Patent Application
20250230477
Present invention relates to a process for the preparation of tetrahydroanthracenes of formula 1 from Streptomyces species [MTCC-25420 and MTCC-25512]. Present invention further relates to tetrahydroanthracenes of formula I for anticancer activity.
Particularly, present invention relates to 9,9′-bianthrylanthracene antibiotic, Setomimycin of formula 1, having activity against Gram positive bacteria has also been demonstrated to have significant anticancer activity against several cancer cell lines.
Biaryl preanthraquinones, represented by the julichromes, spectomycins, and setomimycins, are antibiotics isolated from bacteria (doi.org/10.1021/acs.jnatprod.0c00805). In nature, intermolecular oxidative phenol coupling is the main process for the formation of a troposelective biaryl compounds. The monomeric subunits of these biarylic pre-anthraquinones are derived from a common polyketide precursor, yet the coupling reaction proceeds in a regioselective manner, with the position of attachment of the two subunits depending on the specific streptomycete strain (doi.org/10.1021/ja501630w).
Anthracene derivatives have hydroxyl groups such as 1-hydroxyanthracene and 2-hydroxyanthracene, homologous to phenol or naphthols and hydroxyanthracene are pharmacologically active. Many Anthracenes can also be found with multiple hydroxyl groups such as 9, 10-dihydroxyanthracene.
Setomimycin, a rare 9,9′-bianthrylanthracene antibiotic, was first isolated from Streptomyces pseudovenezuelae in 1978 (doi.org/10.7164/antibiotics.31.1091). It was reported to be active against Gram positive bacteria i.e. S. aureus, B. subtilis, B. cereus, and M. smegmatis, including mycobacteria. It was also shown to reduce tumor growth in a Sarcoma-180 mouse solid tumor model when administered at a dose of 200 mg/kg per day for seven days.
One of the studies showed that under certain conditions Streptomyces lincolnensis ACM-4234 produce a new series of bianthracenes, leading to the isolation and structure elucidation of the Lincolnenins A-D natural products. Staley in 1994 discovered three new tetrahydroanthracene antibiotics that are isolated from culture broths of Streptomyces spectabilis (doi.org/10.1021/acs.joc.0c02492).
Spectomycins A1, A2 and B1, have been reported as symmetrical dimeric forms C38H34O14 (B1) and monomeric C20H20O7 (A1), C19H18O7 (A2). (doi.org/10.7164/antibiotics.47.1425).
Another report revealed Spectomycin B1, a closely related dimeric dihydroanthracene antibiotic from Streptomyces spectabilis UC 2294 (doi.org/10.1021/ja501630w).
Yet another study disclosed four new derivatives of tetrahydroanthracene and antibiotic (A-39183A), from the Streptomyces fumigatiscleroticus HDN10255. (doi.org/10.1021/acs.jnatprod.0c00805 and doi.org/10.7164/antibiotics.47.1425)
Another study revealed Antibiotic A-39183 complex, comprising microbiologically active, related factors A, B, C, D, and E, produced by submerged, aerobic fermentation of a new microorganism Streptomyces sp., NRRL 12049. The A-39183 factors were demonstrated to have antibacterial activity, against Staphylococcus and Streptococcus species that are penicillin resistant. The A-39183 factor ionophores were found active against both Gram-positive and Gram-negative anaerobic bacteria (U.S. Pat. Nos. 4,283,390, 4,452,741, 4,410,628).
Till date, only four strains namely Streptomyces pseudovenezuelae (doi.org/10.7164/antibiotics.31.1091), Streptomyces sp., NRRL 12049 (U.S. Pat. No. 4,283,390), Streptomyces afghaniensis, Streptomyces auranticus JA 4570 (doi.org/10.1021/ja501630w) have been reported for tetrahydroanthracenes and Setomimycin (9,9′-bianthracene antibiotic) production. The available prior art demonstrates the production of tetrahydroanthracenes by submerged aerobic fermentation with the yields upto 150 mg/L setomimycin production, thus, limiting its commercial exploitation. Further, none of the reports have demonstrated anticancer activity of the same.
Main objective of the present invention is to provide an improved and efficient process for the preparation of tetrahydroanthracenes of Formula I from new source i.e. Streptomyces sp. MTCC-25420 and Streptomyces sp. MTCC-25512 isolated from lower Shivalik region of North-Western Himalayas, Jammu, India.
Another objective of the present invention is to provide an improved and efficient process for the preparation of tetrahydroanthracenes of Formula I in terms of time of production as well as yields.
Still another objective of the present invention is to demonstrate the effect of Compounds 1-3 on a panel of anticancer cell lines.
Yet another objective of the present invention is to provide the mode of action of Setomimycin (Compound 3) on anticancer target proteins that regulate cell proliferation, differentiation and apoptosis.
Tetrahydroanthracene producing strains Streptomyces sp. MTCC-25420 and Streptomyces sp. MTCC-25512 used herein, were isolated from soil sample collected from Shivalik region of North-Western Himalayas, Jammu 32.7266° N, 74.8570° E, India.
All other biological raw materials used as media components in the invention was procured from HIMEDIA, Laboratories Pvt. Ltd 23, Vadhani Ind. Est., LBS Marg, Mumbai-400086, India.
Accordingly, present invention provides a process for the preparation of tetrahydroanthracene compounds of Formula I
In yet another embodiment of the present invention, carbon source is selected from the group comprising monosaccharides, disaccharides and polysaccharides like Glucose, Fructose, Sucrose, Mannitol, Glycerol, gluconic acid, Pectin, Lactose, Maltose, Mannose, Chitosan, Dextrin, molasses, Starch, Xylose, Molasses, Corn steep liquor, Inositol, Chitin, Sorbitol either alone or combination thereof.
In yet another embodiment of the present invention, nitrogen sources is selected from the group comprising Beef extract, Meat solubles, corn meal, Casein, Soyabean Meal, Yeast extract, N/Z amine A, N/Z amine B, Casein hydrolysate, enzyme-hydrolyzed casein, Peptone, Sodium nitrate, Valine, Ammonium nitrate, Urea, Arginine, Asparagine, Ammonium phosphate, Potassium nitrate, Ammonium sulphate, nitrate salts either alone or combination thereof.
In yet another embodiment of the present invention, trace salts are selected from the group comprising Calcium carbonate, Magnesium sulphate, Di-potassium hydrogen phosphate, Ferrous sulphate, Potassium chloride and Sodium chloride, Ammonium sulphate/nitrate.
In yet another embodiment of the present invention, Compounds 1-3 have potent anticancer activity in various cancer cell lines such as breast cancer (MCF-7, MDA-MB231), Human lung cancer (A-549), human pancreatic cancer (MiaPaca-2), Human prostate cancer (PC-3), Colon cancer cell line (HCT-116, HT 29).
In yet another embodiment of the present invention, Setomimycin i.e. Compound 3 significantly abrogated cancer cell proliferation and inhibited cancer cell migration and invasion in highly metastatic cancer cells (MiaPaca-2, MCF-7, HT-29 and HCT-116) of Pancreatic, Breast and Colorectal origin.
In yet another embodiment of the present invention, Setomimycin i.e. Compound 3 down-regulates ERK and MEK proteins which are the major regulators of cell proliferation, differentiation and apoptosis, alongside it upregulates pro-apoptotic protein Par-4 and downregulated anti-apoptotic protein BCL-2 in Colon as well as breast cancer cells HCT-116 and MCF-7 respectively, and molecular docking studies and western blot analysis further support strong affinity of setomimycin towards MEK protein.
In yet another embodiment of the present invention, Compounds 1-3 have potent antimicrobial activity against a panel of Gram positive human pathogens such as Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Micrococcus luteus and MRSA/MDRs.
Present invention provides a process for the preparation of tetrahydroanthracenes of Formula I from Streptomyces sp. MTCC-25420 and Streptomyces sp. MTCC-25512 and anticancer activity thereof, which comprises specific fermentation conditions in combination for production of tetrahydroanthracenes of Formula I upto 5.0 g/L and upto 800 mg/L yields of setomimycin of formula 3 or even higher in presence of certain elicitors/precursors, wherein, Setomimycin (Compound-3) significantly abrogated cancer cell proliferation and inhibited cancer cell migration and invasion in highly metastatic cancer cells (MiaPaca-2, MCF-7, HT-29 and HCT-116) of Pancreatic, Breast and Colorectal origin.
Present invention provides a process for the preparation of Tetrahydroanthracenes of Formula I, including rare 9,9′-bianthrylanthracene antibiotic Setomimycin (Compound-3) from new source i.e. Streptomyces sp. MTCC-25420 and Streptomyces sp. MTCC-25512 from NW Himalayan region.
The process for the production of tetrahydroanthracenes of Formula I involves solid state as well as submerged fermentation, wherein consistent extracellular as well as intracellular production takes place in various fermentation media in shake flask as well as in bioreactor upto 500 L size, under various fermentation conditions (either alone or in combination).
Essential trace elements salts like Calcium carbonate, Magnesium sulphate, Di-potassium hydrogen phosphate, Ferrous sulphate, Potassium chloride and Sodium chloride, Ammonium sulphate/nitrate etc. are necessary in combination with various constituents of fermentation media for the growth of the organism and production of tetrahydroanthracenes.
The tetrahydroanthracene producers, i.e. Streptomyces sp. MTCC-25420 and Streptomyces sp. MTCC-25512 are capable to grow at pH 4.0 to pH 10.0 with Setomimycin production at wide pH range i.e. pH 5.0, 6.0, 7.0, 8.0, and 9.0 with preferred pH of 6.0 to 8.0.
Streptomyces sp. MTCC-25420 and Streptomyces sp. MTCC-25512 grow at temperatures ranging from 15° C. to 45° C. with the preferred temperature for Formula-1 production at 20° C. to 40° C.
Streptomyces sp. MTCC-25420 and Streptomyces sp. MTCC-25512 provide the production of tetrahydroanthracenes (Formula-1) within 2-10 days depending on the production medium and fermentation conditions with the yield of crude extract ranging from 200 mg/L to 5.0 g/L or even higher in presence of certain elicitors/precursors e.g. Valine, Arginine, Asparagine, Sodium acetate and Glycerol.
Antibiotic (Compounds 1-3) has potent antimicrobial activity against a panel of Gram positive human pathogens such as Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Micrococcus luteus and Methicillin-resistant Staphylococcus aureus (MRSA)/Multi drug resistant bacteria (MDRs).
Compounds 1-3 have shown potent anticancer activity against various cancer cell lines such as breast cancer (MCF-7, MDA-MB231), Human lung cancer (A-549), human pancreatic cancer (MiaPaca-2), Human prostate cancer (PC-3), Colon cancer cell line (HCT-116, HT-29). Wherein, Setomimycin (Compound-3) significantly abrogated cancer cell proliferation and inhibited cancer cell migration and invasion in highly metastatic cancer cells (MiaPaca-2, MCF-7, HT-29 and HCT-116) of Pancreatic, Breast and Colorectal origin.
The culture was maintained on glass test tube of 18×150 mm2 size having agar slants (Table 1) by transferring a loopful of culture from matured slant and kept for incubation at 28° C.±2° C. for about one week with subsequent sub-culturing after every two weeks. The Pre-inoculum was prepared in a narrow mouth 500 mL Erlenmeyer flask containing 150 mL medium (Table 2) by transferring loopful of freshly grown culture.
The inoculated medium was incubated at about 28° C.±2° C. for about 48-72 hours on a rotating shaker at 250 rpm. 5-10% of pre inoculum was transferred to 1000 mL Erlenmeyer flasks containing 300 mL seed medium (Table 3). The inoculum was prepared by incubating the flasks at 28° C.±2° C. for about 48-72 hours on a rotating shaker at 250 rpm.
Preferably 5-10% of two days old seed culture (inoculum) was transferred to 1000 mL Erlenmeyer flasks containing 300 mL production media (Table 4) and incubated at 15° C.-45° C. for 2-10 days on a rotating shaker at 100-300 rpm followed by liquid-liquid extraction using methanol-ethyl acetate or ethyl acetate-hexane or ethyl acetate-toluene, acetone-toluene or methanol-dichloromethane or methanol-chloroform or ethyl acetate-acetone or combination of these.
Monosaccharides, disaccharides and polysaccharides including Glucose, Fructose, Sucrose, Mannitol, Glycerol, Pectin, Lactose, Maltose, Mannose, Gluconic acid, Chitosan, Dextrin, Starch, Xylose, Molasses, Corn steep liquor, Inositol, Chitin, Sorbitol etc. are useful for the production of tetrahydroanthracenes; wherein, preferred carbon sources are glucose, starch, dextrin, chitosan, molasses, gluconic acid and glycerol either alone or combinations of these nutrients. Among these, glycerol and starch is most preferable carbon sources for antibiotic production or in combinations with, soyabean meal, sodium acetate, glycerol and glucose.
Organic as well as inorganic nitrogen sources like Beef extract, Casein, Soya Meal, Oatmeal, Yeast extract, N/Z amine A, N/Z amine B, Casein hydrolysate, Peptone, Sodium nitrate, Valine, Ammonium nitrate, Urea, Arginine, Asparagine, Ammonium phosphate, Potassium nitrate, Ammonium sulphate support tetrahydroanthracenes production; wherein preferred nitrogen sources are soybean meal, yeast extract, peptone, sodium/potassium nitrate, casein, casein hydrolysate and ammonium nitrate or combinations of these nutrients. Meat solubles, peptone, Beef extract, corn meal, enzyme-hydrolyzed casein, and nitrate salts are also useful.
Trace elements salts like Calcium carbonate, Magnesium sulphate, Di-potassium hydrogen phosphate, Ferrous sulphate, Potassium chloride and Sodium chloride, Ammonium sulphate/nitrate etc. are helpful in combination for the growth of the organism and production of tetrahydroanthracenes.
Setomimycin (Compound-3) has potent antimicrobial activity against a panel of Gram positive human pathogens such as Staphylococcus aureus, Bacillus, subtilis, Bacillus cereus, Micrococcus luteus and Methicillin-resistant Staphylococcus aureus (MRSA)/Multi drug resistant bacteria (MDRs).
The antibiotic Setomimycin (Compound-3) has potent anticancer activity against various cancer cell lines viz. breast cancer (MCF-7, MDA-MB231), Human lung cancer (A-549), human pancreatic cancer (MiaPaca-2), Human prostate cancer (PC-3), Colon cancer cell line (HCT-116, HT 29), wherein, Setomimycin significantly abrogated cancer cell proliferation and inhibited cancer cell migration and invasion in highly metastatic cancer cells (MiaPaca-2, MCF-7, HT-29 and HCT-116) of Pancreatic, Breast and Colorectal origin.
Setomimycin (Compound-3) demonstrates downregulation of ERK and MEK proteins which are the major regulators of cell proliferation, differentiation and apoptosis. Interestingly setomimycin upregulated pro-apoptotic protein Par-4 and down regulated ant-apoptotic protein BCL-2 in Colon as well as breast cancer cells HCT-116 and MCF-7 respectively.
Setomimycin (Compound-3) interacts with MEK1 by forming hydrogen bond with Lys 97, Asp 190 and Ser212 as per molecular docking studies, wherein strong affinity of Setomimycin towards MEK protein was confirmed by Western blot analysis.
The following examples are given by way of illustration of the working of the invention in actual practice and should not be construed to limit the scope of the present invention.
The actinobacteria strains Streptomyces sp. MTCC-25420 and Streptomyces sp. MTCC-25512 were isolated from soil sample collected from Shivalik foothills 32.72660 N, 74.85700 E, Jammu, India on Starch-Casein agar (SCA) medium. The pure cultures were maintained and preserved on same medium. The morphological characteristics were investigated on ISP2 and SCA, respectively at 28° C.±2° C. The mycelial organization and sporulation were observed by light microscopy and scanning electron microscopy (SEM). The cultural characteristics of isolate grown on SCA were observed as powdery white colony with circular and smooth ends. Aerial colonies appear white during early growth and yellow pigmentation starts appearing with time.
Molecular characterization was performed by 16S rRNA gene sequencing using universal primers 27F (5′AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′GGCTACCTTGTTACGACTT-3′) using Emerald Amp GT master mix. The 1500 bp amplicon was purified by Gel elution/SAP. The purified product was sequenced by Sanger sequencing and the partial sequence was assembled and submitted to NCBI with Accession numbers MW199142 and OM540363. The culture has been submitted to Microbial Type Culture Collection, India with Accession no. MTCC-25420 and MTCC-25512.
The cultures Streptomyces sp. MTCC-25420 and Streptomyces sp. MTCC-25512 were maintained on glass test tubes of 18×150 mm2 size having agar slants (Table 1) by transferring a loopful of culture from matured slant and kept for incubation at 28° C.±2° C. for about one week with subsequent sub-culturing after two weeks. The glycerol stocks and lyophilized cultures were kept for long term storage of the culture for future purpose.
Pre-inoculum was prepared in a narrow mouth 500 mL Erlenmeyer flask containing 150 ml medium (Table 2) by transferring loopful of culture from freshly grown culture. The inoculated medium was incubated at about 28° C.±2° C. for about 48-72 hours on a rotating shaker at 250 rpm.
Preferably, 5-10% of pre-inoculum was transferred to 1000 mL Erlenmeyer flasks containing 300 ml seed medium (Table 3). The inoculum was prepared by incubating the flasks at 28° C.±2° C. for about 48-72 hours on a rotating shaker at 250 rpm.
Preferably, 5-10% of two days old seed culture (inoculum) was transferred to 1000 mL Erlenmeyer flasks containing 300 ml production media as shown in (Table 4) and incubated at 28° C.±2° C. for 2-10 days on a rotating shaker at 250 rpm followed by liquid-liquid extraction using methanol-ethyl acetate.
The solvent extracts were evaluated by weight as well as their HPLC/LCMS profiling for tetrahydroanthracenes production.
The culture was grown over a broad pH range from pH 5.0 to 9.0. Inoculum was added in 500 ml Erlenmeyer flask containing 100 mL of production medium with varied pH. Setomimycin production was highest at pH 6.5-7.5, although production was observed at wide pH range (pH 5.0 to 9.0).
Streptomyces sp. MTCC-25420 and Streptomyces sp. MTCC-25512 can be grown over a broad temperature range from 10° C. to about 45° C. The inoculum was added in 500 mL Erlenmeyer flask containing 100 ml of production medium and flasks were incubated at varied temperatures i.e. 10° C., 20° C., 30° C., 40° C. and 50° C. Optimum production of tetrahydroanthracenes appears to occur at a temperature of about 20° to 40° C.
Carbon sources that are essential component for the growth and production of tetrahydroanthracenes and Setomimycin were evaluated. All types of carbon sources i.e. Monosaccharides, Disaccharides and Polysaccharides such as Glucose, Fructose, Sucrose, Mannitol, Glycerol, Pectin, Lactose, Maltose, Mannose, Chitosan, Dextrin, Starch, Xylose, Mollases, Corn steep liquor, Inositol, Chitin, Sorbitol.
Various nitrogen sources such as Beef extract, Casein, Soya Meal, Yeast extract, Oatmeal, N/Z amine A, N/Z amine B, Casein hydrolysate, Peptone, Sodium nitrate, Valine, Ammonium nitrate, Urea, Arginine, Asparagine, Ammonium phosphate, Potassium nitrate, Ammonium sulphate support the production of tetrahydroanthracenes.
To study the effect of agitation the Streptomyces sp. MTCC-25420 and Streptomyces sp. MTCC-25512 was grown on selected production medium with agitation range from 50 to 400 rpm and it has been observed that agitation from 100 rpm to 300 rpm supports maximum production of tetrahydroanthracenes with 10% seed inoculum.
The time course of tetrahydranthracenes production was evaluated by transferring the freshly grown cultures in Seed inoculum and further to the production medium. The growth and production were evaluated by sampling the fermentation broth after regular intervals. Antibiotic complex formation starts after 24 hours and optimum production was found after 4-8 days of incubation. Tetrahydroanthracene antibiotics were produced in 5 L to 500 L stirred tank bioreactor using modified production medium PM-7 (Table 4) with combination of other ingredients providing additional carbon/nitrogen/minerals and elicitations. The inoculum was prepared using freshly grown culture of vegetative stage in conical flask using PM-2 medium (Table 4). The 5-10% of 2-4 days old inoculum was transferred to the fermenter. For the efficient growth the volume of air used was 0.05 to 1.5 (vvm) with agitation speed of 50 to 400 rpm at 28° C.±2° C. for 2-10 days. Further, the fermented broth obtained after 6 days of incubation was homogenised with 10% methanol for about 2 hours. The homogenised broth was then extracted at least thrice with ethyl acetate in the ratio of (1:1 v/v). The organic phase, thus obtained was concentrated on a rotary evaporator at 50° C. and pure setomimycin was quantified by HPLC. Tetrahydroanthracene formation starts within the 24 hours of incubation and highest production occurs between 4 to 8 days fermentation, wherein Setomimycin yields range from 30 mg/L to 800 mg/L in combination with various carbon and nitrogen sources along with trace elements, elicitors and precursors (Table 5).
Further, various carbon & nitrogen sources were used in combination with different trace elements (Table 6) to produce tetrahydroanthracenes from pH 4.0 to 10.0
All the biological materials used in the invention were procured from HIMEDIA, Laboratories Pvt. Ltd 23, Vadhani Ind. Est., LBS marg, Mumbai-400086, India.
For the extraction of bioactives, the fermented broth obtained after 6 days of incubation was homogenised with 10% methanol for about 2 hours. The homogenised broth was then extracted thrice with ethyl acetate in ratio of 1:1 (v/v). The organic phase thus obtained was concentrated on a rotary evaporator at 50° C. LC-MS profile of the crude extract indicated the presence of tetrahydroanthracenes compounds in the extract, which were purified by column chromatography/preparatory TLC/preparatory HPLC.
The isolation of compounds was carried out through open glass gravity column chromatography. Slurry of the crude extract was made using silica 60-120 (5.0 g) which was loaded on a glass column packed with silica 60-120. For purification, elution was carried out by ethyl acetate and thereafter, polarity was increased step by step by adding ethyl acetate to toluene in the order of 5%, 10%, 15% and 20%. A small fraction of 25 mL each was collected and monitored through TLC after charring with anisaldehyde. Fractions were pooled together based on similarity of Rf values of the spots as well as the purity of the isolated fractions. Total seven sub-fractions were collected, wherein fractions 1-3 were further mixed and subjected to silica open glass gravity column and Compounds 1 and 3 was purified, while, Compound 2 was purified from fraction 3-7. The pure sub fractions thus obtained were pooled together and concentrated on rotary evaporator at 30-35° C. under reduced pressure which were further confirmed by 1H NMR and 13C NMR. The purity of compounds was checked by HPLC chromatography.
Compound 1 was obtained as brown solid having molecular mass of 300.0 as obtained by LCMS fragmentation.
1H NMR (400 MHz, CDCl3) δ 16.28 (s, 1H), 9.76 (s, 1H), 7.51 (t, J=8.0 Hz, 1H), 7.17 (d, J=7.6 Hz, 1H), 7.08 (s, 1H), 6.89 (dd, J=7.9, 0.8 Hz, 1H), 4.18 (s, 1H), 3.50 (d, J=18.3 Hz, 1H), 2.73 (dd, J=18.3, 1.7 Hz, 1H), 2.41 (s, 3H), 1.48 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 205.6, 202.5, 166.6, 158.3, 139.0, 133.1, 132.0, 119.3, 118.4, 113.3, 112.0, 108.2, 71.8, 63.0, 47.5, 32.1, 27.5.
The PMR spectra suggest that the compound contains 16 protons: 6 protons from 2 methyl groups, 2 protons from one methylene group, 4 protons from olefinic and/or aromatic moieties, 1 protons a C—H, and 3 protons from 3 hydroxyl groups, and in CMR 17 peaks of carbons were found, 2 in carbonyl region, 2 in aromatic with substitution, 1 in hydroxylated region, 8 in aromatic, 1 in methylene and 2 in methyl region, further, molecular mass of 300.0 as obtained by LCMS fragmentation confirmed the structure of compound 1 as a monomer which is in accordance with literature reports. Two IR spectra revealed that the compound contains C═C and hydroxyl groups. By calculating the 21 degrees of unsaturation and UV absorptions of the compound indicates that it contains naphthocyclinone or anthracyclinone skeleton as a chromophore and it contains no sugar moiety.
Compound 2 was obtained as brown powder with the molecular mass 598.0 as obtained by LCMS fragmentation.
1H NMR (400 MHz, CDCl3) δ 9.98 (s, 2H), 7.20 (dd, J=9.3, 6.8 Hz, 2H), 6.82 (d, J=7.8 Hz, 2H), 6.34 (d, J=8.2 Hz, 2H), 4.16 (s, 2H), 2.93 (d, J=18.5 Hz, 2H), 2.59 (d, J=18.5 Hz, 2H), 1.21 (s, 6H), 1.17 (s, 6H).
13C NMR (101 MHz, CDCl3) δ 205.2, 202.1, 164.7, 157.6, 137.8, 132.5, 132.4, 124.0, 116.6, 111.9, 111.3, 108.6, 70.9, 58.7, 44.7, 32.0, 27.8.
The PMR spectra suggest that the compound contains 30 protons: 12 protons from 4 methyl groups, 4 protons from two methylene group, 6 protons from olefinic and/or aromatic moieties, 2 protons from two C—H, and 6 protons from 6 hydroxyl groups, and in CMR 17 peaks of carbons were found, 2 in carbonyl region, 2 in aromatic with substitution, 1 in hydroxylated region, 8 in aromatic, 1 in methylene and 2 in methyl region, further, molecular mass 580.0 as obtained by LCMS fragmentation confirmed the structure of compound 2 which is in accordance with literature reports. Two IR spectra revealed that the compound contains C═C and hydroxyl groups. By calculating the 21 degrees of unsaturation and UV absorptions of the compound indicates that it contains naphthocyclinone or anthracyclinone skeleton as a chromophore and it contains no sugar moiety.
Compound 3 (Setomimycin) was obtained as red crystalline solid with the molecular mass 580.0 as obtained by LCMS fragmentation.
1H NMR (400 MHz, CDCl3) δ 10.04 (s, 1H), 9.93 (s, 1H), 7.33 (t, J=8.0 Hz, 1H), 7.22-7.15 (m, 2H), 6.93 (d, J=7.8 Hz, 1H), 6.83 (d, J=7.9 Hz, 1H), 6.59 (d, J=8.3 Hz, 1H), 6.21 (d, J=7.5 Hz, 2H), 3.94 (s, 1H), 3.63 (s, 1H), 3.12 (d, J=17.6 Hz, 1H), 2.58 (d, J=17.8 Hz, 1H), 1.95 (s, 3H), 1.63 (s, 3H), 1.34 (s, 3H), 1.22 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 208.5, 203.3, 199.6, 191.1, 166.5, 164.8, 158.8, 158.5, 158.1, 138.7, 137.0, 134.3, 133.9, 133.5, 133.2, 125.8, 125.1, 124.3, 117.5, 116.5, 114.1, 113.4, 113.0, 112.6, 112.5, 109.3, 108.6, 72.1, 60.9, 60.6, 47.2, 33.9, 29.4, 28.2, 23.0.
The PMR spectra suggest that the compound contains 28 protons: 12 protons from 4 methyl groups, 2 protons from a methylene group, 2 proton from two C—H, 7 protons from olefinic and/or aromatic moieties, and 5 protons from 5 hydroxyl groups, and in CMR, 34 carbons were found, 4 in carbonyl region, 5 in aromatic with substitution, 17 in aromatic, 3 in oxygenated region, 1 in methylene and 4 in methyl region. IR spectra revealed that the compound contains C═C and hydroxyl groups. By calculating the 21 degrees of unsaturation and UV absorptions of the compound indicates that it contains naphthocyclinone or anthracyclinone skeleton as a chromophore and it contains no sugar moiety.
The minimum inhibitory concentrations (MIC) of purified compound showing significant zones of inhibition in the preliminary antimicrobial assay using agar well diffusion method were evaluated. MIC assay was carried out by a microtiter broth dilution method with some modifications. Stock solution of each pathogen was prepared in normal saline solution i.e. 0.85% NaCl (w/v) at a concentration of 108 cells/ml and finally diluted with respective growth media to give approximately 105 CFU/ml for all organisms. Different dilutions of compound was prepared (2-500 μg/ml) by serial dilutions. 100 μl of each concentration (2-500 μg/ml) of the compound was loaded onto each well and was mixed with 100 μl of diluted pathogens containing 105 CFU/ml. Each plate had a set of controls: a column with broad-spectrum antibiotics (ciprofloxacin) as positive controls, a column without compound, and one without the relevant test organism. The microtitre plates were then incubated overnight at 37° C. for bacterial pathogens and 28° C. for fungal pathogens and were analysed after incubation. The lowest inhibitory dilution, at which there was no visible growth, was considered as MIC as shown in Table 7.
Staphylococcus aureus
Bacillus cereus
Bacillus subtilis
Micrococcus luteus
Compound Setomimycin showed potent anticancer properties against diverse range of cancer cells of different origin as shown in Table 8. 5×103 cells were seeded/well in a 96 well plate and treated with Compounds (1-3) for 48 hours with 100 μM concentration range to a panel of cancer cell lines. After 44 hour MTT with final concentration of 0.5 mg/ml were added to cells and incubated for 4 hour at 37° C. until formazon crystals are formed. MTT was removed and formazon crystals were lysed by DMSO for 30 minutes at 37° C., the absorbance was measured at 570 nM and the IC50 values were calculated using graph pad prism.
Colony formation assay was performed to further certify the antiproliferative properties of setomimycin in four different cancer cell lines viz. HT-29, MiaPaca-2, HCT-116 and MCF-7. Interestingly, setomimycin showed promising results near the IC50 values across all four cell lines as shown in
The above results depict significant antiproliferative effects of setomimycin in different cell lines near IC50 values. Transwell invasion assay was performed to check the anti-invasive properties of setomimycin. Interestingly the invasion of the cells from the upper chamber of insert were significantly inhibited to the lower chamber where growth medium with fetal bovine serum was present acting as the chemoattractant with increasing concentration of setomimycin in HCT-116 as well as MCF-7 as shown in
(A-B) 1.2×106 HCT-116 and MCF-7 cells were seeded in the upper chamber of inserts in serum free medium and treated with indicated concentration of setomimycin for 48 hours. (After 48 hours the invaded cells from lower insert were fixed with ice cold methanol and stained with 0.1% crystal violet for 20-30 minutes. After staining the inserts were washed with dH2O thoroughly and cells from three different fields in each treated condition were counted and photographed under an inverted microscope (20× magnifications). (C-D) Bar graph representing the number of invaded cells (n=3, error bars indicate ±SD).
Scratch assay was performed in the panel of cancer cell lines with varying degree of invasive and migratory capabilities as shown in
5×105 cells were seeded onto 6 well plate and grown upto more than 90% confluency. After the proper attachment of cells wounds were created with the help of a 20-200 μL sterile pipette tip. Subsequently the cells were treated with indicated concentration of setomimycin and vehicle (DMSO). The images were taken under inverted microscope at 20× magnification.
The molecular docking studies were performed using GLIDE module of Schrödinger software. For molecular docking crystal structure of V600E oncogenic mutant BRAF (4XV2), ERK1 (4QTB), ERK2 (6GDQ), MEK1 (3SLS), Src (4MXO) and STAT3 (6NUQ) was downloaded from RCSB protein data bank. Docking of co-crystalised ligands to its respective target using XP precision generated poses with similar orientation and less RMSD with respect to co-crystalised ligand. Thus, further studies were carried out using Glide XP mode. Among all the targets, the antibiotic setomimycin showed the highest docking score (−6.808 kcal/mol) with the target MEK1 (Table 9). Moreover, the dock score of setomimycin, in comparison to the known inhibitors of the selected targets, was the highest in case of MEK1. The rescoring of the top docking pose of setomimycin was done by calculating DG-binding with MMGBSA and it showed the highest binding affinity (−43.77 kcal/mol) with MEK1. It was further observed that setomimycin interacts with MEK1 by forming hydrogen bond with Lys 97, Asp 190 and Ser212. Lys 97 helps in the binding of ATP to MEK1. Asp 190 is an important catalytic residue involves in the abstraction of a proton of a threonine or tyrosine on ERK1/2. Most of the reported inhibitors act by binding with these residues. Therefore, setomimycin may exhibit anti-proliferative properties by inhibiting MEK1.
Strong active potential of setomimycin against tumor cell proliferation and migration at nearby concentration of IC50 values was depicted by MTT data. Further, molecular docking studies predicted the strong affinity of setomimycin targeting MEK pathway. ERK and MEK are the major signaling pathways regulating cancer cell proliferation, differentiation and apoptosis. Therefore, Western blotting was carried out to check the effects of Setomimycin on ERK and MEK proteins. Phospho MEK was found to be down regulated with increasing concentration of setomimycin validating it as antitumor candidate. Interestingly, Par-4 which is a pro-apoptotic protein was upregulated and BCL-2 an antiapoptotic protein was downregulated by Setomimycin. These results demonstrate the strong antitumor capability of Setomimycin regulating proliferation and metastasis, which further induces pro-apoptotic marker as well, diminishing the expression of anti-apoptotic proteins as shown in
The main advantages of the present invention are:
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111047805 | Oct 2021 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IN2022/050812 | 9/12/2022 | WO |
