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WO2019130346A1 - Nouveaux composés phénoliques de nerium indicum ayant une activité anti-cancer du sein - Google Patents

Nouveaux composés phénoliques de nerium indicum ayant une activité anti-cancer du sein Download PDF

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WO2019130346A1
WO2019130346A1 PCT/IN2018/050876 IN2018050876W WO2019130346A1 WO 2019130346 A1 WO2019130346 A1 WO 2019130346A1 IN 2018050876 W IN2018050876 W IN 2018050876W WO 2019130346 A1 WO2019130346 A1 WO 2019130346A1
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phenyl
compound
ppm
compounds
cells
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SYED ZAMEER Ahmed K
Thanga Kumar A
Sidhra S
Syed Tajudeen S
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/24—Apocynaceae (Dogbane family), e.g. plumeria or periwinkle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C07C233/24—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
    • C07C233/29—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring having the carbon atom of the carboxamide group bound to an acyclic carbon atom of a carbon skeleton containing six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/32—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C07C235/38—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring

Definitions

  • Natural products like plant extracts either in pure isolated compound or as a standardized extracts provides enormous ways for the discovery of new drugs because of the presence of unlimited bioactive compound diversity.
  • These compounds contain wide variety substances that can be used in the treatment of chronic and infectious diseases with favorable biological activities like anticancer, antimicrobial, antioxidant, anti-inflammatory, antiarthritic, analgesic, wound healing and cytotoxic properties.
  • Nerium indicum the most abundant plant from the family of Apocynaceae with enormous biological activities reported by various scientists.
  • MCF 7 breast cancer cell line
  • eleven cardenolide diglycosides including one new compound was isolated and evaluated against normal human fibroblast cells (WI-38), malignant tumour cells induced from WI-38 (VA-13) and human liver tumour cells (HepG2) representing effective cytotoxic effect against tumour cell line.
  • New ursane-type triterpene, oleanane-type triterpene, and dammar ane -type triterpene were isolated from the leaves of Nerium oleander together with 12 known triterpenes by Liwei Fu et al., (2005).
  • the anti-inflammatory and anticancer activity of the isolated compounds demonstrated effective cell growth inhibitory activity against lung carcinoma cell lines.
  • Two new taraxasterane-type triterpenes 20 b 28-epoxy-28 a-methoxytaraxasteran-3 b ol and 20 b, 28-epoxytaraxaster- 2l-en-3 b -ol, were isolated from an ethyl acetate extract of the leaves of Nerium oleander, together with ursane-type triterpenes, 28-nor-urs-l2-ene-3 b, 17 b -diol and 3 b -hydroxyurs-l2-en-28-aldehyde.
  • the cell growth inhibitory activity was evaluated on WI- 38 cells (normal human fibroblast cells induced from lung cells), VA-13 cells (malignant lung tumour cells induced from WI-38), and HepG2 cells (human liver cancer cells).From the results it was evident that the compounds showed mild to moderate cell growth inhibitory activity against the tested cell lines. (Ming Zhao et ah, 2006).
  • the main object of the present invention is to isolate novel phenolic compounds for the treatment of cancer
  • Yet another object of the invention is to elucidate the structure of isolated pure compounds and to screen against human breast cancer cell lines (MCF-7)
  • the major aim of the invention is to find a novel and efficient protocol which overcomes the drawbacks of prior isolation procedures for processing of crude extract covers fractionation, isolation and purification of the bioactive phenolic compounds from Nerium indicum. Further this invention provides the anticancer effects of phenolic compounds against breast cancer cell line
  • the present invention reveals for the first time the methods of isolation; fractionation and characterization of two phenolic compounds from Nerium indicum.
  • Nerium indicum is commonly known as Arali in Tamil found throughout India.
  • the plant parts are widely used for the treatment of cancer, cardiotonic, leprosy and skin diseases. It is interesting that the biological activity exposed by crude extracts of the plants is maintained in the purified phenolic compounds also. This pays a way for the further discovery towards the potential use of Nerium indicum for treatment of various ailments.
  • the invention offer an easy and reliable protocols for the extraction of methanolic crude extracts of Nerium indicum. It also discloses the methods related to fractionation and purification of bioactive compounds from the above said plant.
  • the invention is also concerned with the spectral identification of the phenolic compounds N-(4-Hydroxy-phenyl)-2- methoxy-2-phenyl-acetamide (Table 1 , Fig. 1) and N-(4-Hydroxy-phenyl)-2-phenyl-N- phenyl acetyl-acetamide (Table 2, Fig. 2 ) and to elucidate its structure.
  • the isolated compounds were tested for its in vitro anticancer activity using MCF-7 breast cancer cell line and found to have significant inhibition of cell proliferation and induced apoptosis with nuclear condensation effect on the cells studied using Acridine orange /Ethidium bromide (AO/EtBr) staining method (Fig. 3).
  • the invention delivers a procedure for extracting and purifying biologically active molecules from Nerium indicum by using the following steps
  • ii Fractionating and isolating pure compound using conventional column chromatography with different proportion of solvents (Example II) iii. Characterizing the pure compounds using HPLC, FT-IR, 1 H NMR, 13 C NMR, DEPT NMR spectroscopy, COSY, HMBC NMR spectroscopy and LC-MS analysis.
  • the FT-IR spectrum of the isolated compound I from fraction NB4fl shows characteristic band at 3355 cm 1 which we assign to -NH stretching frequency.
  • the strong band at 1666 cm 1 is attributable to the carbonyl stretching vibration.
  • the NMR data’s of the isolated compound is represented in Table 1.
  • the X H NMR spectrum of compound showed the -NH protons (H-3) as a singlet at d 8.44 ppm, aromatic protons (H-5 & H-9) as a doublet at d 7.33 ppm , with coupling constant of 8.0 and 1.6 ppm, the H-6, H-7 & H-8 protons appears at the range of 7.27 to 7.22 ppm with multiplicity.
  • the H-2’ & H-6’ shows doublet at 7.19 ppm with 8.8 ppm
  • the proton H- 3’ & H-5’ appears doublet at 6.57 ppm with 8.8 ppm of coupling constant.
  • the H-2 proton was showed as singlet at 4.61 ppm and the methoxy proton (H-10) appeared as singlet at 3.29 ppm.
  • the carbonyl C-l signal from 13 C NMR spectrum was found at d 168.07 ppm, the carbon C-4’showed at 152.73 ppm due to directly attached to hydroxyl proton.
  • the C-l’ carbon appeared at 135.43 ppm due to direct attachment to the secondary amine group.
  • the carbons C-5 and C-9 showed at 127.67 ppm with high intensity.
  • the carbons C-6 and C-8 , C-2’ and C-6’ were appeared at 126.24 and 121.15 ppm respectively with high intensity, the carbons C-3’and C-5’ are showed at 114.79 ppm, the C-2 carbon showed at 82.70 ppm and the methoxy carbon signal ( C-10) appeared at 56.17 ppm.
  • compound I was accounted to have 11 carbons comprising four quaternary carbons and seven methine.
  • the 2D spectra the correlation between the H-H coupling shows that the H-3(-OH) protons does not correlate with other protons.
  • the H-5 & H-9 protons correlates with protons of H-7, H-6 and H-8.
  • the protons H-2’ and H-6’ are in correlation with the protons of H-3’ and H-5’ whereas, no correlation was found in COSY spectra.
  • the HSQC spectrum represents the heteronuclear associations of protons with 13 C atoms, thus the 1 H chemical shifts are plotted on one axis and the 13 C chemical shifts on the other.
  • the single bond present in the C-H molecules can be analyzed with HSQC spectrum and hence it provides evidence between the bonding of protons with carbon. Large frequency of Multiplets protons often overlap in 1 H NMR spectra which makes difficulty in identification, so, HSQC spectra supports and helps to identify these protons.
  • the hydrogen and carbon correlation was given in the Table 1.
  • the FT-IR spectrum of the isolated compound II from fraction NCl3bl displays characteristic band at 3275 cm 1 which we consign as -OH stretching frequency.
  • the strong band detected at 1655 cm 1 is observed to the carbonyl stretching vibration.
  • Table 2 demonstrates the NMR data of the isolated compound II, the X H NMR spectrum of compound II represents the -OH protons (H-7) as a singlet at d 10.25 ppm, aromatic protons (H-2 & H-6) as a singlet at d 7.62 ppm attached with phenolic aromatic group with 8.8 as coupling constant, the H-5’& H-7’ & H-5” & H-7” all the protons are equivalent protons appeared as doublet at 7.37 to 7.36 ppm, the H-4’& H-8’ & H-4” & H-8”protons showed as multiple at 7.34 - 7.29 ppm, the H-6’ & H-6” two protons appeared as multiple at 7.26 - 7.23 ppm, the protons H-3
  • the 13 C NMR spectrum showed the carbonyl C-T & C-l” signals at d 170.17 and 169.02 ppm, the carbon C-4 signal showed at 145.79 ppm, this carbon was directly attached to hydroxyl group, the C-3’ & C-3” carbon signal showed at 136.84 and 135.89 ppm, the carbon signal at 133.91 ppm belongs to C-l, the C-2 & C-6 signals at 121.79.
  • the C-4’ & C-8’, C-4” & C-8” carbon signals appeared at 129.42 and 129.03 ppm
  • the carbon signal of C-5’ & C-7’, C-5” & C- 7” appeared at 128.43 and 128.26 ppm
  • the C-6’ & C-6” carbon signals showed at 126.97 and 126.49 ppm
  • the above all the carbon signals are chemically equivalent but special arrangement may be different so that these carbons are in slight different chemical shift values.
  • the carbons C- 2 & C-6 appeared at 121.79 ppm
  • the C-3 & C-5 carbons showed at 119.99 ppm
  • the aromatic methylene carbons C-2’ & C-2” appeared at 43.22 and 40.09 ppm.
  • compound II was accounted to have 16 carbons signals comprising of two methylene, fourteen methine and six quaternary carbons.
  • H-7 (-OH) protons are not correlated with other protons whereas, H-2 & H-6 protons are correlated with H-3 & H-5, the H-5’ & H-7’ , H-5” & H-7” protons are correlated with H-6’ & H-6”, H-4’ & H-8’ , H-4” & H-8” , the H-4’ & H-4” , H-8’ & H-8” proton was correlation with H-5’ & H-7’, H-5” & H-7” protons, the proton H-6’ & H-6” were correlated with H-4’ & H-8’, H-4”& H-8” protons.
  • the hydrogen and the carbon correlation are represented in Table 1.
  • the structure of the compound II (Fig. 2) was predicted and confirmed as N-(4-Hydroxy- phenyl)-2-phenyl-N-phenylacetyl-acetamide with a molecular formula C22H19NO3 and identified molecular weight 345.4.
  • the present study aims to identify the potential and a right candidate from Nerium indicum a traditional herb and a cure against cancer.
  • Fig.3 illustrates the in vitro cytotoxicity activity of compounds I and II ( 10- 100 pg concentrations) against selected cancer cells with dose dependent destruction.
  • the IC50 values were 27 ⁇ 1.0 m g/m L (compound I), 24 ⁇ 1.5 m g/m L (compound II) and the results shows significant inhibition of cell proliferation in in vitro condition.
  • Fluorescence microscopic analysis was carried out to identify the apoptotic activity of isolated phenolic compounds N-(4-Hydroxy-phenyl)-2-methoxy-2-phenyl-acetamide (Compound I) and N-(4-Hydroxy-phenyl)-2-phenyl-N-phenylacetyl-acetamide (Compound II) on MCF-7 breast cancer cell line.
  • the results of fluorescence microscopic images of MCF-7 cells both control (Fig. 4 a) and compounds I and II treated cells are presented in Fig.4 (b-g).
  • the untreated MCF-7 cancer cells did not show any characteristic changes when compared to compounds treated cancer cells (Fig.4 b-g).
  • FIG. 5 shows the results of DAPPI stained breast cancer cells after 24h taken in the absence and presence of compounds using Fluorescence microscope. It is seen from the Fig. 5 (b-g) addition of compounds to the cell line demonstrates higher level of nuclear fragmentation than the untreated cells which didn’t show any significant changes in the nuclear appearance. Compounds treated MCF-7 cancer cells shows bright fetches which indicates the condensed chromatins and nuclear fragmentations in the cancer cells and it comparable with the control cells (Fig. 5 a).
  • Apoptotic cells were detected with DAPI (4'-6'-diamidino-2-phenyl indole) nuclear staining technique and is known to form fluorescent complexes with double- stranded DNA. Morphological change of typical apoptosis, such as cell shrinkage, rounding and detachment of the cells from the flask, as observed by fluorescence microscopy.
  • Fig 4. AO/EtBr analysis of treated cells a) Control untreated cells, (b-d) Compound I treated cells, (e-g) Compound II treated cells at the concentration of 10pg/ml, 2hpg/ml and 30pg/ml; showing nuclear fragmentation and chromatin condensation.
  • FIG. 5 DAPI apoptotic analysis of treated cells(a) Control cells (b-d) Compound I treated cells, (e-g) Compound II treated cells at the concentration of 10pg/ml, 20pg/ml and 30pg/ml; the arrows indicate apoptotic cells.

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Abstract

La présente invention concerne un nouveau procédé efficace pour l'isolement de deux nouveaux composés phénoliques : le composé I N-(4-hydroxy-phényl)-2-méthoxy-2-phényl-acétamide de formule moléculaire C15H15NO3 et le composé II N-(4-hydroxy-phényl)-2-phényl-N-phénylacétyl-acétamide de formule moléculaire C22H19NO3. L'efficacité thérapeutique des nouveaux composés a été évaluée sur des lignées cellulaires du cancer du sein humain (MCF-7) et les résultats ont présenté une cytotoxicité significative confirmant le processus d'apoptose induite.
PCT/IN2018/050876 2017-12-27 2018-12-25 Nouveaux composés phénoliques de nerium indicum ayant une activité anti-cancer du sein Ceased WO2019130346A1 (fr)

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IN201741046767 2017-12-27

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011103563A1 (fr) * 2010-02-22 2011-08-25 Arno Therapeutics, Inc Procédés et compositions pour inhiber et prévenir la croissance de mastocytes malins
WO2011143314A1 (fr) * 2010-05-11 2011-11-17 Arno Therapeutics, Inc Compositions et procédés de réduction de la prolifération et de la viabilité de cellules lymphoblastoïdes
US8318808B2 (en) * 2003-12-02 2012-11-27 The Ohio State University Research Foundation Zn2+chelating motif-tethered short-chain fatty acids as a novel class of histone deacetylase inhibitors

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8318808B2 (en) * 2003-12-02 2012-11-27 The Ohio State University Research Foundation Zn2+chelating motif-tethered short-chain fatty acids as a novel class of histone deacetylase inhibitors
WO2011103563A1 (fr) * 2010-02-22 2011-08-25 Arno Therapeutics, Inc Procédés et compositions pour inhiber et prévenir la croissance de mastocytes malins
WO2011143314A1 (fr) * 2010-05-11 2011-11-17 Arno Therapeutics, Inc Compositions et procédés de réduction de la prolifération et de la viabilité de cellules lymphoblastoïdes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE CAS 14 July 2015 (2015-07-14), retrieved from STN Database accession no. 1799374-96-1 *

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