Synthesis and characterization of β-carboline fatty alcohol hybrids as potential biological active and antioxidant molecules

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Introduction
Organic molecules synthesized from naturally available renewable materials for various applications especially, biological and pharmaceutical applications are an emerging trend in modern synthetic chemistry [1,2]. During the last two decades, numerous kinds of alkaloids were isolated from the natural sources and studied their importance in biological and pharmaceutical applications [3,4]. Clinically natural alkaloids are famous to treat central nervous system (CNS) disorders for instance atropine, physostigmine morphine, anatabine and papaverine etc [5]. Studies have revealed that wide range of natural alkaloids are biologically active molecules and have been using to treat many pathogenic diseases [6]. β-carboline alkaloids (βCAs; 9 H-pyrido- (3,4-β)indole) are naturally available plant-derived indole alkaloids from Peganum harmala (Zygophillaceae), structurally βCAs are constituted with five and six member ring structures along with two nitrogen atoms as a part of aromatic ring [7,8]. βCAs have been found to be associated with diverse biological and pharmaceutical activities including antialzheimer, antimicrobial, anti-inflammatory, antidepressant, neuroprotective and antioxidant [9][10][11][12].
In an endeavor to develop new βCA derivatives Lopes-Ortiz et al., synthesized imide βCA and carbomethoxy βCA derivatives and their pharmacological properties were studied [13]. In another study, Sireesha et al., synthesized new benzimidazole/benzoxazole linked β-carbolines by joining two different anti-cancer fragments, these hybrid β-carbolines showed high fold cytotoxic activity against MCF-7 cell lines [14]. The data available on βCAs suggest there is still lot of potential to develop new molecules combining with other bioactive moieties. In this 4 account, combining of βCA with other bio active molecules such as fatty acid (FA) derivatives is the aim of our present work. Structurally fatty acids are long chain hydrocarbons containing polar carboxylic acid moiety, formed from the hydrolysis of oils and fats. Variety of modified fatty acids and fatty acid derivatives are significant molecules act against many pathogenic microbes. Combination of FA with various bio active molecules is called as fatty acid hybrid molecules (FHM). FHMs provide diverse advantages including improved oral bioavailability, improved targeting to the lymphatic system, enhanced tumor targeting and reduced toxicity [15].
1-β-D-Arabinofuranosylcytosine (Ara-C, Cytarabine) well known drug used in the treatment of cancer, due to its lower lipophilicity Ara-C exhibited low bioavailability. Liu et al., further synthesized a series of Ara-C derivatives by combining fatty acid and amino acid in order to enhance lipophilicity and bioavailability [16]. Moreover, FA analogues containing hetrocyclic moieties such as oxadiazole, triazole and thiadiazole were exhibited promising antidepressant and antimicrobial activity [17]. 10-undecenoic acid (UDA) is a versatile fatty acid containing terminal double bond derived from renewable feed stock castor oil (pyrolysis of ricinoleic acid ((9Z, 12R)-12-hydroxy-9-octadecenoic acid). There have been many reports on 10-undecenoic acid derived compounds synthesis and utilization for diverse applications mainly pharmaceutical and polymer applications [18,19].
Pharmacophore conjugation is an efficient technique for covalently adjoining two biological potent moieties in to one conjugate molecule [20]. The conjugated molecules demonstrate divergent action mechanism, might lead to synergistic effect with high affinity and selectivity [21]. Combined with the benefits of βCA, and fatty acid derivatives we report the synthesis, characterization, biological-evaluation and antioxidant performance of fatty alcohol hybrid βcarboline derivatives. Herein, we synthesized β-carboline by taking natural amino acid L-5 tryptophan and various aldehydes and hybridized with 10-undecenol (a derivative of 10undecenoic acid) by ester linkage.

Synthesis of β-carboline derivatives
Synthesis of novel β-carboline derivatives was carried out in a six step reaction procedure as outlined in scheme 1. Synthesized products were analyzed in each step by NMR and mass spectroscopy. Initially, the amine group present on L-tryptophan (1) was protected with BOC anhydride (2) and the structure of the product 2 was confirmed by spectral studies. The signals appeared at 1.38 and 26.4 ppm in the 1 H and 13 C NMR spectrum indicate the presence of methyl groups. In the next step, the carboxylic group present on BOC tryptophan was esterified with 10undecenol (3). The characteristic peaks at 4.08, 4.95 and 5.80 ppm in 1 H NMR spectrum indicate the appearance of protons α to the ester functional group and unsaturated protons of 10undecenol. Carbon signals appeared at 115, 163.1 and171.4 ppm indicates the appearance of unsaturated and ester carbons, respectively. The esterified product on acid treatment undergone for deprotection to obtain free amine (4). Product 4 was confirmed by the presence of free -NH2 signal at 5.08 ppm and the carbon α to amine at 56.4 ppm in 1 H and 13 C NMR spectra. In the subsequent step amine group was condensed with various types of aldehydes to attain Schiff bases (5a-i). In 1 H NMR spectra, the characteristic singlet peak appeared at 8.41-8.66 ppm for the Schiff bases (5a-i) were indicates the presence of -N=CH proton and it confirmed the imine coupling. In addition, characteristic carbon peaks in the 13 C NMR appeared at 160.4-166.6 ppm due to N=CH group. In the subsequent step Schiff bases were cyclised to give cyclic products (6a-i). Cyclic products (6a-i) were confirmed by the singlet peak appeared at 5.32-5.62 ppm and the disappearance of the peaks in the range of 8.41-8.66 ppm. In 13 C spectrum disappearance of 6 signal at 160.4-166.6 ppm and appearance of new signals at 56.8-62.2 ppm indicate cyclisation.
The cyclic products finally aromatized to obtain titled products (7a-i). The signals disappeared at 5.32-5.62 ppm and the appearance of new signals at 7.64-7.84 ppm in the 1 H NMR spectra and the peaks observed at 144.5-148.0 ppm and110. 5-113.4 ppm in 13 C NMR spectra confirmed the formation of products 7a-i. Further, the expected molecular weights observed in mass spectra positively confirm the synthesis of β-carboline derivatives.

Scheme 1:
Synthetic procedure for the preparation of fatty alcohol β-carboline hybrids.

Antimicrobial activity
Antimicrobial activity of the β-carboline derivatives were evaluated using well diffusion method  [23]. The synthesized six β-carboline derivatives which are potent as antimicrobial were evaluated for their MBC and it was found that the compound 7d and 7f showed good MBC values of 2. 8, 7.8, 14.6, 7.8 and 4.7, 15.6, 28.3, 17.8  standard drug which had MBC values ranged between 0.9 and 1.9 µg/mL. Table 1: Antimicrobial activity of β-carboline derivatives 7 (a-i).

S. No Test compounds
Minimum inhibitory concentration (µg/mL)  These six compounds were further considered for evaluation of biofilm inhibition assay according to a reported protocol [24]. According to the report the bacterial strain which caused biofilm formation were appeared to tolerate antibiotics which posed a major threat in the treatment of bacterial infections [25]. The compounds synthesized in the current study were Compounds 7d and 7g were showed activity against all the strains whereas, compound 7a and 7h showed moderate activity followed by 7i.

Structure-activity relationship
β-carboline is a naturally occurring versatile alkaloid having many diverse biological applications. The fatty alkyl chain (undecenyl (C-11)) in the β-carboline derivatives participate in increasing the lipophilicity. The enhancement in liphophilicity further increases the biological activity for instance antibacterial and antifungal activities. Amino acids especially, a tryptophan derivative with high antimicrobial activity and low toxicity makes the molecules highly effective as novel candidates for both topical and systemic treatment of bacterial infections [26]. βcarboline derivative (7d) with a hydroxyl group on the phenyl ring exhibited promising antibacterial and antifungal activities. Compounds bearing two -OH groups (7g) and compound with -OH, -OCH3 (7h) were exhibited good antibacterial activity and also compound bearing -OCH3 7f showed good antifungal activity. Remaining β-carboline derivatives 7a, 7b, 7c and 7e bearing H, -F, -NO2 and -CN were did not showed considerable activity. The above discussion conclude that the β-carboline derivatives containing electron donating groups like -OH, -OCH3 showed superior activity against antibacterial and antifungal screening compared than those with electron withdrawing groups such as -F, -NO2 and -CN. Whereas, compound 7i did not showed activity even though consisting two -OCH3 groups.

Cytotoxic activity
Fatty alcohol conjugated β-carboline moiety was expected to show cytotoxic activity, βcarboline derivatives were further screened for their anticancer activity [27]. The compounds 7a to 7i including doxorubicin as positive control were screened against five cell lines and the results obtained were presented in

Antioxidant activity
The anti-oxidant activity of the synthesized β-carboline derivatives were screened by the wellestablished DPPH radical scavenging assay. The DPPH is widely using stable free radical to estimate the free radical scavenging capability of the compounds. The results obtained from the test were presented in table 6 and the obtained results were compared with the reference antioxidants α-tocopherol (α-TP) and tert-butylhydroquinone (TBHQ). Among all the tested βcarboline derivatives, the compounds 7d, 7g and 7h exhibited antioxidant activity. Specifically, compound 7g exhibited excellent free radical scavenging activity (FRSA) of 85%, which is nearly close to control α-TP. Compounds 7d and 7h exhibited good free radical scavenging activity (FRSA) of 79 and 82%, respectively. From the above results compounds with phenolic hydroxyl functional groups were active in DPPH assay. This could be due to the radical scavenging efficiency of phenolic hydroxyl moieties. The superior FRSA of compound 7g is due to the presence of more than one phenolic hydroxyl moiety [28]. 0.8 ± 0.15 0.7 ± 0.14 0.7 ± 0.16 0.8 ± 0.14 0.8 ± 0.12 Table 6: DPPH radical scavenging activity of β-carboline derivatives 7 (a-i).

Conclusion
In the present study, β-carboline hybrids were synthesized by joining 10-undecenyl alcohol chain and evaluated their biological activities.