Patent Grant
9206201
The present invention relates to boronic acid bearing liphagane compounds. The present invention particularly relates to boronic acid bearing meroterpenoid liphagane scaffold based compounds. The compounds have been designed, synthesized and their biological evaluation results for anticancer activity by inhibiting PI3K pathway are presented in this invention. The field of invention for this work relates and covers the development of novel PI3K-α/β inhibitors based on meroterpenoid liphagane scaffold for anticancer activity.
PI3Ks are a family of related intracellular signal transducer capable of phosphorylating the 3 position hydroxyl group of the inositol ring of Phosphatidylinositol (PtdIns). They are also known as phosphatidylinositol-3-kinases. The pathway, with oncogene PIK3 and tumor suppressor (PTEN) gene is implicated in insensitivity of cancer tumors to insulin and IGF1, in calorie restriction. 3-kinase (PI3K) signaling pathway is a newly identified strategy for the discovery and development of certain therapeutic agents. Among the various subtypes of PI3K, class IA PI3K-alpha has gained increasing attention as a promising drug target for the treatment of cancer due to its frequent mutations and amplifications in various human cancers. In contrast with cytotoxic agents that do not differentiate between normal proliferating and tumour cells, targeted therapies primarily exert their action in cancer cells. Initiation and maintenance of tumours are due to genetic alterations in specific loci. The identification of the genes in these alterations occurs has opened new opportunities for cancer treatment. The PI3K (phosphoinositide 3-kinase) pathway is often overactive in human cancers and various genetic alteration have been found to cause this. In all cases, PI3K inhibition is considered to be one of the most promising targeted therapies for cancer treatment.
Owing to its widespread activation in inflammation and cancer, a growing appreciation of the therapeutic potential of inhibitors of the phosphoinositide 3-kinase (PI3K) pathway has stimulated intense interest in compounds with suitable pharmacological profiles. These are primarily directed toward PI3K itself. However, as class I PI3Ks are also essential for a range of normal physiological processes, broad spectrum PI3K inhibition could be poorly tolerated.
In recent years, patents describing a new generation of PI3K inhibitors have started to appear, with a particular focus on the development of compounds with enhanced isoform selectivity for use as anti-cancer and anti-inflammatory therapies. However, challenges remain for the efforts to pharmacologically target this enzyme family in a successful manner.
Rationale for the Selection of Phosphoinositide 3-Kinase-α (PI3K-α/β) Inhibitors:—
At cellular level, phosphoinositide-3-kinase signaling contributes to many processes, including cell cycle progression, cell growth, survival and migration and intracellular vesicular transport. The PI3K represents the family of lipid kinases that can be classified into three subfamilies according to structure and substrate specificity viz., class I, class II and class III. The class I PI3Ks are the most extensively studied among lipid kinases, are heterodimeric proteins; each containing a smaller regulatory domain and a larger 110 kDa catalytic domain, which occur in four isoforms differentiated as p110α, p110β, p110γ, and p110δ. Although, there are natural product based small molecules reported in the literature which inhibit the PI3-kinases having the IC50 value in nano-gram range (viz., Wortmannin isolated from Penicillium wortmanni, LY294002 a synthetic analogue of the flavonoid quercetin, etc) but these molecules did not reach to market because of low potency, poor isoform or kinase selectivity, limited stability and unacceptable pharmacological and pharmacokinetic properties. However, PI3 kinase inhibitors having isoform selectivity and promising drug-like properties have now begun to emerge that show promise for the treatment of cancer and other disease indications. In cancer, evidence suggests that inhibition of the class 1A PI3 kinases p110α and p110β appear to be the most appropriate to target. Recently, Andersen et al., in 2006 reported the potential isoform selective PI3K-alpha inhibitor from marine sponge Aka coralliphaga under the collaborative program to screen marine invertabrates against human PI3K-alpha keeping in mind that natural products from marine resources have emerged as a copious repository of molecular diversity and hold considerable promise as a rich source of lead structures in drug discovery. Liphagal (Joshua J. Day, Ryan M. McFadden; The catalytic enantioselective total synthesis of (+)-Liphagal; Angew. Chem. Int. Ed. 2011, 50, 6814-6818; Enrique Alvarez-Manzaneda, RachidChahboun; Enantioselectivetotal synthesis of the selective PI3-kinase inhibitor Liphagal; Org. Lett., 2010, 12 (20), pp 4450-4453; Jonathan H. George, Jack E. Baldwin; Enantiospecific biosynthetically inspired formal total synthesis of (+)-Liphagal, Org. Lett., 2010, 12 (10), pp 2394-2397; Alban R. Pereira, Wendy K. Strangman, Synthesis of phosphatidylinositol 3-kinase (PI3K) inhibitory analogues of the sponge meroterpenoid Liphagal; J. Med Chem., 2010, 53 (24), pp 8523-8533; Dima A. Sabbah, Jonathan L. Vennerstrom; Docking studies on isoform-specific inhibition of phosphoinositide-3-kinases; J. Chem. Inf. Model., 2010, 50 (10), pp 1887-1898; Ram Vishwakarma and Sanjay Kumar; Efficient Synthesis of key intermediate toward Liphagal synthesis; Synthetic Communications; 2010, 41(2), pp 177-183; Frederic Marion, David E. Williams, Liphagal, a selective inhibitor of PI3 kinase-α isolated from the sponge Aka coralliphaga: Structure elucidation and biomimetic synthesis; Org. Lett., 2006, 8 (2), pp 321-324; Goverdhan Mehta, Nachiket S. Likhite, C. S. Ananda Kumar A concise synthesis of the bioactive meroterpenoid natural product (±)-liphagal, a potent PI3K inhibitor, Tet. Lett, 2009, vol. 50, no. 37, pp 321-324) was ˜10-fold more potent against PI3K-α than against PI3K-γ. We have synthesized boron containing analog of liphagal by rational modification on this molecule following diversity oriented synthesis approach for the discovery of lead molecules.
The main object of the present invention is to provide boronic acid bearing liphagane compounds. Another object of the invention provides a process for preparation of boronic acid functional group containing liphagane compounds.
Yet another object of the present invention is to provide process for the preparation for step A6 to A7 and A7 to A by the synthetic route mentioned in the claims of this invention document.
Still another object of the present invention is to evaluate biological activity of the boronic acid based liphagal compounds as anticancer agents.
Yet another object of the present invention is to identify isoform selectivity of these compounds for PI3K inhibition as alpha or beta specific when studied for enzyme specificity.
Yet another object of the invention is to explore the mechanism of action and growth inhibition of the liphagal boronic acid bearing compound by Annexin-V or immunofluroscent assay and by cell cycle analysis.
Accordingly the present invention provides a compound of general formula 1, and pharmaceutically acceptable salts thereof,
wherein,
In another embodiment of the invention, the compound of general formula 1 is represented by compounds of formula A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, Z, AA, AB, AC, AD, AE, AF, AG, AH, AI, AJ, AK, AL, AM, AN, AO, AP, AQ, AR, AS, AT, AU, AV, AW, AX, AY and AZ comprising the following structural formula:
In another embodiment of the invention, the compound is useful as specific inhibitor of PI3K-α or β isoform in cancer treatment.
Yet another embodiment of the invention provides a process for preparation of compounds of general formula 1 and pharmaceutically acceptable salts thereof
wherein,
In yet another embodiment of the invention, the ether solvent used in step (i) and (v) is selected from a group consisting of tetrahydrofuran, dichloromethane, diethyl ether, diisopropyl ether and isopropyl ether.
In yet another embodiment of the invention, the base in step (i) is selected from a group consisting of tetramethyl ethylene diamine, triethyl amine, trimethyl amine and diisopropyl ethyl amine.
In yet another embodiment of the invention, reaction in step (i) is carried out at a temperature in the range of −78° C. to 35° C. for a period ranging between 5 to 10 min.
In yet another embodiment of the invention, reaction in step (ii) is carried out at a temperature in the range of 0-5° C., for a period ranging between 1 to 2 h.
In yet another embodiment of the invention, the water immiscible solvent in step (iii) and (v) is selected from a group consisting of ethylacetate, dichloromethane, ether or chloroform.
In still another embodiment of the invention, reaction in step (iv) is carried out at a temperature ranging between −78° C. to 35° C. for a period ranging between 1 to 3 h,
In still another embodiment of the invention, the compound of general formula 1 obtained in step (v) is converted into a pharmaceutically acceptable salt.
In still another embodiment of the invention, the compound of general formula 1 is converted into a pharmaceutically acceptable salt by a process comprising the steps of mixing the compound of general formula 1 with a base in a ratio 1:1 proportion, wherein the base is selected from a group consisting of sodium hydroxide, potassium hydroxide and ammonium hydroxide in water, stirring the reaction mixture for 1-2 h followed by drying to obtain the pharmaceutically acceptable salt of the compound of general formula 1.
Yet another embodiment of the invention provides a pharmaceutical composition comprising an effective amount of the compound of formula 1, optionally along with a pharmaceutically acceptable carrier, salt, excipients or diluents.
In still another embodiment of the invention, the pharmaceutically acceptable carrier is selected from a group consisting of water, buffered saline, glycols, glycerols, olive oil and liposomes.
Still another embodiment of the invention provides a method of treatment of cancer by specific inhibition of PI3K-α or β isoform in a human cancer cell line using a compound of general formula 1,
wherein,
In another embodiment of the invention, dosage of compound of general formula 1 is in the range of 20 mg/kg to 100 mg/kg.
In another embodiment of the invention, the representative compound A has a GI50 concentration in the range of 2.4 μM-2.6 μM when used for in vitro activity against colon and breast cancer cell lines.
In another embodiment of the invention, the representative compound A demonstrates >74% optimal growth inhibition in human cancer cell lines at a concentration of 10 μM.
In another embodiment of the invention, the representative compound E when used for in vitro activity against colon cancer cell lines increases sub-G1/G0 population and shows concentration dependent growth arrest in G1/G0 population and late apoptosis in colon cancer cell lines.
ACN: acetonitrile
Ac: acetyl
CDCl3: deuterated chloroform
CHCl3: chloroform
13CNMR: carbon nuclear magnetic resonance
DCM or CH2Cl2: dichloromethane
DIPEA: diisopropyl ethyl amine
DMF: dimethylformamide
DMSO: dimethylsulfoxide
EtOAc: ethylacetate
h or hr: hour
1HNMR: proton nuclear magnetic resonance
IC50: 50% inhibitory concentration
IR: infrared
J: coupling constant (Hz)
MeOH: methanol
MHz: Megahertz
mg: milli gram
μg: microgram
μL: micro liter
Mmol: milli mole
MTT: mitochondrial membrane potential
m/z: mass-to-charge ratio
PI3-K: phosphatidylinositol-3-kinase
TEA: triethyl amine
TFA: trifluoroacetic acid
THF: tetrahydrofuran
TLC: thin layer chromatography
TMA: trimethyl amine
TMEDA: tetramethyl ethylene diamine
In an embodiment of the present invention the Formula 1 represents different compounds of meroterpenoid based liphagane scaffold having boronic acid functionality at 6th position of phenyl ring, wherein, R1 to R3 are independently selected from H, OH, OR, COR, CHO, CO2R, OCOR, NH2, NHR, NRR′, NO2, F, Cl, Br, I, OSO3H, SO2R, CN, SiRR′R″, OCF3, CF3 and R. Wherein, R, R′, R″ may be alkyl, aryl, heteroaryl or any cyclic aliphatic ring with different substitutions.
In yet another embodiment, ‘Y’ is O, S, NH, and NR, wherein, R-may be substituted with alkyl, aryl, heteroaryl moiety or any cyclic aliphatic or aromatic system.
In an embodiment of the present invention, n and n1 are selected carbon chain length from 0, 1 and 2.
In an another embodiment, wherein, R4 is H or OR or SR or SO2R or OSO3R or SiRR′R″ or NH2 or NHR or NRR′ or a one to ten carbon chain either linear or branched, saturated or unsaturated alkyl group optionally substituted with OH, H, OH, ═O, ═S, OR, COR, CHO, CO2R, OCOR, NH2, NHR, NRR′, NO2, F, Cl, Br, I, OSO3H, SO2R′, CN, SiRR′R″ and R. Wherein, R, R′, R″ are alkyl, aryl, heteroaryl or cyclic aliphatic ring having substitutions with varying chain length.
In an embodiment, wherein, the substituent R4 is also selected from a group consisting of hydrogen, alkyl substituents viz., methyl, ethyl, propyl and the higher homologues either linear of branched, including alicyclic such as cyclopentane, cyclohexane or higher membered rings, fused rings, aryl/heteroaryl substituted alkyl groups including benzlic or its higher homologues that might include unsaturated alkyl groups such as cinnamul, crotyl and prenyl substituents.
In yet another embodiment of the present invention, wherein, R5, R6 and R7 are H or one to ten carbon chain either linear or branched, saturated or unsaturated at any position, alkyl group optionally substituted with OH, H, OH, ═O, ═S, OR, COR, CHO, CO2R, OCOR, NH2, NHR′, NR′, NR′R″, NO2, F, Cl, Br, I, OSO3H, SO2R, CN, SiRR′R″ and R. Here R, R′, R″ may be alkyl, aryl, heteroaryl or any cyclic aliphatic ring with different substitutions.
In embodiment of the present invention, R is independently selected from H or one to ten carbon chain either linear or branched, saturated or unsaturated, alkyl group optionally substituted with OH, H, OH, ═O, ═S, OR, COR, CHO, CO2R, OCOR, NH2, NHR, NRR′, NO2, F, Cl, Br, I, OSO3H, SO2R, CN, SiRR′R″ and R. Here R, R′, R″ may be alkyl, aryl, heteroaryl or any cyclic aliphatic ring with different substitutions.
Wherein, further the substituent R at various positions is also selected from a group consisting of hydrogen, alkyl substituents viz., methyl, ethyl, propyl and the higher homologues either linear or branched, including alicyclic such as cyclopentane, cyclohexane or higher membered rings, fused ringsm aryl/heteroaryl substituted alkyl groups including benzlic or its higher homologues that might include unsaturated alkyl groups such as cinnamul, crotyl and prenyl substituents.
In an embodiment in the present invention, the routine method was used for the in silico bioinformatics study of liphagal and its boronic acid based compounds, it is as mentioned below: all the computational studies were carried out in the Schrodinger suite 2010 molecular modeling software. The 2D structures of all the molecules were built in the maestro window. All the molecules were then converted to their respective 3D structure, with various conformers, tautomers and ionization states using the Ligprep and Confgen modules. The molecules were then minimized using the OPLS—2005 force field. The 3D crystal structure of PI3Kα reported in Protein Data Bank (PDB) was used as receptor for docking studies (PDB ID: 3HHM). The protein was downloaded from the PDB and was prepared for docking using the Protein Preparation wizard. Hydrogen's were added to the protein and the missing loops were built. Bond length and bond order correction was also carried out for preparing the protein for docking studies. The active site grid was generated based on the already co-crystallised ligand of the receptor using receptor grid generation module. The ligands were docked on to the receptor through this grid using Glide module and flexible docking was carried out for all the conformers in order to find out the binding mode of these ligands. The extra precision (XP) scoring function of Glide was used for carrying out these studies. In yet another embodiment of the present invention, wherein, the results obtained in the in silico studies of liphagal and its boronic acids based compounds are as: based on the docking studies, it was found that the boronic acid analogues of liphagal bind with better affinity to PI3Kα than liphagal. The interaction studies show that boronic acid (OH) are involved in strong H-bond interactions with Val851 and Gln859, whereas liphagal is involved in H-bond interaction at one place only with Gln859. Also the dock score of boronic acid based compound was about −10 and that of liphagal was about −8.5, which shows a stronger affinity of boronic acid analogues towards PI3Kα.
The invention is further described by reference to following examples which are intended to illustrate and should not be construed to limit the scope of the present invention.
Materials and Method:
Chemistry:
General: Solvents were purified according to the standard procedures, and reagents used were of highest purity available. All reactions were performed in flame-dried glass apparatus under argon/nitrogen atmosphere unless mentioned otherwise. Anhydrous solvents like CH2Cl2, Et2O, THF, CH3OH, CH3CN, DMF, pyridine, Et3N were freshly dried using standard methods. NMR measurements (1H and 13C) were recorded on either 400 or 500 MHz spectrometer (Bruker) fitted with pulse-field gradient probe, and trimethylsilane (TMS) or residual resonance of deuterated solvent were used as internal reference. Chemical shifts are expressed in (δ) parts per million and coupling constants J in hertz. Mass spectra were recorded on ESI MS or MALDI-TOF/TOF MS/MS-MS spectrophotometer using 2,5-Dihydroxy benzoic acid/α-Cyano-4-hydroxy benzoic acid/Sinapinic acid (Sigma-Aldrich) as matrix in acetonitrile:water containing 0.01% TFA. Optical rotations were measured on a digital PerkinElmer-241 polarimeter. Analytical TLC was performed on Merck 60 F254 plates, and compounds were visualized by spraying and charring with phosphomolybdic acid or 20% H2SO4 in MeOH as developing reagent. Preparative TLC was performed on pre-coated silica gel 60 F254 plates (20×20 cm) purchased from Merck. Silica column chromatography was carried out with silica gel (100-200 mesh) or flash silica gel (230-400 mesh) purchased from Merck.
For steps 1 to 6 (Ref: Mehta, G; Likhite, N. S.; Ananda Kumar, C. S. Tet. Lett. 2009, 50, 5260.) The steps involved for the synthesis of compound A are described as below—
To solution of 3,4,5-trimethoxybenzaldehyde (5 g, 25.5 mmol) in CH2Cl2 (125 ml) at 0° C. was added BBr3 (6.39 g, 25.5 mmol). The resulting dark mixture was stirred at 0° C. for 10 hrs after completion of the reaction checked by TLC H2O (100 mL) was then added and the mixture was stirred for 10 min and the aqueous phase was extracted by CH2Cl2. The organic phase was dried over Na2SO4, and evaporated under reduced pressure. The resulting residue was purified by silica gel (CH2Cl2) afforded the 2-hydroxy 4,5dimethoxybenzaldehyde A1 (4.3 g) in 87% yield isolated yellow solid: 1H NMR (500 MHz, CDCl3) δ 11.33 (s, 1H), 9.63 (s, 1H), 6.83 (s, 1H), 6.40 (s, 1H), 3.87 (s, 3H), 3.81 (s, 3H) ppm. Mass: ESI [M+Na]+: 225.06; Elemental anal. calcd. for C9H10O4; C, 59.34; H, 5.53; O, 35.13. found C, 59.14; H, 5.13; O, 34.90.
To a solution of 2-hydroxy 4,5-dimethoxybenzaldehyde (2 g, 10.98 mmol) in butane-2-one (15 ml) added K2CO3 (6.07 g, 43.95 mmol) and then stirred at 0° C. for 10 min added bromoacetone (2.24 g, 16.47 mmol) and refluxed at 90° C. for 4 hr. After completion of the reaction butane-2-one was distilled off and water was added and extracted by ethylacetate twice. The EtOAc phase was dried over Na2SO4, Chromatography of the residue on silica gel (3:7 EtOAc/hexane) afforded the ketone A2 (1.69 g) in 70% yield pale yellow solid: 1H NMR (500 MHz, CDCl3) δ 7.29 (s, 1H), 7.04 (s, 1H), 6.98 (s, 1H), 3.94 (s, 3H), 3.90 (s, 3H), 2.30 (s, 3H) ppm. Mass: ESI [M+Na]+: 243.07; Elemental anal. calcd. for C12H12O4; C, 65.45; H, 5.49. found C, 65.20; H, 5.25.
To a solution of 1-(5,6 dimethoxy benzofuran-2-yl)ethanone A2 (1 g, 4.545 mmol) in anhydrous Toluene added tBuOK (0.51 g, 4.545 mmol) at 0° C. under argon atmosphere and then stirred at the same temperature for 15 min to this reaction mixture geranyl bromide added drop wise. Resulting suspension stirred at the same temperature for 2 hrs. 50 ml water was added to the reaction mixture and layers were separated. Aqueous layer was extracted with ethylaectate. The combined organic extract were washed with brine and dried over Na2SO4, evaporated under reduced pressure. Chromatography of the residue on silica gel (5% EtOAc/hexanes) afforded the ketone A3 (1.69 g) in 70% yield pale yellow liquid: 1H NMR (500 MHz, CDCl3) δ 7.425 (s, 1H), 7.365 (s, 1H), 7.06 (s, 1H), 5.12-5.16 (m, 1H), 5.08-5.04 (m, 1H), 3.96 (s, 3H), 3.93 (s, 3H), 2.97-2.89 (t, 2H), 2.50-2.40 (m, 2H), 2.17-2.00 (m, 4H), 1.66 (s, 3H), 1.63 (s, 3H), 1.64 (s, 3H), 1.58 (s, 3H), ppm. Mass: ESI [M+Na]+: 379.2; Elemental anal. calcd. for C22H28O4; C, 74.13; H, 7.92. found C, 74.0; H, 5.25.
Triphenylphos-phenemethyl iodide (wittig salt, 2.247 g 5.6 mmol) has taken in a dry RBF kept in ice-salt mixture to this added dry THF (6 mL) to the resulting mixture nBuLi (2.5 mol in hexane 3.370, 8.4 mmol), was added drop wise until reaction mixture converted to yellow suspension. To the reaction mixture a solution of ketone (1 g, 2.8 mmol) in THF was added drop wise. Resulting suspension was stirred for 2 hrs. After completion of the reaction 10% ammonium chloride solution 30 ml was added and extracted by EtOAc. Chromatography of the residue on silica gel (3% EtOAc/Hexanes) afforded the compound A4 (0.79 g) in 80% yield colorless liquid: 1H NMR (500 MHz, CDCl3) δ 6.95 (s, 1H), 6.63 (s, 1H), 5.86 (s, 1H), 5.31-5.30 (d, 1H), 5.20-5.18 (d, 2H), 5.11-5.08 (t, 1H), 3.90 (s, 3H), 3.90 (s, 3H), 2.46-2.43 (t, 2H), 2.32-2.28 (q, 2H), 2.08-2.04 (q, 2H), 2.01-1.98 (t, 2H), 1.68 (s, 3H), 1.59 (s, 3H), 1.56 (s, 3H), ppm. Mass: ESI [M+Na]+: 377.22; Elemental anal. calcd. for C23H30O3; C, 77.93; H, 8.53. found C, 77.55; H, 8.35.
To a solution of compound A4 (0.7 g 1.9 mmol) in MeOH was added 10 Mol % Pd/C and reaction was shacked at 40 psi pressure for 0.5 hrs. After completion of the reaction (monitored by TLC) filtered the reaction mixture and evaporated the methanol completely. Chromatography of the residue on silica gel (3% EtOAc/Hexanes) afforded the compound A5 (0.633 g) in 90% yield colorless liquid: 1H NMR (500 MHz, CDCl3) δ 7.04 (s, 1H), 6.98 (s, 1H), 6.294 (s, 1H), 5.15-5.12 (m, 4H), 3.940 (s, 6H), 2.94-2.91 (m, 1H), 2.10-1.98 (m, 4H), 1.87-1.82 (m, 2H), 1.74 (s, 3H), 1.634 (s, 3H), 1.59 (s, 3H), 1.34-1.32 (d, 3H), ppm. Mass: ESI [M+Na]+: 356.24; Elemental anal. calcd. for C23H32O3; C, 77.49; H, 9.05. found C, 77.49; H, 9.05.
To a solution of the benzofuran A5 (0.6 g, 1.6 mmol) in 2-nitropropane (25 mL), at −85° C. was added chlorosulfonic acid (0.977 g, 8.42 mmol). The resulting mixture was allowed to stir at −78° C. for 30 min. An aqueous solution of NaHCO3 was then added and the aqueous phase was extracted with EtOAc. The EtOAc phase was dried over Na2SO4, filtered and evaporated under reduced pressure. Chromatography of the residue on silica gel (3% EtOAc/Hexanes) afforded the compounds A6 (0.3 g) in 50% yield colorless liquid with racemic mixture 1H NMR (CDCl3, 500 MHz) δ 7.13 (s, 1H), 6.85 (s, 1H), 3.89 (s, 3H), 3.88 (s, 3H), 2.56 (br d, J=14.1 Hz, 1H), 2.15 (m, 1H), 1.82 (m, 1H), 1.71 (qt, J=13.7, 3.5 Hz, 1H), 1.69 (m, 1H), 1.64-1.41 (m, 8H), 1.40 (d, J=7.2 Hz, 3H), 1.36 (s, 3H), 1.25 (ddd, J=13.3, 13.3, 3.5 Hz, 1H), 0.99 (s, 3H), 0.95 (s, 3H). Mass: ESI [M+Na]+: 356.24; Elemental anal. calcd. for C23H32O3; C, 77.49; H, 9.05. found C, 77.49; H, 9.05.
For the purpose of this application, compound A7 has been interchangeably referred to as compound E.
To a solution of benzofuran A6 (0.1 g, 0.281 mmol) in THF (1.5 mL) at 0° C. was added nBuLi (2.5 M in hexane). After stirring at this temperature for 20 min triethylborate was added. The mixture was stirred at rt for 1 hr. Aqueous NH4Cl was added and the aqueous phase was extracted with EtOAc dried over Na2SO4, and evaporated under reduced pressure. Chromatography of the residue on silica gel (8% EtOAc/Hexanes) afforded the compounds A7 (0.05 g) in 50% white solid racemic mixture 1H NMR (500 MHz, CDCl3) δ 6.83 (s, 1H), 3.94 (s, 3H), 3.92 (s, 3H), 2.56-2.49 (m, 1H), 1.56-1.52 (m, 4H), 1.48 (s, 3H), 1.37 (s, 3H), 0.99 (s, 3H), 0.96 (d, 3H) ppm. Mass: ESI [M+Na]+: 400.24; Elemental anal. calcd. for C23H33BO5; C, 69.01; H, 8.31; B, 2.70. found C, 69.10; H, 8.20; B, 2.50.
The solution of BI3 in DCM was added slowly and drop wise in the round bottom flask containing solution of compound A7 in DCM at −78° C. The mixture of this was stirred at same temperature for half an hour the slowly raised to rt. The progress of reaction was monitored by TLC. The reaction mixture was neutralized using potassium thiosulphate solution and extracted with DCM solution and separated the organic layer, dried over sodium sulphate, concentrated in vaccuo. The crude was purified by column chromatography using hexane/EtOAc as eluent. 1H NMR (500 MHz, CDCl3) δ 6.83 (s, 1H), 2.56-2.49 (m, 1H), 1.56-1.52 (m, 4H), 1.48 (s, 3H), 1.37 (s, 3H), 0.99 (s, 3H), 0.96 (d, 3H) ppm. Mass: ESI [M+Na]+: 372.211; Elemental anal. calcd. for C21H29BO5; C, 67.75; H, 7.85; B, 2.90. found C, 67.65; H, 7.61; B, 2.30.
All the compounds disclosed in formula 1, are prepared by employing the similar method containing different substitutions at R1, R2, R3 and R4 positions, as described for the preparation of compound A. The details of reaction conditions are depicted in the table given below—
Compound B: 1H NMR (500 MHz, CDCl3) δ6.91 (s, 1H), 3.18 (m, 1H), 1.82 (t, 2H), 1.78 (t, 1H), 1.71 (q, 2H), 1.45 (m, 2H), 1.42 (t, 2H), 1.44 (s, 3H), 1.38 (q, 2H), 1.34 (d, 3H), 1.12 (s, 6H), ppm. Mass: ESI [M+Na]+: 531.186; Elemental anal. calcd for C23H27BF6O7; C, 54.35; H, 5.31; B, 2.10; F, 22.44. found C, 54.33; H, 5.34; B, 2.11; F, 22.41.
Compound C: 1H NMR (500 MHz, CDCl3) δ7.60 (s, 1H), 3.18 (m, 1H), 1.82 (t, 2H), 1.78 (t, 1H), 1.66 (q, 2H), 1.49 (m, 2H) 1.44 (s, 3H), 1.48 (t, 2H), 1.36 (m, 2H), 1.34 (d, 3H) 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 499.215; Elemental anal. calcd for C23H27BF6O3; C, 58.01; H, 5.31; F, 23.97; B, 2.27. found C, 58.06; H, 5.34; F, 23.99; B, 2.25.
Compound D: 1H NMR (500 MHz, CDCl3) δ7.20 (s, 1H), 3.17 (m, 1H), 2.35 (s, 6H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H), 1.48 (t, 2H), 1.43 (s, 3H), 1.38 (m, 2H) 1.34 (d, 3H) 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 391.215; Elemental anal. calcd for C23H33BO3; C, 75.1; H, 9.03; B, 2.94. found C, 75.16; H, 9.06; B, 2.89.
Compound E: 1H NMR (500 MHz, CDCl3) δ6.9 (s, 1H), 3.73 (s, 6H), 3.17 (m, 1H), 1.82 (t, 2H), 1.71 (m, 2H), 1.49 (m, 2H), 1.48 (t, 2H), 1.43 (s, 3H), 1.37 (t, 2H), 1.34 (d, 3H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 423.25; Elemental anal. calcd for C23H33BO5; C, 69.01; H, 8.32; B, 2.74. found C, 69.05; H, 8.33; B, 2.77.
Compound F: 1H NMR (500 MHz, CDCl3) δ7.1 (s, 1H), 3.17 (m, 1H), 2.59 (q, 4H), 1.82 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.34 (d, 3H), 1.24 (t, 6H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 419.29; Elemental anal. calcd for C25H37BO3; C, 75.75; H, 9.42; B, 2.65. found C, 75.72; H, 9.44; B, 2.66.
Compound G: 1H NMR (500 MHz, CDCl3) δ6.9 (s, 1H), 5.90 (s, 2H), 3.17 (m, 1H), 1.82 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H), 1.44 (s, 3H), 1.42 (t, 2H), 1.38 (m, 2H), 1.34 (d, 3H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 407.21; Elemental anal. calcd for C22H29BO5; C, 68.76; H, 7.63; B, 2.85. found C, 68.77; H, 7.65; B, 2.84.
Compound H: 1H NMR (500 MHz, CDCl3) δ6.67 (s, 1H), 4.2 (bs, 2H). 3.7 (t, 4H), 3.17 (m, 1H) 2.9 (t, 4H), 1.86 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H), 1.46 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.34 (d, 3H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 463.28 Elemental anal. calcd for C25H37BN2O4; C, 68.16; H, 8.43; B, 2.44; N, 6.46. found C, 68.16; H, 8.44; B, 2.41.
Compound I: 1H NMR (500 MHz, CDCl3) δ6.4 (s, 1H), 4.15 (bs, 4H). 3.17 (m, 1H), 1.82 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H), 1.46 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.34 (d, 3H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 463.28 Elemental anal. calcd for C21H31BN2O3; C, 68.14; H, 8.44; B, 2.94; N, 7.56. found C, 68.14; H, 8.42; N, 7.55.
Compound J: 1H NMR (500 MHz, CDCl3) δ7.8 (s, 1H), 8.01 (bs, 2H), 3.17 (m, 1H), 2.06 (s, 6H), 1.85 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H), 1.46 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.34 (d, 3H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 477.28 Elemental anal. calcd for C25H35BN2O5; C, 66.04; H, 7.74; B, 2.38; N, 6.17. found C, 66.02; H, 7.74; B, 2.36; N, 6.16.
Compound K: 1H NMR (500 MHz, CDCl3) δ6.4 (s, 1H), 4.02 (bs, 2H), 3.17 (m, 1H), 2.78 (d, 6H), 1.85 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H), 1.46 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.34 (d, 3H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 421.27 Elemental anal. calcd for C23H35BN2O3; C, 69.50; H, 8.83; B, 2.75; N, 7.05. found C, 69.51; H, 8.82; B, 2.77; N, 7.04.
Compound L: 1H NMR (500 MHz, CDCl3) δ7.56 (s, 1H), 8.03 (s, 1H), 5.07 (bs, 1H), 3.17 (m, 1H), 1.85 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H), 1.46 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.34 (d, 3H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 403.23 Elemental anal. calcd for C22H29BN2O3; C, 69.47; H, 7.66; B, 2.83; N, 7.35. found C, 69.47; H, 7.65; B, 2.82; N, 7.33.
Compound M: 1H NMR (500 MHz, CDCl3) δ6.8 (s, 1H), 5.2 (bs, 2H), 3.17 (m, 1H), 1.85 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H), 1.46 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.34 (d, 3H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 553.16 Elemental anal. calcd for C21H31BN2O9S2; C, 47.55; H, 5.88; B, 2.05; N, 5.24; S, 12.09. found C, 47.54; H, 5.89; B, 2.04; N, 5.24; S, 12.08.
Compound N: 1H NMR (500 MHz, CDCl3) δ7.3 (s, 1H), 3.17 (m, 1H), 1.85 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H), 1.46 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.34 (d, 3H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 555.12 Elemental anal. calcd for C21H29BO11S2; C, 47.45; H, 5.46; B, 2.05; S, 12.09. found C, 47.44; H, 5.45; B, 2.06; S, 12.1.
Compound O: 1H NMR (500 MHz, CDCl3) δ6.8 (s, 1H), 3.17 (m, 1H), 1.85 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H), 1.46 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.34 (d, 3H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 658.96 Elemental anal. calcd for C21H27BCl6O5Si2; C, 39.45; H, 4.25; B, 1.66; Si, 8.79; Cl, 33.29. found C, 39.44; H, 4.25; B, 1.68; Si, 8.78; Cl, 33.30.
Compound P: 1H NMR (500 MHz, CDCl3) δ6.9 (s, 1H), 5.89 (m, 2H), 5.24 (m, 2H), 5.23 (m, 2H) 4.65 (d, 4H), 3.17 (m, 1H), 1.82 (t, 2H), 1.71 (m, 2H), 1.49 (m, 2H), 1.48 (t, 2H), 1.43 (s, 3H), 1.37 (t, 2H), 1.34 (d, 3H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 475.27; Elemental anal. calcd for C27H37BO5; C, 71.69; H, 8.29; B, 2.34. found C, 71.68; H, 8.28; B, 2.33.
Compound Q: 1H NMR (500 MHz, CDCl3) δ8.2 (s, 1H), 3.18 (m, 1H), 1.82 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H) 1.44 (s, 3H), 1.48 (t, 2H), 1.36 (m, 2H), 1.34 (d, 3H) 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 453.19; Elemental anal. calcd for C21H27BN2O7; C, 58.69; H, 6.31; N, 6.55; B, 2.57. found C, 58.69; H, 6.33; N, 6.54; B, 2.56.
Compound R: 1H NMR (500 MHz, CDCl3) δ8.1 (s, 1H), 3.18 (m, 1H), 1.82 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H) 1.44 (s, 3H), 1.48 (t, 2H), 1.36 (m, 2H), 1.34 (d, 3H) 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 313.21; Elemental anal. calcd for C23H27BN2O3; C, 70.78; H, 6.92; N, 7.18; B, 2.77. found C, 70.77; H, 6.92; N, 7.16; B, 2.78.
Compound S: 1H NMR (500 MHz, CDCl3) 67.4 (s, 1H), 3.18 (m, 1H), 1.82 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H) 1.44 (s, 3H), 1.48 (t, 2H), 1.36 (m, 2H), 1.34 (d, 3H) 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 399.21; Elemental anal. calcd for C21H27BF2O3; C, 67.64; H, 7.23; F, 10.12; B, 2.83. found C, 67.65; H, 7.22; F, 10.12; B, 2.84.
Compound T: 1H NMR (500 MHz, CDCl3) δ7.2 (s, 1H), 3.18 (m, 1H), 1.82 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H) 1.44 (s, 3H), 1.48 (t, 2H), 1.36 (m, 2H), 1.34 (d, 3H) 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 431.143; Elemental anal. calcd for C21H27BCl2O3; C, 61.64; H, 6.56; Cl, 17.33; B, 2.65. found C, 61.66; H, 6.56; Cl, 17.32; B, 2.64.
Compound U: 1H NMR (500 MHz, CDCl3) δ6.59 (s, 1H), 9.1 (s, 2H), 8.03 (s, 1H), 3.18 (m, 1H), 1.78 (t, 1H), 1.75 (t, 2H), 1.64 (m, 2H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.34 (d, 3H) 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 394.226; Elemental anal. calcd for C21H30BNO4; C, 67.95; H, 8.14; N, 3.77; B, 2.91. found C, 67.98; H, 8.15; N, 3.76; B, 2.91.
Compound V: 1H NMR (500 MHz, CDCl3) 6.89 (s, 1H), 9.1 (s, 2H), 8.03 (s, 1H), 2.94 (m, 1H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.34 (d, 3H) 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 401.188 Elemental anal. calcd for C21H29BO4S; C, 64.95; H, 7.65; S, 8.24; B, 2.73. found C, 64.96; H, 7.66; S, 8.23; B, 2.72.
Compound W: 1H NMR (500 MHz, CDCl3) δ6.54 (s, 1H), 9.1 (s, 2H), 3.34 (s, 2H), 2.34 (m, 1H), 1.53 (t, 2H), 1.49 (m, 2H), 1.46 (t, 2H), 1.43 (t, 1H) 1.41 (m, 2H), 1.38 (m, 2H), 1.26 (s, 3H) 1.17 (d, 3H) 1.12 (s, 6H) ppm. Mass: ESI [M+Na]+: 393.236; Elemental anal. calcd. For C22H31BO4; C, 71.36; H, 8.44; B, 2.92. found C, 71.36; H, 8.43; B, 2.93.
Compound X: 1H NMR (500 MHz, CDCl3) δ6.59 (s, 1H), 9.1 (s, 2H), 3.62 (s, 3H), 2.95 (m, 1H), 1.78 (t, 1H), 1.75 (t, 2H), 1.64 (m, 2H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.34 (d, 3H) 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 408.242; Elemental anal. calcd for C22H32BNO4; C, 68.59; H, 8.34; N, 3.65; B, 2.81. found C, 68.58; H, 8.35; N, 3.65; B, 2.83.
Compound Y: 1H NMR (500 MHz, CDCl3) δ6.7 (s, 1H), 3.73 (s, 6H). 3.62 (s, 3H), 2.94 (m, 1H), 1.78 (t, 1H), 1.75 (t, 2H), 1.64 (m, 2H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.34 (d, 3H) 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 436.27; Elemental anal. calcd for C24H36BNO4; C, 69.73; H, 8.76; N, 3.38; B, 2.64. found C, 69.75; H, 8.77; N, 3.38; B, 2.65.
Compound Z: 1H NMR (500 MHz, CDCl3) δ6.9 (s, 1H), 2.98 (m, 1H), 2.21 (m, 1H), 1.82 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H), 1.01 (d, 6H) ppm. Mass: ESI [M+Na]+: 423.242; Elemental anal. calcd for C23H33BO5; C, 69.24; H, 8.31; B, 2.70. found C, 69.26; H, 8.35; B, 2.72.
Compound AA: 1H NMR (500 MHz, CDCl3) δ7.3 (s, 1H), 3.18, (m, 1H), 2.08 (s, 6H), 1.82 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.36 (m, 2H), 1.34 (d, 3H) 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 479.236; Elemental anal. calcd for C25H33BO7; C, 65.76; H, 7.29; B, 2.37. found C, 65.77; H, 7.30; B, 2.38.
Compound AB: 1H NMR (500 MHz, CDCl3) δ7.1 (s, 1H), 8.01 (bs, 2H), 10.1 (bs, 1H), 2.94 (m, 1H), 2.06 (s, 6H), 1.85 (t, 2H), 1.78 (t, 1H), 1.61 (m, 2H), 1.49 (m, 2H), 1.46 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.34 (d, 3H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 476.28 Elemental anal. calcd for C25H36BN3O4; C, 66.24; H, 8.04; B, 2.38; N, 9.17. found C, 66.25; H, 8.02; B, 2.39; N, 9.16.
Compound AC: 1H NMR (500 MHz, CDCl3) δ6.66 (s, 1H), 3.17 (m, 1H), 2.83 (s, 12H), 1.82 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H), 1.46 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.34 (d, 3H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 449.302 Elemental anal. calcd for C25H39BN2O3; C, 70.42; H, 9.22; B, 2.54; N, 6.52. found C, 70.43; H, 9.21; B, 2.55; N, 6.53.
Compound AD: 1H NMR (500 MHz, CDCl3) δ6.85 (s, 1H), 9.1 (s, 2H), 2.75 (t, 3H), 1.83 (t, 2H) 1.78 (t, 1H), 1.56 (m, 2H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 395.201; Elemental anal. calcd for C20H25BO6; C, 64.55; H, 6.83; B, 2.91. found C, 64.54; H, 6.84; B, 2.92.
Compound AE: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H), 4.33 (t, 1H), 1.89 (m, 2H) 1.83 (t, 2H), 1.78 (t, 1H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 465.51; Elemental anal. calcd for C21H26BF3O6; C, 57.05; H, 5.91; B, 2.39; F, 12.89. found C, 57.06; H, 5.92; B, 2.40; F, 12.88.
Compound AF: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H), 3.83 (t, 1H), 1.83 (t, 2H), 1.78 (t, 1H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 449.184; Elemental anal. calcd for C21H26BF3O5; C, 59.18; H, 6.15; B, 2.59; F, 13.35. found C, 59.16; H, 6.16; B, 2.58; F, 13.35.
Compound AG: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H), 4.13 (m, 1H), 2.61 (bs, 2H), 1.93 (m, 2H), 1.83 (t, 2H), 1.78 (t, 1H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 396.206; Elemental anal. calcd for C20H28BNO5; C, 64.36; H, 7.56; B, 2.9; N, 3.75. found C, 64.34; H, 7.54; B, 2.89; N, 3.77.
Compound AH: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H), 4.76 (m, 1H), 3.5 (bs, 1H), 1.87 (m, 2H), 1.83 (t, 2H), 1.78 (t, 1H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 397.20; Elemental anal. calcd for C20H27BO6; C, 64.26; H, 7.26; B, 2.89. found C, 64.25; H, 7.24; B, 2.88.
Compound AI: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H), 5.43 (m, 1H), 2.25 (s, 3H), 1.93 (m, 2H), 1.83 (t, 2H), 1.78 (t, 1H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 439.210; Elemental anal. calcd for C22H29BO7; C, 63.26; H, 7.02; B, 2.62. found C, 63.26; H, 7.04; B, 2.63.
Compound AJ: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H), 8.10 (bs, 1H), 5.13 (m, 1H), 2.15 (s, 3H), 1.97 (m, 2H), 1.83 (t, 2H), 1.78 (t, 1H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 438.214; Elemental anal. calcd for C22H30BNO6; C, 63.64; H, 7.28; O, 23.26; B, 2.60; N, 3.35. found C, 63.65; H, 7.30; N, 3.34.
Compound AK: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H), 4.13 (m, 1H), 3.05 (m, 1H), 2.45 (d, 3H), 1.83 (m, 2H), 1.82 (t, 2H), 1.78 (t, 1H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 410.21; Elemental anal. calcd for C21H30BNO5; C, 65.15; H, 7.84; B, 2.76; N, 3.96. found C, 65.14; H, 7.85; B, 2.76; N, 3.95.
Compound AL: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H), 4.13 (m, 1H), 3.05 (m, 1H), 2.25 (d, 6H), 1.82 (t, 2H), 1.80 (m, 2H) 1.78 (t, 1H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 424.235; Elemental anal. calcd for C22H32BNO5; C, 65.85; H, 8.05; B, 2.68; N, 3.49. found C, 65.82; H, 8.02; B, 2.67; N, 3.51.
Compound AM: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H), 6.47 (d, 1H), 5.03 (t, 1H), 4.18 (dd, 1H), 4.04 (dd, 1H), 2.05 (m, 2H, 1.83 (t, 2H), 1.78 (t, 1H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 423.26; Elemental anal. calcd for C22H29BO6; C, 66.21; H, 7.35; B, 2.69. found C, 66.23; H, 7.36; B, 2.68.
Compound AN: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H), 4.13 (t, 1H), 2.24 (d, 4H), 1.82 (t, 2H), 1.80 (m, 2H) 1.78 (t, 1H), 1.50 (m, 6H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 464.235; Elemental anal. calcd for C25H36BNO5; C, 68.05; H, 8.25; B, 2.48; N, 3.19. found C, 68.10; H, 8.22; B, 2.47; N, 3.21.
Compound AO: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H), 4.13 (t, 1H), 3.65 (t, 4H) 2.34 (d, 4H), 1.82 (t, 2H), 1.80 (m, 2H) 1.78 (t, 1H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 466.35; Elemental anal. calcd for C24H34BNO6; C, 65.08; H, 7.69; B, 2.24; N, 3.17. found C, 65.09; H, 7.71; B, 2.25; N, 3.18.
Compound AP: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H), 4.13 (t, 1H), 2.4 (q, 4H), 1.82 (t, 2H), 1.80 (m, 2H) 1.78 (t, 1H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H), 1.05 (t, 6H) ppm. Mass: ESI [M+Na]+: 452.28; Elemental anal. calcd for C24H36BNO5; C, 67.18; H, 8.49; B, 2.51; N, 3.28. found C, 67.19; H, 8.47; B, 2.53; N, 3.27.
Compound AQ: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.76 (s, 1H), 9.1 (s, 2H) 3.89 (t, 1H), 2.08 (m, 2H) 1.83 (t, 2H), 1.78 (t, 1H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 409.34; Elemental anal. calcd for C21H27BO6; C, 65.37; H, 7.08; B, 2.91. found C, 65.36; H, 7.09; B, 2.92.
Compound AR: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H) 2.93 (m, 1H), 1.83 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H) 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H), 0.59 (m, 1H), 0.34 (m, 4H) ppm. Mass: ESI [M+Na]+: 409.34; Elemental anal. calcd for C21H27BO6; C, 65.37; H, 7.08; B, 2.91. found C, 65.34; H, 7.09; B, 2.92.
Compound AS: 1H NMR (500 MHz, CDCl3) δ7.3-7.5 (m, 5H), 6.83 (s, 1H), 6.59 (d, 1H) 9.1 (s, 2H) 2.8 (m, 1H), 1.83 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H) 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 484.23; Elemental anal. calcd for C27H32BNO5; C, 70.81; H, 6.72; B, 2.31; N, 3.04. found C, 70.84; H, 6.73; B, 2.32; N, 3.06.
Compound AT: 1H NMR (500 MHz, CDCl3) δ7.05 (dd, 2H), 6.83 (s, 1H), 6.59 (t, 1H), 6.43 (d, 2H) 9.1 (s, 2H), 3.48 (t, 2H), 3.28 (m, 1H), 1.83 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H) 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 486.253; Elemental anal. calcd for C27H34BNO5; C, 70.01; H, 7.43; B, 2.32; N, 3.04. found C, 70.06; H, 7.44; B, 2.35; N, 3.06.
Compound AU: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H), 5.62 (d, 1H), 4.98 (d, 1H), 2.23 (t, 2H), 1.83 (t, 2H), 1.78 (t, 1H), 1.71 (m, 2H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.41 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 393.17; Elemental anal. calcd for C21H27BO5 C, 68.17; H, 7.38; B, 2.91. found C, 68.16; H, 7.35; B, 2.94.
Compound AV: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H), 10.05 (bs, 1H), 2.73 (m, 1H), 1.73 (t, 2H), 1.78 (t, 1H), 1.63 (m, 2H) 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H), 0.59 (m, 1H), 0.34 (m, 4H) ppm. Mass: ESI [M+Na]+: 420.24; Elemental anal. calcd for C23H32BNO4; C, 69.57; H, 8.18; B, 2.71; N, 3.56. found C, 69.55; H, 8.13; B, 2.71; N, 3.58.
Compound AW: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H), 2.74 (m, 1H), 1.73 (t, 2H), 1.78 (t, 1H), 1.63 (m, 2H) 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H), 0.59 (m, 1H), 0.34 (m, 4H) ppm. Mass: ESI [M+Na]+: 437.02; Elemental anal. calcd for C23H31BO4S; C, 66.57; H, 7.58; B, 2.61; S, 7.78. found C, 66.55; H, 7.59; B, 2.62; S, 7.77.
Compound AX: 1H NMR (500 MHz, CDCl3) δ6.83 (s, 1H), 9.1 (s, 2H), 10.1 (bs, 1H), 3.53 (t, 1H), 1.83 (t, 2H), 1.78 (t, 1H), 1.64 (m, 2H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 448.194; Elemental anal. calcd for C21H27BF3O4N; C, 59.38; H, 6.45; B, 2.59; F, 13.40; N, 3.29. found C, 59.34; H, 6.43; B, 2.58; F, 13.41; N, 3.28.
Compound AY: 1H NMR (500 MHz, CDCl3) δ7.95 (d, 2H), 7.47 (t, 1H), 7.37 (dd, 2H), 6.83 (s, 1H), 9.1 (bs, 2H), 5.48 (t, 1H), 2.16 (m, 2H), 1.83 (t, 2H), 1.78 (t, 1H), 1.49 (m, 2H), 1.48 (t, 2H), 1.44 (s, 3H), 1.38 (m, 2H), 1.11 (s, 6H) ppm. Mass: ESI [M+Na]+: 501.218; Elemental anal. calcd for C27H31BO7; C, 67.71; H, 6.59; B, 2.26. found C, 67.74; H, 6.58; B, 2.26.
Compound AZ: For step 1 to 7 Ref: (Pereira A. R.; Strangman, W. K.; Marion, F.; Feldberg, L.; Roll, D.; Mallon, R.; Hollander, I.; Andersen, R. J. J. Med. Chem. 2010, 53, 8523)
Step 1: Synthesis of compound 13 (2-hydroxy-4,5-dimethoxybenzaldehyde): To a solution of 3,4,5-trimethoxybenzaldehyde (5 g, 25.510 mmol) in CH2Cl2 (125 ml) at 0° C., BBr3 (6.39 g, 25.510 mmol) was added. The resulting dark mixture was stirred at rt for 9 h. Water (100 mL) was charged and the mixture was stirred for 10 min, the aqueous phase was extracted by CH2Cl2. Organic phase was dried over Na2SO4, and evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography using plain dichloromethane as eluent, afforded the 2-hydroxy-4,5-dimethoxybenzaldehyde 13 (4.3 g, 87%) isolated as yellow solid. Mp 105-107° C.; 1H NMR (CDCl3, 400 MHz): δ 11.40 (br. s, 1H), 9.70 (s, 1H), 6.91 (s, 1H), 6.48 (s, 1H), 3.94 (s, 3H), 3.88 (s, 3H) ppm. 13C NMR (CDCl3. 125 MHz): 194.0, 159.3, 157.3, 142.9, 113.3, 112.9, 100.1, 56.4, 56.3. HRMS (ESI) m/z: [M+H]+ calcd for C9H10O4+H+ 183.0657. Found 183.0653.
A solution of 5 (1 g, 5.49 mmol) in anhydrous CH2Cl2 under nitrogen was cooled to 0° C., to it diisopropyl ethylamine (DIPEA) (1.77 g, 13.736 mmol) and MOMCl (0.66 g, 8.241 mmol) were added. The reaction mixture was stirred at room temperature for 1 h. After completion of the reaction, water was added extracted with dichloromethane. The organic extract was washed with brine, dried over anhydrous Na2SO4 and evaporated under reduced pressure, the resultant product 14 (2.15 g, 98%) as colorless liquid was used for further reaction without purification. 1H NMR (CDCl3, 400 MHz): δ 10.34 (s, 1H), 7.30 (s, 1H), 6.77 (s, 1H), 5.26 (s, 2H), 3.95 (s, 3H), 3.88 (s, 3H), 3.54 (s, 3H) ppm. 13C NMR (CDCl3, 125 MHz): 188.0, 156.4, 155.5, 144.4, 118.1, 108.2, 99.9, 95.4, 56.4, 56.2, 56.1. HRMS (ESI) m/z: [M+H]+ calcd for C11H14O5+H+ 227.0919. Found 227.0897
The solution of compound 14 (1 g, 4.423 mmol) and sodium hydroxide (0.177 g, 4.423 mmol) in MeOH was taken in round bottom flask, to it NaBH4 (0.25 g, 6.635 mmol) was added. The reaction mixture was stirred for half hour at room temperature. The reaction mixture was concentrated under reduced pressure to remove MeOH, added water and was extracted with ethyl acetate, washed with brine, dried over anhydrous Na2SO4 and evaporated under reduced pressure afforded colorless liquid 15 (0.99 g, 98%), the resultant product was used for further reaction without purification. 1H NMR (200 MHz, CDCl3): δ 6.86 (s, 1H), 6.75 (s, 1H), 5.16 (s, 2H), 4.65-4.62 (d, J=5.29 Hz, 2H), 3.87 (s, 3H), 3.86 (s, 3H), 3.52 (s, 3H) ppm. 13C NMR (CDCl3, 100 MHz): 149.15, 149.0, 144.1, 122.1, 112.56, 101.3, 96.0, 60.7, 56.3, 56.1, 55.9. HRMS (ESI) m/z: [M+Na]+ calcd for C11H16O5+Na+ 251.0896. Found 251.0874
Step 4: Synthesis of compound 16 (2-hydroxy-4,5-dimethoxybenzyl)triphenylphosphonium hydrogen bromide salt): A solution of compound 15 (1 g, 4.384 mmol) in acetonitrile was taken, to this PPh3HBr (1.8 g, 5.260 mmol) was added at room temperature and refluxed for about 2 h. After completion of the reaction solvent was removed under reduced pressure and washed with ether, gave compound 16 (1.69 g, 90%) as a white amorphous solid. Mp 240-242° C.; 1H NMR (CDCl3, 400 MHz): δ 8.62 (br. s, 1H), 7.75-7.71 (m, 3H), 7.60-7.52 (m, 12H), 7.03 (s, 1H), 6.44-6.43 (d, J=12.8 Hz, 1H), 4.48-4.45 (d, J=12 Hz, 2H), 3.69 (s, 3H), 3.48 (s, 3H) ppm. 13CNMR (CDCl3, 125 MHz): 134.7, 134.7, 134.3, 130.0, 129.9, 113.9, 113.90, 101.9, 101.9, 56.3, 55.9, 25.3, 24.8. HRMS (ESI) m/z: [M]+ calcd for C27H26O3P 429.1620 (—HBr). Found 429.1615 (—HBr).
Step 5: Synthesis of compound 18 a starting material 3-(2,6,6-trimethylcyclohex-1-enyl)propanoic acid: 17 g (425.001 mmol) of NaOH was dissolved in water to make a 70 ml solution in a 250 ml conical flask with a magnetic stirrer. The alkali solution was then cooled in an ice bath and 17 g (106.25 mmol) of bromine was added to the solution after stirring for 1 h, 4.5 g (23.19 mmol) of dihydro-β-ionone 17 in 10 ml of dioxane was dropped into the solution, the stirring was continued at rt for 4 h. The excess of hypobromite was neutralized with 10% sodium bisulfite and solution was extracted with diethylether to remove remaining impurities. Acidification of the alkaline solution with conc. hydrochloric acid was done under usual conditions and workup gave 18 (4.1 g, 90.1%) as a colorless liquid. 1H NMR (CDCl3, 400 MHz): δ 2.44-2.39 (m, 2H), 2.37-2.31 (m, 2H), 1.93-1.89 (t, J=8 Hz, 2H), 1.61 (s, 3H), 1.58-1.54 (s, 2H), 1.44-1.41 (m, 2H), 1.00 (s, 6H). 13CNMR (CDCl3, 100 MHz): 180.3, 135.4, 128.5, 39.7, 34.9, 34.7 (multiple merged peaks), 28.4, 23.5, 19.6, 19.4. HRMS (ESI) m/z: [M+H]+ calcd for C12H20O2+H+ 197.1541. Found 197.1530.
Step 6: Synthesis of compound 19 (5,6-dimethoxy-2-(2-(2,6,6-trimethylcyclohex-1-enyl)ethyl)benzofuran): The intermediate 16 (2 g, 4.651 mmol) was taken in dry DCM along with dihydro-β-ionicacid 18 (0.91 g, 4.65 mmol) in round bottom flask, in dry conditions and cooled to 0° C. To it DCC (2.87 g, 13.953 mmol) and DMAP (0.56 g, 4.651 mmol) were added and stirred at room temperature for 18 h. DCM was evaporated under reduced pressure and the crude reaction mixture was dissolved in THF and to it was added triethylamine and refluxed for 3 h. THF was evaporated under reduced pressure and purified by column chromatography 5% ethyl acetate: hexane afforded 19 (1.42 g, 93%) as colorless oil. 1H NMR (400 MHz, CDCl3): δ 6.93 (s, 1H), 6.85 (s, 1H), 6.20 (d, J=0.6 Hz, 1H), 3.81 (d, 6H), 2.70-2.66 (m, 2H), 2.34-2.30 (m, 2H), 1.87-1.85 (t, J=6.1 Hz, 2H), 1.58 (s, 3H), 1.54-1.44 (m, 2H), 1.51-1.49 (m, 2H), 0.96 (s, 6H). 13CNMR (CDCl3, 100 MHz): 158.8, 149.1, 146.9, 146.1, 136.2, 128.1, 102.7, 101.0, 95.3, 56.4, 56.2, 39.7 (multiple merged peaks), 35.0, 32.7, 29.3, 28.5, 27.2, 19.8, 19.4 (merged peaks). HRMS (ESI) m/z: [M+H]+ calcd for C21H28O3+H+ 329.2117. Found 329.2099.
Step 7: Synthesis of compound 20: The solution of compound 19 (1 g, 3.049 mmol) was prepared in 2-nitro propane and cooled to 78° C., to it chlorosulfonic acid (1.06 g, 9.146 mmol) was added under inert atmosphere. The reaction mixture was stirred for 30 min. Then quenched with NaHCO3 and extracted by ethyl acetate. The organic layer was dried over Na2SO4 and evaporated under reduced pressure and purified by column chromatography using 5% ethyl acetate:hexane afforded 20 (0.9 g, 90% yield) as colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.00 (s, 1H), 6.97 (s, 1H), 3.91 (s, 3H), 3.89 (s, 3H), 2.79-2.74 (m, 1H), 2.72-2.65 (m, 1H), 2.42 (d, J=13.0 Hz, 1H), 2.06-1.91 (m, 2H), 1.87-1.76 (m, 2H), 1.50-1.63 (m, 2H), 1.45-1.39 (m, 2H), 1.31 (s, 3H), 0.97 (s, 3H), 0.95 (s, 3H). 13CNMR (CDCl3, 125 MHz): 151.2, 149.2, 146.4, 145.4, 124.3, 118.8, 102.4, 95.5, 56.6, 56.2, 52.6, 41.8, 37.6, 35.9, 33.5, 33.1, 24.9, 21.8, 21.3 (merged peaks), 18.8. HRMS (ESI) m/z: [M+H]+ calcd for C21H28O3+H+ 329.2117. Found 329.2105, [M+Na]+ calcd for C21H28O3+Na+ 351.1936. Found 351.1931
Step 8: Synthesis of compound 21: The solution of compound 20 (1 g, 3.048 mmol) was prepared in dry THF, and cooled to 0° C. under dry condition. To it n-BuLi (0.195 g, 3.048 mmol) was added and the reaction mixture was kept for 20 minutes stirring, to it triethyl borate (0.45 g, 3.048 mmol) was added and continued stirring for another 1 h at rt. Quenched with ammonium chloride solution and extracted by ethyl acetate. Concentrated under reduced pressure and purified by column chromatography using 6% ethyl acetate: hexane afforded 21 (1.043 g, 92%) as a colorless liquid. 1H NMR (400 MHz, CDCl3) δ 7.18 (s, 1H), 6.83 (s, 2H), 3.96 (s, 3H), 3.93 (s, 3H), 2.80-2.73 (m, 2H), 2.42 (d, J=12.0 Hz, 1H), 2.03 (m, 2H, merged signals), 1.89-1.76 (m, 2H), 1.73-1.66 (m, 2H), 1.45-1.40 (m, 2H), 1.32 (s, 3H), 0.99 (s, 3H), 0.96 (s, 3H). 13C NMR (CDCl3, 100 MHz) 153.0, 152.9, 151.0, 148.4, 124.3, 122.4, 107.1, 61.9, 56.7, 52.7, 41.8, 36.1, 33.5, 33.2 (merged peaks), 25.1, 21.9, 21.4, 18.9, 18.8. HRMS (ESI) m/z: [M+H]+ calcd for C21H29BO5+H+ 372.2217. Found 372.2231.
Step 9: Synthesis of compound 22: To (0.5 g, 4.301 mmol) of dry aluminium chloride, 5 ml of dichloromethane was poured, then (0.245 g, 3.225 mmol) of crystalline thiourea was added in small portions and stirred for 20 minutes. The reaction mixture becomes transparent oily solution. Then compound 21 (0.1 g, 0.268 mmol) dissolved in dichloromethane was added to this over a period of 5 minutes and stirred for 2 h at rt. The excess of AlCl3 was removed by quenching with ice and extracted by dichloromethane and then purified by column chromatography using 15% ethylacetate: hexane afforded 22 (0.077 g, 85% yield) as colorless liquid. 1H NMR (500 MHz, CDCl3): δ 7.10 (s, 1H), 6.74 (s, 1H), 6.48 (br.s, 1H), 3.94 (s, 2H), 2.80-2.71 (m, 2H), 2.39 (d, J=10 Hz, 1H), 2.20-2.03 (m, 2H), 1.80-1.70 (m, 2H), 1.54-1.51 (m, 2H), 1.44-1.37 (m, 2H), 1.30 (s, 3H), 0.98 (s, 3H), 0.95 (s, 3H). 13C NMR (CDCl3, 125 MHz): 153.4, 151.1, 147.9, 142.9, 124.3, 118.1, 114.1, 105.1, 52.6, 41.8, 37.7, 36.0, 33.5, 29.5, 25.0, 21.9, 21.4, 18.9, 18.8; HRMS (ESI) m/z: [M+]: calcd for C19H25BO5 344.1795. Found 344.1761.
Biology:
1. Cytotoxic Assay
The MTT assay (MTT Assay (Legrier M E, Yang C P, Yan H G et al. Targeting protein translation in human non small lung cancer via combined MEK and mammalian target of rapamycin suppression. Cancer Res 67:11300-8(2007).) is useful for measuring the effect of a wide range of compounds on the in vitro growth of either normal or cancer cell lines. The assay was set up in a 96-well, flat-bottomed polystyrene microtiter plate. 3-5000 cells were suspended per well in appropriate growth medium, and the cells were added to replicate wells (triplicates were preferred). It was preferable to add the cells to the required number of wells in the plate prior to adding the drugs or the test agents. After the cells were added to the plate, it was placed on the incubator for overnight incubation, while the agents to be tested were being prepared. After overnight incubation drugs or test compounds were added at defined concentrations to each set of replicate wells and incubated for 48 hrs in CO2 incubator. Most of these compounds were dissolved in dimethyl sulfoxide (DMSO) for the final addition. After 48 hr incubation, diluted the MTT stock solution (2.5 μg/ml) with an equal volume of tissue culture medium and added 20 μl of this solution directly to each well with a multichannel pipette. As with the adherent-cell method, return the plates to the incubator for a period of at least 4 h. After 4 hr incubation centrifuge the plates at 1000 g for 10 min at ambient temperature, followed by inversion of the plates and blotting of excess medium. Add 150 μl of working DMSO to solubilize the MTT formazan product. A standard micro plate reader with adjustable wavelength across the visible spectrum was used. The OD values at 570 nm obtained for each set of triplicates corresponding to a specific concentration of a test agent was then transferred into a spreadsheet program.
Results: Cytotoxicity assay based on MTT was performed on the panel of cancer cell lines using compound A and compound E as a test material. In order to determine the effect of compound A and compound E on cell proliferation and in relative IC50 values, MCF-7, caco-2 & HCT-115 were treated with compound A and compound E at indicated concentrations (0.01, 0.1, 1, 10 μM) for 48 h. In the present study, compound A and compound E produced concentration dependent inhibition of cell proliferation. From the MTT based inhibition in cell proliferation, the calculated cell based IC50 value of 2.6 μM and 2.4 μM in breast (MCF-7) and colon (caco-2) cell line were observed for compound A and 5.6, 3.7 and 3.1 μM in colon (caco-2, HCT-115) and breast (MCF-7) for compound E was calculated (
2. PI3K Inhibition Assays:
PI3K inhibition assay (PI3K Assay (Emmanuelle M, Huang Y, Yan H G et al. Targeting Protein Translation in Human Non-Small Cell Lung Cancer via Combined MEK and Mammalian Target of Rapamycin Suppression. Cancer Res 67:(23). (2007).) was carried out by PI3 Kinase activity/inhibitor assay kit, where PI3 kinase reaction was set up in Glutathione-coated strips/plate for inhibitor reaction. Kinase and inhibitors were pre-incubated for 10 minutes prior to the addition of PIP2 substrate. 5 μL of 5× kinase reaction buffer were added in each well followed by the further addition of 5 μL/well of PIP2 substrate. Then distilled H2O was added to each well so as to make up a final volume of 25 μL/well. Incubation was done at rt for 1 hour which was followed by washing the wells 3 times with 200 μL of 1×TBST per well and then 2 times with 200 μL of IX TBS per well. Then 100 μL of the Substrate TMB per well was added and then to keep for colour development in the dark for 5-20 minutes. However, appearance of the blue color to avoid over-development were monitored. 100 μL of the stop solution per well was used to stop the reaction. Readings were recorded at 450 nm.
Results:
The IC50 value of a drug measures the effectiveness of a compound in inhibiting biological or biochemical function. Drug molecules can be categorized as low, active or highly active based on IC50 values. The determination of enzyme based IC50 values helps in early analysis and estimation of the drug activities in order to narrow down drug candidates for further experimental purpose. The liphagal, compound A and compound E used in the present study inhibited PI3Kα enzyme activity in dose dependent pattern with varying concentration i.e 20, 40, 80, 160, 320 and 640 nM respectively. Moreover, an IC50 of 108, 140 and 102 nM for liphagal, compound A and compound E against PI3Kα was observed and 100 nM for compound A against PI3Kβ was also determined (
3. Cell Cycle Analysis:
Analysis (Cell cycle (Waxman D J, Schwartz P S, Harnessing apoptosis for improved anti-cancer gene therapy, Cancer Res. 63:8563-8572(2003).) of a population of cells replication state can be achieved by fluorescence labeling of the nuclei of cells in suspension and then analyzing the fluorescence properties of each cell in the population. The experiment was performed using caco-2, colon human cancer cell line. Cells were seeded in 6 well plates at the concentration of 3×105 cells/ml/well. Plate was incubated in CO2 incubator for overnight. After overnight incubation test sample(s) were added at desired concentration, sparing wells for negative and positive control and incubated for 24 hrs. After 24 hr incubation, cell were trypsinized along with test sample from each well was extracted using a micropippete and separately transferred into 15 ml centrifuge tubes. Tubes were centrifuged at 3000 rpm for 5 min. The supernatant was discarded and pellet was resuspended in 1 ml filtered PBS and centrifuged at 2000 rpm for 5 min. After 5 mins supernatant was discarded and pellet was resuspended in 70% ethanol. Cells were fixed for at least 1 hour at 4° C. (cells may be stored in 70% ethanol at −20° C. for several weeks prior to PI staining and flow cytometric analysis). Cells were again centrifuged at 2000 rpm for 5 minutes and washed twice in filtered PBS by centrifuging at 2000 rpm for 5 min. Supernatant was discarded and tubes were placed in inverted position over tissue paper till all the supernatant drained over the paper. 1 ml of cell cycle reagent (CCR) was added in each acquisition tube in dark. Reading was taken on flow cytometer (BD Biosciences).
Results: Cell cycle is the life cycle of a cell. Each stage of the cell cycle i,e. G1 (Gap1), S, G2 (Gap 2), & M (mitosis) have unique events that occur within each of them. Two of the most popular flow cytometric applications are the measurement of cellular DNA content and the analysis of the cell cycle which are fundamental processes of cell survival. In the present study, the effect of compound E on the DNA content by cell cycle phase distribution was assessed by using colon (caco-2) cell line. In addition to determining the relative cellular DNA content, flow cytometry also enables the identification of the cell distribution during the various phases of the cell cycle. Cells (2×106/ml/6-well plate), exposed to different concentrations of compound E were stained with propidium iodide (PI) to determine DNA fluorescence and cell cycle phase distribution. The percentage of compound E treated sub-G0 cells with 1, 5, 7 and 9 μM for 24 h was found to be 62.5%, 64.3%, 65.6% and 70.2% respectively. Under similar conditions, Liphagal treated cultures showed 64.9% cells in sub-G0 phase. Further, the cell cycle at G2/M phase was not affected indicating that compound E treatments does not produce any mitotic block or cause delay in cell cycle. Overall, each treatment with an increase in concentration led to an increase in sub-G0 after 24 h treatment. Thus, it is clear that compound E induced early cell cycle arrest with concentration dependent manner (
4. Annexin-V Apoptotic Assay:
The cell death status was analysed using Annexin-V (Annexin-V apoptotic assay (Yunqing Li, FadilaGuessous, SherwinKwon, Manish Kumar. PTEN Has Tumor-Promoting Properties in the Setting of Gain-of-Function p53 Mutations, 2008 Cancer Res; 68: (6) (2008).) Flow cytometery. The experiment was performed using caco-2 colon human cancer cell line. Cells were seeded in 6 well plates at the concentration of 2×105 cells/ml/well. Plates were incubated in CO2 incubator for overnight. After overnight incubation test sample(s) were added at desired concentration, sparing wells for negative and positive control and incubated for 48 hrs. After 48 hr incubation, cell were trypsinized and separately transferred into 15 ml centrifuge tubes. Tubes were centrifuged at 3000 rpm for 5 min. The supernatant was discarded and pellet was resuspended in 1 ml filtered PBS and centrifuged at 2000 rpm for 5 min. After 5 mins supernatant was discarded and pellet was resuspended in 400 ml of 1× binding buffer to make cell suspension. From this suspension, 100 μl of cells is transferred in falcon tube and then 10 μl of propidium iodide (PI) and 5 μl Annexin-V antibody were added and incubated for 30 min in dark. After 30 min incubation in dark, apoptosis were analysed by flow cytometer (BD Biosciences).
Results: In the present study, the percentage of compound E treated late apoptotic cells with 1, 5, 7 and 9 μM for 48 h was found to be 36.3%, 34.8%, 38.5% and 56.8% respectively. Under similar conditions, Liphagal treated cultures showed 42.7% cells late apoptotic phase and reverse was found in early apoptotic phase with cell population decreasing 21.9%/o, 23.4%, 21.4% and 14.7% in early apoptotic phase. Further, there were not so much population of cell in necrotic phase indicating that compound E, treatments does not produce any early apoptosis and necrosis. Overall, there was a concentration dependent net increase in late apoptotic cell population (
Materials & Methods:
For immunofluorescence microscopic analysis, 4×104 CACO-2 cells/ml were seeded on 18-mm coverslips in 6-well plates, one day before experiment. Cells were serum starved overnight and treated with liphagal and compound E, 4 and 3 μM respectively for 24 hr. Following treatment, cells were washed in PBS, followed by fixation in absolute methanol at −20° C. for 5 min19. The fixed cells were blocked with 10% goat serum in PBS for 20 min at room temperature to eliminate non-specific binding of secondary antibody. Cells were incubated with polyclonal rabbit pAKT (serine 473) primary antibody (1:100 in 0.5% BSA in PBS; Santa Cruz Biotechnology) for 1 h at 25° C. in moist chamber, then washed and incubated with secondary antibody. The cells were washed and incubated for 45 min with a Texas red-conjugated goat antirabbit antibody (1:500 in 0.5% BSA in PBS; Santa Cruz Biotechnology) at 25° C. The coverslips were mounted on glass slides with 4′,6-diamidino-2-phenylindole-containing ProLong Gold Antifade mounting medium (Invitrogen) and visualized by fluorescence microscope (Olympus, IX81) under an Olympus 60× oil immersion objective lens. The negative controls were also used in which incubation of cells with primary antibody was omitted.
Results: Phosphorylation (activation) of Akt is associated with protection of cells from apoptosis20 (K. Nicholson, N. Anderson. 2002. The protein kinase B/Akt signaling pathway in human malignancy. Cell signal 14: 381-395). In the present studies it was observed that treatment of CACO-2 cells with liphagal and compound E, 4 and 3 M respectively for 24 hr caused the inhibition of pAkt (Ser 473). The inhibition of Akt consequently leads to apoptosis. The untreated cells showed the pAKT in the cytoplasm (
Advantages of introducing boronic acid functionality: Recent report on synthetic analog of liphagal (Alban R. Pereira, Wendy K. Strangman, Synthesis of phosphatidylinositol 3-kinase (PI3K) inhibitory analogues of the sponge meroterpenoid Liphagal; J. Med. Chem., 2010, 53 (24), pp 8523-8533) with an IC50 of 66 nM and selectivity towards PI3K-α, suggests that this analog possess greater chemical structure stability and gives opportunity for developing this skeleton into lead preclinical candidate. As a part of our ongoing program on developing isoform selective PI3K inhibitors, it occurred to us that it would be interesting to embark a program on the preparation of compounds based on this modified structure, leveraging the evidence of biological activity exhibited by this molecule. In this direction, we initiated our efforts, and planed to replace aldehyde functionality with boronic acid. Further, the 14-formyl-15,16-dihydroxy substitution pattern in the aromatic ring of liphagal is required to achieve nanomolar potency. It is also demonstrated that the absence of the C-14 formyl group appears to destabilize the liphagane heterocyclic ring system, making it more susceptible to air oxidation and skeletal rearrangements involving ring B contraction. This evidence suggests that the C-8 desmethyl analog with contracted B ring to six-membered, must be ultimately responsible for the activity, which supports our envision. Therefore, instead of formyl at the C-14, we designed a contracted B ring analog without formyl functionality having boronic acid in this place, assuming that this analog would offer more rigidity to the structure. Also, using a boronic acid instead of an aldehyde could circumvent the associated drawbacks. Moreover, boron has ability to biomimic carbon and forms the covalent adducts with the serine or histidine residues of the active site ((a) Adams, J. A.; Behnke, M.; Chen, S.; Cruichshank, A. A.; Dick, L. R.; Grenier, L.; Klunder, J. M.; Ma, Y. T.; Plamondon, L.; Stein, R. L. Bioorg. Med. Chem. Lett. 1998, 98, 333. (b) Paramore, A.; Frantz, S. Nat. Rev. Drug Discovery 2003, 2, 611).
Keeping in view the role of boron, the importance of boronic acid bearing compounds of liphagal are visualized as potential PI3K inhibitor. The evidence from the computational in silico docking of this boronic acid bearing liphagal compounds PI3K showed excellent H-bonding interactions with key amino acids, which are also previously reported as a key amino acid to be involved in inhibitory interactions in the p110α active site of PI3K-α with improved docking score of −8.08 over 1 and 2.12 The biological potential of boronic acid as PI3K inhibitor was also examined, which has shown PI3K-α isoform selectivity and excellent inhibitory activity (IC50 23 nM) for one of the compound i.e. compound-AZ.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0794/DEL/2012 | Mar 2012 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IN2013/000169 | 3/18/2013 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2013/140417 | 9/26/2013 | WO | A |
| Entry |
|---|
| International Search Report, Council of Scientific & Industrial Research, PCT/IN2013/000169, Aug. 2, 2013. |
| Written Opinion of the International Searching Authority, Council of Scientific & Industrial Research, PCT/IN2013/000169, Aug. 2, 2013. |
| Marion, Frederic et al., Liphagal, a Selective Inhibitor of P13 Kinase α Isolated from the Sponge Aka coralliphaga: Structure Elucidation and Biomimetic Synthesis, Orginal Letters., vol. 8, No. 2, 2006, pp. 321-324. |
| Mehta, Goverdhan et al., A concise synthesis of the bioactive meroterpenoid natural product (±)-liphagal, a potent P13K inhibitor, Tetrahedron Letters 50 (2009), pp. 5260-5262. |
| Kim, Yeong Gil et al., Aromatic hole injecting or transportingmaterial for organic electroluminescent device, C:\EPOPROGS\SEA\.\..\..\epodata\sea\eplogf\SA1202753.1og, XP-002704039, 2 pgs. Jul. 19, 2013. |
| Kawamura, Masahiro et al., Anthracene derivative for organic electroluminescent device, C:\EPOPROGS\SEA\.\..\..\epodata\sea\eplogf\SA1202753.log, XP-002704040, 3 pgs. Jul. 19, 2013. |
| Number | Date | Country | |
|---|---|---|---|
| 20150051173 A1 | Feb 2015 | US |
