecular ion peak in compound 11a-11j at m/z = ~601 to ~668 in mass spectra; molecular ion peak was in agreement with the proposed molecular weight and elemental analysis.
In the entire chemistry, the simple and traditional methods were used to derived novel scaffolds and were further characterized by various spectroscopic techniques. The initial results provoked us to explore the synthetic applicability of 11a-11j to attain new derivatives of gabapentin-amino acid-based hybrid peptide molecules of expected anticancer activity. Thus, introducing a gabapentin core into the peptide framework may provide products with potential anticancer activity from a structure-activity perspective.

Anticancer Evaluation
MCF-7 cancer cell line is valuable for in vitro breast cancer studies because of several ideal characteristics, particularly the mammary epithelium, including estrogen process and sensitivity to cytokeratin. Herein, we observed that five molecules (11a, 11b, 11h, 11i, and 11j) are more active than their corresponding peptides (11c, 11d, 11e, 11f, 11g). The biological effect is dependent on the substituent present on R 1 and R 2 (C & N terminal sides). However, a clear structure-activity relationship between the size/substitutions of the moiety and the antiproliferative effect of the MCF-7 human breast cancer cell line is not observed. The most active compound (11j), which presents a methoxybenzene and fluorobenzyl group as a substituent at two terminals of peptide core, shows significant results with % cell proliferation, 25.62% and antiproliferation/cell death, 74.38%. The following two more active compounds, 11a, and 11h, present substituents as the 2-methyl pyridine (R 2 in both) and 4-CF 3 benzene and 4-F benzyl (as R 1 , respectively) showed antiproliferative activity nearly 71%. Compounds 11b, which feature a 2pyridine (R 2 ) heterocycle and 4-CF 3 benzene, exhibited similar activity with the 11i, featured with 4-F benzyl (as R 1 & R 2 ) with cell proliferation nearly 38% and 34% respectively (Table 1 and Fig 2).
Generally, in all derivatives, the three powerful combinations: a) pyridine and 4-CF 3 benzene, b) 4-F benzyl and 4-F benzyl, and c) 4methoxybenzene and 4-F benzyl are essential for the antitumor activity of similar kinds of molecules. Furthermore, if we compare derivatives 11a and 11b with 11f, the substitution of the benzene ring by its isosteric "3-CF 3 " and "4-CF 3 ", the latter one has led to an improvement in the activity.

SAR (Structure-Activity Relationship) for used Substituents
Literature-based findings directed us to choose specific -R 1 and -R 2 groups to enhance the potency of derived peptides. The epidermal growth factor receptor (EGFR) is one of the transmembrane receptor tyrosine kinase ErbB family. The EGFR also stimulates vascular endothelial growth factor (VEGF), which helps to induce tumour angiogenesis and these both are important targets in cancer therapy [53]. It plays a crucial role in regulating cell proliferation, apoptosis, and migration [54]. The unique properties of fluoro organic molecules may arise due to some factors to affect the pharmacological properties of the fluorinated molecules such as electronegativity, strength in the carbon-fluorine bond, low polarizability, the smallest atomic radius of the fluorine atom, in well studied anticancer agents (for example, 5-fluorouracil (5-FU) and 5-fluoro-2-deoxyuridine) [55][56][57][58]. The bioavailability of the pyridine ring has been evaluated and was found to be most active against two human BC cell lines and was explained the cytotoxic potency to induce the apoptosis of MCF-7 cells (for example, Trovirdin hydrochloride as anticancer agent contains two pyridine rings) [59,60]. Previous research has suggested contrasting effects of methoxy substitutions in chemical entities: it has been reported that they may have unfavorable steric effects and the enhanced potency of some of the lead scaffold is most likely the result of methoxylation leading to improved pharmacokinetic properties and increased stability [61]. Moreover, it has been reported that upon delivery, methoxylated compounds are targeted by tumour-specific O-demethylases that provide free hydroxyl groups and hence an increase in redox properties [62]. Therefore, it seems reasonable that methoxylated novel compounds could make promising candidates as potential chemotherapeutic agents, providing improved pharmacological attributes, including cancer-specific activation [63].

CONCLUSIONS
In this study, ten peptide derivatives having Gabapentin and chiral amino acid as core were synthesized, purified, and characterized by various analysis methods. In vitro cytotoxic activities were screened against breast (MCF-7) cancer cell lines by MTT assay. Five of the ten compounds showed high cytotoxic activity against tested cell lines. It was summarized that the compounds with benzylic and trifluoromethyl functionality enhanced the potency with comparable %cell proliferation and cell death. Based on the findings in this work and their easily accessible molecular structures, compounds 11a and 11j are worthy of further biological investigations.

SUPPLEMENTARY INFORMATION
Supplementary files contain 1 H NMR, 13 C NMR, IR, and mass of 11a-11j and required NMR of intermediate compounds.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by the personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.