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Synthesis of new secretory phospholipase A2-inhibitory indole containing isoxazole derivatives as anti-inflammatory and anticancer agents
Accepted Manuscript Synthesis of new secretory phospholipase A2-inhibitory indole containing isoxazole derivatives as anti-inflammatory and anticancer Agents Srinivasa Rao Pedada, Nagendra Sastry Yarla, Pawan J. Tambade, Dhananjaya Bhadrapura Lakkappa, Anuapam Bishayee, Kalle M. Arunasree, Gundala Harold Philip, Gangappa Dharmapuri, Gjumrach Aliev, Swathi Putta, Gururaja Rangaiah PII:
S0223-5234(16)30095-2
DOI:
10.1016/j.ejmech.2016.02.025
Reference:
EJMECH 8376
To appear in:
European Journal of Medicinal Chemistry
Received Date: 2 September 2015 Revised Date:
7 February 2016
Accepted Date: 8 February 2016
Please cite this article as: S.R. Pedada, N.S. Yarla, P.J. Tambade, D.B. Lakkappa, A. Bishayee, K.M. Arunasree, G.H. Philip, G. Dharmapuri, G. Aliev, S. Putta, G. Rangaiah, Synthesis of new secretory phospholipase A2-inhibitory indole containing isoxazole derivatives as anti-inflammatory and anticancer Agents, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.02.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical abstract N O
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N Boc
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N H
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10o (R1=F, R2=CF3, R=CH3) GIIA sPLA2 (IC50 = 10.23±0.91 µM)
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Synthesis of new secretory phospholipase A2-inhibitory indole containing isoxazole derivatives as anti-inflammatory and anticancer Agents
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Running title: sPLA2-inhibitory indole containing isoxazoles Srinivasa Rao Pedadaa, +, δ, Nagendra Sastry Yarlab, δ, Pawan J. Tambadec, Dhananjaya Bhadrapura Lakkappad, Anuapam Bishayeee, Kalle M. Arunasreef, Gundala Harold Philipg, Gangappa
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Dharmapurif, g, Gjumrach Alievh, Swathi Puttai, Gururaja Rangaiah j,*
Centre for Research and development,PRIST University, Thanjavur-613 403, Tamilnadu, India
b
Department of Biochemistry, Institute of Science, GITAM University, Visakhapatnam-530 045,
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a
Andhra Pradesh, India c
Department of Chemistry, Arts, Commerce and Science College, Nandgaon, Maharashtra, India
d
Toxinology/Toxicology and Drug Discovery Unit, Center for Emerging Technologies, Jain Global
Campus, Jain University, Ramanagara-562 112, Karnataka, India
Department of Pharmaceutical Sciences, College of Pharmacy, Larkin Health Sciences Institute,
Miami, Florida 33169, USA f
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e
Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad-500
046, Telangana, India
Department of Biotechnology, Sri Krishnadevaraya University, Anantapuramu-515 003, Andhra
Pradesh, India h
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g
School of Health Sciences and Healthcare Administration,
University of Atlanta, E. Johns
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Crossing, Johns Creek, GA 30097, USA i
Pharmacology Division, University College of Pharmaceutical Sciences, Andhra University,
Visakhapatnam-530003, Andhra Pradesh, India j
Department of Biochemistry, Sarada Vilas College, Mysore-570004, Karnataka, India
+
the work is part of PhD work
δ equally contributed to this work *corresponding author. E-mail address: [email protected] (G. Rangaiah)
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ABSTRACT Secretory phospholipase A2 (sPLA2) is an important enzyme that plays a key role in various inflammatory diseases including cancer and its inhibitors have been developed as preventive or
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therapeutic agents. In the present study, a series of new indole containing isoxazole derivatives (10a-10o) is synthesized and evaluated for their sPLA2 inhibitory activities. All compounds (10a10o) showed significant sPLA2 inhibition activities both in vitro and in vivo studies which is
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substantiated in in silico studies. Among all the tested compounds, 10o showed potent sPLA2 inhibition activity, that is comparable or more to ursolic acid (positive control). Further studies
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demonstrated that 10o showed in vitro antiproliferative activity when tested against MCF-7 breast and DU145 prostate cancer cells. Furthermore, compounds 10a-10o obeyed lipinsky`s rule of 5 and suggesting druggable properties. The in vitro, in vivo and in silico results are encouraging and warrant pre-clinical studies to develop sPLA2-inhibitory compound 10o as novel therapeutic agent
Key words:
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for various inflammatory disorders and several malignancies.
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Indole, Isooxazole, Secretory phospholipase A2, Anticancer, Anti-inflammatory activity
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1.
Introduction Phospholipase A2s (PLA2s) are group of enzymes, which cleave phospholipids specifically
at sn-2 position to liberate free fatty acid, mostly arachidonic acid (AA) and lysophospholipids
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(LPLs) [1-3]. Inhibition of PLA2 prevents the liberation of AA and LPLs. Hence, researchers have been considering that PLA2s could be good therapeutic target than cyclooxygense-2 and lipoxygenase, which are downstream enzymes [1-3]. Several isoforms of PLA2s exist, which are
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divided into various groups; secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2), and calciumindependent PLA2 (iPLA2), platelet activating factor-acyl hydrolase (PAF-AH), lysosomal PLA2
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(LPLA2), adipose-specific PLA2 (AdPLA2). The secreted PLA2s (13-55 kDa) are the first type of PLA2 enzymes discovered and categorized into several groups [4]. Among all groups of the sPLA2s, group IIA sPLA2 (GIIA sPLA2) is predominantly distributed and plays key role in various inflammatory diseases, including arthritis, inflammatory bowel disease, atherosclerosis, psoriasis,
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and several cancers [1-4].
Realizing the role of sPLA2 in inflammatory diseases and various cancers, considerable efforts are being made by several investigators for the discovery and development of sPLA2
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inhibitors as therapeutic or preventive agents [2]. Several sPLA2 inhibitors have been developed and some of the sPLA2 inhibitors are currently under clinical trials for various inflammatory and
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oncologic diseases [2-4]. During last several decades, various derivatives of indoles have been developed as GIIA sPLA2-inhibitors [5-7]. A series of indole acids, indole amides, indole-3acetamides, indole-3-glyoxamides are developed as sPLA2 inhibitors [5]. Lilly Research Laboratory (Indianapolis, IN, USA) developed a series of indole-3-acetamide derivatives with sPLA2 inhibition activity. Among all compounds, varespladib or LY315920 inhibited group IIA sPLA2 with high potency and it entered into clinical trials for inflammatory diseases [6]. However, LY315920 or varespladib failed phase II clinical trials because of poor anti-inflammatory efficacy. Further, Lilly 3
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Research Laboratory synthesized LY333013 or methyl varespladib, a modified form to LY315920, but it also failed in clinical trials due to its poor anti-inflammatory efficacy [7]. Recently, Anthera Pharmaceuticals (Hayward‚ CA, USA) announced the use of A-001 (varespladib or LY315920) and
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A-002 (LY333013 or methyl varespladib) in treatment of cardiovascular diseases as their efficacy in reducing LDL-C and atherosclerosis in patients with acute coronary syndrome (ACS) along with atorvastastin treatment, according to phase II clinical trial namely, Fewer Recurrent Acute Coronary
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Events With Near Term Cardiovascular Inflammation Suppression (FRANCIS) [8]. In phase III clinical trial of varespladib in patients with ACS, namely Vascular Inflammation Supression to
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Treat Acute Coronary Syndrome for 16 days (VISTA-16), varespladib showed poor efficacy in reduction of ACS and significantly increased the risk of nonfatal myocardial infarction (MI) [9]. Most of the sPLA2 inhibitors failed in clinical trials due to poor efficacy and adverse effects [2]. Therefore, discovery of new sPLA2-inhibitory molecules or optimization of existed sPLA2-
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inhibitory compounds with more potency, safety and selectivity is urgently needed for prevention and treatment of inflammatory disorders and several cancers. Isoxazoles have been reported to possess various biological activities, including PLA2-
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inhibitory activities [10, 11]. Sadashiva et al. [11] synthesized trimethoxyphenyl isoxazolidine derivatives with sPLA2 inhibitory activities. Various derivatives of indoles have been developed as
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sPLA2 inhibitors and some of them are under clinical evaluation for various inflammatory diseases. Both indoles and isoxazoles are attractive chemical scaffolds for development of novel sPLA2 inhibitors [5-7, 10, 11]. Considering this background, the present study aimed to synthesize indole containing isoxazoles (10a-10o) with potent sPLA2-inhibitory activity. Among all compounds, 10o showed substantial sPLA2-inhibitory activity. Further in vivo antiinflammatry studies of 10o were performed using inflammogen (carrageenan)-induced rat paw edema model [12]. In vitro anticancer studies using sPLA2-inhibitory 10o were also performed on MCF-7 breast carcinoma and DU145 4
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prostate cancer cell lines since sPLA2 exhibited pro-tumorigenic role in breast and prostate cancers [13, 14]. 2.
Results
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2.1. Chemistry The target molecules were synthesized in a convergent fashion by coupling indole aldehydes 8(a-c) and isoxazole containing primary amines 5(a-e), which were subsequently converted to their
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hydrochloride salts 10 (a-o). The isoxazole methyl amines were synthesized starting from commercially available aromatic aldehydes. Aldehydes were reacted with hydroxyl amine
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hydrochloride to obtain their corresponding oxime derivatives 2(a-e). They were converted to their corresponding bromo methyl isoxazoles by intermolecular cycloaddition of activated oximes with with propargyl bromide in presence of chloramine-T in ethanol [15]. The bromo methyl isoxazoles were later converted to azides by treating with sodium azide in DMF 4(a-e), which were finally
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reduced to the desired isoxazole methyl amines 5(a-e) by following a mild and efficient Schodinger’s protocol using triphenyl phosphine (TPP) and water in THF as a solvent system [16]. On the other hand indole aldehydes were synthesized by electrophilic acylation of
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unprotected indoles following Vilsmier-Haack method using POCl3/DMF in good to excellent yields 7(a-c). To avoid any unforeseen complications, the indole NH was protected by Boc
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anhydride. The boc protected indole-3-aldehydes 8(a-c) and isoxazole methyl amines 5(a-e) were coupled by reductive amination in the presence of sodium cyanoborohydride and catalytic amount of acetic acid in methanol at ambient temperatures. The isolated yields of the coupled products 9(ao) were moderate to good. Finally, Boc group was deprotected with TFA in DCM, which were subsequently converted to their hydrochloride salts with 20% HCl in ethyl acetate in good yields 10(a-o) (Scheme-1). All the synthesized compounds were well characterized by 1H NMR, 13C NMR, IR and mass spectra. 5
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2.2.
Pharmacology
2.2.1. sPLA2-inhibitory activity of indole containing isoxazole derivatives (10a-10o) All synthesized indole containing isoxazole derivatives showed significant in vitro sPLA2-
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inhibitory activity and good binding affinity on crystal structure of sPLA2 (Table 1). Among all tested compounds, 10o showed substantial sPLA2-inhibitory activity with IC50 of 10.23±0.91 µM in vitro and docked on crystal structure of sPLA2 with binding energy of -129.5 Kcal/mol where as
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ursolic acid (positive control) inhibited with IC50 of 12.59±1.03 µM in vitro and docked with binding energy of -128.8 Kcal/mol (Fig. 1 & Table 1). Both in vitro and in silico results
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demonstrated that 10o showed comparable or more sPLA2-inhibitory activity than ursolic acid (positive control). Further in silico studies demonstrated that 10o binds the vicinity of amino acid residues at active site PHE-5, TYR-21, GLY-22, CYS-28, GLY-29, CYS-44, HIS-47, ASP-48, TYR-51, and PHE-98 (Fig. 2). The strong binding affinity and interactions of 10o with sPLA2 may
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be responsible for its sPLA2-inhibitory activity. From the Table 1 it was evident that compounds containing electron donating group such as CH3 on indole ring (R group) and electron withdrawing groups, such as F, CF3, and NO2 on isoxazole phenyl ring (R1, R2 group) are found to have
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remarkable activities, which were further supported by structure activity relationship (SAR) studies.
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When R1 and R2 = H, R= CH3>H>Br.
2.2.2. Antiiflammatory activity of 10o in wistar albino rats In vivo oral anti-inflammatory efficacy of 10o (10 mg/kg body weight) was tested on group of rats using carrageenan model and paw volumes (ml) of treated 10o and ursolic acid (positive control)) and untreated (vehicle control) (Fig. 3). 10o (10 mg/kg body weight) showed 75.67±4.21%, and 76.54±3.1% edema inhibition (maximum) at 3rd h and 4th h, respectively, where as ursolic acid (10 mg/kg body weight) showed 72.97±3.56% and 75.17±5.70% edema inhibition at 3rd h and 4th h, 6
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respectively. 10o exhibited in vivo anti-inflammatory activity wich was comparable to or more than ursolic acid (positive control).
Antiproliferative activity of 10o against breast and prostate cancer cells
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2.2.3.
Antiproliferative effect of 10o was evaluated against MCF-7 breast and DU145 prostate cancer cell lines by MTT assay. Compound 10o showed significant anti-proliferative effect in
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MCF-7 and DU145 cancer cell lines with IC50 of 10.03±0.86 and 14.15±1.2 µM, respectively, whereas doxorubicin (positive control) exhibited anticancer activity with 8.02±0.69 and 4.75±0.35
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µM on respective cancer cell lines (Fig. 4). The results demonstrate that 10o showed comparable antiproliferative effect against MCF-7 breast cancer cells with that of doxorubicin (positive control).
2.2.4. Drug likeness of compounds
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In addition to sPLA2 inhibition studies, the evaluation of druglikeness of compounds according to Lipinski’s rule of five is done which states that, in general, an orally active drug has not more than 5 hydrogen bond donors (OH and NH groups), not more than 10 hydrogen bond
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acceptors (notably N and O), molecular weigh under 500 g/mol, partition coefficient log P less than
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5. Almost all compounds 10a-10o obey lipinsky`s rule and is of druggable nature (Table 2).
3. Discussion
Several sPLA2 inhibitory molecules have been developed as antinflammaotry and anticaner agents by several investigation and some of them under pre-clinical and clinilcal developmental stage. Mayur et al. [17] synthesized azetidin-2-one derivatives wth sPLA2 inhibitory activity. Further studies demonstrated that azetidin-2-one derivatives showed in vivo anti-inflammatory activity in mice. 4-tetradecyloxybenzamidine (PMS815) derivatives were designed and synthesized 7
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with PLA2 inhibitory activity [18]. Among all compounds, 4,5-dihydro-3-(4-tetradecyloxybenzyl)1,2,4-4H-oxadiazol-5-one (PMS1062) exhibited a micromolar IC50 towards GII sPLA2s, while inactive towards GI and GIII PLA2s in vitro and also blocked the GII sPLA2 activities induced by
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lipopolysaccharides and interleukin-6 in HepG2 cell line and no cytotoxicity was observed in LLCPK1 and A549 cell lines [18]. Several indole derivatives have been developed as sPLA2 inhibitors and some of them are entered into clinical trials for various inflammatory diseases [2, 3, 5-7].
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Sadashiva et al. [11] synthesized trimethoxyphenyl isoxazolidine derivatives with sPLA2 inhibitory activities. Indole and isoxazoles are attractive scaffolds for development of novel sPLA2 inhibitors.
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Considering this background, in the present study, we have synthesized indole containing isoxazole derivatives and evaluated for their sPLA2 inhibitory activity. Among all compounds, 10o showed substantial sPLA2 inhibitory activity. In in vivo anti-inflammatory studies, 10o exhibited antiinflammatory activity wich was comparable to or more than ursolic acid (positive control). In
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anticancer studies, 10o showed comparable antiproliferative effect against MCF-7 breast cancer cells with that of doxorubicin (positive control). Moreover all compounds including 10o obey Lipinsky`s rule of 5 and can be druggable. Further detailed investigations are needed to develop
and in vivo studies. Conclusions
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4.
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sPLA2-inhibitory 10o as antiinflammatory and anticancer agent upon encouraging resutls of in vitro
In the present study, a series of novel indole containing isoxazole derivatives (10a-o) were synthesized and showed significant sPLA2-inhibitory activities. Remarkable sPLA2 inhibition activities were observed when electron donating group like -CH3 on indole ring and electron withdrawing groups like -F, and -CF3 on phenyl ring of isoxazole were present. Among all compounds, 10o showed substantial sPLA2-inhibitory activity. 10o also showed significant in vivo anti-inflammatory activity in carrageenan-induced rat paw edema model as well as good 8
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antiproliferative effect against breast MCF-7 and prostate DU145 cancer cells. Moreover, compounds 10a-10o obey Lipinsky`s rule of 5 and can be druggable. The in vitro, in silico and in vivo results of this study are encouraging and warrant further investigations to develop sPLA2-
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inhibitory compound 10o as novel therapeutic agent for inflammatory disorders and oncologic
5.
Experimental
5.1.
Chemistry
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diseases.
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5.1.1. General information
All chemicals used for the synthesis were of reagent grade and procured from SigmaAldrich, St. Louis, MO, USA. 1H and
13
C NMR spectra were recorded on AS 400 MHz Varian
NMR spectrometer using TMS as an internal standard. IR spectra were recorded by using Perkin-
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Elmer Spectrum 100 Series FT-IR spectrometer. Mass spectra were recorded on Agilent 1200 Series LC/MSD VL system. Melting points were determined by using Buchi melting point B-545 instrument and are uncorrected. All the reactions were monitored by thin layer chromatography
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(TLC) using percolated silica 60 F254, 0.25 mm aluminum plates (Merck). The crude compounds were purified by column chromatography using silica gel (100-200 mesh) and gradient (0-20%)
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ethyl acetate in hexane as the eluent system.
5.1.2. General Procedure for the synthesis of 9(a-o) To the stirred mixture of substituted indole (8(a-c), 0.50 g, 2.0 mmol) and substituted isoxazole amine (5(a-e), 0.35 g, 2.0 mmol) in methanol (5.0 ml) was added 4A° molecular sieves (0.50 g) and catalytic amount of acetic acid. The above reaction mixture was allowed to stir for 1h at room temperature. followed by the portion wise addition of sodium cyanoborohydride (0.32 g, 9
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5.0 mmol) was added portionwise at room temperature and the reaction mixture was allowed to stir for 16 h. Solvent was removed under reduced pressure and then diluted with dichloromethane, which was washed with 10% sodium bicarbonate solution. The obtained DCM layer was dried over
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sodium sulphate and distilled under vacuum to obtain crude 9(a-o), which was further purified by
in 80% yield.
5.1.3. General Procedure for the synthesis of 10(a-o)
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column chromatography using ethyl acetate and hexane mixture (80:20) to obtain pure compounds
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To a solution of compound 9(a-o) (0.5 g, 1.2 mmoles) in ethyl acetate (5.0 ml) was added HCl in ethyl acetate (20%) by drop wise manner and stirred for 16h. The so obtained solid was collected by filtration and washed with ethyl acetate, dried under vacuum to obtain compound 10(a-
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o) in 63 – 79 % yield.
5.1.3.1. N-((1H-indol-3-yl)methyl)(3-phenylisoxazol-5-yl)methanamine hydrochloride(10a) Yield: 79 %; light orange solid; m.p. 185 – 187 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 4.45
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(d, 4H, J = 10 Hz), 7.09 (dd, 1H, , J = 1.2 Hz, J = 8 Hz), 7.16 (dd, 1H, , J = 1.2 Hz, J = 8.0 Hz), 7.21 (s, 1H), 7.44 (d, 1H, J = 8.0 Hz) 7.58 – 7.52 (m, 3H), 7.64 (d, 1H, J = 2.8 Hz), 7.78 (d, 1H, J =
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8.0 Hz) 7.90 – 7.80 (m, 2H), 9.92 (s, 2H), 11.47 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 40.3, 41.8, 104.5, 104.7, 112.2, 119.1, 119.6, 122.1, 127.0 (2C), 127.2, 128.3, 128.5, 129.7 (2C), 131.0, 136.4, 162.5, 165.34; IR (KBr) cm-1 : 3645, 3301, 3116, 2948, 2730, 2603. 2422, 1709, 1612, 1579, 1458, 1409, 1367, 1344, 1279, 1235, 1180, 1097, 1018, 947, 920, 825, 768, 734, 690; MS (ESI) m/z [M-H]-:338.4(HCl salt); Anal. Calcd. for C19H18ClN3O; C, 67.15; H, 5.34; Cl, 10.43; N, 12.37; O, 4.71 %. Found C, 67.45; H, 5.34; N, 12.33 %.
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5.1.3.2. N-((5-bromo-1H-indol-3-yl)methyl)(3-phenylisoxazol-5-yl)methanamine
hydrochloride
(10b) Yield: 70 % , off white solid; m.p. 191 - 193 ˚C ; 1H NMR (400 MHz, DMSO-d6) δ: 4.46 (d,
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4H, J = 15.6 Hz), 7.25 – 7.24, (m, 1H) 7.27 (d, 1H, , J = 4 Hz), 7.41 (s, 1H, , J = 8 Hz), 7.60 – 7.50 (m, 3H) 7.66 (d, 1H, , J = 4 Hz), 7.86 (dd, 2H, , J = 4 Hz, J = 8 Hz), 8.04 (d, 1H, J = 1.6 Hz) 9.78(s, 2H), 11.65 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 40.5, 41.6, 104.5, 104.6, 112.4, 114.2, 121.8,
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124.5, 124.6, 127.0, 128.5, 129.0, 129.7 (2C), 130.0, 130.9, 135.1, 162.5, 165.3; IR (KBr) cm-1 : 2947, 2809, 2697, 1695, 1604, 1526, 1466, 1388, 1348, 1281, 1230, 1172, 1093, 1039, 1017, 988,
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953, 905, 853, 823, 802,740; MS (ESI) m/z [M-H]-: 380.2 Anal. Calcd. for C19H17BrClN3O; C, 54.50; H, 4.09; Br, 19.08; Cl, 8.47; N, 10.04; O, 3.82 %. Found C, 54.32; H, 4.27; N, 10.76 %.
5.1.3.3. N-((5-methyl-1H-indol-3-yl)methyl)(3-phenylisoxazol-5-yl)methanamine
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(10c) :
hydrochloride
Yield: 65 %; light pink solid, m.p. 208 - 210 ˚C ;1H NMR (400 MHz, DMSO-d6) δ: 2.42 (s, 3H), 4.45 (d, 4H, J = 20.8 Hz), 6.96 (dd, 1H, , J = 4.0 Hz, J = 8.0 Hz), 7.26 (s, 1H), 7.32 (d, 1H, , J 13
C NMR (100
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= 8.0 Hz) 7.57 – 7.53 (m, 5H), 7.87 – 7.84, (m, 2H), 9.87 (s, 2H), 11.34 (s, 1H);
MHz, DMSO-d6) δ: 21.7, 40.5, 41.8, 104.0, 104.5, 111.9, 118.6, 123.6, 124.6, 127.0, 127.4, 128.1,
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128.3, 128.5, 129.7 (2C), 130.9, 134.7, 162.5, 165.3; IR (KBr) cm-1 : 2948, 2809, 2697, 1695, 1604, 1526, 1466, 1388, 1348, 1281, 1230, 1172, 1094, 1017, 953, 905, 850, 824, 802,740; MS (ESI) m/z [M-H]-: 351.9 (HCl salt); Anal. Calcd. for C20H20ClN3O; C, 67.89; H, 5.70; Cl, 10.02; N, 11.88; O, 4.52 %. Found C, 67.67; H, 5.83; N, 11.74 %.
5.1.3.4. N-((1H-indol-3-yl)methyl)(3-p-tolylisoxazol-5-yl)methanamine hydrochloride (10d)
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Yield: 70 %; off white solid; m.p. 191 – 193 ˚C;1H NMR (400 MHz, DMSO-d6) δ: 2.42 (s, 3H), 4.45 (d, 4H, J = 20.0 Hz), 7.09 (t, 1H, J = 8.0 Hz), 7.14 (t, 1H, J = 8.0 Hz), 7.22 (s, 1H), 7.34 (d, 2H, J = 8.0 Hz) 7.44 (d, 1H, J = 8.0 Hz) 7.63 (d, 1H, J = 2.0 Hz), 7.74 (d, 2H, J = 8.0 Hz) 7.78
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(d, 1H, J = 8.0 Hz), 9.95(s, 2H), 11.48 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 21.4, 40.5, 41.8, 104.3, 104.6, 112.2, 119.1, 119.6, 122.0, 124.5, 125.7, 126.91, 127.2, 128.3, 130.2 (2C), 136.3, 140.7, 162.4, 165.1; IR (KBr) cm-1 : 3556, 3306, 3112, 2936, 2722, 2608, 2411, 1725, 1613, 1571,
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1464, 1357, 1268, 1224, 1182, 1094, 1016, 986, 946, 816, 754; MS (ESI) m/z [M-H]-; 351.9 (HCl salt); Anal. Calcd. for Anal. Calcd. for C20H20ClN3O; C, 67.89; H, 5.70; Cl, 10.02; N, 11.88; O,
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4.52 %. Found C, 67.67; H, 5.91; N, 11.92 %.
5.1.3.5. N-((5-bromo-1H-indol-3-yl)methyl)(3-p-tolylisoxazol-5-yl)methanaminehydrochloride (10e)
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Yield: 70 %; off white solid; m.p. 197 - 199 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 2.42 (s, 3H), 4.43 (d, 4H, J = 12.0 Hz), 7.21 (s, 1H), 7.25 (dd, 1H, J = 1.6 Hz, J = 8.4 Hz), 7.35 (d, 2H, J = 8.0Hz), 7.41 (d, 1H, J = 8.0 Hz), 7.65 ( d, 1H, J = 4Hz),7.75 (d, 2H, J = 8.0 Hz), 8.04 (d, 1H, J = 4
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Hz) 9.81 (s, 2H), 11.66 (s, 1H); 13C NMR (100 MHz, DMSO) δ: 21.4, 40.6, 41.6, 104.3, 104.6, 112.4, 114.2, 121.8, 124.5, 124.6, 125.7, 126.9, 129.0, 129.9, 130.2 (2C), 135.1, 140.7, 162.4,
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165.1; IR (KBr) cm-1 : 2917, 2756, 2601, 2420, 1742, 1612, 1565, 1455, 1420, 1336, 1245, 1217, 1170, 1100, 1014, 914, 862, 845, 799, 723; MS (ESI) m/z [M-H]- 429.8 (HCl salt) ; Anal. Calcd. for C20H19BrClN3O; C, 55.51; H, 4.43; Br, 18.46; Cl, 8.19; N, 9.71; O, 3.70%. Found C, 55.21; H, 4.73; N, 9.49 %.
5.1.3.6.
N-((5-methyl-1H-indol-3-yl)methyl)(3-p-tolylisoxazol-5-yl)methanaminehydrochloride (10f) 12
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Yield: 63 %; off white solid; m.p. 185 - 187 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 2.49 (s, 6H), 4.43 (d, 4H, J = 16 Hz), 6.97 (dd, 1H, J = 1.6 Hz, J= 8.0 Hz), 7.21 (s, 1H), 7.37 – 7.32 (m, 3H), 7.56 – 7.53 (m, 2H), 7.75 (d, 2H, J = 8.0 Hz), 9.79 (s. 2H), 11.32 (s, 1H);
13
C NMR (100 MHz,
RI PT
DMSO-d6) δ: 21.4, 21.7, 40.5, 41.8, 104.0, 104.4, 111.9, 118.6, 123.6, 124.6, 125.7, 126.9, 127.4, 128.1, 128.3, 130.2 (2C), 134.7, 140.7, 162.4, 165.1; IR (KBr) cm-1 : 3118, 2955, 2718, 2609, 2440, 1709, 1612, 1593, 1463, 1368, 1344, 1282, 1233, 1174, 1091, 1020, 953, 925, 808, 769, 742, 691;
M AN U
N, 11.42; O, 4.35%. Found C, 68.46; H, 6.23; N; 11.53 %.
SC
MS (ESI) m/z [M-H]- 367.1 (HCl salt) ;Anal. Calcd. for C21H22ClN3O; C, 68.56; H, 6.03; Cl, 9.64;
5.1.3.7. N-((1H-indol-3-yl)methyl)(3-(4-methoxyphenyl)isoxazol-5-yl)methanaminehydrochloride (10g)
Yield: 70 % ; off white solid; mp 184 – 186 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 3.82 (s,
TE D
3H), 4.43 (s, 4H), 7.10 (m, 3H), 7.18 (m, 2H), 7.44 (d, 1H, J = 8.0 Hz), 7.63 (d, 1H, J = 2.4 Hz), 7.78 (m, 3H), 9.91 (s, 2H), 11.47 (s, 1H,); 13C NMR (100 MHz, DMSO-d6) δ: 40.5, 41.8, 55.7, 104.2, 104.6, 112.2, 115.1 (2C), 119.1, 119.6, 120.8, 122.0, 127.2, 128.3, 128.5 (2C), 136.4, 161.3,
EP
162.1, 164.9; IR (KBr) cm-1 : 3440, 3131, 2936, 2893, 2746, 2611, 2421, 1614, 1583, 1530, 1457, 1436, 1304, 1252, 1181, 1102, 1029, 982, 933, 824, 746; ; MS (ESI) m/z [M-H]- 367.9(HCl salt) ;
AC C
Anal. Calcd. for C20H20ClN3O2; C, 64.95; H, 5.45; Cl, 9.59; N, 11.36; O, 8.65 %. Found C, 65.11; H, 5.35; N, 11.46 %.
5.1.3.8. N-((5-bromo-1H-indol-3-yl)methyl)(3-(4-methoxyphenyl)isoxazol-5-yl)methanamine hydrochloride (10h) Yield: 73 %; off white solid; mp 202 – 204 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 3.87 (s, 3H), 4.44 (d, 4H), 7.07 (d, 2H, J = 4.8 Hz) 7.18 (s, 1H), 7.26 (dd, 1H, J = 2.0 Hz, J = 8.4 Hz), 7.41 13
ACCEPTED MANUSCRIPT
(d, 1H, J = 8.8 Hz), 7.66 (d, 1H, J = 4.0 Hz), 7.81 (d, 2H, J = 4.0 Hz), 8.04 ( d, 1H, J = 4.0 Hz), 9.79 (d, 1H), 11.66 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ:40.2, 41.6, 55.8, 104.2, 104.6, 112.4, 114.2, 115.0 (2C), 120.8, 121.8, 124.5, 128.5 (2C), 129.0, 129.9, 135.1, 161.3, 162.1, 164.9; IR
RI PT
(KBr) cm-1 : 3208, 3001, 2939, 2743, 2565, 2409, 1613, 1564, 1532, 1432, 1394, 1299, 1259, 1178, 1108, 1030, 979, 948, 904, 886, 838, 802; MS (ESI) m/z [M-H]- 410.5 ;
Anal. Calcd. for
C20H19BrClN3O2; C, 53.53; H, 4.27; Br, 17.81; Cl, 7.90; N, 9.36; O, 7.13 %. Found C, 53.73; H,
SC
4.01; N, 9.76 %.
M AN U
5.1.3.9. (3-(4-methoxyphenyl)isoxazol-5-yl)-N-((5-methyl-1H-indol-3-yl)methyl)methanamine hydrochloride (10i)
Yield: 65 %; off white solid; mp 177 – 179 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 2.39 (s, 3H), 3.82 (s, 3H), 4.41(d, 4H), 6.98 (d, 1H, J = 8.0 Hz), 7.08 (d, 2H, J = 8.8 Hz), 7.18 (s, 1H),
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7.32 ( s, 1H, J = 8.4 Hz), 7.55 – 7.53 (m, 2H), 7.80 (d, 2H, J = 8.8 Hz), 9.75 (s, 2H), 11.31 (s, 1H); 13
C NMR (100 MHz, DMSO-d6) δ: 21.7, 40.5, 41.8, 55.8, 104.1, 104.2, 111.9, 115.0, 118.6, 120.8,
123.6, 124.6, 127.4, 128.1, 128.3, 128.5 (2C), 134.7, 161.3, 162.1, 164.9; IR (KBr) cm-1 : 3125,
EP
3080, 2836, 2709, 1699, 1615, 1576, 1529, 1469, 1387, 1350, 1283, 1257, 1175, 1090, 1020, 919, 900, 837, 808; MS (ESI) m/z [M-H]- 381.9 (HCl salt); Anal. Calcd. for C21H22ClN3O2 C, 65.71; H,
AC C
5.78; Cl, 9.24; N, 10.95; O, 8.34 %. Found C, 65.91; H, 5.58; N, 10.97 %.
5.1.3.10.
N-((1H-indol-3-yl)methyl)(3-(4-nitrophenyl)isoxazol-5-yl)methanaminehydrochloride (10j)
Yield: 70 %; off white solid; mp 212 – 214 ˚C 0C;; 1H NMR (400 MHz, DMSO-d6) δ: 4.48 (d, 4H, J = 28 Hz), 7.18 – 7.08 (m, 2H), 7.39 (s, 1H), 7.44 (d, 1H, J = 8.0 Hz), 7.60 ( d, 1H, J = 4.0 Hz),7.77 (d, 1H, J = 8.0 Hz), 8.17 (d, 2H, J = 4.0 Hz), 8.39 ( d, 2H, J = 8.0Hz), 9.77 ( s, 2H), 14
ACCEPTED MANUSCRIPT
11.43 (s, 1H);
13
C NMR (100 MHz, DMSO-d6) δ: 40.5, 42.03, 104.8, 104.9, 112.2, 119.1, 119.6,
122.1, 124.9 (2C), 127.1, 128.2, 128.4 (2C), 134.4, 136.4, 148.9, 161.2, 166.31; IR (KBr) cm-1 : 3440, 3125, 2946, 2780, 2602, 2422, 1695, 1600, 1522, 1462, 1433, 1347,1290, 1170, 1103, 1015,
RI PT
938, 833, 858, 754; MS (ESI) m/z [M-H]- 383.2 (HCl salt) ; Anal. Calcd. for C19H17ClN4O3; C, 59.30; H, 4.45; Cl, 9.21; N, 14.56; O, 12.47 %. Found C, 59.21; H, 4.35; N, 12.36 %.
SC
5.1.3.11. N-((5-bromo-1H-indol-3-yl)methyl)(3-(4-nitrophenyl)isoxazol-5-yl)methanamine hydrochloride (10k)
M AN U
Yield: 79%; off white solid; mp 188 – 190 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 4.47 (d, 4H, J = 24 Hz), 7.25 (dd, 1H, J = 1.6 Hz, J = 8.8 Hz), 7.43 – 7.40 (m, 2H), 7.69 ( d, 1H, J = 2.0 Hz), 8.04 (s, 1H), 8.15 (d, 2H, J = 8.0 Hz), 8.39 (d, 2H, J = 8.0 Hz), 10.01 (s, 2H), 11.70 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 40.5, 41.7, 104.6, 104.9, 112.4, 114.2, 121.8, 124.5, 124.9 (2C),
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128.4 (2C), 129.0, 129.9, 134.4, 135.1, 161.1, 166.2 (2C); IR (KBr) cm-1 : 3318, 3099, 2943, 2722, 1601, 1522, 1451, 1347, 1290, 1231, 1169, 1111, 1045, 984, 923, 886, 855, 806, 755, 698; MS (ESI) m/z [M-H]- 425.5 ; Anal. Calcd. C19H16BrClN4O3;C, 49.21; H, 3.48; Br, 17.23; Cl, 7.65; N,
EP
12.08; O, 10.35 %.Found C, 49.39; H, 3.23; N, 12.01 %.
AC C
5.1.3.12. N-((5-methyl-1H-indol-3-yl)methyl)(3-(4-nitrophenyl)isoxazol-5-yl)methanamine hydrochloride (10l)
Yield: 70 % ;off white solid; mp 198 – 200˚C; 1H NMR (400 MHz, DMSO-d6) δ: 2.40 (s,
3H), 4.45 (d, 4H, J = 37.2 Hz), 6.98 (d, 1H, J = 8.0 Hz), 7.32 (d, 1H, J = 8.0 Hz), 7.39 (s, 1H), 7.54 (s, 2H), 8.16 (d, 2H, J = 8.0 Hz), 8.39 (d, 2H, J = 8.0 Hz), 9.70 (s, 2H), 11.29 (s, 1H);
13
C NMR
(100 MHz, DMSO-d6) δ: 21.7, 40.3, 40.6, 104.1, 104.9, 118.6, 123.7, 124.9 (2C), 127.3, 128.1, 128.27, 128.4 (2C), 134.4, 134.7, 148.9, 161.6 , 166.2 (2C).;IR (KBr) cm-1 :3399, 3117, 2776, 1703, 15
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1598, 1556, 1526, 1431, 1349, 1291, 1247, 1217, 1169, 1089, 1039, 1016, 975, 856, 836, 808, 758, 714, 699; MS (ESI) m/z [M-H]- 397.2 (HCl salt) ; Anal. Calcd. for C20H19ClN4O3;C, 60.23; H, 4.80;
RI PT
Cl, 8.89; N, 14.05; O, 12.03%.Found C, 60.43; H, 4.70; N, 14.34 %.
5.1.3.13. N-((1H-indol-3-yl)methyl)(3-(4-fluoro-3-(trifluoromethyl)phenyl)isoxazol-5yl)methanamine hydrochloride (10m)
SC
Yield: 70 %; off white solid; mp 165 - 167 ˚C; 1HNMR (400 MHz, DMSO-d6) δ: 4.46 (d, 4H, J = 18.8 Hz), 7.09 (t, 1H, J = 4.0Hz), 7.16 (t, 1H, J = 8.0Hz), 7.43 ( t, 2H, J = 8.0Hz), 7.63 (d,
M AN U
1H, J = 4.0Hz), 7.78 – 7.71 (m, 2H), 8.20 (dd, 1H, J = 4.0Hz, J = 8.0Hz), 8.28 – 8.25 (m, 1H), 9.92 (s, 2H), 11.45 (s, 1H);13C NMR (100 MHz, DMSO-d6) δ: 40.5, 41.8, 104.6, 104.8, 112.2, 117.9, 118.1, 118.9, 119.1, 119.6, 121.3, 122.0, 124.0, 125.7, 127.2, 128.3, 134.1, 136.4, 160.7, 165.9; IR (KBr) cm-1 : 3442, 3127, 2890, 2782, 2610, 2421, 1614, 1522, 1457, 1430, 1384, 1325, 1272, 1245,
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1140, 1105, 1080, 1053, 1015, 968, 937, 907, 833, 750; MS (ESI) m/z [M-H]- 424.4 (HCl salt) ; Anal. Calcd. for C20H16ClF4N3O; C, 56.41; H, 3.79; Cl, 8.33; F, 17.85; N, 9.87; O, 3.76%.Found C,
EP
56.66; H, 3.98; N, 9.66%.
5.1.3.14. N-((5-bromo-1H-indol-3-yl)methyl)(3-(4-fluoro-3-(trifluoromethyl)phenyl)isoxazol-5-
AC C
yl)methanamine hydrochloride (10n) Yield: 73%; off white solid; mp 176 – 178 ˚C;1H NMR (400 MHz, DMSO-d6) δ: 4.47 (d, 4H, J = 23.6 Hz), 7.26 (d,1H, J = 8.0Hz), 7.35 – 7.45 ( d, 2H, J = 4.0Hz), 7.65 ( d, 1H, J = 4.0Hz), 7.75 (t, 1H, J = 8.0Hz), 8.02 ( d, 1H, , J = 4.0Hz), 8.20, (d, 1H, J = 4.0Hz), 8.29 – 8.25 (m, 1H), 9.80 (s, 2H), 11.64 (s, 1H);13C NMR (100 MHz, DMSO-d6) δ: 40.5, 41.6, 104.6, 104.8, 112.4, 114.2, 118.0, 118.9, 119.1, 121.3, 121.8, 124.0, 124.5, 125.7, 129.0, 130.0, 134.0, 135.1, 160.7, 165.9;IR (KBr) cm-1 : 3347, 3111, 2973, 2732, 2606, 2455, 1611, 1524, 1458, 1431, 1400, 1377, 1321, 1270, 1249, 16
ACCEPTED MANUSCRIPT
1170, 1132, 1081, 1053, 968, 945, 905, 871, 835, 798; MS (ESI) m/z [M-H]- 504.2 (HCl salt);Anal. Calcd. for C20H15BrClF4N3O; C, 47.60; H, 3.00; Br, 15.83; Cl, 7.02; F, 15.06; N, 8.33; O,
5.1.3.15.
RI PT
3.17 %.Found C, 47.89; H, 2.71; N, 8,23 %.
(3-(4-fluoro-3-(trifluoromethyl)phenyl)isoxazol-5-yl)-N-((5-methyl-1H-indol-3-
yl)methyl)methanamine (10o)
SC
Yield: 70%; off white solid; mp 155 -156 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 2.39 (s, 3H), 4.40 (s, 2H), 4.48 (s, 2H), 6.98 (d, 1H, J = 8.0 Hz), 7.32 ( d, 1H, J = 8.0 Hz), 7.40 ( s, 1H),
M AN U
7.50 – 7.60 (m , 2H), 7.75 ( t, 1H, J = 4.0 Hz), 8.21, (d, 1H, J = 8.0 Hz), 8.27 (m, 1H), 9.75 (s, 2H), 11.31 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 21.7, 40.5, 41.8, 104.1, 104.8, 111.9, 118.6, 118.9, 119.1,121.5, 123.7,124.5, 125.7, 127.4, 128.1, 128.3, 130.0, 134.1, 134.8, 160.73, 165.9; IR (KBr) cm-1 : 3347, 2949, 2778, 2592, 1742, 1693, 1626, 1523, 1429, 1400, 1321, 1266, 1168, 1134, 1079,
TE D
1054, 833, 771, 704, 679; MS (ESI) m/z [M-H]- 438.3 (HCl salt);Anal. Calcd. for C21H18ClF4N3O; C, 57.35; H, 4.12; Cl, 8.06; F, 17.28; N, 9.55; O, 3.64% Found C, 57.50; H, 4.42; N, 9.35 %.
Pharmacology
EP
5.2.
5.2.1. Chemicals and reagents
AC C
Phosphate buffered saline (PBS), RPMI 1640, fetal calf serum (FCS), pencillin and streptomycin were purchased from Gibco BRL (Life Technologies) Rockville, MD, USA). MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], doxorubicin, λ-carrageenan and ursolic acid were purchased from Sigma-Aldrich Chemical Company, (St. Louis, MO, USA).
5.2.2. In vitro secretory phospholipase A2 assay
17
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sPLA2 assay was performed using type IIA sPLA2 enzyme activity kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. The reaction mixture contained 10 µl of sPLA2, 10 µl of inhibitor (test wells only), 200 µl substrate and was incubated for 15 min.
RI PT
Then, 10 µl of 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) was added to develop the colour and read at a wavelength of 415 nm. sPLA2 enzyme hydrolysed the thioester bond at the sn-2 position of diheptanoyl thio-PC (substrate), free thiols were released and reacted with DTNB to form 5-thio-2-
SC
nitrobenzoic acid, which had absorbance at 415 nm. The control wells contained only sPLA2, substrate and DTNB. Urosolic acid was used as a positive control [19]. The percent inhibition of
M AN U
enzyme activity was calculated using the following formula:
% inhibition = [(Absorbance of control – Absorbance of test/Absorbance of control) ×100].
5.2.3. In vivo anti-inflammatory activity by carrageenan-induced rat paw edema model
TE D
Carrageenan-induced paw edema model is the most widely used for the evaluation of antiinflammatory activity [12]. Male wistar albino rats weighting 150 - 200 g were obtained from M/S Mahavir Enterprises (Hyderabad, Telangana, India). The animals were housed under standard
EP
conditions (temperature of 22±100C with an alternating 12-h light-dark cycle and relative humidity of 60±5%, before and also during the experiment. The animals were fed with standard laboratory which
was
purchased
from
M/S
Rayans
Biotechnology Pvt.
Ltd.
AC C
diet,
(Hyderabad, Telangana, India). During the experiment, the rats were allowed water and food ad libitum. Animal experimental protocol was approved by institutional animal ethical committee (IAEC no. GU/GIS/IAEC/PROTOCOL NO.03/2013). Animal experiments were conducted according to CPCSEA guidelines. The animals were divided into three groups (n = 3-6). Group I served as control and received saline at 1 ml/kg body weight (b.w) per os (p.o). Group II served as standard, received ursolic acid 18
ACCEPTED MANUSCRIPT
at a dose of 5 mg/kg b.w, p.o. and group III served as test and received synthesized compound(s) at a dose of 5 mg/kg b.w, p.o, respectively. The animals were treated with synthesized compounds and ursolic acid before 1 h to inject
RI PT
(subcutaneous) 0.1 ml of 1% λ-carrageenan solution with saline into sub-plantar region of left hind paw of each rat. The right hind paw of same rat was treated with 0.1 ml of saline alone in manner as control. Before induction of edema, the dorsiventral thickness of both the paws of each was
SC
measured using mercury displaced glass plethysmometer and also the measurements were taken at 1st, 2nd, 3rd, 4th and 6th hour after carrageenan injection. The results are expressed in percent edema
M AN U
inhibition. Ursolic acid was used as a positive control. 5.2.4. Anticancer studies 5.2.4.1. Cancer cell lines
MCF-7 breast cancer cell line was purchased from National Cancer Institute (Frederick,
TE D
MD, USA). Human prostate carcinoma cell line DU145 was obtained from American Type Culture Collection (Manassas, VA, USA). These cancer cells were maintained in RPMI medium supplemented with 10% FCS, 100 units pencillin and 100 µg of streptomycin per ml of medium.
EP
The cells were maintained in 95% humidity with CO2 at 37 0C temperature.
AC C
5.2.4.2. Cell proliferation by MTT assay MTT assay was performed to assess the antiproliferative activity of compounds [20]. Cancer cells were treated with isolated compound at various concentrations for 48 h. 20 µl of freshly prepared MTT reagent was added to cells and incubated at 37 °C for 2 h. The supernatant growth medium was removed and replaced with DMSO (100 µl) to dissolve the formazan crystals. The optical density (absorbance) was read at 570 nm with reference wavelength of 620 nm. 5.2.5.
In silico studies 19
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5.2.5.1. Drug like properties The rule was formulated by Christopher A. Lipinski in 1997 [21]. The rule describes the molecular properties important for a drug’s pharmacokinetics in the human body, including their
RI PT
absorption, distribution, metabolism and excretion (ADME). The rule is important for drug development where a pharmacologically active lead structure is optimized stepwise for increased activity and selectivity, as well as druglike properties. Molinspiration tool used to demonstrate the
SC
druglike properties of compounds [22]. 5.2.5.2. Molecular docking studies
M AN U
X-ray crystal structures of human group IIA sPLA2 (PDB ID: 1DB5) protein was obtained from Protein Data Bank and used in docking studies. Co-cystalized ligands and water molecules were removed from target protein using Argus lab. Ligands are prepared using chemoffice (Cambridge, UK). Energy minimization was done using molecular mechanics. The minimization
TE D
was executed until root mean square value reached below 0.001 Kcal/mol. Such energy minimized ligands and receptors were used for docking studies using GEMDOCK (Generic Evolutionary Method for molecular DOCKing), which is a generic evolutionary method with an empirical
EP
scoring function for molecular docking (protein–ligand docking) [12, 23, 24]. A population size of 300 with 70 generations and 3 solutions were used in docking accuracy setting. PyMol was used for
AC C
better visualization of interactions [23-25].
Conflict of Interest: The authors declare no conflict of interest.
Acknowledgements SRP and GR are grateful to Sarada Villas College Mysore for the support and encouragement to higher education. SRP and GR also thankful to CRD Department of Chemistry, PRIST University, 20
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Thanjavur-613403, India. NSY are thankful to GITAM University for providing research facilities. NSY expresses his sincere gratitude to Dr. M.V.V.S. Murthy, Pesident, GITAM University for his support and encouragement by providing necessary research facilities. NSY is very much thankful
RI PT
to Prof. G. Subrahmanyam, Prof. N. Lakshmana Das and Prof. Ch. Ramakrishna for their encouragement and motivation. This work was partly supported by the Russian Sciene Foundation
SC
(www.rscf.ru, Российский Научный Фонд, grant number 14-23-00160 for 2014-2016).
References
E.B. John and A.D. Edward, J. Lipid Res., 50 (2009) S237–S242.
[2]
E.A. Dennis, J. Cao, Y.H. Hsu, V. Magrioti, G. Kokotos, Chem. Rev., 111 (2011) 6130-
M AN U
[1]
6185. [3]
N.S. Yarla, A. Bishayee, L. Vadlakonda, R. Chintala, G.R. Duddukuri, P. Reddanna, D.S.
TE D
Kaladhar. Curr Drug Targets, 16 (2015) 10.2174/1389450116666150727122501 D.Q. Nhat, D.A. Robert, S.C. Bria, Biochem. Pharmacol., 90 (2014) 338–348.
[5]
R.W. Schevitz, N.J. Bach, D.G.Carlson, Nat. Struct. Biol., 2 (1995) 458-465.
[6]
E Abraham, C Naum, V Bandi, Crit. Care Med., 31 (2003) 718-728.
[7]
J.D. Bradley, A.A. Dmitrienko, A.J. Kivitz,. J. Rheumatol., 32 (2005) 417-423.
[8]
http://clinicaltrials.gov/, Identifier: NCT00743925.
[9]
S.J. Nicholls, J.P. Kastelein, G.G.Schwartz, The VISTA-16 Randomized Clinical Trial.
AC C
EP
[4]
JAMA, 311 (2014) 252-262. [10]
Y. Pradeepkumar, M. Ruthu, C.C. Madhusudhana, G. Prasanthi, S.R.V. Jaya, J. Global Trends Pharm. Sci., 2(1) (2011) 55- 62.
[11]
M.P. Sadashiva, A. Nataraju, H. Mallesha, R. Rajesh, B.S. Vishwanath, K.S. Rangappa, Int. J. Mol. Med., 16(5) (2005) 895-904. 21
ACCEPTED MANUSCRIPT
[12]
N.S. Yarla, A. Rajaram, M. Basha, R. Azad, M. Basha, A. Rajack, D.S. Kaladhar, B.K. Allam, R.R. Pragada, K.N. Singh, S.K. Kadiri, R. Pallu, U. Parimi, A. Bishayee, G.R. Duddukuri. Anticancer Agents Med. Chem., 15(8) (2015) 1066 - 1077 S. Yamashita, J. Yamashita, M. Ogawa, Br. J. Cancer, 69 (1994) 1166-1170.
[14]
J. Jiazhong, L.N. Blake, R. Jeremy, Am. J. Pathol., 160 (2002) 667–671.
[15]
A.S. Paniraj. R.K.M Lokanath. Bulgarian Chem. Comm., 41(1) (2009) 80–83.
[16]
V.Y. Maria, W. Jizhen, C. Michael, L.R. Lijun, J.W. Duncan J. Med. Chem., 54(3) (2011)
SC
RI PT
[13]
765–781.
C.Y. Mayur, K.M. Satish, M. Salahuddin, S.K.S Math, K.N. Thimmaiah, B.S. Vishwanath.
M AN U
[17]
Lett. Drug. Des. Discov., 2(4) (2005) 307 – 310. [18]
L. Assogba, A. Ahamada-Himidi, N. M Habich, D. Aoun, L. Boukli, F. Massicot, C.M. Mounier, J. Huet, A.Lamouri, J.E. Ombetta, J.J. Godfroid, C.Z. Dong, F. Heymans. Eur J.
[19]
A. Nataraj, G.C.D Raghavendra. R. Rajesh, B.S. Vishwanath, Cur. Topics in Med. Chem., 7(8) (2007) 801-809.
Y.L. Murthy, K.P. Suhasini, A.S.Pathania, S. Bhushan, Y.S. Nagendra, Eur. J. Med.
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[20]
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Med. Chem., 40(9) (2005) 850–861.
Chem., 62 (2013) 545-55.
C.A. Lipinski, Drug Dis. today: Techn, 1(4) (2004), 337–341.
[22]
M.Q. Zhang, Mol. Biol. 803 (2012) 297-307.
[23]
M.Y. Jinn. J. Comput Chem., 25 (2004) 843–857.
[24]
J.M Yang, C.C Chen, GEMDOCK: A Generic Evolutionary Method for Molecular
AC C
[21]
Docking. Proteins., 55 (2004) 288-304. [25]
D. Seeliger, B.L.DeGroot, Ligand J. Comput. Aided Mol. Des., 24 (2010) 417-422.
22
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Legends to figures: Figure 1. In vitro sPLA2 inhibitory activity of 10o and Ursolic acid (positive control). Compound 10o dose-dependley inhibited sPLA2 activity with IC50 of 10.23±0.91 µM where as ursolic acid
RI PT
(positive control) inhibited with IC50 of 12.59±1.03 µM.
Figure 2. Molecular docking studies of 10o on human GIIA sPLA2 crystal protein. (A) Binding orientation of 10o on crystal structure of human GIIA PLA2 (PDB ID: 1DB5) in ribbon model. (B).
M AN U
GLY-29, CYS-44, HIS-47, ASP-48, TYR-51, PHE-98.
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10o binds vicinity of amino acid residues at active site were PHE-5, TYR-21, GLY-22, CYS-28,
Figure 3. Anti-inflammatory activity of 10o. (A) Represents the rat paw edema volumes (ml) of treated (10o and ursolic acid) and untreated at various time intervals (1, 2, 3, 4 and 6 hrs) after carrageenan injection. Experimental results are expressed as mean (n = 3-6) ± standard error mean
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(S.E.M). The difference between experimental groups was compared by one-way analysis of variance (ANOVA). The P value of results less than 0.05 (P˂0.05) versus control was considered as significant (*represents p<0.05, **p<0.001). (B). 10o showed comparable activity with ursolic acid
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injection.
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(positive control) and exhibited maximum edema inhibition between 3rd and 4th h after carrageena
Figure 4. Antiproliferative effect of 10o against MCF-7 breast and DU145 prostate cancer cells. Compound 10o showed significant anti-proliferative effect in MCF-7 breast and DU145 prostate cancer cell lines with IC50 of 10.03±0.86 and 14.15±1.2 µM, respectively, whereas doxorubicin (positive control) exhibited anticancer activity with 8.02±0.69 and 4.75±0.35 µM, respectively.
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OH N
H
Br
O N
i
iii
ii
R2
R2
R1
R2
R1 NH2.HCl
O N iv
5a=R1=H,R2=H, R2
R R
R H
vi
R 1
R
5(a-e)
10c=R=CH3,R1=H,R2=H,
10d=R=H,R1=CH3,R2=H,
2
1
2
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10a=R=H,R =H,R =H,
10b=R=Br,R1=H,R2=H,
1
1
8b=R=Br,
10f=R=CH3,R =CH3,R =H,
10g=R=H,R1=OCH3,R2=H,
10h=R=Br,R1=OCH3,R2=H,
R1
R2
10i=R=CH3,R =OCH3,R =H,
9(a-o)
viii
10m=R=H,R1=F,R2=CF3,
10n=R=Br,R1=F,R2=CF3,
R
R1
N H
R2 10(a-o)
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10l=R=CH3,R1=NO2,R2=H,
N Boc
HN .HCl
10j=R=H,R1=NO2,R2=H,
10k=R=Br,R1=NO2,R2=H,
R
N O
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8a=R=CH3
HN
2
10e=R=Br,R =CH3,R =H,
1
8a=R=H,
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2
8(a-c)
H
N O
H N Boc
O
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R
NH2.HCl
O N
5e=R1=F,R2 =CF3
N Boc 8(a-c)
N H 7
6
4
5d=R1=NO2,R2=H,
O
v N H
R1
5b=R1=CH3,R2=H,
5c=R1=OCH3,R2=H,
R1
R2
R1 3
2
1
5(a-e)
N3
O N
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O
10o=R=CH3,R1=F,R2=CF3,
(i) NH2OH.HCl, NaOH, H2O, rt, (ii) Chloramino-T, Propargyl bromide, EtOH, 60-65 °C, (iii) NaN3, DMF, 60-65 °C, (iv) PPh3, H2O, THF, rt v) POCl3, DMF, rt, (Vi) NaH, Boc anhydride, DMF, rt. (Vii) NaCNBH3, MeOH (Viii) EtOAc in HCl
Scheme 1: Synthesis of indole containing isoxazole derivatives (10 a-o)
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Table 1
In silico analysis
In vitro IC50 (µM)
10a
Dock score (-Kcal/mol) -107.3
30.63±0.91
10b
-109.6
32.49±1.02
10c
-111.8
27.92±0.86
10d
-115.4
22.68±1.13
10e
-118.4
27.32±1.73
10f
-111.3
10g
-112.3
10h
-103.8
10i
-105.6
10j
-108.2
10m 10n
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10o
Ursolic acid (positive control)
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10l
21.55±1.21 18.22±1.42 32.64±2.38 24.55±2.81 25.61±1.72
-103.7
22.35±2.49
-104.1
19.44±2.1
-108.7
21.5±1.52
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10k
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Compound no
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GIIA sPLA2 inhibitory activities of indole containing isoxazole derivatives.
-110.3
17.18±1.43
-129.5
10.23±0.91
-128.8
12.59±1.03
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Table 2 Drug like properties of indole containing isoxazole derivatives. Log P
Non
10a
303.365
3.442
4
10b
382.261
4.227
4
10c
317.392
3.866
4
10d
317.392
3.89
4
10e
396.28
4.675
4
10f
331.41
4.325
4
2
10g
333.39
3.498
5
2
10h
412.287
4.283
5
2
10i
347.418
3.923
5
2
10j
348.362
3.4
7
2
10k
427.258
4.186
7
2
10l
362.389
3.825
7
2
10m
389.352
4.429
4
2
10n
468.248
5.214
4
2
10o
403.379
4.853
4
2
2 2
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2
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NoHnH
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MW
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Compound
2 2
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Highlights:
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A series of new indole containing isoxazole derivatives (10a-10o) is synthesized. These compounds (10a-10o) showed significant sPLA2-inhibition activity. 10o showed potent sPLA2 inhibition activity among other compounds. 10o exhibited in vivo antinflammatory activity in mice. 10o showed in vitro anticancer activity against MCF-7 breast and DU145 prostate cancer cells.
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• • • • •
S0223-5234(16)30095-2
DOI:
10.1016/j.ejmech.2016.02.025
Reference:
EJMECH 8376
To appear in:
European Journal of Medicinal Chemistry
Received Date: 2 September 2015 Revised Date:
7 February 2016
Accepted Date: 8 February 2016
Please cite this article as: S.R. Pedada, N.S. Yarla, P.J. Tambade, D.B. Lakkappa, A. Bishayee, K.M. Arunasree, G.H. Philip, G. Dharmapuri, G. Aliev, S. Putta, G. Rangaiah, Synthesis of new secretory phospholipase A2-inhibitory indole containing isoxazole derivatives as anti-inflammatory and anticancer Agents, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.02.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphical abstract N O
R2
R
HN R1
N Boc
R2
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HN
N H
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10o (R1=F, R2=CF3, R=CH3) GIIA sPLA2 (IC50 = 10.23±0.91 µM)
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R1
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Synthesis of new secretory phospholipase A2-inhibitory indole containing isoxazole derivatives as anti-inflammatory and anticancer Agents
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Running title: sPLA2-inhibitory indole containing isoxazoles Srinivasa Rao Pedadaa, +, δ, Nagendra Sastry Yarlab, δ, Pawan J. Tambadec, Dhananjaya Bhadrapura Lakkappad, Anuapam Bishayeee, Kalle M. Arunasreef, Gundala Harold Philipg, Gangappa
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Dharmapurif, g, Gjumrach Alievh, Swathi Puttai, Gururaja Rangaiah j,*
Centre for Research and development,PRIST University, Thanjavur-613 403, Tamilnadu, India
b
Department of Biochemistry, Institute of Science, GITAM University, Visakhapatnam-530 045,
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a
Andhra Pradesh, India c
Department of Chemistry, Arts, Commerce and Science College, Nandgaon, Maharashtra, India
d
Toxinology/Toxicology and Drug Discovery Unit, Center for Emerging Technologies, Jain Global
Campus, Jain University, Ramanagara-562 112, Karnataka, India
Department of Pharmaceutical Sciences, College of Pharmacy, Larkin Health Sciences Institute,
Miami, Florida 33169, USA f
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e
Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad-500
046, Telangana, India
Department of Biotechnology, Sri Krishnadevaraya University, Anantapuramu-515 003, Andhra
Pradesh, India h
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g
School of Health Sciences and Healthcare Administration,
University of Atlanta, E. Johns
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Crossing, Johns Creek, GA 30097, USA i
Pharmacology Division, University College of Pharmaceutical Sciences, Andhra University,
Visakhapatnam-530003, Andhra Pradesh, India j
Department of Biochemistry, Sarada Vilas College, Mysore-570004, Karnataka, India
+
the work is part of PhD work
δ equally contributed to this work *corresponding author. E-mail address: [email protected] (G. Rangaiah)
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ABSTRACT Secretory phospholipase A2 (sPLA2) is an important enzyme that plays a key role in various inflammatory diseases including cancer and its inhibitors have been developed as preventive or
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therapeutic agents. In the present study, a series of new indole containing isoxazole derivatives (10a-10o) is synthesized and evaluated for their sPLA2 inhibitory activities. All compounds (10a10o) showed significant sPLA2 inhibition activities both in vitro and in vivo studies which is
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substantiated in in silico studies. Among all the tested compounds, 10o showed potent sPLA2 inhibition activity, that is comparable or more to ursolic acid (positive control). Further studies
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demonstrated that 10o showed in vitro antiproliferative activity when tested against MCF-7 breast and DU145 prostate cancer cells. Furthermore, compounds 10a-10o obeyed lipinsky`s rule of 5 and suggesting druggable properties. The in vitro, in vivo and in silico results are encouraging and warrant pre-clinical studies to develop sPLA2-inhibitory compound 10o as novel therapeutic agent
Key words:
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for various inflammatory disorders and several malignancies.
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Indole, Isooxazole, Secretory phospholipase A2, Anticancer, Anti-inflammatory activity
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1.
Introduction Phospholipase A2s (PLA2s) are group of enzymes, which cleave phospholipids specifically
at sn-2 position to liberate free fatty acid, mostly arachidonic acid (AA) and lysophospholipids
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(LPLs) [1-3]. Inhibition of PLA2 prevents the liberation of AA and LPLs. Hence, researchers have been considering that PLA2s could be good therapeutic target than cyclooxygense-2 and lipoxygenase, which are downstream enzymes [1-3]. Several isoforms of PLA2s exist, which are
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divided into various groups; secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2), and calciumindependent PLA2 (iPLA2), platelet activating factor-acyl hydrolase (PAF-AH), lysosomal PLA2
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(LPLA2), adipose-specific PLA2 (AdPLA2). The secreted PLA2s (13-55 kDa) are the first type of PLA2 enzymes discovered and categorized into several groups [4]. Among all groups of the sPLA2s, group IIA sPLA2 (GIIA sPLA2) is predominantly distributed and plays key role in various inflammatory diseases, including arthritis, inflammatory bowel disease, atherosclerosis, psoriasis,
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and several cancers [1-4].
Realizing the role of sPLA2 in inflammatory diseases and various cancers, considerable efforts are being made by several investigators for the discovery and development of sPLA2
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inhibitors as therapeutic or preventive agents [2]. Several sPLA2 inhibitors have been developed and some of the sPLA2 inhibitors are currently under clinical trials for various inflammatory and
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oncologic diseases [2-4]. During last several decades, various derivatives of indoles have been developed as GIIA sPLA2-inhibitors [5-7]. A series of indole acids, indole amides, indole-3acetamides, indole-3-glyoxamides are developed as sPLA2 inhibitors [5]. Lilly Research Laboratory (Indianapolis, IN, USA) developed a series of indole-3-acetamide derivatives with sPLA2 inhibition activity. Among all compounds, varespladib or LY315920 inhibited group IIA sPLA2 with high potency and it entered into clinical trials for inflammatory diseases [6]. However, LY315920 or varespladib failed phase II clinical trials because of poor anti-inflammatory efficacy. Further, Lilly 3
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Research Laboratory synthesized LY333013 or methyl varespladib, a modified form to LY315920, but it also failed in clinical trials due to its poor anti-inflammatory efficacy [7]. Recently, Anthera Pharmaceuticals (Hayward‚ CA, USA) announced the use of A-001 (varespladib or LY315920) and
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A-002 (LY333013 or methyl varespladib) in treatment of cardiovascular diseases as their efficacy in reducing LDL-C and atherosclerosis in patients with acute coronary syndrome (ACS) along with atorvastastin treatment, according to phase II clinical trial namely, Fewer Recurrent Acute Coronary
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Events With Near Term Cardiovascular Inflammation Suppression (FRANCIS) [8]. In phase III clinical trial of varespladib in patients with ACS, namely Vascular Inflammation Supression to
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Treat Acute Coronary Syndrome for 16 days (VISTA-16), varespladib showed poor efficacy in reduction of ACS and significantly increased the risk of nonfatal myocardial infarction (MI) [9]. Most of the sPLA2 inhibitors failed in clinical trials due to poor efficacy and adverse effects [2]. Therefore, discovery of new sPLA2-inhibitory molecules or optimization of existed sPLA2-
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inhibitory compounds with more potency, safety and selectivity is urgently needed for prevention and treatment of inflammatory disorders and several cancers. Isoxazoles have been reported to possess various biological activities, including PLA2-
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inhibitory activities [10, 11]. Sadashiva et al. [11] synthesized trimethoxyphenyl isoxazolidine derivatives with sPLA2 inhibitory activities. Various derivatives of indoles have been developed as
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sPLA2 inhibitors and some of them are under clinical evaluation for various inflammatory diseases. Both indoles and isoxazoles are attractive chemical scaffolds for development of novel sPLA2 inhibitors [5-7, 10, 11]. Considering this background, the present study aimed to synthesize indole containing isoxazoles (10a-10o) with potent sPLA2-inhibitory activity. Among all compounds, 10o showed substantial sPLA2-inhibitory activity. Further in vivo antiinflammatry studies of 10o were performed using inflammogen (carrageenan)-induced rat paw edema model [12]. In vitro anticancer studies using sPLA2-inhibitory 10o were also performed on MCF-7 breast carcinoma and DU145 4
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prostate cancer cell lines since sPLA2 exhibited pro-tumorigenic role in breast and prostate cancers [13, 14]. 2.
Results
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2.1. Chemistry The target molecules were synthesized in a convergent fashion by coupling indole aldehydes 8(a-c) and isoxazole containing primary amines 5(a-e), which were subsequently converted to their
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hydrochloride salts 10 (a-o). The isoxazole methyl amines were synthesized starting from commercially available aromatic aldehydes. Aldehydes were reacted with hydroxyl amine
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hydrochloride to obtain their corresponding oxime derivatives 2(a-e). They were converted to their corresponding bromo methyl isoxazoles by intermolecular cycloaddition of activated oximes with with propargyl bromide in presence of chloramine-T in ethanol [15]. The bromo methyl isoxazoles were later converted to azides by treating with sodium azide in DMF 4(a-e), which were finally
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reduced to the desired isoxazole methyl amines 5(a-e) by following a mild and efficient Schodinger’s protocol using triphenyl phosphine (TPP) and water in THF as a solvent system [16]. On the other hand indole aldehydes were synthesized by electrophilic acylation of
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unprotected indoles following Vilsmier-Haack method using POCl3/DMF in good to excellent yields 7(a-c). To avoid any unforeseen complications, the indole NH was protected by Boc
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anhydride. The boc protected indole-3-aldehydes 8(a-c) and isoxazole methyl amines 5(a-e) were coupled by reductive amination in the presence of sodium cyanoborohydride and catalytic amount of acetic acid in methanol at ambient temperatures. The isolated yields of the coupled products 9(ao) were moderate to good. Finally, Boc group was deprotected with TFA in DCM, which were subsequently converted to their hydrochloride salts with 20% HCl in ethyl acetate in good yields 10(a-o) (Scheme-1). All the synthesized compounds were well characterized by 1H NMR, 13C NMR, IR and mass spectra. 5
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2.2.
Pharmacology
2.2.1. sPLA2-inhibitory activity of indole containing isoxazole derivatives (10a-10o) All synthesized indole containing isoxazole derivatives showed significant in vitro sPLA2-
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inhibitory activity and good binding affinity on crystal structure of sPLA2 (Table 1). Among all tested compounds, 10o showed substantial sPLA2-inhibitory activity with IC50 of 10.23±0.91 µM in vitro and docked on crystal structure of sPLA2 with binding energy of -129.5 Kcal/mol where as
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ursolic acid (positive control) inhibited with IC50 of 12.59±1.03 µM in vitro and docked with binding energy of -128.8 Kcal/mol (Fig. 1 & Table 1). Both in vitro and in silico results
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demonstrated that 10o showed comparable or more sPLA2-inhibitory activity than ursolic acid (positive control). Further in silico studies demonstrated that 10o binds the vicinity of amino acid residues at active site PHE-5, TYR-21, GLY-22, CYS-28, GLY-29, CYS-44, HIS-47, ASP-48, TYR-51, and PHE-98 (Fig. 2). The strong binding affinity and interactions of 10o with sPLA2 may
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be responsible for its sPLA2-inhibitory activity. From the Table 1 it was evident that compounds containing electron donating group such as CH3 on indole ring (R group) and electron withdrawing groups, such as F, CF3, and NO2 on isoxazole phenyl ring (R1, R2 group) are found to have
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remarkable activities, which were further supported by structure activity relationship (SAR) studies.
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When R1 and R2 = H, R= CH3>H>Br.
2.2.2. Antiiflammatory activity of 10o in wistar albino rats In vivo oral anti-inflammatory efficacy of 10o (10 mg/kg body weight) was tested on group of rats using carrageenan model and paw volumes (ml) of treated 10o and ursolic acid (positive control)) and untreated (vehicle control) (Fig. 3). 10o (10 mg/kg body weight) showed 75.67±4.21%, and 76.54±3.1% edema inhibition (maximum) at 3rd h and 4th h, respectively, where as ursolic acid (10 mg/kg body weight) showed 72.97±3.56% and 75.17±5.70% edema inhibition at 3rd h and 4th h, 6
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respectively. 10o exhibited in vivo anti-inflammatory activity wich was comparable to or more than ursolic acid (positive control).
Antiproliferative activity of 10o against breast and prostate cancer cells
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2.2.3.
Antiproliferative effect of 10o was evaluated against MCF-7 breast and DU145 prostate cancer cell lines by MTT assay. Compound 10o showed significant anti-proliferative effect in
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MCF-7 and DU145 cancer cell lines with IC50 of 10.03±0.86 and 14.15±1.2 µM, respectively, whereas doxorubicin (positive control) exhibited anticancer activity with 8.02±0.69 and 4.75±0.35
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µM on respective cancer cell lines (Fig. 4). The results demonstrate that 10o showed comparable antiproliferative effect against MCF-7 breast cancer cells with that of doxorubicin (positive control).
2.2.4. Drug likeness of compounds
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In addition to sPLA2 inhibition studies, the evaluation of druglikeness of compounds according to Lipinski’s rule of five is done which states that, in general, an orally active drug has not more than 5 hydrogen bond donors (OH and NH groups), not more than 10 hydrogen bond
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acceptors (notably N and O), molecular weigh under 500 g/mol, partition coefficient log P less than
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5. Almost all compounds 10a-10o obey lipinsky`s rule and is of druggable nature (Table 2).
3. Discussion
Several sPLA2 inhibitory molecules have been developed as antinflammaotry and anticaner agents by several investigation and some of them under pre-clinical and clinilcal developmental stage. Mayur et al. [17] synthesized azetidin-2-one derivatives wth sPLA2 inhibitory activity. Further studies demonstrated that azetidin-2-one derivatives showed in vivo anti-inflammatory activity in mice. 4-tetradecyloxybenzamidine (PMS815) derivatives were designed and synthesized 7
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with PLA2 inhibitory activity [18]. Among all compounds, 4,5-dihydro-3-(4-tetradecyloxybenzyl)1,2,4-4H-oxadiazol-5-one (PMS1062) exhibited a micromolar IC50 towards GII sPLA2s, while inactive towards GI and GIII PLA2s in vitro and also blocked the GII sPLA2 activities induced by
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lipopolysaccharides and interleukin-6 in HepG2 cell line and no cytotoxicity was observed in LLCPK1 and A549 cell lines [18]. Several indole derivatives have been developed as sPLA2 inhibitors and some of them are entered into clinical trials for various inflammatory diseases [2, 3, 5-7].
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Sadashiva et al. [11] synthesized trimethoxyphenyl isoxazolidine derivatives with sPLA2 inhibitory activities. Indole and isoxazoles are attractive scaffolds for development of novel sPLA2 inhibitors.
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Considering this background, in the present study, we have synthesized indole containing isoxazole derivatives and evaluated for their sPLA2 inhibitory activity. Among all compounds, 10o showed substantial sPLA2 inhibitory activity. In in vivo anti-inflammatory studies, 10o exhibited antiinflammatory activity wich was comparable to or more than ursolic acid (positive control). In
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anticancer studies, 10o showed comparable antiproliferative effect against MCF-7 breast cancer cells with that of doxorubicin (positive control). Moreover all compounds including 10o obey Lipinsky`s rule of 5 and can be druggable. Further detailed investigations are needed to develop
and in vivo studies. Conclusions
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4.
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sPLA2-inhibitory 10o as antiinflammatory and anticancer agent upon encouraging resutls of in vitro
In the present study, a series of novel indole containing isoxazole derivatives (10a-o) were synthesized and showed significant sPLA2-inhibitory activities. Remarkable sPLA2 inhibition activities were observed when electron donating group like -CH3 on indole ring and electron withdrawing groups like -F, and -CF3 on phenyl ring of isoxazole were present. Among all compounds, 10o showed substantial sPLA2-inhibitory activity. 10o also showed significant in vivo anti-inflammatory activity in carrageenan-induced rat paw edema model as well as good 8
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antiproliferative effect against breast MCF-7 and prostate DU145 cancer cells. Moreover, compounds 10a-10o obey Lipinsky`s rule of 5 and can be druggable. The in vitro, in silico and in vivo results of this study are encouraging and warrant further investigations to develop sPLA2-
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inhibitory compound 10o as novel therapeutic agent for inflammatory disorders and oncologic
5.
Experimental
5.1.
Chemistry
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diseases.
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5.1.1. General information
All chemicals used for the synthesis were of reagent grade and procured from SigmaAldrich, St. Louis, MO, USA. 1H and
13
C NMR spectra were recorded on AS 400 MHz Varian
NMR spectrometer using TMS as an internal standard. IR spectra were recorded by using Perkin-
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Elmer Spectrum 100 Series FT-IR spectrometer. Mass spectra were recorded on Agilent 1200 Series LC/MSD VL system. Melting points were determined by using Buchi melting point B-545 instrument and are uncorrected. All the reactions were monitored by thin layer chromatography
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(TLC) using percolated silica 60 F254, 0.25 mm aluminum plates (Merck). The crude compounds were purified by column chromatography using silica gel (100-200 mesh) and gradient (0-20%)
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ethyl acetate in hexane as the eluent system.
5.1.2. General Procedure for the synthesis of 9(a-o) To the stirred mixture of substituted indole (8(a-c), 0.50 g, 2.0 mmol) and substituted isoxazole amine (5(a-e), 0.35 g, 2.0 mmol) in methanol (5.0 ml) was added 4A° molecular sieves (0.50 g) and catalytic amount of acetic acid. The above reaction mixture was allowed to stir for 1h at room temperature. followed by the portion wise addition of sodium cyanoborohydride (0.32 g, 9
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5.0 mmol) was added portionwise at room temperature and the reaction mixture was allowed to stir for 16 h. Solvent was removed under reduced pressure and then diluted with dichloromethane, which was washed with 10% sodium bicarbonate solution. The obtained DCM layer was dried over
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sodium sulphate and distilled under vacuum to obtain crude 9(a-o), which was further purified by
in 80% yield.
5.1.3. General Procedure for the synthesis of 10(a-o)
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column chromatography using ethyl acetate and hexane mixture (80:20) to obtain pure compounds
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To a solution of compound 9(a-o) (0.5 g, 1.2 mmoles) in ethyl acetate (5.0 ml) was added HCl in ethyl acetate (20%) by drop wise manner and stirred for 16h. The so obtained solid was collected by filtration and washed with ethyl acetate, dried under vacuum to obtain compound 10(a-
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o) in 63 – 79 % yield.
5.1.3.1. N-((1H-indol-3-yl)methyl)(3-phenylisoxazol-5-yl)methanamine hydrochloride(10a) Yield: 79 %; light orange solid; m.p. 185 – 187 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 4.45
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(d, 4H, J = 10 Hz), 7.09 (dd, 1H, , J = 1.2 Hz, J = 8 Hz), 7.16 (dd, 1H, , J = 1.2 Hz, J = 8.0 Hz), 7.21 (s, 1H), 7.44 (d, 1H, J = 8.0 Hz) 7.58 – 7.52 (m, 3H), 7.64 (d, 1H, J = 2.8 Hz), 7.78 (d, 1H, J =
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8.0 Hz) 7.90 – 7.80 (m, 2H), 9.92 (s, 2H), 11.47 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 40.3, 41.8, 104.5, 104.7, 112.2, 119.1, 119.6, 122.1, 127.0 (2C), 127.2, 128.3, 128.5, 129.7 (2C), 131.0, 136.4, 162.5, 165.34; IR (KBr) cm-1 : 3645, 3301, 3116, 2948, 2730, 2603. 2422, 1709, 1612, 1579, 1458, 1409, 1367, 1344, 1279, 1235, 1180, 1097, 1018, 947, 920, 825, 768, 734, 690; MS (ESI) m/z [M-H]-:338.4(HCl salt); Anal. Calcd. for C19H18ClN3O; C, 67.15; H, 5.34; Cl, 10.43; N, 12.37; O, 4.71 %. Found C, 67.45; H, 5.34; N, 12.33 %.
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5.1.3.2. N-((5-bromo-1H-indol-3-yl)methyl)(3-phenylisoxazol-5-yl)methanamine
hydrochloride
(10b) Yield: 70 % , off white solid; m.p. 191 - 193 ˚C ; 1H NMR (400 MHz, DMSO-d6) δ: 4.46 (d,
RI PT
4H, J = 15.6 Hz), 7.25 – 7.24, (m, 1H) 7.27 (d, 1H, , J = 4 Hz), 7.41 (s, 1H, , J = 8 Hz), 7.60 – 7.50 (m, 3H) 7.66 (d, 1H, , J = 4 Hz), 7.86 (dd, 2H, , J = 4 Hz, J = 8 Hz), 8.04 (d, 1H, J = 1.6 Hz) 9.78(s, 2H), 11.65 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 40.5, 41.6, 104.5, 104.6, 112.4, 114.2, 121.8,
SC
124.5, 124.6, 127.0, 128.5, 129.0, 129.7 (2C), 130.0, 130.9, 135.1, 162.5, 165.3; IR (KBr) cm-1 : 2947, 2809, 2697, 1695, 1604, 1526, 1466, 1388, 1348, 1281, 1230, 1172, 1093, 1039, 1017, 988,
M AN U
953, 905, 853, 823, 802,740; MS (ESI) m/z [M-H]-: 380.2 Anal. Calcd. for C19H17BrClN3O; C, 54.50; H, 4.09; Br, 19.08; Cl, 8.47; N, 10.04; O, 3.82 %. Found C, 54.32; H, 4.27; N, 10.76 %.
5.1.3.3. N-((5-methyl-1H-indol-3-yl)methyl)(3-phenylisoxazol-5-yl)methanamine
TE D
(10c) :
hydrochloride
Yield: 65 %; light pink solid, m.p. 208 - 210 ˚C ;1H NMR (400 MHz, DMSO-d6) δ: 2.42 (s, 3H), 4.45 (d, 4H, J = 20.8 Hz), 6.96 (dd, 1H, , J = 4.0 Hz, J = 8.0 Hz), 7.26 (s, 1H), 7.32 (d, 1H, , J 13
C NMR (100
EP
= 8.0 Hz) 7.57 – 7.53 (m, 5H), 7.87 – 7.84, (m, 2H), 9.87 (s, 2H), 11.34 (s, 1H);
MHz, DMSO-d6) δ: 21.7, 40.5, 41.8, 104.0, 104.5, 111.9, 118.6, 123.6, 124.6, 127.0, 127.4, 128.1,
AC C
128.3, 128.5, 129.7 (2C), 130.9, 134.7, 162.5, 165.3; IR (KBr) cm-1 : 2948, 2809, 2697, 1695, 1604, 1526, 1466, 1388, 1348, 1281, 1230, 1172, 1094, 1017, 953, 905, 850, 824, 802,740; MS (ESI) m/z [M-H]-: 351.9 (HCl salt); Anal. Calcd. for C20H20ClN3O; C, 67.89; H, 5.70; Cl, 10.02; N, 11.88; O, 4.52 %. Found C, 67.67; H, 5.83; N, 11.74 %.
5.1.3.4. N-((1H-indol-3-yl)methyl)(3-p-tolylisoxazol-5-yl)methanamine hydrochloride (10d)
11
ACCEPTED MANUSCRIPT
Yield: 70 %; off white solid; m.p. 191 – 193 ˚C;1H NMR (400 MHz, DMSO-d6) δ: 2.42 (s, 3H), 4.45 (d, 4H, J = 20.0 Hz), 7.09 (t, 1H, J = 8.0 Hz), 7.14 (t, 1H, J = 8.0 Hz), 7.22 (s, 1H), 7.34 (d, 2H, J = 8.0 Hz) 7.44 (d, 1H, J = 8.0 Hz) 7.63 (d, 1H, J = 2.0 Hz), 7.74 (d, 2H, J = 8.0 Hz) 7.78
RI PT
(d, 1H, J = 8.0 Hz), 9.95(s, 2H), 11.48 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 21.4, 40.5, 41.8, 104.3, 104.6, 112.2, 119.1, 119.6, 122.0, 124.5, 125.7, 126.91, 127.2, 128.3, 130.2 (2C), 136.3, 140.7, 162.4, 165.1; IR (KBr) cm-1 : 3556, 3306, 3112, 2936, 2722, 2608, 2411, 1725, 1613, 1571,
SC
1464, 1357, 1268, 1224, 1182, 1094, 1016, 986, 946, 816, 754; MS (ESI) m/z [M-H]-; 351.9 (HCl salt); Anal. Calcd. for Anal. Calcd. for C20H20ClN3O; C, 67.89; H, 5.70; Cl, 10.02; N, 11.88; O,
M AN U
4.52 %. Found C, 67.67; H, 5.91; N, 11.92 %.
5.1.3.5. N-((5-bromo-1H-indol-3-yl)methyl)(3-p-tolylisoxazol-5-yl)methanaminehydrochloride (10e)
TE D
Yield: 70 %; off white solid; m.p. 197 - 199 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 2.42 (s, 3H), 4.43 (d, 4H, J = 12.0 Hz), 7.21 (s, 1H), 7.25 (dd, 1H, J = 1.6 Hz, J = 8.4 Hz), 7.35 (d, 2H, J = 8.0Hz), 7.41 (d, 1H, J = 8.0 Hz), 7.65 ( d, 1H, J = 4Hz),7.75 (d, 2H, J = 8.0 Hz), 8.04 (d, 1H, J = 4
EP
Hz) 9.81 (s, 2H), 11.66 (s, 1H); 13C NMR (100 MHz, DMSO) δ: 21.4, 40.6, 41.6, 104.3, 104.6, 112.4, 114.2, 121.8, 124.5, 124.6, 125.7, 126.9, 129.0, 129.9, 130.2 (2C), 135.1, 140.7, 162.4,
AC C
165.1; IR (KBr) cm-1 : 2917, 2756, 2601, 2420, 1742, 1612, 1565, 1455, 1420, 1336, 1245, 1217, 1170, 1100, 1014, 914, 862, 845, 799, 723; MS (ESI) m/z [M-H]- 429.8 (HCl salt) ; Anal. Calcd. for C20H19BrClN3O; C, 55.51; H, 4.43; Br, 18.46; Cl, 8.19; N, 9.71; O, 3.70%. Found C, 55.21; H, 4.73; N, 9.49 %.
5.1.3.6.
N-((5-methyl-1H-indol-3-yl)methyl)(3-p-tolylisoxazol-5-yl)methanaminehydrochloride (10f) 12
ACCEPTED MANUSCRIPT
Yield: 63 %; off white solid; m.p. 185 - 187 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 2.49 (s, 6H), 4.43 (d, 4H, J = 16 Hz), 6.97 (dd, 1H, J = 1.6 Hz, J= 8.0 Hz), 7.21 (s, 1H), 7.37 – 7.32 (m, 3H), 7.56 – 7.53 (m, 2H), 7.75 (d, 2H, J = 8.0 Hz), 9.79 (s. 2H), 11.32 (s, 1H);
13
C NMR (100 MHz,
RI PT
DMSO-d6) δ: 21.4, 21.7, 40.5, 41.8, 104.0, 104.4, 111.9, 118.6, 123.6, 124.6, 125.7, 126.9, 127.4, 128.1, 128.3, 130.2 (2C), 134.7, 140.7, 162.4, 165.1; IR (KBr) cm-1 : 3118, 2955, 2718, 2609, 2440, 1709, 1612, 1593, 1463, 1368, 1344, 1282, 1233, 1174, 1091, 1020, 953, 925, 808, 769, 742, 691;
M AN U
N, 11.42; O, 4.35%. Found C, 68.46; H, 6.23; N; 11.53 %.
SC
MS (ESI) m/z [M-H]- 367.1 (HCl salt) ;Anal. Calcd. for C21H22ClN3O; C, 68.56; H, 6.03; Cl, 9.64;
5.1.3.7. N-((1H-indol-3-yl)methyl)(3-(4-methoxyphenyl)isoxazol-5-yl)methanaminehydrochloride (10g)
Yield: 70 % ; off white solid; mp 184 – 186 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 3.82 (s,
TE D
3H), 4.43 (s, 4H), 7.10 (m, 3H), 7.18 (m, 2H), 7.44 (d, 1H, J = 8.0 Hz), 7.63 (d, 1H, J = 2.4 Hz), 7.78 (m, 3H), 9.91 (s, 2H), 11.47 (s, 1H,); 13C NMR (100 MHz, DMSO-d6) δ: 40.5, 41.8, 55.7, 104.2, 104.6, 112.2, 115.1 (2C), 119.1, 119.6, 120.8, 122.0, 127.2, 128.3, 128.5 (2C), 136.4, 161.3,
EP
162.1, 164.9; IR (KBr) cm-1 : 3440, 3131, 2936, 2893, 2746, 2611, 2421, 1614, 1583, 1530, 1457, 1436, 1304, 1252, 1181, 1102, 1029, 982, 933, 824, 746; ; MS (ESI) m/z [M-H]- 367.9(HCl salt) ;
AC C
Anal. Calcd. for C20H20ClN3O2; C, 64.95; H, 5.45; Cl, 9.59; N, 11.36; O, 8.65 %. Found C, 65.11; H, 5.35; N, 11.46 %.
5.1.3.8. N-((5-bromo-1H-indol-3-yl)methyl)(3-(4-methoxyphenyl)isoxazol-5-yl)methanamine hydrochloride (10h) Yield: 73 %; off white solid; mp 202 – 204 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 3.87 (s, 3H), 4.44 (d, 4H), 7.07 (d, 2H, J = 4.8 Hz) 7.18 (s, 1H), 7.26 (dd, 1H, J = 2.0 Hz, J = 8.4 Hz), 7.41 13
ACCEPTED MANUSCRIPT
(d, 1H, J = 8.8 Hz), 7.66 (d, 1H, J = 4.0 Hz), 7.81 (d, 2H, J = 4.0 Hz), 8.04 ( d, 1H, J = 4.0 Hz), 9.79 (d, 1H), 11.66 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ:40.2, 41.6, 55.8, 104.2, 104.6, 112.4, 114.2, 115.0 (2C), 120.8, 121.8, 124.5, 128.5 (2C), 129.0, 129.9, 135.1, 161.3, 162.1, 164.9; IR
RI PT
(KBr) cm-1 : 3208, 3001, 2939, 2743, 2565, 2409, 1613, 1564, 1532, 1432, 1394, 1299, 1259, 1178, 1108, 1030, 979, 948, 904, 886, 838, 802; MS (ESI) m/z [M-H]- 410.5 ;
Anal. Calcd. for
C20H19BrClN3O2; C, 53.53; H, 4.27; Br, 17.81; Cl, 7.90; N, 9.36; O, 7.13 %. Found C, 53.73; H,
SC
4.01; N, 9.76 %.
M AN U
5.1.3.9. (3-(4-methoxyphenyl)isoxazol-5-yl)-N-((5-methyl-1H-indol-3-yl)methyl)methanamine hydrochloride (10i)
Yield: 65 %; off white solid; mp 177 – 179 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 2.39 (s, 3H), 3.82 (s, 3H), 4.41(d, 4H), 6.98 (d, 1H, J = 8.0 Hz), 7.08 (d, 2H, J = 8.8 Hz), 7.18 (s, 1H),
TE D
7.32 ( s, 1H, J = 8.4 Hz), 7.55 – 7.53 (m, 2H), 7.80 (d, 2H, J = 8.8 Hz), 9.75 (s, 2H), 11.31 (s, 1H); 13
C NMR (100 MHz, DMSO-d6) δ: 21.7, 40.5, 41.8, 55.8, 104.1, 104.2, 111.9, 115.0, 118.6, 120.8,
123.6, 124.6, 127.4, 128.1, 128.3, 128.5 (2C), 134.7, 161.3, 162.1, 164.9; IR (KBr) cm-1 : 3125,
EP
3080, 2836, 2709, 1699, 1615, 1576, 1529, 1469, 1387, 1350, 1283, 1257, 1175, 1090, 1020, 919, 900, 837, 808; MS (ESI) m/z [M-H]- 381.9 (HCl salt); Anal. Calcd. for C21H22ClN3O2 C, 65.71; H,
AC C
5.78; Cl, 9.24; N, 10.95; O, 8.34 %. Found C, 65.91; H, 5.58; N, 10.97 %.
5.1.3.10.
N-((1H-indol-3-yl)methyl)(3-(4-nitrophenyl)isoxazol-5-yl)methanaminehydrochloride (10j)
Yield: 70 %; off white solid; mp 212 – 214 ˚C 0C;; 1H NMR (400 MHz, DMSO-d6) δ: 4.48 (d, 4H, J = 28 Hz), 7.18 – 7.08 (m, 2H), 7.39 (s, 1H), 7.44 (d, 1H, J = 8.0 Hz), 7.60 ( d, 1H, J = 4.0 Hz),7.77 (d, 1H, J = 8.0 Hz), 8.17 (d, 2H, J = 4.0 Hz), 8.39 ( d, 2H, J = 8.0Hz), 9.77 ( s, 2H), 14
ACCEPTED MANUSCRIPT
11.43 (s, 1H);
13
C NMR (100 MHz, DMSO-d6) δ: 40.5, 42.03, 104.8, 104.9, 112.2, 119.1, 119.6,
122.1, 124.9 (2C), 127.1, 128.2, 128.4 (2C), 134.4, 136.4, 148.9, 161.2, 166.31; IR (KBr) cm-1 : 3440, 3125, 2946, 2780, 2602, 2422, 1695, 1600, 1522, 1462, 1433, 1347,1290, 1170, 1103, 1015,
RI PT
938, 833, 858, 754; MS (ESI) m/z [M-H]- 383.2 (HCl salt) ; Anal. Calcd. for C19H17ClN4O3; C, 59.30; H, 4.45; Cl, 9.21; N, 14.56; O, 12.47 %. Found C, 59.21; H, 4.35; N, 12.36 %.
SC
5.1.3.11. N-((5-bromo-1H-indol-3-yl)methyl)(3-(4-nitrophenyl)isoxazol-5-yl)methanamine hydrochloride (10k)
M AN U
Yield: 79%; off white solid; mp 188 – 190 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 4.47 (d, 4H, J = 24 Hz), 7.25 (dd, 1H, J = 1.6 Hz, J = 8.8 Hz), 7.43 – 7.40 (m, 2H), 7.69 ( d, 1H, J = 2.0 Hz), 8.04 (s, 1H), 8.15 (d, 2H, J = 8.0 Hz), 8.39 (d, 2H, J = 8.0 Hz), 10.01 (s, 2H), 11.70 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 40.5, 41.7, 104.6, 104.9, 112.4, 114.2, 121.8, 124.5, 124.9 (2C),
TE D
128.4 (2C), 129.0, 129.9, 134.4, 135.1, 161.1, 166.2 (2C); IR (KBr) cm-1 : 3318, 3099, 2943, 2722, 1601, 1522, 1451, 1347, 1290, 1231, 1169, 1111, 1045, 984, 923, 886, 855, 806, 755, 698; MS (ESI) m/z [M-H]- 425.5 ; Anal. Calcd. C19H16BrClN4O3;C, 49.21; H, 3.48; Br, 17.23; Cl, 7.65; N,
EP
12.08; O, 10.35 %.Found C, 49.39; H, 3.23; N, 12.01 %.
AC C
5.1.3.12. N-((5-methyl-1H-indol-3-yl)methyl)(3-(4-nitrophenyl)isoxazol-5-yl)methanamine hydrochloride (10l)
Yield: 70 % ;off white solid; mp 198 – 200˚C; 1H NMR (400 MHz, DMSO-d6) δ: 2.40 (s,
3H), 4.45 (d, 4H, J = 37.2 Hz), 6.98 (d, 1H, J = 8.0 Hz), 7.32 (d, 1H, J = 8.0 Hz), 7.39 (s, 1H), 7.54 (s, 2H), 8.16 (d, 2H, J = 8.0 Hz), 8.39 (d, 2H, J = 8.0 Hz), 9.70 (s, 2H), 11.29 (s, 1H);
13
C NMR
(100 MHz, DMSO-d6) δ: 21.7, 40.3, 40.6, 104.1, 104.9, 118.6, 123.7, 124.9 (2C), 127.3, 128.1, 128.27, 128.4 (2C), 134.4, 134.7, 148.9, 161.6 , 166.2 (2C).;IR (KBr) cm-1 :3399, 3117, 2776, 1703, 15
ACCEPTED MANUSCRIPT
1598, 1556, 1526, 1431, 1349, 1291, 1247, 1217, 1169, 1089, 1039, 1016, 975, 856, 836, 808, 758, 714, 699; MS (ESI) m/z [M-H]- 397.2 (HCl salt) ; Anal. Calcd. for C20H19ClN4O3;C, 60.23; H, 4.80;
RI PT
Cl, 8.89; N, 14.05; O, 12.03%.Found C, 60.43; H, 4.70; N, 14.34 %.
5.1.3.13. N-((1H-indol-3-yl)methyl)(3-(4-fluoro-3-(trifluoromethyl)phenyl)isoxazol-5yl)methanamine hydrochloride (10m)
SC
Yield: 70 %; off white solid; mp 165 - 167 ˚C; 1HNMR (400 MHz, DMSO-d6) δ: 4.46 (d, 4H, J = 18.8 Hz), 7.09 (t, 1H, J = 4.0Hz), 7.16 (t, 1H, J = 8.0Hz), 7.43 ( t, 2H, J = 8.0Hz), 7.63 (d,
M AN U
1H, J = 4.0Hz), 7.78 – 7.71 (m, 2H), 8.20 (dd, 1H, J = 4.0Hz, J = 8.0Hz), 8.28 – 8.25 (m, 1H), 9.92 (s, 2H), 11.45 (s, 1H);13C NMR (100 MHz, DMSO-d6) δ: 40.5, 41.8, 104.6, 104.8, 112.2, 117.9, 118.1, 118.9, 119.1, 119.6, 121.3, 122.0, 124.0, 125.7, 127.2, 128.3, 134.1, 136.4, 160.7, 165.9; IR (KBr) cm-1 : 3442, 3127, 2890, 2782, 2610, 2421, 1614, 1522, 1457, 1430, 1384, 1325, 1272, 1245,
TE D
1140, 1105, 1080, 1053, 1015, 968, 937, 907, 833, 750; MS (ESI) m/z [M-H]- 424.4 (HCl salt) ; Anal. Calcd. for C20H16ClF4N3O; C, 56.41; H, 3.79; Cl, 8.33; F, 17.85; N, 9.87; O, 3.76%.Found C,
EP
56.66; H, 3.98; N, 9.66%.
5.1.3.14. N-((5-bromo-1H-indol-3-yl)methyl)(3-(4-fluoro-3-(trifluoromethyl)phenyl)isoxazol-5-
AC C
yl)methanamine hydrochloride (10n) Yield: 73%; off white solid; mp 176 – 178 ˚C;1H NMR (400 MHz, DMSO-d6) δ: 4.47 (d, 4H, J = 23.6 Hz), 7.26 (d,1H, J = 8.0Hz), 7.35 – 7.45 ( d, 2H, J = 4.0Hz), 7.65 ( d, 1H, J = 4.0Hz), 7.75 (t, 1H, J = 8.0Hz), 8.02 ( d, 1H, , J = 4.0Hz), 8.20, (d, 1H, J = 4.0Hz), 8.29 – 8.25 (m, 1H), 9.80 (s, 2H), 11.64 (s, 1H);13C NMR (100 MHz, DMSO-d6) δ: 40.5, 41.6, 104.6, 104.8, 112.4, 114.2, 118.0, 118.9, 119.1, 121.3, 121.8, 124.0, 124.5, 125.7, 129.0, 130.0, 134.0, 135.1, 160.7, 165.9;IR (KBr) cm-1 : 3347, 3111, 2973, 2732, 2606, 2455, 1611, 1524, 1458, 1431, 1400, 1377, 1321, 1270, 1249, 16
ACCEPTED MANUSCRIPT
1170, 1132, 1081, 1053, 968, 945, 905, 871, 835, 798; MS (ESI) m/z [M-H]- 504.2 (HCl salt);Anal. Calcd. for C20H15BrClF4N3O; C, 47.60; H, 3.00; Br, 15.83; Cl, 7.02; F, 15.06; N, 8.33; O,
5.1.3.15.
RI PT
3.17 %.Found C, 47.89; H, 2.71; N, 8,23 %.
(3-(4-fluoro-3-(trifluoromethyl)phenyl)isoxazol-5-yl)-N-((5-methyl-1H-indol-3-
yl)methyl)methanamine (10o)
SC
Yield: 70%; off white solid; mp 155 -156 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 2.39 (s, 3H), 4.40 (s, 2H), 4.48 (s, 2H), 6.98 (d, 1H, J = 8.0 Hz), 7.32 ( d, 1H, J = 8.0 Hz), 7.40 ( s, 1H),
M AN U
7.50 – 7.60 (m , 2H), 7.75 ( t, 1H, J = 4.0 Hz), 8.21, (d, 1H, J = 8.0 Hz), 8.27 (m, 1H), 9.75 (s, 2H), 11.31 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 21.7, 40.5, 41.8, 104.1, 104.8, 111.9, 118.6, 118.9, 119.1,121.5, 123.7,124.5, 125.7, 127.4, 128.1, 128.3, 130.0, 134.1, 134.8, 160.73, 165.9; IR (KBr) cm-1 : 3347, 2949, 2778, 2592, 1742, 1693, 1626, 1523, 1429, 1400, 1321, 1266, 1168, 1134, 1079,
TE D
1054, 833, 771, 704, 679; MS (ESI) m/z [M-H]- 438.3 (HCl salt);Anal. Calcd. for C21H18ClF4N3O; C, 57.35; H, 4.12; Cl, 8.06; F, 17.28; N, 9.55; O, 3.64% Found C, 57.50; H, 4.42; N, 9.35 %.
Pharmacology
EP
5.2.
5.2.1. Chemicals and reagents
AC C
Phosphate buffered saline (PBS), RPMI 1640, fetal calf serum (FCS), pencillin and streptomycin were purchased from Gibco BRL (Life Technologies) Rockville, MD, USA). MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], doxorubicin, λ-carrageenan and ursolic acid were purchased from Sigma-Aldrich Chemical Company, (St. Louis, MO, USA).
5.2.2. In vitro secretory phospholipase A2 assay
17
ACCEPTED MANUSCRIPT
sPLA2 assay was performed using type IIA sPLA2 enzyme activity kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. The reaction mixture contained 10 µl of sPLA2, 10 µl of inhibitor (test wells only), 200 µl substrate and was incubated for 15 min.
RI PT
Then, 10 µl of 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) was added to develop the colour and read at a wavelength of 415 nm. sPLA2 enzyme hydrolysed the thioester bond at the sn-2 position of diheptanoyl thio-PC (substrate), free thiols were released and reacted with DTNB to form 5-thio-2-
SC
nitrobenzoic acid, which had absorbance at 415 nm. The control wells contained only sPLA2, substrate and DTNB. Urosolic acid was used as a positive control [19]. The percent inhibition of
M AN U
enzyme activity was calculated using the following formula:
% inhibition = [(Absorbance of control – Absorbance of test/Absorbance of control) ×100].
5.2.3. In vivo anti-inflammatory activity by carrageenan-induced rat paw edema model
TE D
Carrageenan-induced paw edema model is the most widely used for the evaluation of antiinflammatory activity [12]. Male wistar albino rats weighting 150 - 200 g were obtained from M/S Mahavir Enterprises (Hyderabad, Telangana, India). The animals were housed under standard
EP
conditions (temperature of 22±100C with an alternating 12-h light-dark cycle and relative humidity of 60±5%, before and also during the experiment. The animals were fed with standard laboratory which
was
purchased
from
M/S
Rayans
Biotechnology Pvt.
Ltd.
AC C
diet,
(Hyderabad, Telangana, India). During the experiment, the rats were allowed water and food ad libitum. Animal experimental protocol was approved by institutional animal ethical committee (IAEC no. GU/GIS/IAEC/PROTOCOL NO.03/2013). Animal experiments were conducted according to CPCSEA guidelines. The animals were divided into three groups (n = 3-6). Group I served as control and received saline at 1 ml/kg body weight (b.w) per os (p.o). Group II served as standard, received ursolic acid 18
ACCEPTED MANUSCRIPT
at a dose of 5 mg/kg b.w, p.o. and group III served as test and received synthesized compound(s) at a dose of 5 mg/kg b.w, p.o, respectively. The animals were treated with synthesized compounds and ursolic acid before 1 h to inject
RI PT
(subcutaneous) 0.1 ml of 1% λ-carrageenan solution with saline into sub-plantar region of left hind paw of each rat. The right hind paw of same rat was treated with 0.1 ml of saline alone in manner as control. Before induction of edema, the dorsiventral thickness of both the paws of each was
SC
measured using mercury displaced glass plethysmometer and also the measurements were taken at 1st, 2nd, 3rd, 4th and 6th hour after carrageenan injection. The results are expressed in percent edema
M AN U
inhibition. Ursolic acid was used as a positive control. 5.2.4. Anticancer studies 5.2.4.1. Cancer cell lines
MCF-7 breast cancer cell line was purchased from National Cancer Institute (Frederick,
TE D
MD, USA). Human prostate carcinoma cell line DU145 was obtained from American Type Culture Collection (Manassas, VA, USA). These cancer cells were maintained in RPMI medium supplemented with 10% FCS, 100 units pencillin and 100 µg of streptomycin per ml of medium.
EP
The cells were maintained in 95% humidity with CO2 at 37 0C temperature.
AC C
5.2.4.2. Cell proliferation by MTT assay MTT assay was performed to assess the antiproliferative activity of compounds [20]. Cancer cells were treated with isolated compound at various concentrations for 48 h. 20 µl of freshly prepared MTT reagent was added to cells and incubated at 37 °C for 2 h. The supernatant growth medium was removed and replaced with DMSO (100 µl) to dissolve the formazan crystals. The optical density (absorbance) was read at 570 nm with reference wavelength of 620 nm. 5.2.5.
In silico studies 19
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5.2.5.1. Drug like properties The rule was formulated by Christopher A. Lipinski in 1997 [21]. The rule describes the molecular properties important for a drug’s pharmacokinetics in the human body, including their
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absorption, distribution, metabolism and excretion (ADME). The rule is important for drug development where a pharmacologically active lead structure is optimized stepwise for increased activity and selectivity, as well as druglike properties. Molinspiration tool used to demonstrate the
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druglike properties of compounds [22]. 5.2.5.2. Molecular docking studies
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X-ray crystal structures of human group IIA sPLA2 (PDB ID: 1DB5) protein was obtained from Protein Data Bank and used in docking studies. Co-cystalized ligands and water molecules were removed from target protein using Argus lab. Ligands are prepared using chemoffice (Cambridge, UK). Energy minimization was done using molecular mechanics. The minimization
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was executed until root mean square value reached below 0.001 Kcal/mol. Such energy minimized ligands and receptors were used for docking studies using GEMDOCK (Generic Evolutionary Method for molecular DOCKing), which is a generic evolutionary method with an empirical
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scoring function for molecular docking (protein–ligand docking) [12, 23, 24]. A population size of 300 with 70 generations and 3 solutions were used in docking accuracy setting. PyMol was used for
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better visualization of interactions [23-25].
Conflict of Interest: The authors declare no conflict of interest.
Acknowledgements SRP and GR are grateful to Sarada Villas College Mysore for the support and encouragement to higher education. SRP and GR also thankful to CRD Department of Chemistry, PRIST University, 20
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Thanjavur-613403, India. NSY are thankful to GITAM University for providing research facilities. NSY expresses his sincere gratitude to Dr. M.V.V.S. Murthy, Pesident, GITAM University for his support and encouragement by providing necessary research facilities. NSY is very much thankful
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to Prof. G. Subrahmanyam, Prof. N. Lakshmana Das and Prof. Ch. Ramakrishna for their encouragement and motivation. This work was partly supported by the Russian Sciene Foundation
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(www.rscf.ru, Российский Научный Фонд, grant number 14-23-00160 for 2014-2016).
References
E.B. John and A.D. Edward, J. Lipid Res., 50 (2009) S237–S242.
[2]
E.A. Dennis, J. Cao, Y.H. Hsu, V. Magrioti, G. Kokotos, Chem. Rev., 111 (2011) 6130-
M AN U
[1]
6185. [3]
N.S. Yarla, A. Bishayee, L. Vadlakonda, R. Chintala, G.R. Duddukuri, P. Reddanna, D.S.
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Kaladhar. Curr Drug Targets, 16 (2015) 10.2174/1389450116666150727122501 D.Q. Nhat, D.A. Robert, S.C. Bria, Biochem. Pharmacol., 90 (2014) 338–348.
[5]
R.W. Schevitz, N.J. Bach, D.G.Carlson, Nat. Struct. Biol., 2 (1995) 458-465.
[6]
E Abraham, C Naum, V Bandi, Crit. Care Med., 31 (2003) 718-728.
[7]
J.D. Bradley, A.A. Dmitrienko, A.J. Kivitz,. J. Rheumatol., 32 (2005) 417-423.
[8]
http://clinicaltrials.gov/, Identifier: NCT00743925.
[9]
S.J. Nicholls, J.P. Kastelein, G.G.Schwartz, The VISTA-16 Randomized Clinical Trial.
AC C
EP
[4]
JAMA, 311 (2014) 252-262. [10]
Y. Pradeepkumar, M. Ruthu, C.C. Madhusudhana, G. Prasanthi, S.R.V. Jaya, J. Global Trends Pharm. Sci., 2(1) (2011) 55- 62.
[11]
M.P. Sadashiva, A. Nataraju, H. Mallesha, R. Rajesh, B.S. Vishwanath, K.S. Rangappa, Int. J. Mol. Med., 16(5) (2005) 895-904. 21
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[12]
N.S. Yarla, A. Rajaram, M. Basha, R. Azad, M. Basha, A. Rajack, D.S. Kaladhar, B.K. Allam, R.R. Pragada, K.N. Singh, S.K. Kadiri, R. Pallu, U. Parimi, A. Bishayee, G.R. Duddukuri. Anticancer Agents Med. Chem., 15(8) (2015) 1066 - 1077 S. Yamashita, J. Yamashita, M. Ogawa, Br. J. Cancer, 69 (1994) 1166-1170.
[14]
J. Jiazhong, L.N. Blake, R. Jeremy, Am. J. Pathol., 160 (2002) 667–671.
[15]
A.S. Paniraj. R.K.M Lokanath. Bulgarian Chem. Comm., 41(1) (2009) 80–83.
[16]
V.Y. Maria, W. Jizhen, C. Michael, L.R. Lijun, J.W. Duncan J. Med. Chem., 54(3) (2011)
SC
RI PT
[13]
765–781.
C.Y. Mayur, K.M. Satish, M. Salahuddin, S.K.S Math, K.N. Thimmaiah, B.S. Vishwanath.
M AN U
[17]
Lett. Drug. Des. Discov., 2(4) (2005) 307 – 310. [18]
L. Assogba, A. Ahamada-Himidi, N. M Habich, D. Aoun, L. Boukli, F. Massicot, C.M. Mounier, J. Huet, A.Lamouri, J.E. Ombetta, J.J. Godfroid, C.Z. Dong, F. Heymans. Eur J.
[19]
A. Nataraj, G.C.D Raghavendra. R. Rajesh, B.S. Vishwanath, Cur. Topics in Med. Chem., 7(8) (2007) 801-809.
Y.L. Murthy, K.P. Suhasini, A.S.Pathania, S. Bhushan, Y.S. Nagendra, Eur. J. Med.
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[20]
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Med. Chem., 40(9) (2005) 850–861.
Chem., 62 (2013) 545-55.
C.A. Lipinski, Drug Dis. today: Techn, 1(4) (2004), 337–341.
[22]
M.Q. Zhang, Mol. Biol. 803 (2012) 297-307.
[23]
M.Y. Jinn. J. Comput Chem., 25 (2004) 843–857.
[24]
J.M Yang, C.C Chen, GEMDOCK: A Generic Evolutionary Method for Molecular
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[21]
Docking. Proteins., 55 (2004) 288-304. [25]
D. Seeliger, B.L.DeGroot, Ligand J. Comput. Aided Mol. Des., 24 (2010) 417-422.
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Legends to figures: Figure 1. In vitro sPLA2 inhibitory activity of 10o and Ursolic acid (positive control). Compound 10o dose-dependley inhibited sPLA2 activity with IC50 of 10.23±0.91 µM where as ursolic acid
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(positive control) inhibited with IC50 of 12.59±1.03 µM.
Figure 2. Molecular docking studies of 10o on human GIIA sPLA2 crystal protein. (A) Binding orientation of 10o on crystal structure of human GIIA PLA2 (PDB ID: 1DB5) in ribbon model. (B).
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GLY-29, CYS-44, HIS-47, ASP-48, TYR-51, PHE-98.
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10o binds vicinity of amino acid residues at active site were PHE-5, TYR-21, GLY-22, CYS-28,
Figure 3. Anti-inflammatory activity of 10o. (A) Represents the rat paw edema volumes (ml) of treated (10o and ursolic acid) and untreated at various time intervals (1, 2, 3, 4 and 6 hrs) after carrageenan injection. Experimental results are expressed as mean (n = 3-6) ± standard error mean
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(S.E.M). The difference between experimental groups was compared by one-way analysis of variance (ANOVA). The P value of results less than 0.05 (P˂0.05) versus control was considered as significant (*represents p<0.05, **p<0.001). (B). 10o showed comparable activity with ursolic acid
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injection.
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(positive control) and exhibited maximum edema inhibition between 3rd and 4th h after carrageena
Figure 4. Antiproliferative effect of 10o against MCF-7 breast and DU145 prostate cancer cells. Compound 10o showed significant anti-proliferative effect in MCF-7 breast and DU145 prostate cancer cell lines with IC50 of 10.03±0.86 and 14.15±1.2 µM, respectively, whereas doxorubicin (positive control) exhibited anticancer activity with 8.02±0.69 and 4.75±0.35 µM, respectively.
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OH N
H
Br
O N
i
iii
ii
R2
R2
R1
R2
R1 NH2.HCl
O N iv
5a=R1=H,R2=H, R2
R R
R H
vi
R 1
R
5(a-e)
10c=R=CH3,R1=H,R2=H,
10d=R=H,R1=CH3,R2=H,
2
1
2
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10a=R=H,R =H,R =H,
10b=R=Br,R1=H,R2=H,
1
1
8b=R=Br,
10f=R=CH3,R =CH3,R =H,
10g=R=H,R1=OCH3,R2=H,
10h=R=Br,R1=OCH3,R2=H,
R1
R2
10i=R=CH3,R =OCH3,R =H,
9(a-o)
viii
10m=R=H,R1=F,R2=CF3,
10n=R=Br,R1=F,R2=CF3,
R
R1
N H
R2 10(a-o)
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10l=R=CH3,R1=NO2,R2=H,
N Boc
HN .HCl
10j=R=H,R1=NO2,R2=H,
10k=R=Br,R1=NO2,R2=H,
R
N O
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8a=R=CH3
HN
2
10e=R=Br,R =CH3,R =H,
1
8a=R=H,
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2
8(a-c)
H
N O
H N Boc
O
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O
R
NH2.HCl
O N
5e=R1=F,R2 =CF3
N Boc 8(a-c)
N H 7
6
4
5d=R1=NO2,R2=H,
O
v N H
R1
5b=R1=CH3,R2=H,
5c=R1=OCH3,R2=H,
R1
R2
R1 3
2
1
5(a-e)
N3
O N
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O
10o=R=CH3,R1=F,R2=CF3,
(i) NH2OH.HCl, NaOH, H2O, rt, (ii) Chloramino-T, Propargyl bromide, EtOH, 60-65 °C, (iii) NaN3, DMF, 60-65 °C, (iv) PPh3, H2O, THF, rt v) POCl3, DMF, rt, (Vi) NaH, Boc anhydride, DMF, rt. (Vii) NaCNBH3, MeOH (Viii) EtOAc in HCl
Scheme 1: Synthesis of indole containing isoxazole derivatives (10 a-o)
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Table 1
In silico analysis
In vitro IC50 (µM)
10a
Dock score (-Kcal/mol) -107.3
30.63±0.91
10b
-109.6
32.49±1.02
10c
-111.8
27.92±0.86
10d
-115.4
22.68±1.13
10e
-118.4
27.32±1.73
10f
-111.3
10g
-112.3
10h
-103.8
10i
-105.6
10j
-108.2
10m 10n
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10o
Ursolic acid (positive control)
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10l
21.55±1.21 18.22±1.42 32.64±2.38 24.55±2.81 25.61±1.72
-103.7
22.35±2.49
-104.1
19.44±2.1
-108.7
21.5±1.52
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10k
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Compound no
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GIIA sPLA2 inhibitory activities of indole containing isoxazole derivatives.
-110.3
17.18±1.43
-129.5
10.23±0.91
-128.8
12.59±1.03
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Table 2 Drug like properties of indole containing isoxazole derivatives. Log P
Non
10a
303.365
3.442
4
10b
382.261
4.227
4
10c
317.392
3.866
4
10d
317.392
3.89
4
10e
396.28
4.675
4
10f
331.41
4.325
4
2
10g
333.39
3.498
5
2
10h
412.287
4.283
5
2
10i
347.418
3.923
5
2
10j
348.362
3.4
7
2
10k
427.258
4.186
7
2
10l
362.389
3.825
7
2
10m
389.352
4.429
4
2
10n
468.248
5.214
4
2
10o
403.379
4.853
4
2
2 2
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2
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NoHnH
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MW
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Compound
2 2
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Highlights:
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A series of new indole containing isoxazole derivatives (10a-10o) is synthesized. These compounds (10a-10o) showed significant sPLA2-inhibition activity. 10o showed potent sPLA2 inhibition activity among other compounds. 10o exhibited in vivo antinflammatory activity in mice. 10o showed in vitro anticancer activity against MCF-7 breast and DU145 prostate cancer cells.
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• • • • •