Synthesis and immunomodulatory activity of novel amino acid analogues of fluoxetine

Abstract Immunomodulators are assigned as a robust tool for the treatment of various medical conditions like AIDS, cancer and autoimmune diseases. In this work, we have synthesized a series of fluoxetine-based small-molecule immunomodulators. Molecules were synthesized by a convergent strategy, where mitsunobu reaction was conducted to synthesize the common starting material. The novel analogues displayed immunomodulatory potential as indicated by the anti-inflammatory effect derived through the compound’s T cell proliferation inhibitory property. The synthesized novel analogues manifested encouraging biological responses, assure their application as immunomodulators. GRAPHICAL ABSTRACT


Introduction
Eli-Lily and company in 1976 have discovered originally Fluoxetine hydrochloride (Prozac V R ,(N-methyl-3-phenyl-3-[4-(trifuoromethyl) phenoxy] propan-1-amine hydrochoride) an selective serotonin-reuptake inhibitor (SSRI), which was later approved by the FDA in 1987 for therapeutic management of depression. [1] It augments the serotoninergic tone by enhancing the concentration of the neurotransmitter in the synaptic cleft through restraining the serotonin transporter. The clinically used drug is a racemic mixture. From a stereochemical point of view, it is, as individual enantiomer show a moderate difference in terms of their serotonin reuptake inhibitory activity. Being a pharmacologically active entity on a wide spectrum of mood disorders fluoxetine has been a clinically accepted drug all over the world for the treatment of major depression, [2] further research on fluoxetine indicated the possible protection aligned with the adverse effects of various types of stress factors of immune system, oxidative damage through a permutation of mechanisms. This is supposed to be a key role player in neuroprotection, since brain being a high energy demanding organ susceptible to oxidative stress. Abnormally increase in generation of reactive oxygen species (ROS) results in oxidative stress which might have implications in the pathogenesis of many neuro degenerative and psychiatric and disorders. Antidepressant therapy has been inseparably linked with immune disorder. Investigational research findings specify that lymphocytes express the serotonin transporter and ability of fluoxetine to alter the immune functioning through a serotonin-dependent pathway and through a novel independent ways. Research shows that fluoxetine and thapsigargin deplete the intracellular Ca 2þ levels thereby causing a stress on the endoplasmic reticulum, thereby altering the T cell immune response. [3,4] In addition, fluoxetine has ability to modify the tumor cell viability as confirmed through in vivo experiments that tumor growth is inhibited through chronic fluoxetine treatment by enhancing the antitumor T-cell activity, indirectly by antagonizing the inhibition of Ca 2þ -ATPase (sarco-endoplasmic reticulum Ca 2þ -ATPase) in tumor cells. [5,6] Thus, there has been ample evidence in literature regarding the ability of fluoxetine to amend the functionality of the immune system. These research findings point to the significance of the novel pharmacological action of fluoxetine and its analogues as immunomodulator aiding to treat several disorders related with immune deficiency and it's deregulation.
Furthermore, Fluoxetine displayed potent inhibitory effect on enterovirus replication as revealed in drug repurposing screens in mice and in addition to this, fluoxetine has been used to successfully treat immunocompromised children suffering from severe and lethal enterovirus encephalitis. [7] Together it is evident that structural modification of fluoxetine offers a potential strategy for evolving effective immunomodulators and antiviral therapeutics for clinical use. Here in, we report a preliminary investigation of a series of fluoxetine analogues, in which we introduce amino acids on the N terminus of the original scaffold, to gain insight into the structure-activity relationships of fluoxetine vis-a-vis the antiviral and immunomodulatory potentials. Previously reports pointed that the structural features of the trifluoro-phenoxy moiety and the amino moiety are essential for the antiviral potency whereas the 3-phenyl moiety seems expendable. The para-trifluoro-phenoxy moiety is critical for the SSRI activity as changes of the substituent lowers the affinity toward the serotonin transporter (SERT) as shown in Figure 1. Hence, fluoxetine analogues with alterations on the CF 3 -substituent positions on the phenoxy ring were synthesized. Rather the second moiety of choice was the methylamine group. The wellestablished pan-enterovirus inhibitor guanidine hydrochloride (GuaHCl) has been shown to target AAAþ ATPase protein 2C. [8] We designed fluoxetine analogues substituting the methylamine group with various Alpha aminoacids, to explore the need of modification across the basic nitrogen. The newly synthesized compounds have been characterized spectroscopically and tested for their immunomodulatory potential.
Immunomodulators belong to the class of medicine able to modify the immune responses either by escalating or suppressing the synthesis of serum antibodies and are classified as an immunostimulant or immunosuppressant respectively. [9] Immunostimulants are utilized to trigger immune responses against existing infections, cancer, and in other immuno-deficient conditions, while immunosuppressants are prescribed for graft rejection and autoimmune disease. [10][11][12] There is significant evidence demonstrating the unbroken communication between the brain and the immune system. [13] Hence, findings on the anti-inflammatory action of antidepressants are of attention in the field of immunotherapy. [14] Fluoxetine, first launched by Eli Lilly and company in 1988 with the brand name Prozac, [15] is a selective serotonin reuptake inhibitor (SSRI), often utilized to treat obsessive-compulsive disorder, panic disorder and depression because of its tolerability and safety. [16][17][18] Clinically fluoxetine is utilized in a racemic form. [19] It is postulated that this first-line anti-depressant drug can decrease the level of pro-inflammatory cytokines. [20] Available works of literature demonstrate the anti-inflammatory characteristic of fluoxetine. Investigations revealed that fluoxetine noticeably reduces the IFN c/IL-10, [21] suppresses TNF-a release, [22] and increases the formation of anti-inflammatory cytokine IL-10. [23] Literature clearly indicates a remarkable increment in immunological diseases thus great attention is required for the development of immunomodulators. There is a huge worldwide demand of efficient immunomodulatory/anti-inflammatory drugs for the treatment of viral infections. 24 , 25 In this endeavor, herein we have demonstrated the synthesis and immunological evaluation of designer focused library of novel fluoxetine analogues bearing different alpha amino acids.

Results and discussion
We started preparing novel amino acid analogues of fluoxetine following convergent synthesis strategy. In one hand, esters of amino acid were synthesized as per the general procedure mentioned as in Figure 2. Thionyl chloride was the reagent of choice to convert amino acid into corresponding acid chloride. Methanol and ethanol were used as the solvents as well as reactant to produced methyl ester 2(a-q) and ethyl esters 2(r-s), respectively. Reactions were performed at 0 C to room temperature. On the other hand, synthesis of 4-substituted 1-(3-chloro-1-phenylpropoxy)-4-benzene 5(a-c) were achieved by using well-known name reaction; Mitsunobu Reaction. Initially diisopropyl azodicarboxylate (DIAD) (17.58 mmol) and triphenylphosphine (17.58 mmol) were reacted together in THF, under nitrogen atmosphere followed by addition of substituted phenol 4(a-c) into the reaction pot and stirred for 4 hours. A solution of 3-chloro-1phenylpropan-1-ol 3 in THF was poured into the reaction pot and further stirred for 16 hours. Purification of crude obtained from reaction mixture afford pure product 5(ac) as oil, in moderate to good yield. Both the fragments; esters 2(a-s) and 1-(3-chloro-1-phenylpropoxy)-4-benzene 5(a-c) were couple together by N-alkylation reaction, where compound 5(a-c) (0.32 mmol.) was treated with sodium iodide (0.63 mmol.) in DMF for 30 minutes. In a separate flask, esters (0.79 mmol.) 2(a-s) were treated with diisopropylethylamine (DiPEA) (0.95 mmol.) in DMF and stirred for 30 minute at room temperature. Mixture of 2(a-s) and diisopropylethylamine (DiPEA) in DMF, was transferred into the solution of 5 (a-c) and sodium iodide at room temperature. This reaction mixture was refluxed at 130 C for 5 hours. Crude obtained from reaction mixture was subjected for column chromatography purification to afford compound 6(a-u) in good yield. Fluoxetine was prepared as per the protocols mentioned, where 1-(3-chloro-1-phenylpropoxy)-4-(trifluoromethyl) benzene (0.32 mmol.) reacted with excess of aqueous MeNH 2 (aqueous 40%) in ethanol under sealed tube. All the synthesized analogues and standard molecule (Table 1) were structurally confirmed by 1 H-NMR, 13 C-NMR, 19 F-NMR, IR spectroscopy and HRMS spectrometry techniques and subjected for biological evaluation.
The fluoxetine analogues were preliminarily screened for T cell or B cell proliferation using splenocytes. Cells were treated with different concentration of compounds along with Con A and LPS to estimate the type of cell proliferation. From the data obtained we infer that most of the analogues elicited an LPS mediated B cell proliferation and no cytotoxicity of the analogues was observed in any of the analogues. This goes in parity with the parent fluoxetine property to inhibit T cell responses. [26] After confirming the non-cytotoxicity (Supplementary Fig. 1) we now evaluate the immunomodulatory potency of the novel analogues using quantification of pro and anti-inflammatory cytokines IL-6, TNF-a and IL-10 from stimulated macrophages. From the results ( Table 2) we observe that all the analogues have elicited a very sub optimal IL-6 and TNF-a pro inflammatory cytokines in comparison to positive control LPS but a significant enhancement in the IL-10 cytokine was observed. From the above preliminary data generated we can conclude that the analogues 6a, 6b, 6c, 6d, 6g, 6i, 6l, 6m, 6o, and 6q had a significant IL-10 response and these compounds were carried forward to evaluate their ability to inhibit or proliferate T cell population. Mouse T cells were isolated from the spleens and cultured using RPMI 1640 media. The cells were plated at an density of 2 Ã 10 5/well in 24 well plate and treated with the down selected analogues at a optimal 10 mg/mL concentration along with standard Con A. After 48 hrs proliferation was read using flowcytometer and from the data in Figure 3 we can conclude that compounds 6c, 6g, 6i, 6o, and 6q had significant inhibition of T cell proliferation as that of standard fluoxetine.  From all the above data we can infer that the novel analogues have elicited a significant IL-10 response-anti-inflammatory cytokine and were successful in suppressing the T cell proliferation.

Experimental section
Chemistry General for synthetic experiments All commercially available reagents and chemicals were purchased from sigma Aldrich and used without further purification. All organic solvents were dried and freshly distilled before use wherever necessary, by using available standard protocols. All the reaction progress was monitored by using Thin Layer Chromatography (TLC) Merck Silica Gel 60 F254 plates (0.25 mm). TLC visualization was done under UV lamp. Staining was developed with Iodine or by dipping TLC in Ninhydrin solution in ethanol or 10% phosphomolybdic acid solution in ethanol and then charred on a hot plate. Column chromatography was carried out by using silica gel (Merck 60-120 mesh) with technical grade solvent. FT-IR was recorded as neat from Bruker FTIR Alpha spectrometer, wave numbers in cm À1 . NMR spectra were recorded with a Bruker AC-300 MHz, a Bruker AM-400MHz and a Bruker AMX-500MHz spectrometer instruments in CDCl 3 with (CH 3 ) 4 Si is an internal standard for 1 H NMR spectra and solvent signals as internal standard for 13 C NMR spectra at ambient temperature. Chemical shifts (d) are reported in ppm. Coupling constants J were reported in Hz. NMR signal splitting patterns were designated as follows: s-singlet, d-doublet, dd-doublet of doublet, dt-doublet of triplet, td-triplet of doublet, t-triplet, q-quartet, p-pentet, bs-broad singlet, m-multiplet, brbroad. For ESI-MS, m/z values are reported in atomic mass units and recorded in Shimadzu instrument.
General procedure for the synthesis of fluoxetine analogues In a double neck round bottom flask, 5 (a-c) (0.32 mmol.) and sodium iodide (0.63 mmol.) were taken, dissolved in DMF and stirred for 30 minute at room temperature. In a separate flask, aliphatic ester of amino acid (0.79 mmol.) and diisopropylethylamine (DiPEA) (0.95 mmol.) were dissolved in DMF and stirred for 30 minute at room  temperature. This mixture was poured into the solution of 5 (a-c) and sodium iodide at room temperature. This reaction mixture was refluxed at 130 C for 5 hours. Reaction progress was monitored on TLC plate. After completion of reaction, reaction mixture was cool down at room temperature, quenched with ice cold water and extracted with ethyl acetate (2 Â 25 mL). Organic layers were combined, washed with brine solution, dried with anhydrous sodium sulfate and evaporated under reduced pressure to obtained crude product. The crude was purified by column chromatography to give final product. [27] Methyl (2S)-2-phenyl-2-((3-phenyl-3-(4-(trifluoromethyl)phenoxy)propyl)amino)acetate (6a) Pale yellow oil. 120 mg, 85% yield. IR (

Experimental section
Biology Proliferation Proliferation of the novel analogues was assessed using splenocytes from mice. 1 Â 10 5 cells/well were seeded in a 96-well plate with RPMI 1640 medium and were treated with novel fluoxetine analogues in combination with Con A (2 mg/mL) and LPS (10 mg/mL) and incubated for 48 hours. Cells were added with 20 mL/well (5 mg/mL) of 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) reagent and further incubated for 2 hours. Untransformed MTT was removed by aspirating the supernatant and DMSO was added. Plate was read at 630 nm after 15 min. Proliferation of the analogues was calculated considering cell controls as 100% viability. [28] Peritoneal macrophage isolation Dweller macrophages were accessed by peritoneal bath of crude mice. 2% starch solution was prepared by adding starch to the sterile Millipore water and making it clear solution by sonication. The starch solution was stored for 2-3 weeks at room temperature and then was used at same concentration. 2 mL of 2% starch injection was given to mice at intra peritoneal site. After 4-5 days of immunization, the mice were sacrificed by CO 2 asphyxiation and 5 mL PBS injection was given to same site. After 5 min the fluid was collected from peritoneal site either through syringe or dropper. The suspension was centrifuged at 2000 rpm, 621 g for 10 min at RT. The pellets so collected were cultured in high glucose DMEM and stored at 37 C. [29] The macrophages were treated with the analogues at four different concentrations (1000-1 mg/mL) in tenfold dilution and pro inflammatory cytokines were estimated.
Cytokine estimation-sandwich ELISA Quantification of proinflammatory cytokines (TNF-a and IL-6) from cell supernatant of splenocytes was carried out by Opti Elisa Kit (BD) according to manufacturer's protocol. The supernatant was collected from treated well separately for cytokine estimation by coating ELISA plates with purified anti-mouse antibody diluted with bicarbonatecarbonate buffer and incubated at 4 C overnight. Plates were washed thrice with PBST 20 and blocked with 100 mL of 1% BSA. After 1 hour incubation at RT, plates were again washed thrice with same washing buffer. 100 mL of cell supernatant sample was added along with serially diluted recombinant antibody. Plates were incubated for 3 hours at 37 C. Post incubation was followed by four times washing and then addition of detection antibody prepared in BSA with 1 hour incubation at RT. After four times washing, HRP streptavidin (1:3000) in 1% BSA was added 100 mL each and kept for incubation for 30 min at RT. TMB substrate (TMBA:TMBB; 1:1) was added after thrice washing and kept for incubation till development of color or for 15 min. The reaction was stopped by adding 50 mL of 2 N H 2 SO 4 and the absorbance was measured at 450 nm in ELISA reader (Tecan, Infinite pro). [30] T cell isolation The Mouse T Cell Isolation Kit, is a fast enrichment kit for untouched isolation of all T cell populations. Isolation assay was performed as per the manufacturer's instructions. Briefly, cells were prepared (1 Â 10 8 cells/mL) in the range of 0.1-2.0 mL and sample was added to 5.0 mL tubes. Enrichment cocktail (50 mL/mL) was added to the cells, which was mixed well and incubated at RT for 10 min. The cells were then washed with the recommended medium and centrifuged at 300 g for 10 min. Supernatant was discarded and was resuspended in the original volume of 0.1-2.0 mL with recommended medium. This was followed by adding the Rapid spheres (75 mL/mL) to the cells, mixed well and was incubated at RT for 5 min. Recommended medium was added to the sample, mixed gently by pipetting up and down. The tubes, without lid was inserted into the magnet and incubated at RT for 5 min. The magnet was lifted and at once inverted the magnet with tube, pouring the entire enriched cell suspension into a new tube. The above mentioned step was repeated one more time and the isolated cells were ready for use. [31] CFSE staining Cells were liquated in to 15 mL centrifuge tubes and added with 5 mM staining solution each and incubated for 20 minutes in at 37 C. After incubation cells were centrifuged 5 minutes at 300 Â g and resuspended the cell pellet in pre-warmed RPMI 1640 media. The cells were treated with 10 mg of compounds and incubated for 48 hr. The proliferation of cells was quantified using FACSVerse. % proliferation was calculated considering the un stimulated cells as 100% proliferation. [32]

Conclusion
In conclusion, we have constructed a library of novel amino acid analogues of fluoxetine and confirmed their structures by using various spectroscopic techniques like 1 H-NMR, 13 C-NMR, 19 F-NMR, IR spectroscopy and HRMS spectrometry. The in-vitro immunomodulatory activity of these analogues was carried out using peritoneal macrophages and isolated T cells. From the results obtained, we conclude that the novel fluoxetine derivatives have elicited a significant level of anti-inflammatory IL-10 response and were successful in suppressing the T cell proliferation similar to parent molecule fluoxetine. These novel analogues can find a potential application in the fields of inflammatory diseases where potential immunomodulators are required to suppress the inflammatory responses and also in auto immune disorders where the T cell activity can be marginalized to control the self-generated responses.
Supplementary data associated with this article can be found as a separate file.