WO2023187830A1 - Piperic acid-conjugate with homochiral and heterochiral dipeptides containing phenylalanine and process for preparation thereof - Google Patents

Piperic acid-conjugate with homochiral and heterochiral dipeptides containing phenylalanine and process for preparation thereof Download PDF

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WO2023187830A1
WO2023187830A1 PCT/IN2023/050304 IN2023050304W WO2023187830A1 WO 2023187830 A1 WO2023187830 A1 WO 2023187830A1 IN 2023050304 W IN2023050304 W IN 2023050304W WO 2023187830 A1 WO2023187830 A1 WO 2023187830A1
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conjugate
group
cells
skin cancer
piperic acid
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PCT/IN2023/050304
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French (fr)
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Rajkishor RAI
Anindya GOSWAMI
Junaid RAHIM UR
Mir Mohd FAHEEM
Shah NAWAZ
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Council Of Scientific & Industrial Research
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent

Definitions

  • the present invention relates to conjugates of piperic acid and small peptides and its application in skin cancer.
  • This invention particularly relates to piperic acid dipeptide conjugate containing L Phe and D Phe in the first part, its synthesis and composition comprising the same.
  • the present invention also explores the biological activity of these conjugates.
  • Piperic acid (PA) and 4-ethyl piperic acid (EPA) conjugates with non-protein amino acids have been reported as potent anti -metastatic agents in Pancreatic Ductal Adenocarcinoma (PDAC) cells via the abrogation of MMP-9 expression (Wani et al., 2016);(Amin et al., 2015). Recently, cyclodipeptide conjugates with piperic acid as cytotoxic agents against metastatic breast and prostate cancers have been investigated (Shankar et al., 2018).
  • W02002/057260A1 has described the piperic acid amides as anticancer agents against skin cancer with mild activity.
  • Several therapeutically relevant applications are being uncovered for peptide-conjugate systems, which drive intense research activities in this emerging field. Rationally, our efforts focus on the synthesis of piperic acid conjugates with homochiral and heterochiral dipeptides containing phenylalanine and their application in skin cancer.
  • FC-3 Piperic acid- D Phe- D Phe (FC-3) conjugate is identified as a potential lead against skin cancer (melanoma) in vitro by delineating the anti-proliferative and anti- migratory potential together with its anti-inflammatory potential against a range of pro- inflammatory interleukins (IL-ip, IL-6, and IL-8).
  • IL-ip pro-inflammatory interleukins
  • IL-6 pro-inflammatory interleukins
  • IL-8 pro-inflammatory interleukins
  • the main objective of the invention is to provide piperic acid and small peptides conjugates.
  • Another objective of the invention is to synthesize the said conjugates.
  • Yet another objective of the invention is to characterize and study the self-assembling properties, and in vitro cytotoxic screening of piperic acid-peptide conjugates.
  • Yet another objective of the invention is to evaluate the anti-proliferative, anti-migratory, and anti-inflammatory properties of the piperic acid dipeptide conjugates.
  • Yet another objective of the invention is to conduct In vivo validation of lead conjugate in the mouse two-step chemically induced skin cancer model.
  • the present invention provides piperic acid-peptide conjugate of general Formula I, hydrogen and aryl;
  • R5 is selected from the groups consisting of OH, O-alkyl, HN-CH2PI1, HN- CH 2 CH 2 -Ph, and HN-CH2CF3.
  • Ri, R2, R3 and R4 in the piperic acid-peptide conjugate of general Formula I are selected from group consisting of
  • R5 in the piperic acid-peptide conjugate of general Formula I are selected from group consisting of
  • the compound is selected from the group consisting
  • the present invention provides a process for the preparation of piperic acid-peptide conjugate of general Formula I, comrising the steps of; i. coupling of amino acids, Boc-AAi and H-AA2-0Me (1:1) ratiousing EDC.HC1, HOBt in the presence of NMM followed by deprotection of Boc group with 30% TFA in dry CH2CI2 at 0°C to obtain dipeptide H-AAi-AA2-0Me;
  • the present invention provides a pharmaceutical composition
  • the pharmaceutically acceptable excipient are selected from the group consisting of diethylene glycol, cycloaliphatic and/or aromatic-aliphatic alcohols and pyrrolidones/polyol ethers/polyols.
  • the conjugate and the pharmaceutical composition thereof abrogates skin cancer cells proliferation, attenuates the levels of pro-inflammatory interleukins and decreases tumor growth.
  • the compound of Formula I effectively abrogates skin cancer cells proliferation and migration.
  • the compound of Formula I strongly attenuates the levels of pro-inflammatory interleukins.
  • the compound of Formula I decreases the cumulative number of tumors and results in attenuation of tumor growth.
  • Fig. 1 displays post gelation SEM images of FC-3 and FC-4
  • Fig. 2 represents the Circular Dichroism (CD) curve of peptides FC-l-FC-4.
  • Fig. 3 shows the cytotoxic potential of piperic acid-peptide conjugates, FC-1-FC4, as determined by MTT assay against various melanoma cell lines (human and murine) and normal skin cells.
  • Fig. 4 is a diagram reflecting on the anti -proliferative and anti-migratory properties of conjugate FC-3.
  • Fig. 5 illustrates the abrogation of Stat-3 expression and nuclear accumulation by Conjugate FC-3.
  • Fig. 6 is a diagram representing the down-modulation of pro-inflammatory interleukins (IL- 1 P, IL-6, and IL-8) in the conditional media collected from FC-3 treated cells.
  • pro-inflammatory interleukins IL- 1 P, IL-6, and IL-8
  • Fig. 7 depicts the in vivo efficacy of Conjugate FC-3 against the two-step carcinogen-induced skin cancer model.
  • Fig. 8 is a diagram representing the down-modulation of pro-inflammatory interleukins (IL- 1 P, IL-6, and IL-8) in the serum collected from FC-3 treated mice cohorts.
  • Fig. 9 describes preparation of piperic acid-peptide conjugate of general Formula I
  • the present invention describes the synthesis, characterization, self-assembling properties, and in vitro cytotoxic screening of piperic acid-peptide conjugates.
  • Piperic acid- D Phe- D Phe (FC-3) conjugate is identified as a potential lead against skin cancer (melanoma) in vitro by delineating the anti-proliferative and anti -migratory potential together with its anti-inflammatory potential against a range of pro-inflammatory interleukins (IL-ip, IL-6, and IL-8).
  • IL-ip pro-inflammatory interleukins
  • IL-6 pro-inflammatory interleukins
  • IL-8 pro-inflammatory interleukins
  • PA Refers to piperic acid
  • EDC.HC1 l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • Conjugates are compounds formed by the linking of two or more chemical compounds.
  • Peptides are short chains of amino acids connected through amide bonds or peptide bonds.
  • Self-assembly Spontaneous association of individual molecules in a well-organized manner.
  • Skin cancer refers to the uncontrolled and abnormal growth of skin cells, most often caused due to UV irradiation or exposure to chemical carcinogens. There are two main types of skin cancer: melanoma and non-melanoma
  • Melanoma refers to a form of skin cancer that begins in melanocytes (cells that control the pigment in our skin).
  • cytotoxic refers to the property of specific molecules being toxic to cells.
  • ICso half maximal inhibitory concentration
  • anti-proliferative, anti-migratory, and anti-inflammatory refer to the inhibition in cell growth, migration, and inflammation of cells, respectively, in response to specific molecules.
  • wound healing refers to the capacity of cancer cells to replenish any wound created by external agents by activating cellular migration.
  • Interleukins are a group of cytokines (secreted proteins and signal molecules) that perform a myriad of cellular functions like proliferation, inflammation, chemotaxis, angiogenesis, etc.
  • Angiogenesis is the formation of new blood vessels. This process involves the migration, growth, and differentiation of endothelial cells, which line the inside wall of blood vessels.
  • metastasis used herein refers to the ability of cancer cells to migrate from their primary niches and colonize distant sites resulting in secondary tumors.
  • the two-step chemically induced skin cancer model is an experimental inflammatory cancer model.Tumor formation is induced in this model by the topical application of two different chemicals, 7,12-dimethylbenz[a]anthracene (DMBA) and 12- O-tetradecanoyl phorbol-13- acetate (TPA), that together cause papilloma formation in the skin.
  • DMBA 7,12-dimethylbenz[a]anthracene
  • TPA 12- O-tetradecanoyl phorbol-13- acetate
  • Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
  • the present invention provides piperic acid-peptide conjugate of general Formula I, wherein n is 0-2; Ri, R2, R3, and R4 are independently selected from the group consisting of hydrogen and aryl; R5 is selected from the groups consisting of OH, O-alkyl, HN-CH2PI1, HN- CH2-CH2-PI1, and HN-CH2CF3.
  • This invention demonstrates the synthesis and characterization of conjugates of piperic acid with dipeptides containing L Phe and D Phe in the first part.
  • the piperic acid-peptide conjugate of general Formula I is prepared according to Figure 9. wherein n is 0-2; Ri, R2, R3, R4 and R5 are as defined above.
  • the process comprises the steps of: i. coupling of amino acids, Boc-AAl and H-AA2-0Me, using EDC.HC1, HOBt in the presence of NMM followed by deprotection of Boc group with 30% TFA in CH2C12 at 00C to obtain dipeptide H-AAl-AA2-0Me; ii. coupling of piperic acid with dipeptide H-AAl-AA2-0Me obtained in step-i using EDC.HC1, HOBt in presence of NMM followed by the saponification using 2N-NaOH/MeOH (1: 1) at room temperature.
  • the present invention also describes their application in skin cancer as anti-proliferative, anti- migratory and anti-inflammatory agents.
  • conjugate FC-3 exhibited promising cytotoxic activity against melanoma cell lines and potent anti -proliferative and anti-migratory properties.
  • FC-3 significantly attenuated the levels of pro-inflammatory interleukins (IL-ip, IL-6, and IL-8) in the conditional media of melanoma cell lines.
  • Conjugate FC-3 effectively abolished the expression and nuclear translocation of Stat-3 (a critical downstream effector of interleukins) in a dose and time-dependent manner in vitro.
  • FC-l-FC-4 The synthesized peptide conjugates, FC-l-FC-4, were characterized by HRMS/HNMR spectroscopy. All the peptide conjugates were subjected to selfassembly in DMSO/buffer solutions. Among all, the conjugates FC-3 and FC-4 formed the hydrogels. Descriptive Figure Legends
  • Fig 1 Post-gelation (lOOOx) Scanning electronic microscopy (SEM) pictographs of (A) FC-3 (B) FC-4
  • Fig 2 Circular Dichroism (CD) of peptides FC-l-FC-4 in MeOH.
  • FC-land FC-4 showing a positive cotton effect
  • FC-2 and FC-3 showing a negative cotton effect.
  • Fig3 Bar graphs depicting the percent cell viability of A375 and B16F10 cells upon treatment with FC series of conjugates at 0.1, 1, 10 and 100 pM concentrations.
  • Fig 4 (A) Colony formation assay in A375 cells treated with either vehicle or FC-3 (1,2,3 and 5pM). Camptothecin (1 pM) was used as a positive control. (B) Corresponding bar graph of colony formation assay depicting the average number of colonies/field. (C) Wound healing assay depicting pictographs at successive time points of 0 h and 48 h after treatment with either vehicle or FC-3 (1, 2 and 3 pM). (D) Corresponding bar graph of wound healing assay illustrating the % wound closure of A375 cells treated with either vehicle or FC-3. The data represent three independent experiments performed separately.Statistical analysis -*P ⁇ 0.05. **P ⁇ 0.01, ***P ⁇ 0.001.
  • Fig 5 (A) Dose-dependent inhibition of Stat-3 by FC-3 in A375 cells at 48 h by western blotting. Corresponding bar representing the relative expression of Stat-3 expression in a dosedependent fashion. (B) Time-dependent inhibition of Stat-3 by FC-3 (5 pM) in A375 cells by western blotting. P- Actin expression was considered to ensure equal loading. Corresponding bar representing the relative expression of Stat-3 expression in a time-dependent fashion. (C) Fractionation study as determined by western blot analysis to evaluate the nuclear translocation of Stat-3 upon treatment with FC-3 (5 pM, 48 h). P-Actin was used as the cytosolic loading control and Histone 3 was used as the nuclear loading control.
  • Fig 6 Bar graphs depicting the CM levels (pg/ml of CM) of pro-inflammatory interleukins (IL-ip, IL-6 and IL-8) as determined by specific ELISA assays in the vehicle (DMSO) or FC-3 (3 and 5 pM) treated A375 cells at 24 h. LPS (IpM) was used as positive control 4 h prior to the treatment for priming the cells.
  • Fig 7 (A) Schematic representation of the timeline of the two-step (DMBA/TPA) skin cancer model indicating the various time -points for tumor initiation, tumor promotion, data acquisition and sampling.
  • (B) Graph representing the cumulative number of tumors per cohort (vehicle, FC- 3 10 mg/kg b.wt. and FC-3 30mg/kg b.wt.) over the course of multiple weeks (0-24 weeks).
  • Fig 8 (A-B) Graphs depicting the serum levels (pg/ml of serum) of pro-inflammatory interleukins (IL-ip and IL-6) as determined by specific ELISA assays in the vehicle or FC-3 (10 and 30 mg/kg b.wt.) treated groups at 8, 16 and 24 weeks.
  • Example 1 Synthesis of PA- D Phe- L Phe-OH, FC-1 Boc- D Phe-OH (3mmol) dissolved in CH2CI2, was added with NMM (9mmol), EDC.HC1 (3.6mmol), HOBt (3 mmol) and H- L Phe-OMe (3.6 mmol) at 0°C. The reaction mixture was allowed to attain room temperature and stirred for 12 hours. After completion of the reaction, the solvent was evaporated, and the residue was extracted with ethyl acetate (3x15ml). The ethyl acetate layers were combined and washed with 2N-HC1 (1x20 ml), 2M-NaHCOs (1x20 ml), and brine solution (1x20 ml).
  • PA- L Phe- D Phe-OH (FC-2): 1 H NMR (400 MHz, DMSO- d 6 ) 5 8.44 (b, 1H), 8.09 (b, 1H), 7.43 - 7.06 (m, 13H), 7.05 - 6.79 (m, 3H), 6.26 - 6.03 (m, 3H), 4.65 (s, 1H), 4.38 (s, 1H), 3.13 (b, 2H), 2.89 (b, 2H).HRESIMS m/z 513.2013[M+H] + (calcd.for C30H28N2O6 512. 19).
  • IC50 Cytotoxicity (IC50) of all synthesized conjugates of the invention against various skin cancer cell lines.
  • IC50 (pM) values are calculated as mean ⁇ standard deviation based on three independent experiments.
  • FC-3 was the most potent conjugate against melanoma cell lines.
  • the IC50 values of 5.6 ⁇ 0.95 pM and 8.4 ⁇ 1.3 pM were observed in A375 and B16F10 cells, respectively.
  • Conjugate FC-3 has an IC50 value of 31.3 ⁇ 2.7 pM, indicating less toxicity against normal cells, thus signifying a higher therapeutic index.
  • Conjugate FC-3 has higher cytotoxic potential against metastatic melanoma cell line-A375 than less aggressive melanoma cells.
  • Conjugates FC-1, FC-2, and FC-4 showed IC50 values of 49.2 ⁇ 4.3, 21.0 ⁇ 2.7, 43 ⁇ 3.9 pM against A375 cells, and 47.4 ⁇ 3.8, 19.6 ⁇ 2.4 and 39.0 ⁇ 3.2 pM against B16F10 cells, respectively.
  • Cell viability assay The cell viability was determined by the standard MTT assay method (Riss et al., 2016). Briefly, A37F (human), B16F10 (murine), and primary murine keratinocyte cells were seeded in 96 well tissue culture plates (Nunc) at a density of 4 x 10 3 cells per well and treated with varying concentrations of the test compounds in triplicates. DMSO was employed as a vehicle, and its final concentration was maintained at 0.2 % in the culture medium. Doxorubicin and camptothecin were used as positive controls. After 44 h of incubation, MTT dye solution was added to the medium, and cells were incubated for another 4 h at 37 °C in 5 % CO2.
  • the amount of colored formazan derivatives formed was measured by taking optical density (OD) using a microplate reader (TECAN, Infinite M200 Pro) at 570 nm, and the percentage of cell viability was determined.
  • IC50 values were calculated using GraphPad Prism software (Version 5.1).
  • Camptothecin (IpM) was used as a positive control for the study. Interestingly, besides the reduction in the size of colonies, fewer number of cells per colony in the FC-3 treated cells were observed. In conclusion, our results indicate that FC-3 is a potent anti-proliferative agent against A375 melanoma cells.
  • FC-3 showed the most prominent anti -migratory properties at 2 and 3 pM concentrations in A375 cells.
  • IpM dose the percent wound closure was determined to be non-significant.
  • 2 and 3 pM doses of FC-3 showed 63.72 and 45.92% wound closure, respectively, indicating a profound reduction in the number of migratory A375 cells at these doses.
  • Cancer-related inflammation has been suggested as the seventh hallmark of cancer (Hanahan & Weinberg, 2011). Inflammation affects all the critical aspects of cancer, such as the proliferation and survival of cancer cells, tumor response to chemotherapeutic drugs and hormones, metastasis, and angiogenesis. Inflammation is associated with different stages of skin cancer development, including initiation, promotion, malignant conversion, invasion, and metastasis. Two pathways connect cancer and inflammation: the intrinsic and extrinsic pathways (Dep Prete et al., 2011). The intrinsic pathway is activated by genetic events. In contrast, the extrinsic pathway represents inflammatory leukocytes and soluble mediators, leading to conditions, which increase cancer risk.
  • transcription factors mainly nuclear factor kappa B (NF-KB), signal transducer and activator of transcription-3 (Stat-3) and hypoxia-inducible factor-1 alpha (HIF-la) in tumor cells (Kany, Vollrath, & Relja, 2019; Pedranzini, Leitch, & Bromberg, 2004).
  • NF-KB nuclear factor kappa B
  • Stat-3 signal transducer and activator of transcription-3
  • HIF-la hypoxia-inducible factor-1 alpha
  • These transcription factors coordinate the production of pro-inflammatory mediators, including cytokines (TNF-a, IL-6, IL-1), as well as the production of cyclooxygenase-2 (Cox-2) (Wojdasiewicz, Poniatowski, & Szukiewicz, 2014).
  • cytokines Pro-inflammatory cytokines, the most important mediators of inflammation, have distinguished roles in skin cancer development. Along with nitric oxide (NO), these cytokines act as cell-to- cell messengers and help in the activation of NF-KB and Stat-3. Rationally, it was sought to quantify the effect of FC-3 treatment on pro-inflammatory interleukins (a class of cytokines) viz: IL-ip, IL-6, and IL-8 and anti-inflammatory cytokine - IL- 10 in A375 cells.
  • CM Conditioned media
  • FC-3 was collected from the vehicle or FC-3 (3 and 5 pM) treated cells and subjected to specific ELISA analysis. Additionally, LPS (1 pM) was used as a positive control to drive the expression of interleukins.
  • FC-3 treatment was also evaluated.
  • 3 pM dose had an insignificant effect on IL-ip and IL-8 levels.
  • both 3 and 5 pM doses of FC-3 caused significant down-regulation of IL-ip, IL-6 and IL-10 levels compared to LPS alone primed cells.
  • Example 10 Quantification of IL-ip, IL-6, IL-8, and IL-10 by ELISA assay.
  • A375 cells were grown in DMEM media containing 10% FBS. 3xl0 5 cells were plated in 6 well plates for 24 h. Cells were primed with 1 pM of LPS (lipopolysaccharide) for 4 h, followed by treatment with either vehicle in the media already constituted with LPS for 24 h.
  • Conditional media in the form of supernatants were collected and analyzed for pro-inflammatory interleukins - IL-ip, IL- 6, and IL-8 and the anti-inflammatory interleukin - IL- 10 by ELISA using specific ELISA kits according to manufacturer’s instructions (R&D Systems; Cat. No. DLB50, D6050, D8000C, and D1000B). The absorbance was measured at 450 nm (Tecan Multi-mode Plate Reader), and optical density values were normalized with respective controls. All experiments were performed in triplicate.
  • the signal transducer and activator of transcription-3 belong to the Stat family of transcription factors.
  • Stats are proteins that are activated by extracellular signaling proteins, growth factors such as epidermal growth factor receptor (EGFR), cytokines (TNF-a, IL-ip, IL-6, IL-10 IL-17, IL-22), and various peptides (Aggarwal et al., 2009; Pedranzini et al., 2004).
  • Stat-3 regulates the expression of genes that mediate survival (survivin, BcLxl, myeloid cell leukemia sequence 1 [mcl-1], c-FLIP), proliferation (c-fos, c-myc, cyclin DI), invasion (MMP-2), and angiogenesis (VEGF) (Macias, Rao, & DiGiovanni, 2013; Messina et al., 2008).
  • BcLxl myeloid cell leukemia sequence 1 [mcl-1], c-FLIP
  • proliferation c-fos, c-myc, cyclin DI
  • invasion MMP-2
  • VEGF angiogenesis
  • Stat-3 In response to cytokines and growth factors, Stat-3 is phosphorylated by receptor-associated Janus kinases (JAK), form homo- or heterodimers, and translocate to the cell nucleus where they act as transcription activators.
  • JNK receptor-associated Janus kinases
  • Stat-3 mediates the expression of a variety of genes in response to cell stimuli, and thus plays a key role in many cellular processes, such as cell growth, inflammation, angiogenesis, and evasion from apoptosis (Xin et al., 2020).
  • A375 cells were seeded in 60 mm petri-dishes and maintained up to 70% confluency; afterward, the cells were treated with either vehicle or Conjugate FC-3 for indicated time-points and concentrations. After treatment, the cells were harvested, washed thrice with ice-cold PBS, and subjected to protein lysis with lysis buffer (HEPES 1 mM/L, KC1 60 mM/L, NP-40 0.3%, EDTA 1 mM/L, DTT 1 mM/L, sodium orthovanadate 1 mM/L, PMSF 0.1 mM/L and cocktail protease inhibitor).
  • lysis buffer HEPBS 1 mM/L, KC1 60 mM/L, NP-40 0.3%
  • EDTA 1 mM/L DTT 1 mM/L
  • sodium orthovanadate 1 mM/L sodium orthovanadate 1 mM/L
  • PMSF 0.1 mM/L and cocktail protease inhibitor
  • the lysis product was centrifuged at 12000 rpm for 45 min at 4°C to remove the cellular debris, and the supernatants were collected. Total protein was estimated with the help of the Bradford assay. An equal amount of protein (30 pg for Stat-3) was subjected to SDS-PAGE. The proteins were transferred onto the PVDF membrane (Millipore) by wet transfer. The membrane was blocked with 5% BSA and incubated in a primary antibody prepared in 5% BSA (dilution ranging from 1: 1000-1:2000) for 2-3 h.
  • the membrane was then washed with TBST buffer (137 mM NaCl, 2.7 mM KC1, 19 Mm Tris base, and 0.1 % Tween-20, pH 7.6) for 30 min (three times for 10 min each) and incubated with species-specific secondary antibodies (CST, Cat. No. 7074S, 7076S) tagged with horseradish peroxide.
  • CST species-specific secondary antibodies
  • the protein expression was analyzed by adding a chemiluminescent substrate (Millipore, Cat No. WBULUF0100) to the membrane.
  • the signal obtained was captured on x-ray films (ThermoFisher, Cat. No. 34090). Densitometry analyses of the captured bands on X-ray film were performed using ImageJ software(version Fiji) and Graph pad prism (version 5.1).
  • cytoplasmic and nuclear fractions for immunoblotting A375 cells treated with vehicle control (DMSO) or FC-3 (5.0 pM, 48 h) were washed with ice-cold PBS and centrifuged. All steps of fractionation were carried out at 4 °C. Cell pellets were homogenized in 200 pl of ice-cold hypotonic buffer (5 mM HEPES, pH 7.9, 10 mM KC1, 1 mM DTT, 0.5 mM PMSF, 1.5 mM MgCh, 1 mM NaVCM, 10% protease cocktail inhibitor, 0.02 volumes of 10% NP-40) with mild vortexing by keeping on ice for 30 min. The cell suspension was accordingly centrifuged at 10000 g for 1 min.
  • DMSO vehicle control
  • FC-3 5.0 pM, 48 h
  • the resulting cytosolic supernatants were stored at -80 °C.
  • the pellets obtained were washed in 100 pl of hypotonic buffer and resuspended in 100 pl of hypertonic buffer (5 mM HEPES, pH 7.9, 0.25% NP-40, 25% glycerol, 500 mM NaCl, 1.5 mM MgC12, 0.2 mM EDTA, 0.5 mM PMSF, 1 mM NaVC , and 10% protease cocktail inhibitor), incubated in the end to end rotor for 2 h at 4 °C following centrifugation at 10000 g for 30 min at 4 °C.
  • the supernatants obtained were stored at -80°C (Thermo Fischer) for analysis of nuclear protein.
  • the cells were incubated with corresponding primary antibodies for 1 h and accordingly washed three times with PBS. The cells were then incubated with Alexa Fluor 555 (red channel) conjugated secondary antibody for 1 h. Post incubation, the cells were washed, and mounted with an ultracruz mounting medium containing DAPI (blue channel). The prepared slides were then analyzed with EVOS FLoid Cell Imaging Station(Thermo Fisher Scientific) under 20 X magnification.
  • FC-3 treated groups (10 and 30 mg/kg b.wt.).
  • the reduction was more profound in the 30 mg/kg b.wt. FC-3 treated group when compared with the 10 mg/kg b.wt. FC-3 treated group.
  • the cumulative number of tumors/group was 34, 20 and 16 for the vehicle and FC-3 (10 and 30 mg/kg b.wt) treated groups, respectively.
  • the tumor volume of animals in the three groups at three different points (8 th , 16 th and 24 th week) was calculated.
  • mice Two-step chemically induced mouse skin cancer model. All the in vivo experimental protocols were approved by the Institutional Animal Ethics Committee, CPCSEA, of the Indian Institute of Integrative Medicine, Jammu. The animals were maintained at 22 °C with a 12 h light-dark cycle and free access to feed and water inside the institutional animal house. Proper care was taken to maintain them in a healthy condition and to avoid any risk of possible pathogenic contaminations. C57BL/6 sex-matched female mice were chosen for the study. The mice were 7-9 weeks of age because the skin in most mice is in telogen (the resting phase) around that age (Vahatupa et al., 2019). The mice were weighed twice a week for any potential weight loss, and the readings were recorded.
  • mice The back skin of the mice was shaved with a trimmer at the beginning of the study and bi-weekly during the entire course of the study, specifically two days prior to any chemical exposure.
  • DMBA and TPA both diluted in acetone
  • the working concentration of DMBA was 250 g/1.
  • a dose for one animal was 50 pg of DMBA in 200 pl of acetone.
  • DMBA was given topically for 4 weeks, twice a week, followed by topical TPA application (again twice a week for 4 weeks).
  • the stock concentration of TPA was 125 g/1, and the treatment concentration 25 g/1.
  • a TPA dose for one animal was 5 pg in 200 pl of acetone.
  • the papillomas started developing post 7 th week. At the end of the 8 th week, discrete papillomas were observed in 28 mice out of 30. A palpable mass greater than 1 mm in diameter was considered a papilloma and taken into account. Tumor dimension was measured with a digital vernier caliper (Safeseed, Cat. No. DIGICALPLBK001). Tumor volume was calculated by the formula [length x (width) 2 /2], where length was taken as the diameter of the largest side. The tumor-bearing animals were divided into three groups- vehicle, FC-3 (10 mg/kg b.wt. and 30 mg/kg b.wt.) in a randomized fashion. FC-3 (10 mg/kg b.wt.
  • mice were treated with acetone only.
  • the papillomas were counted, recorded, and photographed every week.
  • the animals were sacrificed at the end of the 24 th week, and tumors were isolated and stored for downstream applications.
  • FC-3 comprising about 0.01% to about 5%w/w FC-3 + diethylene glycol + cycloaliphatic and/or aromatic-aliphatic alcohols + pyrrolidones/polyol ethers/polyols could be constituted.
  • the present invention leads to the discovery of a novel potent piperic acid- D Phe- D Phe conjugate (FC-3) as an anticancer agent against diverse skin cancer cell lines.
  • Compound FC-3 has shown the tendency to form self-assembled hydrogels.
  • Conjugate FC-3 inhibits the proliferation and migration ability of human melanoma cancer cells in vitro.
  • FC-3 significantly attenuates the expression of pro-inflammatory cytokines- IL-ip, IL-6, and IL-8 in vitro.
  • Conjugate FC-3 significantly down-regulates the Stat-3 expression and impedes its nuclear translocation.
  • Stat-3 serves as a critical effector in the signaling of interleukins and other cytokines and is implicated in angiogenesis and metastasis of cancer.
  • Conjugate FC-3 is an effective inhibitor of tumor growth in vivo against the two-step chemically induced skin cancer model.
  • the present invention leads to the development of an anticancer drug-like molecule from the conjugation of homo- and heterochiral peptides containing phenylalanine with piperic acid that can potentially halt the proliferation, migration, and angiogenesis in skin cancer by impeding the interleukin/Stat-3 axis.

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Abstract

The present invention unleashes the development of novel conjugates of piperic acid and small peptides pertinent to its application in skin cancer. This invention demonstrates the synthesis and characterization of conjugates of piperic acid with dipeptides containing LPhe and DPhe in the first part and later explores the biological activity of these said conjugates. Piperic acid-DPhe- DPhe (FC-3) conjugate is thus identified as a potential lead against skin cancer (melanoma) in vitro by delineating the anti-proliferative and anti-migratory potential together with its anti- inflammatory potential against a range of pro-inflammatory interleukins (IL-1β, IL-6, and IL-8). The in vivo efficacy of FC-3 is validated in the two-step (DMBA/TPA) chemically induced mouse skin cancer model.

Description

PIPERIC ACID-CONJUGATE WITH HOMOCHIRAL AND HETEROCHIRAL DIPEPTIDES CONTAINING PHENYLALANINE AND PROCESS FOR PREPARATION THEREOF
FIELD OF THE INVENTION
The present invention relates to conjugates of piperic acid and small peptides and its application in skin cancer. This invention particularly relates to piperic acid dipeptide conjugate containing LPhe and DPhe in the first part, its synthesis and composition comprising the same. The present invention also explores the biological activity of these conjugates.
BACKGROUND OF THE INVENTION
Environmental factors are equally responsible for cancer as much as genetic predisposition (Anand et al., 2008). Among various cancers, skin cancer (melanoma and non-melanoma) is highly susceptible to environmental factors (Woodhead, Setlow, & Tanaka, 1999).
Globally, there were nearly 300,000 new cases of skin cancer in 2018 (Wright, du Preez, Millar, & Norval, 2020). Epidemiological and experimental evidence suggests "chronic inflammation" to be one of the hallmarks in disseminating skin cancer.
Solar ultraviolet radiation and several other environmental factors, including toxins, pesticides, and heavy metals, are the primary causal agents of skin cancers. The identification of transcription factors, mainly nuclear factor-kappa B (NF-KB), signal transducer and activator of transcription 3 (Stat3), hypoxia-inducible factor- 1 alpha (HIF-la) and their gene products, i.e., prostaglandins, cyclooxygenase-2 (COX-2), interleukins (IL-ip, IL-6, IL-8, IL-10) cytokines [tumor necrosis factor-alpha (TNF-a)], chemokines [CXC-chemokine ligand (CXCL)] and chemokine receptors suggest the critical role of inflammation in skin tumorigenesis (Lazennec & Richmond, 2010; Maru, Gandhi, Ramchandani, & Kumar, 2014; Mollica Poeta, Massara, Capucetti, & Bonecchi, 2019).
Considering the potential role of inflammation in tumor initiation, progression, angiogenesis, and metastasis, inflammatory pathways are attractive targets for skin cancer prevention and therapeutics. A series of piperine-amino acid ester conjugates were thoroughly explored for their cytotoxic activities against a panel of human cancer cell lines (Rao et al., 2012).
Piperic acid (PA) and 4-ethyl piperic acid (EPA) conjugates with non-protein amino acids have been reported as potent anti -metastatic agents in Pancreatic Ductal Adenocarcinoma (PDAC) cells via the abrogation of MMP-9 expression (Wani et al., 2016);(Amin et al., 2015). Recently, cyclodipeptide conjugates with piperic acid as cytotoxic agents against metastatic breast and prostate cancers have been investigated (Shankar et al., 2018).
W02002/057260A1 has described the piperic acid amides as anticancer agents against skin cancer with mild activity. Several therapeutically relevant applications are being uncovered for peptide-conjugate systems, which drive intense research activities in this emerging field. Rationally, our efforts focus on the synthesis of piperic acid conjugates with homochiral and heterochiral dipeptides containing phenylalanine and their application in skin cancer.
In the present invention, Piperic acid-DPhe-DPhe (FC-3) conjugate is identified as a potential lead against skin cancer (melanoma) in vitro by delineating the anti-proliferative and anti- migratory potential together with its anti-inflammatory potential against a range of pro- inflammatory interleukins (IL-ip, IL-6, and IL-8). The in vivo efficacy of FC-3 is validated in the two-step (DMBA/TPA) chemically induced mouse skin cancer model.
OBJECTIVE OF THE INVENTION
The main objective of the invention is to provide piperic acid and small peptides conjugates.
Another objective of the invention is to synthesize the said conjugates.
Yet another objective of the invention is to characterize and study the self-assembling properties, and in vitro cytotoxic screening of piperic acid-peptide conjugates.
Yet another objective of the invention is to evaluate the anti-proliferative, anti-migratory, and anti-inflammatory properties of the piperic acid dipeptide conjugates.
Yet another objective of the invention is to conduct In vivo validation of lead conjugate in the mouse two-step chemically induced skin cancer model.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides piperic acid-peptide conjugate of general Formula I,
Figure imgf000004_0005
hydrogen and aryl; R5 is selected from the groups consisting of OH, O-alkyl, HN-CH2PI1, HN- CH2 CH2-Ph, and HN-CH2CF3.
In a preferred embodiment of the present invention Ri, R2, R3 and R4 in the piperic acid-peptide conjugate of general Formula I are selected from group consisting of
Figure imgf000004_0001
In a preferred embodiment of the present invention R5 in the piperic acid-peptide conjugate of general Formula I are selected from group consisting of
Figure imgf000004_0002
In a preferred embodiment of the present invention the compound is selected from the group consisting
Figure imgf000004_0003
Figure imgf000004_0004
The present invention provides a process for the preparation of piperic acid-peptide conjugate of general Formula I, comrising the steps of; i. coupling of amino acids, Boc-AAi and H-AA2-0Me (1:1) ratiousing EDC.HC1, HOBt in the presence of NMM followed by deprotection of Boc group with 30% TFA in dry CH2CI2 at 0°C to obtain dipeptide H-AAi-AA2-0Me;
Figure imgf000005_0001
Boc-AArOH H-AA2-OMe H-AArAA2-OMe wherein AAi and AA2 are amino acids, Ri, R2, R3, and R4 are independently selected from the group consisting of hydrogen and aryl; ii. coupling of piperic acid with the dipeptide H-AAi-AA2-0Me obtained in step-i in (1:1) ratio using EDC.HC1, HOBt in presence of NMM followed by the saponification using 2N- NaOH/MeOH (1: 1) at room temperature, wherein R5 is selected from the groups consisting of OH, O-alkyl, HN-CH2PI1, HN-CH2 CH2-PI1, and HN-CH2CF3.
Figure imgf000005_0002
The present invention provides a pharmaceutical composition comprising the piperic acid- peptide conjugate of general Formula I,
Figure imgf000006_0001
wherein n = 0-2, wherein Ri, R2, R3, and R4 are independently selected from the group consisting of hydrogen and aryl; R5 is selected from the groups consisting of OH, O-alkyl, HN-CH2PI1, HN- CH2-CH2-PI1, and HN-CH2CF3 and a pharmaceutically acceptable excipient.
In an embodiment the pharmaceutically acceptable excipient are selected from the group consisting of diethylene glycol, cycloaliphatic and/or aromatic-aliphatic alcohols and pyrrolidones/polyol ethers/polyols.
In an embodiment of the present invention the conjugate and the pharmaceutical composition thereof abrogates skin cancer cells proliferation, attenuates the levels of pro-inflammatory interleukins and decreases tumor growth.
In a preferred embodiment of the present invention the compound of Formula I effectively abrogates skin cancer cells proliferation and migration.
In a preferred embodiment of the present invention the compound of Formula I strongly attenuates the levels of pro-inflammatory interleukins.
In a preferred embodiment of the present invention the compound of Formula I decreases the cumulative number of tumors and results in attenuation of tumor growth.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 displays post gelation SEM images of FC-3 and FC-4
Fig. 2 represents the Circular Dichroism (CD) curve of peptides FC-l-FC-4.
Fig. 3 shows the cytotoxic potential of piperic acid-peptide conjugates, FC-1-FC4, as determined by MTT assay against various melanoma cell lines (human and murine) and normal skin cells. Fig. 4 is a diagram reflecting on the anti -proliferative and anti-migratory properties of conjugate FC-3.
Fig. 5 illustrates the abrogation of Stat-3 expression and nuclear accumulation by Conjugate FC-3.
Fig. 6 is a diagram representing the down-modulation of pro-inflammatory interleukins (IL- 1 P, IL-6, and IL-8) in the conditional media collected from FC-3 treated cells.
Fig. 7 depicts the in vivo efficacy of Conjugate FC-3 against the two-step carcinogen-induced skin cancer model.
Fig. 8 is a diagram representing the down-modulation of pro-inflammatory interleukins (IL- 1 P, IL-6, and IL-8) in the serum collected from FC-3 treated mice cohorts.
Fig. 9 describes preparation of piperic acid-peptide conjugate of general Formula I
DETAILED DESCRIPTION OF THE INVENTION
The invention is now being described in detail in the following sections with certain preferred and optional embodiments so that various aspects thereof may be more thoroughly understood.
The present invention describes the synthesis, characterization, self-assembling properties, and in vitro cytotoxic screening of piperic acid-peptide conjugates. Piperic acid-DPhe-DPhe (FC-3) conjugate is identified as a potential lead against skin cancer (melanoma) in vitro by delineating the anti-proliferative and anti -migratory potential together with its anti-inflammatory potential against a range of pro-inflammatory interleukins (IL-ip, IL-6, and IL-8). The in vivo efficacy of FC-3 is validated in the two-step (DMBA/TPA) chemically induced mouse skin cancer model.
For convenience, before further describing the present disclosure, specific terms employed in the specification and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art; however, particular terms and their meanings are set forth below for convenience and completeness.
The articles "a", "an" and "the" are used to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as "consists of only".
Throughout this specification, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.
Abbreviations:
PA: Refers to piperic acid
LPhe: L-Phenylalanine
DPhe: D-Phenylalanine
DCM: Dichloromethane
NMM: N-Methylmorpholine
EDC.HC1: l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
TFA: Trifluoroacetic acid
HOBt: Hydroxybenzotriazole
°C: degree Celsius
DMSO: Dimethylsulfoxide
HEPES: 4-(2-hydroxyethyl)-l -piperazine ethanesulfonic acid
'HNMR: Proton Nuclear Magnetic Resonance
6: Chemical shift in parts per million (ppm)
HRESIMS: High-resolution electron spray ionization mass spectroscopy
SEM: Scanning electron microscopy
TLC: Thin layer chromatography
CD: CircularDichroism
Conjugates are compounds formed by the linking of two or more chemical compounds. Peptides are short chains of amino acids connected through amide bonds or peptide bonds.
Self-assembly: Spontaneous association of individual molecules in a well-organized manner.
Skin cancer refers to the uncontrolled and abnormal growth of skin cells, most often caused due to UV irradiation or exposure to chemical carcinogens. There are two main types of skin cancer: melanoma and non-melanoma
Melanoma refers to a form of skin cancer that begins in melanocytes (cells that control the pigment in our skin).
The term cytotoxic refers to the property of specific molecules being toxic to cells.
The termICso(half maximal inhibitory concentration)measures the potency of a substance in inhibiting a specific biological or biochemical function.
The terms anti-proliferative, anti-migratory, and anti-inflammatory refer to the inhibition in cell growth, migration, and inflammation of cells, respectively, in response to specific molecules.
The term wound healing used herein refers to the capacity of cancer cells to replenish any wound created by external agents by activating cellular migration.
Interleukins (ILs) are a group of cytokines (secreted proteins and signal molecules) that perform a myriad of cellular functions like proliferation, inflammation, chemotaxis, angiogenesis, etc.
Angiogenesis is the formation of new blood vessels. This process involves the migration, growth, and differentiation of endothelial cells, which line the inside wall of blood vessels.
The term metastasis used herein refers to the ability of cancer cells to migrate from their primary niches and colonize distant sites resulting in secondary tumors.
The two-step chemically induced skin cancer model is an experimental inflammatory cancer model.Tumor formation is induced in this model by the topical application of two different chemicals, 7,12-dimethylbenz[a]anthracene (DMBA) and 12- O-tetradecanoyl phorbol-13- acetate (TPA), that together cause papilloma formation in the skin.
Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
The present disclosure is not limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.
The present invention provides piperic acid-peptide conjugate of general Formula I,
Figure imgf000010_0002
wherein n is 0-2; Ri, R2, R3, and R4 are independently selected from the group consisting of hydrogen and aryl; R5 is selected from the groups consisting of OH, O-alkyl, HN-CH2PI1, HN- CH2-CH2-PI1, and HN-CH2CF3. This invention demonstrates the synthesis and characterization of conjugates of piperic acid with dipeptides containing LPhe and DPhe in the first part.
In a preferred embodiment of the present invention describes the synthesis and characterization of piperic acid conjugates with homochiral and heterochiral dipeptides containing phenylalanine, FC- 1 -FC-4.
Figure imgf000010_0001
The piperic acid-peptide conjugate of general Formula I, is prepared according to Figure 9. wherein n is 0-2; Ri, R2, R3, R4 and R5 are as defined above.
The process comprises the steps of: i. coupling of amino acids, Boc-AAl and H-AA2-0Me, using EDC.HC1, HOBt in the presence of NMM followed by deprotection of Boc group with 30% TFA in CH2C12 at 00C to obtain dipeptide H-AAl-AA2-0Me; ii. coupling of piperic acid with dipeptide H-AAl-AA2-0Me obtained in step-i using EDC.HC1, HOBt in presence of NMM followed by the saponification using 2N-NaOH/MeOH (1: 1) at room temperature.
The present invention also describes their application in skin cancer as anti-proliferative, anti- migratory and anti-inflammatory agents. Among all, conjugate FC-3 exhibited promising cytotoxic activity against melanoma cell lines and potent anti -proliferative and anti-migratory properties. Further, FC-3 significantly attenuated the levels of pro-inflammatory interleukins (IL-ip, IL-6, and IL-8) in the conditional media of melanoma cell lines. Interestingly, Conjugate FC-3 effectively abolished the expression and nuclear translocation of Stat-3 (a critical downstream effector of interleukins) in a dose and time-dependent manner in vitro. Stat-3 controls the process of angiogenesis by imposing a tight regulation of various angiogenic factors like VEGF-R and Hif-la signaling. Our results also demonstrated a pronounced down -regulation of VEGF-R (a primary angiogenic regulator). Of note, the efficacy of the conjugate FC-3 was further validated in vivo in the mouse two-step (DMBA-initiator/TPA-promoter) skin cancer model. The FC-3 treated cohorts of mice unveiled a significant decrease in the cumulative number of tumors and resulted in attenuation of tumor growth compared to vehicle-treated mice. In corroboration with our in vitro findings, serum collected from mice groups at various intervals during the treatment regimen demonstrated abrogation of IL-ip, IL-6, and IL-8 levels in FC-3 treated groups compared to the vehicle-treated group. Hence, the results collectively indicate that Conjugate FC-3 possesses potent cytotoxic activity against skin cancer and alleviates the pro- inflammatory interleukin landscape, resulting in abrogation of Stat-3 mediated angiogenesis and tumor growth.
Herein describe tis he conjugation of piperic acid with the dipeptides containing D- &L- phenylalanineamino acid residues. The synthesized peptide conjugates, FC-l-FC-4, were characterized by HRMS/HNMR spectroscopy. All the peptide conjugates were subjected to selfassembly in DMSO/buffer solutions. Among all, the conjugates FC-3 and FC-4 formed the hydrogels. Descriptive Figure Legends
Fig 1 : Post-gelation (lOOOx) Scanning electronic microscopy (SEM) pictographs of (A) FC-3 (B) FC-4
Fig 2:Circular Dichroism (CD) of peptides FC-l-FC-4 in MeOH. FC-land FC-4 showing a positive cotton effect, FC-2 and FC-3 showing a negative cotton effect.
Fig3: Bar graphs depicting the percent cell viability of A375 and B16F10 cells upon treatment with FC series of conjugates at 0.1, 1, 10 and 100 pM concentrations.
Fig 4: (A) Colony formation assay in A375 cells treated with either vehicle or FC-3 (1,2,3 and 5pM). Camptothecin (1 pM) was used as a positive control. (B) Corresponding bar graph of colony formation assay depicting the average number of colonies/field. (C) Wound healing assay depicting pictographs at successive time points of 0 h and 48 h after treatment with either vehicle or FC-3 (1, 2 and 3 pM). (D) Corresponding bar graph of wound healing assay illustrating the % wound closure of A375 cells treated with either vehicle or FC-3. The data represent three independent experiments performed separately.Statistical analysis -*P < 0.05. **P < 0.01, ***P < 0.001.
Fig 5: (A) Dose-dependent inhibition of Stat-3 by FC-3 in A375 cells at 48 h by western blotting. Corresponding bar representing the relative expression of Stat-3 expression in a dosedependent fashion. (B) Time-dependent inhibition of Stat-3 by FC-3 (5 pM) in A375 cells by western blotting. P- Actin expression was considered to ensure equal loading. Corresponding bar representing the relative expression of Stat-3 expression in a time-dependent fashion. (C) Fractionation study as determined by western blot analysis to evaluate the nuclear translocation of Stat-3 upon treatment with FC-3 (5 pM, 48 h). P-Actin was used as the cytosolic loading control and Histone 3 was used as the nuclear loading control. Corresponding bar graph for densitometric analysis ofnuclear and cytosolic Stat-3. (D) Immunocytochemistry images depicting the down-modulation of Stat-3 by FC-3 (5pM) at 48 h in A375 cells. (E) Immunocytochemistry images depicting the down-modulation of VEGF-R by FC-3 (5pM) at 48 h in A375 cells. Scale bar 10 pm, magnification-20X. The data represent three independent experiments performed separately. Fig 6: (A-C) Bar graphs depicting the CM levels (pg/ml of CM) of pro-inflammatory interleukins (IL-ip, IL-6 and IL-8) as determined by specific ELISA assays in the vehicle (DMSO) or FC-3 (3 and 5 pM) treated A375 cells at 24 h. LPS (IpM) was used as positive control 4 h prior to the treatment for priming the cells. (D) Histogram depicting the serum levels of anti-inflammatory interleukin - IL- 10 in the vehicle or FC-3 treated A375 cells at 24 h by ELISA assay. The data represent three independent experiments performed separately. Statistical analysis -*P < 0.05. **P < 0.01, ***P < 0.001.
Fig 7: (A) Schematic representation of the timeline of the two-step (DMBA/TPA) skin cancer model indicating the various time -points for tumor initiation, tumor promotion, data acquisition and sampling. (B) Graph representing the cumulative number of tumors per cohort (vehicle, FC- 3 10 mg/kg b.wt. and FC-3 30mg/kg b.wt.) over the course of multiple weeks (0-24 weeks). (C) Histogram representing the average tumor volume (mm3) per cohort (vehicle, FC-3 10 mg/kg b.wt. and FC-3 30 mg/kg b.wt.) at the start of treatment regimen (8 weeks), mid-point (16 weeks) and end-point (24 weeks). (D) Images of tumor-bearing mice (n=5) in the vehicle or FC- 3 treated (10 and 30 mg/kg b.wt.) at 24 weeks post the start of the study.
Fig 8: (A-B) Graphs depicting the serum levels (pg/ml of serum) of pro-inflammatory interleukins (IL-ip and IL-6) as determined by specific ELISA assays in the vehicle or FC-3 (10 and 30 mg/kg b.wt.) treated groups at 8, 16 and 24 weeks. (C) Histogram depicting the serum levels of anti-inflammatory interleukin - IL- 10 in the vehicle or FC-3 (10 and 30 mg/kg b.wt.) treated groups at 8, 16 and 24 weeks by ELISA assay. The data represent three independent experiments performed separately. Statistical analysis -*P < 0.05. **P < 0.01, ***P < 0.001.
Fig 9: Scheme describing the preparation of piperic acid-peptide conjugate of general Formula I
Examples
Following are the examples given to illustrate the invention further and should not be construed to limit the scope of the present invention.
Example 1: Synthesis of PA-DPhe-LPhe-OH, FC-1
Figure imgf000013_0001
Boc-DPhe-OH (3mmol) dissolved in CH2CI2, was added with NMM (9mmol), EDC.HC1 (3.6mmol), HOBt (3 mmol) and H-LPhe-OMe (3.6 mmol) at 0°C. The reaction mixture was allowed to attain room temperature and stirred for 12 hours. After completion of the reaction, the solvent was evaporated, and the residue was extracted with ethyl acetate (3x15ml). The ethyl acetate layers were combined and washed with 2N-HC1 (1x20 ml), 2M-NaHCOs (1x20 ml), and brine solution (1x20 ml). The organic layer was dried over anhydrous Na2SC>4 and evaporated under a vacuum to yield Boc-DPhe-LPhe-OMe.Piperic acid dissolved in 2 ml CH2CI2 was added with EDC.HC1 (0.412 g, 2.16 mmol), NMM (0.6 ml, 6 mmol), and H-DPhe-Phe-OMe, which was obtained by deprotection of Boc-DPhe-LPhe-OMe in 2.0 ml 25% TFA/CfEChat 0°C. The reaction mixture was stirred for 12 hours. After monitoring the reaction progress by TLC, the workup was performed as described above to yield PA-DPhe-LPhe-OMe, which was further saponified in 4 ml NaOH/MeOH (1:1). After completion of the reaction, the solvent was evaporated. The residue was dissolved in water (5 ml) and extracted with EtOAc. The aqueous layer was acidified using 2N-HC1 and extracted with EtOAc (3x 15 ml). The organic layer was evaporated under a vacuum to afford PA-DPhe-LPhe-OH (FC-1).
PA-DPhe-LPhe-OH (FC-l):1!! NMR (400 MHz, DMSO-d6): 5 8.44 (b, 1H), 8.09 (b, 1H), 7.43 - 7.06 (m, 13H), 7.05 - 6.79 (m, 3H), 6.26 - 6.03 (m, 3H), 4.65 (s, 1H), 4.38 (s, 1H), 3.13 (b, 2H), 2.89 (b, 2H).HRESIMS m/z513.2013[M+H]+(calcd. for C30H28N2O6 512. 19).
A similar synthetic procedure was followed for FC-2 - FC-3.
Example 2
PA-LPhe-DPhe-OH (FC-2):1H NMR (400 MHz, DMSO- d6) 5 8.44 (b, 1H), 8.09 (b, 1H), 7.43 - 7.06 (m, 13H), 7.05 - 6.79 (m, 3H), 6.26 - 6.03 (m, 3H), 4.65 (s, 1H), 4.38 (s, 1H), 3.13 (b, 2H), 2.89 (b, 2H).HRESIMS m/z 513.2013[M+H]+(calcd.for C30H28N2O6 512. 19).
Example 3
PA-DPhe-DPhe-OH (FC-S^H NMR (400 MHz, DMSO-d6) 5 8.44 (b, 1H), 8.09 (b, 1H), 7.43 - 7.06 (m, 13H), 7.05 - 6.79 (m, 3H), 6.26(b, 1H), 6.03 (S, 2H), 4.65 (s, 1H), 4.38 (s, 1H), 3.13 (b, 2H), 2.89 (b, 2H).HRESIMSm/z513.2017[M+H]+(calcd. for C30H28N2O6 512. 19).
Example 4 PA-LPhe-LPhe-OH (FC-^H NMR (400 MHz, DMSO-d6) 5 8.44 (b, 1H), 8.09 (b, 1H), 7.43 - 7.06 (m, 13H), 7.05 - 6.79 (m, 3H), 6.26 (b, 1H), 6.03 (s, 2H), 4.65 (s, 1H), 4.38 (s, 1H), 3.13 (b, 2H), 2.89 (b, 2H).HRESIMSm/z 513.2017[M+H]+(calcd. for C30H28N2O6 512. 19).
Example 5
Preparation of hydrogels. FC-l-FC-4 (2mg) were dissolved in DMSO (10 ml) in glass vials. After that, aqueous buffer HEPES(pH 7.4), 200 pl] was added. The mixtures were heated till a complete homogenous mixture was obtained. The solution was cooled to room temperature, and the gelation was evaluated by an inverted tube test (Figure 2). FC-3 and FC-4 formed the hydrogels, which were further analyzed by SEM images (Figure 3).
Example 6 Circular dichroism (CD) spectroscopy.CD measurements were recorded on JASCO CD (J-1500) at fixed temperature 25°C with 4 scans between 190-260 nm using 1mm optical path, data integration Isec, and scanning speed 20nm/min. Baseline correction was done with methanol, and then CD spectra raw data of peptides FC-1 to FC-4 were collected, and the plot was drawn on GraphPad PRISM 5 (Figure 4).
Assessment of the cytotoxic potential of phenylalanine peptide-conjugates with piperic acid
Table 1:
Figure imgf000016_0001
Cytotoxicity (IC50) of all synthesized conjugates of the invention against various skin cancer cell lines. IC50 (pM) values are calculated as mean ± standard deviation based on three independent experiments.
The conjugates were examined against various skin cancer (melanoma) cell lines (human and murine) and normal skin cells by MTT assay. Among all the conjugates in the series, FC-3 was the most potent conjugate against melanoma cell lines. The IC50 values of 5.6 ± 0.95 pM and 8.4 ± 1.3 pM were observed in A375 and B16F10 cells, respectively. Notably, against normal skin cells (primary murine keratinocytes), Conjugate FC-3 has an IC50 value of 31.3 ± 2.7 pM, indicating less toxicity against normal cells, thus signifying a higher therapeutic index.
Further, upon diligent examination of the screening data, it was evident that Conjugate FC-3 has higher cytotoxic potential against metastatic melanoma cell line-A375 than less aggressive melanoma cells. In contrast, Conjugates FC-1, FC-2, and FC-4 showed IC50 values of 49.2 ± 4.3, 21.0 ± 2.7, 43 ± 3.9 pM against A375 cells, and 47.4 ± 3.8, 19.6 ± 2.4 and 39.0 ± 3.2 pM against B16F10 cells, respectively.
Example 7
Measurement of cytotoxicity
Cell viability assay. The cell viability was determined by the standard MTT assay method (Riss et al., 2016). Briefly, A37F (human), B16F10 (murine), and primary murine keratinocyte cells were seeded in 96 well tissue culture plates (Nunc) at a density of 4 x 103 cells per well and treated with varying concentrations of the test compounds in triplicates. DMSO was employed as a vehicle, and its final concentration was maintained at 0.2 % in the culture medium. Doxorubicin and camptothecin were used as positive controls. After 44 h of incubation, MTT dye solution was added to the medium, and cells were incubated for another 4 h at 37 °C in 5 % CO2. The amount of colored formazan derivatives formed was measured by taking optical density (OD) using a microplate reader (TECAN, Infinite M200 Pro) at 570 nm, and the percentage of cell viability was determined. The IC50 values were calculated using GraphPad Prism software (Version 5.1).
Assessment of anti-proliferative properties of Conjugate FC-3 (Figure 4)
The colony formation assay to check the effect of Conjugate FC-3 on the proliferation of A375 cells was used. Our results unveiled that Conjugate FC-3 attenuated the clonogenic capacity of A375 cells. There was a slight reduction in the number of colonies at 1 pM dose of FC-3, which was deemed statistically non-significant. However, as shown in Figure 4, FC-3 attenuated the colony formation ability of A375 cells at 2,3 and 5 pM concentrations in a statistically significant manner (Figure 4). Compared to the vehicle (DMSO) treated cells, the average number of colonies per field was reduced by 10.71, 53.57, 85.71, and 92.85 % at 1, 2, 3, and 5 pM concentrations of FC-3. Camptothecin (IpM) was used as a positive control for the study. Interestingly, besides the reduction in the size of colonies, fewer number of cells per colony in the FC-3 treated cells were observed. In conclusion, our results indicate that FC-3 is a potent anti-proliferative agent against A375 melanoma cells.
Example 8 Colony formation assay. A375 cells were plated at a seeding density of (1 x 103 cells/well) in 60 mm tissue culture grade plates. After 24 h, the culture medium was replenished, fresh medium was added, and cells were exposed to vehicle or FC-3 (1, 2, 3, and 5 pM concentrations) or camptothecin (1 pM) for five days at 37 °C incubator in 5% CO2. Later, the obtained colonies were fixed with 4% paraformaldehyde for 15 min and were stained with 0.5% crystal violet solution for 1 h. The colonies (on the plates) were counted and randomly taken as an average from the observed fields (n= 5) and photographed under Nikkon D3100.
Assessment of anti-migratory properties of Conjugate FC-3 (Figure 6)
Our cytotoxicity and anti-proliferation analysis of FC-3 unveiled a substantial effect on A375 cells, which are characterized as malignant melanoma cells with a high propensity for invasion and migration. Accordingly, a wound healing assay was performed to understand more comprehensively about the anti-migratory property of Conjugate FC-3 (Figure 4). The results implied that FC-3 showed the most prominent anti -migratory properties at 2 and 3 pM concentrations in A375 cells. At IpM dose, the percent wound closure was determined to be non-significant. Compared to vehicle-treated cells (100 % wound closure), 2 and 3 pM doses of FC-3 showed 63.72 and 45.92% wound closure, respectively, indicating a profound reduction in the number of migratory A375 cells at these doses.
Example 9
Wound healing assay. A375 cells (IX 106) were seeded onto six-well plates and grown up to more than 90% confluency, and wounds were created with a sterile pipette tip (20-200pl), and the media was replaced with reduced serum media. Multiple bright-field images (n=5, 10 X magnification) were captured under a microscope, accounting for a zero-hour time -point. Subsequently, the cells were treated either with vehicle (DMSO) or Conjugate FC-3 (1, 2, and 3pM concentrations) for 48 h. Following termination, the cells were washed with PBS, and multiple fields per wound were captured under a microscope (Nikon D3100) at 10 X magnification (Figure 4). The images were processed by Image J software using the ‘wound healing plugin,’ and the percent wound closure was calculated in GraphPad Prism software (version 5.1).
Assessment of the anti-inflammatory potential of Conjugate FC-3 (Figure 5) Cancer-related inflammation has been suggested as the seventh hallmark of cancer (Hanahan & Weinberg, 2011). Inflammation affects all the critical aspects of cancer, such as the proliferation and survival of cancer cells, tumor response to chemotherapeutic drugs and hormones, metastasis, and angiogenesis. Inflammation is associated with different stages of skin cancer development, including initiation, promotion, malignant conversion, invasion, and metastasis. Two pathways connect cancer and inflammation: the intrinsic and extrinsic pathways (Dep Prete et al., 2011). The intrinsic pathway is activated by genetic events. In contrast, the extrinsic pathway represents inflammatory leukocytes and soluble mediators, leading to conditions, which increase cancer risk. The two pathways converge, resulting in the activation of transcription factors, mainly nuclear factor kappa B (NF-KB), signal transducer and activator of transcription-3 (Stat-3) and hypoxia-inducible factor-1 alpha (HIF-la) in tumor cells (Kany, Vollrath, & Relja, 2019; Pedranzini, Leitch, & Bromberg, 2004). These transcription factors coordinate the production of pro-inflammatory mediators, including cytokines (TNF-a, IL-6, IL-1), as well as the production of cyclooxygenase-2 (Cox-2) (Wojdasiewicz, Poniatowski, & Szukiewicz, 2014). Pro-inflammatory cytokines, the most important mediators of inflammation, have distinguished roles in skin cancer development. Along with nitric oxide (NO), these cytokines act as cell-to- cell messengers and help in the activation of NF-KB and Stat-3. Rationally, it was sought to quantify the effect of FC-3 treatment on pro-inflammatory interleukins (a class of cytokines) viz: IL-ip, IL-6, and IL-8 and anti-inflammatory cytokine - IL- 10 in A375 cells. Conditioned media (CM) was collected from the vehicle or FC-3 (3 and 5 pM) treated cells and subjected to specific ELISA analysis. Additionally, LPS (1 pM) was used as a positive control to drive the expression of interleukins. The effect of FC-3 treatment on cells with prior priming by LPS was also evaluated. Our results depicted that 5 pM FC-3 profoundly abrogated the IL-ip, IL-6 and IL-10 levels in the CM of A375 cells (Figure 5). Apart from IL-6, 3 pM dose had an insignificant effect on IL-ip and IL-8 levels. However, in cells with prior LPS priming, both 3 and 5 pM doses of FC-3 caused significant down-regulation of IL-ip, IL-6 and IL-10 levels compared to LPS alone primed cells. Further, the effect of FC-3 treatment on the anti-inflammatory interleukin -IL- 10 (Figure 5) was also evaluated.However, the results mainly were deemed insignificant, with no apparent change by FC-3 treatment on IL- 10 levels. Only a slight decrease in the IL-10 expression in the LPS-primed FC-3(5 pM) treated A375 cells was observed. Taken together, our ELISA-based results demonstrate a profound abrogation of pro-inflammatory interleukins - IL-ip, IL-6, and IL- 10.
Example 10. Quantification of IL-ip, IL-6, IL-8, and IL-10 by ELISA assay. A375 cells were grown in DMEM media containing 10% FBS. 3xl05 cells were plated in 6 well plates for 24 h. Cells were primed with 1 pM of LPS (lipopolysaccharide) for 4 h, followed by treatment with either vehicle in the media already constituted with LPS for 24 h. Conditional media in the form of supernatants were collected and analyzed for pro-inflammatory interleukins - IL-ip, IL- 6, and IL-8 and the anti-inflammatory interleukin - IL- 10 by ELISA using specific ELISA kits according to manufacturer’s instructions (R&D Systems; Cat. No. DLB50, D6050, D8000C, and D1000B). The absorbance was measured at 450 nm (Tecan Multi-mode Plate Reader), and optical density values were normalized with respective controls. All experiments were performed in triplicate.
Conjugate FC-3 abrogates Stat-3 expression and it’s nuclear translocation (Figure 6)
The signal transducer and activator of transcription-3 (Stat-3) belong to the Stat family of transcription factors. Stats are proteins that are activated by extracellular signaling proteins, growth factors such as epidermal growth factor receptor (EGFR), cytokines (TNF-a, IL-ip, IL-6, IL-10 IL-17, IL-22), and various peptides (Aggarwal et al., 2009; Pedranzini et al., 2004). Stat-3 regulates the expression of genes that mediate survival (survivin, BcLxl, myeloid cell leukemia sequence 1 [mcl-1], c-FLIP), proliferation (c-fos, c-myc, cyclin DI), invasion (MMP-2), and angiogenesis (VEGF) (Macias, Rao, & DiGiovanni, 2013; Messina et al., 2008). Along with NF- KB, Stat-3 is a point of convergence for numerous oncogenic signaling pathways, particularly those concerning inflammations like cytokine and interleukin signaling. In response to cytokines and growth factors, Stat-3 is phosphorylated by receptor-associated Janus kinases (JAK), form homo- or heterodimers, and translocate to the cell nucleus where they act as transcription activators. Stat-3 mediates the expression of a variety of genes in response to cell stimuli, and thus plays a key role in many cellular processes, such as cell growth, inflammation, angiogenesis, and evasion from apoptosis (Xin et al., 2020).
Keeping in purview the backdrop of Stat-3 ’s role as an effector node for interleukin-mediated inflammation, it was sought to check the effect of FC-3 treatment on Stat-3 expression and localization in A375 cells. The dose-dependent immunoblotting results at a 48-hour time-point showed a gradual decrease in the expression of Stat-3 at 2.5, 5.0, and 10.0 pM doses (eA). Timedependent analysis at 5 p M FC-3 concentration revealed a steady decline in Stat- 3 levels post 12. hours of treatment (Figure 6). Notably, fractionation analysis of A375 cells treated with 5 pM FC-3 unveiled a profound decline of Stat-3 expression in the nuclear fraction, indicating an impediment of Stat-3 translocation from cytoplasm to the nucleus upon FC-3 treatment (Figure 8C). Immunofluorescence analysis of FC-3 treated cells to corroborate our fractionation analysis further was performed. Our results depicted a diffused localization of Stat-3 with a profound decrease in nuclear levels of Stat-3 in FC-3 treated cells compared to vehicle-treated cells (Figure 6). Stat- 3 is a key transcription factor regulating angiogenesis. Therefore, the effect of FC-3 treatment in A375 cells on VEGF-R expression by immunofluorescence was evaluated. As with Stat-3 expression, FC-3 treatment resulted in a significant down-regulation of the VEGF receptor, indicating a halt in angiogenesis (Figure 6). Collectively, these results demonstrate the inhibitory potential of FC-3 against Stat-3 activation and functionality.
Example 11
Western Blot analysis for the detection of protein expression. A375 cells were seeded in 60 mm petri-dishes and maintained up to 70% confluency; afterward, the cells were treated with either vehicle or Conjugate FC-3 for indicated time-points and concentrations. After treatment, the cells were harvested, washed thrice with ice-cold PBS, and subjected to protein lysis with lysis buffer (HEPES 1 mM/L, KC1 60 mM/L, NP-40 0.3%, EDTA 1 mM/L, DTT 1 mM/L, sodium orthovanadate 1 mM/L, PMSF 0.1 mM/L and cocktail protease inhibitor). The lysis product was centrifuged at 12000 rpm for 45 min at 4°C to remove the cellular debris, and the supernatants were collected. Total protein was estimated with the help of the Bradford assay. An equal amount of protein (30 pg for Stat-3) was subjected to SDS-PAGE. The proteins were transferred onto the PVDF membrane (Millipore) by wet transfer. The membrane was blocked with 5% BSA and incubated in a primary antibody prepared in 5% BSA (dilution ranging from 1: 1000-1:2000) for 2-3 h. The membrane was then washed with TBST buffer (137 mM NaCl, 2.7 mM KC1, 19 Mm Tris base, and 0.1 % Tween-20, pH 7.6) for 30 min (three times for 10 min each) and incubated with species-specific secondary antibodies (CST, Cat. No. 7074S, 7076S) tagged with horseradish peroxide. After washing thrice with TBST, the protein expression was analyzed by adding a chemiluminescent substrate (Millipore, Cat No. WBULUF0100) to the membrane. The signal obtained was captured on x-ray films (ThermoFisher, Cat. No. 34090). Densitometry analyses of the captured bands on X-ray film were performed using ImageJ software(version Fiji) and Graph pad prism (version 5.1).
Example 12
Preparation of cytoplasmic and nuclear fractions for immunoblotting: A375 cells treated with vehicle control (DMSO) or FC-3 (5.0 pM, 48 h) were washed with ice-cold PBS and centrifuged. All steps of fractionation were carried out at 4 °C. Cell pellets were homogenized in 200 pl of ice-cold hypotonic buffer (5 mM HEPES, pH 7.9, 10 mM KC1, 1 mM DTT, 0.5 mM PMSF, 1.5 mM MgCh, 1 mM NaVCM, 10% protease cocktail inhibitor, 0.02 volumes of 10% NP-40) with mild vortexing by keeping on ice for 30 min. The cell suspension was accordingly centrifuged at 10000 g for 1 min. The resulting cytosolic supernatants were stored at -80 °C. The pellets obtained were washed in 100 pl of hypotonic buffer and resuspended in 100 pl of hypertonic buffer (5 mM HEPES, pH 7.9, 0.25% NP-40, 25% glycerol, 500 mM NaCl, 1.5 mM MgC12, 0.2 mM EDTA, 0.5 mM PMSF, 1 mM NaVC , and 10% protease cocktail inhibitor), incubated in the end to end rotor for 2 h at 4 °C following centrifugation at 10000 g for 30 min at 4 °C. The supernatants obtained were stored at -80°C (Thermo Fischer) for analysis of nuclear protein.
Example 13
Immunocytochemistry analysis following FC-3 treatment. A375 cells were seeded in 4-well chamber slides (Lab-Tek chamber slides, Thermo Scientific, Boston, MA, USA) at a density of 3 x 104 cells and treated with either vehicle control (DMSO) or FC-3 (5.0 pM, 48 h). Posttreatment, immunocytochemistry was performed. Briefly, cells were pelleted down and washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature, then permeabilized with 0.1% Triton X-100 in PBS for 5 min and successively blocked with 1% BSA for 1 h. To detect Stat-3 and VEGF-R expression/ localization, the cells were incubated with corresponding primary antibodies for 1 h and accordingly washed three times with PBS. The cells were then incubated with Alexa Fluor 555 (red channel) conjugated secondary antibody for 1 h. Post incubation, the cells were washed, and mounted with an ultracruz mounting medium containing DAPI (blue channel). The prepared slides were then analyzed with EVOS FLoid Cell Imaging Station(Thermo Fisher Scientific) under 20 X magnification.
In vivo efficacy of Conjugate FC-3 against murine skin cancer model (Figure 7) One of the most commonly used experimental inflammatory cancer models is the DMBA-TPA two-stage skin carcinogenesis model(Vahatupa, Pemmari, Junttila, Pesu, & Jarvinen, 2019). Tumor formation is induced in this model by the topical application of two different chemicals, 7, 12-dimethylbenz[ a] anthracene (DMBA) and 12-O-tetradecanoyl phorbol-13-acetate (TPA), that together cause papilloma formation in the skin. As the primary outcome is papilloma formation in the skin, the model is an ideal, reliable, and reproducible way to address both tumor initiation (tumor-free survival) and tumor progression (number and size of visible tumors). The effects of the DMBA-TPA (Sigma, #D3254; Sigma, #P1585) treatment are transmitted via an inflammatory mechanism, which makes this model especially suitable for our study. Rationally, this model to validate the in vivo potential of FC-3 against skin cancer was employed. The experimental design for the study is given in Figure 7. Our results unveiled a robust increase in the cumulative number of tumors in the vehicle-treated group from the 8th week onwards through the 24th week (Figure 7). However, it was observed a significant reduction in the FC-3 treated groups (10 and 30 mg/kg b.wt.). The reduction was more profound in the 30 mg/kg b.wt. FC-3 treated group when compared with the 10 mg/kg b.wt. FC-3 treated group. The cumulative number of tumors/group was 34, 20 and 16 for the vehicle and FC-3 (10 and 30 mg/kg b.wt) treated groups, respectively. Further, the tumor volume of animals in the three groups at three different points (8th, 16th and 24th week) was calculated. While the animals in all the groups had comparable tumor volumes at the start of FC-3 treatment (8th week), a significant augmentation in the tumor volumes over the 16th and 24th week in the vehicle-treated group (Figure 7) was observed. However, in the FC-3 treated cohorts, there was robust tumor growth inhibition compared with the vehicle-treated cohort at the respective time points. The results particularly showed appreciable tumor growth inhibition at the 24th-week sampling time-point. The average tumor volume/animal at the end-time sampling point (24th week) was recorded as 147.67, 32.39, and 16.57 mm3 for the vehicle, FC-3 10 mg/kg b.wt. and FC-3 30 mg/kg b.wt. treated groups, respectively. It is evident from the above data that Conjugate FC-3 is a potent anti-tumorigenic agent against skin cancer in vivo.
Example 14
Two-step chemically induced mouse skin cancer model. All the in vivo experimental protocols were approved by the Institutional Animal Ethics Committee, CPCSEA, of the Indian Institute of Integrative Medicine, Jammu. The animals were maintained at 22 °C with a 12 h light-dark cycle and free access to feed and water inside the institutional animal house. Proper care was taken to maintain them in a healthy condition and to avoid any risk of possible pathogenic contaminations. C57BL/6 sex-matched female mice were chosen for the study. The mice were 7-9 weeks of age because the skin in most mice is in telogen (the resting phase) around that age (Vahatupa et al., 2019). The mice were weighed twice a week for any potential weight loss, and the readings were recorded. The back skin of the mice was shaved with a trimmer at the beginning of the study and bi-weekly during the entire course of the study, specifically two days prior to any chemical exposure. For tumor initiation and promotion, DMBA and TPA, both diluted in acetone, were respectively used. The working concentration of DMBA was 250 g/1. A dose for one animal was 50 pg of DMBA in 200 pl of acetone. DMBA was given topically for 4 weeks, twice a week, followed by topical TPA application (again twice a week for 4 weeks). The stock concentration of TPA was 125 g/1, and the treatment concentration 25 g/1. A TPA dose for one animal was 5 pg in 200 pl of acetone.
The papillomas started developing post 7th week. At the end of the 8th week, discrete papillomas were observed in 28 mice out of 30. A palpable mass greater than 1 mm in diameter was considered a papilloma and taken into account. Tumor dimension was measured with a digital vernier caliper (Safeseed, Cat. No. DIGICALPLBK001). Tumor volume was calculated by the formula [length x (width)2/2], where length was taken as the diameter of the largest side. The tumor-bearing animals were divided into three groups- vehicle, FC-3 (10 mg/kg b.wt. and 30 mg/kg b.wt.) in a randomized fashion. FC-3 (10 mg/kg b.wt. and 30 mg/kg b.wt. for the respective groups) was given topically twice a week for 16 weeks. The vehicle group mice were treated with acetone only. The papillomas were counted, recorded, and photographed every week. The animals were sacrificed at the end of the 24th week, and tumors were isolated and stored for downstream applications.
Example 15
Assessment of the inflammatory landscape from the serum of tumor-bearing mice (Figure 8)
As discussed in the preceding sections about the relevance of interleukins (a class of cytokines) in skin cancer, it was sought to quantify the effect of FC-3 treatment on pro-inflammatory interleukins viz: IL-ip and IL-6, and anti-inflammatory cytokine - IL- 10 in our two-step chemically induced skin cancer model. Serum was collected from vehicle (acetone) or FC-3 (10 and 30 mg/kg b.wt.) treated cohorts and subjected to specific ELISA analysis. Serum collection was done at three different time points during the course of our study (the 8th, 16th’ and 24th week). Our results depicted that compared to the 8th week (start of FC-3 treatment), the levels of IL-ip and IL-6 augmented steadily through the 16th to 24th week in the vehicle cohort of mice. However, in cohorts treated with FC-3 (10 and 30 mg/kg b.wt.), a sharp attenuation in IL-ip and IL-6 expression with respect to the corresponding vehicle-treated mice at the 16th and 24th week (Figure 10A-10B) was observed. The decrease was more profound in the cohort with 30 mg/kg b.wt treatment than in the cohort with 10 mg/kg b.wt. treatment. Further, the effect of FC-3 treatment on the anti-inflammatory interleukin -IE- 10 (Figure 10C) was evaluated. While there was no apparent difference in the vehicle or the FC-3 (10 and 30 mg/kg b.wt.) treated groups at the 8th" week time-point, our results revealed a gradual augmentation of IE- 10 levels at the 16th and 24th-week time -point with respect to the corresponding vehicle group at these time -points. Taken together, our EEISA-based in vivo results demonstrate a profound abrogation of pro- inflammatory interleukins - IF-ip and IE-6 with concomitant up-regulation of anti-inflammatory cytokine-IL-10. These results are in accordance with our in vitro findings as well.
Example 16
Formulation: Since, excellent papilloma inhibition by the acetonic solution of FC-3(10 mg/kg b.wt. and 30 mg/kg b.wt.), was found therefore, the formulation of FC-3 comprising about 0.01% to about 5%w/w FC-3 + diethylene glycol + cycloaliphatic and/or aromatic-aliphatic alcohols + pyrrolidones/polyol ethers/polyols could be constituted.
Quantification of serum levels of IL-ip, IL-6 and IL-10 by ELISA assay. Three sampling time-points were determined for the study - the 8th week (at the start of the treatment), the 16th week (mid-point in the treatment course), and the 24th week (end-point of the treatment). For serum-based experiments, blood was collected from the optical vein of mice with a capillary without causing any discomfort to the mice. Serum was immediately isolated from the whole blood and analyzed for pro-inflammatory interleukins - IL-ip, IL-6, and the anti-inflammatory interleukin - IL- 10 by ELISA assay using specific ELISA kits according to manufacturer’s instructions (R&D Systems, Cat. No. MLB00C, M6000B M1000B). The absorbance was measured at 450 nm, and optical density values were normalized with respective controls. ADVANTAGES OF THE INVENTION
• The present invention leads to the discovery of a novel potent piperic acid-DPhe-DPhe conjugate (FC-3) as an anticancer agent against diverse skin cancer cell lines.
• Compound FC-3 has shown the tendency to form self-assembled hydrogels.
• Conjugate FC-3 inhibits the proliferation and migration ability of human melanoma cancer cells in vitro.
• FC-3 significantly attenuates the expression of pro-inflammatory cytokines- IL-ip, IL-6, and IL-8 in vitro.
• Conjugate FC-3 significantly down-regulates the Stat-3 expression and impedes its nuclear translocation. Stat-3 serves as a critical effector in the signaling of interleukins and other cytokines and is implicated in angiogenesis and metastasis of cancer.
• Conjugate FC-3 is an effective inhibitor of tumor growth in vivo against the two-step chemically induced skin cancer model.
In a nutshell, the present invention leads to the development of an anticancer drug-like molecule from the conjugation of homo- and heterochiral peptides containing phenylalanine with piperic acid that can potentially halt the proliferation, migration, and angiogenesis in skin cancer by impeding the interleukin/Stat-3 axis.

Claims

We claim:
1. A piperic acid-peptide conjugate of Formula I,
Figure imgf000027_0001
wherein n is 0-2, Ri, R2, R3, and R4 are independently selected from the group consisting of hydrogen and aryl; R5 is selected from the groups consisting of OH, O-alkyl, HN-CH2PI1, HN- CH2 CH2-Ph, and HN-CH2CF3.
2. The conjugate as claimed in claim 1, wherein Ri, R2, R3 and R4 are selected from the group consisting of;
Figure imgf000027_0002
3. The conjugate as claimed in claim 1, wherein R5 is selected from the group consisting of
Figure imgf000027_0003
4. The conjugate as claimed in claim 1, wherein the compound is selected from the group consisting of;
Figure imgf000028_0001
5. A process for preparation of piperic acid-peptide conjugate of Formula I,
Figure imgf000028_0002
wherein n is 0-2, Ri, R2, R3, and R4 are independently selected from the group consisting of hydrogen and aryl; R5 is selected from the groups consisting of OH, O-alkyl, HN-CH2PI1, HN- CH2-CH2-PI1, and HN-CH2CF3, said method comprising the steps of; i). coupling of amino acids, Boc-AAi and H-AA2-0Me (1: 1) ratio, using EDC.HC1, HOBt in the presence of NMM followed by deprotection of Boc group with 30% TFA in dry CH2CI2 at 0°C to obtain dipeptide H-AAi-AA2-0Me;
Figure imgf000029_0001
wherein AAi and AA2 are amino acids, Ri, R2, R3, and R4 are independently selected from the group consisting of hydrogen and aryl; ii). coupling of piperic acid with the dipeptide H-AAi-AA2-0Me obtained in step-i in (1;1) ratio using EDC.HC1, HOBt in presence of NMM followed by the saponification using 2N-NaOH/MeOH (1 : 1) at room temperature,
Figure imgf000029_0002
wherein Ri, R2, R3, and R4 are as defined above and R5 is selected from the groups consisting of OH, O-alkyl, HN-CH2Ph, HN-CH2.CH2-Ph, and HN-CH2CF3.
6. The process as claimed in claim 5 , wherein Ri, R2, R3 and R4 are selected from combinations consisting of;
Figure imgf000029_0003
7. The process as claimed in claim 5 , wherein R5 is selected from the group consisting of
Figure imgf000030_0001
8. A pharmaceutical composition comprising the piperic acid-peptide conjugate of Formula I,
Figure imgf000030_0002
wherein n = 0-2, wherein Ri, R2, R3, and R4 are independently selected from the group consisting of hydrogen and aryl; R5 is selected from the groups consisting of OH, O-alkyl, HN-CH2PI1, HN- CH2 CH2-Ph, and HN-CH2CF3. and pharmaceutically acceptable excipient.
9. The pharmaceutical composition as claimed in claim 8, wherein the pharmaceutically acceptable excipient is selected from the group consisting of diethylene glycol , cycloaliphatic and/or aromatic-aliphatic alcohols and pyrrolidones/polyol ethers/polyols.
10. The conjugate as claimed in claim 3, for use as a medicament for abrogating skin cancer cells proliferation, attenuating the levels of pro-inflammatory interleukins and decreasing tumor growth.
11. A method for treating skin cancer by administering conjugate of claim 1, by halting proliferation, migration and angiogenesis in skin cancer by impeding the interlukin/Stat-3 axis.
12. Attenuating IL-ip, IL-6, and IL-8 expression by conjugate of claim 1
PCT/IN2023/050304 2022-03-29 2023-03-29 Piperic acid-conjugate with homochiral and heterochiral dipeptides containing phenylalanine and process for preparation thereof WO2023187830A1 (en)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SHANKAR SUDHA, FAHEEM MIR MOHD, NAYAK DEBASIS, WANI NAIEM AHMAD, FAROOQ SALEEM, KOUL SURRINDER, GOSWAMI ANINDYA, RAI RAJKISHOR: "Cyclodipeptide c(Orn-Pro) Conjugate with 4-Ethylpiperic Acid Abrogates Cancer Cell Metastasis through Modulating MDM2", BIOCONJUGATE CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 29, no. 1, 17 January 2018 (2018-01-17), US , pages 164 - 175, XP093098764, ISSN: 1043-1802, DOI: 10.1021/acs.bioconjchem.7b00670 *
V. RAMA SUBBA RAO ET AL.: "Synthesis of piperine-amino acid ester conjugates and study of their cytotoxic activities against human cancer cell lines", MED CHEM RES, vol. 21, 2012, pages 38 - 46, XP019992711, DOI: 10.1007/s00044-010-9500-5 *

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