Patent Application
20230234920
The present invention relates to an azirine containing compound of formula I, an enantiomer, diastereoisomer, or a pharmaceutically acceptable salt thereof, to be useful as anti-angiogenesis agents.
wherein
R1 is selected from alkyl (C1-C12); alkenyl (C1-C12), hydroxy, nitro, halogen, amino, cyano, aryl, heteroaryl, cycloalkyl (C1-C7), cyclo ether (C1-C6), heteroalkyl, alkoxy, alkylamino, aryl amino, alkyl (C1-C12) ester, (hetero)aryl ester, alkyl phosphonate, (hetero)aryl phosphonate, tetrazole;
R2 and R3 are either same or different and selected from the group consisting of H, COOR4, cyano, tetrazole, phosphonate, alkoxybenzyl, hydroxy, nitro, halogen, amino, alkyl (C1-C12), aryl, heteroaryl, cycloalkyl (C1-C7), heteroalkyl, alkoxy, alkylamino, aryl amino, (hetero)aryl ester, alkyl phosphonate, (hetero)aryl phosphonate or tetrazole;
wherein each of these groups [R1, R2 and R3] may further substituted with one or more substituents selected from the group consisting of hydrogen, hydroxy, halogen, cyano, alkyl amino, aryl amino, alkoxy, amino, nitro, aldehyde, carboxylic acid, ester or phosphoester, (hetero)aryl,
or 5-7 membered hetrocyclic ring wherein heteroatom is O, N or S.
R4 is selected from the group consisting of alkyl (C1-C12), allyl, methoxyethyl and benzyl;
R5, R6 and R7 are either same or different and selected from the group consisting of H, OMe, H, CH3, t-butyl; Halogen (Cl, Br, F, I), OCF3, CN, OBu, vinyl; dimethylamino or
wherein R5 and R6 may be joined to form a 5 to 6 membered hetrocyclic ring.
Particularly, the present invention relates to a process for the preparation of azirine containing compounds of formula I.
Azirine ring is the smallest nitrogen-containing heterocycle, and its existence in the natural products is remarkable. The first azirine containing natural product, azirinomicine, was isolated from a strain of the soil bacterium Streptomyces aureus (J. Antibiot. 1971, 24, 42-47; J. Antibiot. 1971, 24, 48-50). The long chain azirine carboxylic acid methyl ester, dysidazirines (J. Org. Chem. 1988, 53, 2103-2105, J. Nat. Prod. 1995, 58, 1463-1466) and terminal halogenated compounds, antazirines (J. Nat. Prod. 1995, 58, 1463-1466) were isolated from Dysidea fragilis. Recently, motualevic acid F along with antazirine were isolated from Siliquariaspongia sp. (Org. Lett. 2009, 11, 1087-1090). Apart from being in the natural products, azirine is also the reactive intermediate and serves as electrophile and nucleophile. Thus, it is widely used in the synthesis of several heterocycles such as pyrroles, indoles, and isoxazoles. The versatility of azirine ring prompted several research groups to develop synthetic methods, and the conventional methods such as thermolysis, photolysis of vinyl azides and Neber rearrangement of ketoxime tosylates are more often employed to access their synthesis. Although extensive efforts in developing methods for azirines and converting them into other heterocycles, the activity profiles of azirines are not up to far with other heterocycles, this is presumably the azirine ring erroneously assumed as an inherently unstable molecule. However, a very few numbers of azirine derivatives were synthesized and evaluated for various biological activities and found to exhibit prominent activities. The following are the references related to the invention. EP3053905, CH619706A5, CH619461A5, US20030027769; US20090317456; U.S. Pat. Nos. 7,872,029; 7,709,031; 6,664,272; 3,772,284; KR20050097494; EP0713393; EP0684820; US20070185069, U.S. Pat. No. 3,772,284, WO2013081549, U.S. Pat. No. 7,030,134, J. Am. Chem. Soc. 1996, 118, 8491-8492; U.S. Pat. Nos. 5,576,330; 6,025,353; Chem. Commun., 2012, 48, 3996-3998; J. Am. Chem. Soc. 2012, 134, 4104-4107; Org. Lett., 13, 24, 2011, 6374-6377; Angew. Chem. Int. Ed. 2014, 53, 4959-4963; Angew. Chem. Int. Ed. 2013, 52, 2212-2216; Tetrahedron 71, 2015, 1058-1067. The existence of several natural products and their limited anti-microbial and cytotoxicity activity screening data prompted us to synthesize azirine containing compounds and evaluate their anti-angiogenesis activity. Angiogenesis is the formation of new blood vessels from pre-existing blood vessels which has a crucial role in wound healing and several diseases states such as diabetic retinopathy, rheumatoid arthritis, cancers, etc. Inhibition of blood vessel growth through anti-angiogenesis technique is a promising strategy to cure diseases like cancers. Over last few decades, researchers have been developing several drug inhibitors and monoclonal antibodies to stop the angiogenesis process in cancer. However, the emergence of drug resistance, low bioavailability, and other factors limit their use for effective treatment. Therefore, potent anti-angiogenic molecules are currently in demand to cure malignant tumors. Cancer research always requires the discovery of new drugs that inhibit tumor growth by attenuation of blood vessel formation. In this context a library of azirine containing compounds with diverse structural features have been synthesized and evaluated for their anti-angiogenesis potential. We have found highly potent lead molecule for anti-angiogenic activity based on in vitro and in vivo experiments. In primary studies, the compound shows better efficacy as compared to Sunitinib.
The main objective of the present invention is to provide an azirine containing compound of formula I.
Another objective of the present invention is to provide a process for the preparation of azirine containing compound of formula I.
Yet another objective of the present invention is to provide azirine containing compound of formula I, useful as anti-angiogenesis agents.
Accordingly, the present invention provides an Azirine containing compound of formula I, an enantiomer, diastereoisomer, a pharmaceutically acceptable salt or a mixture thereof,
wherein
R1 is selected from alkyl (C1-C12); alkenyl (C1-C12), hydroxy, nitro, halogen, amino, cyano, aryl, heteroaryl, cycloalkyl (C1-C7), cyclo ether (C1-C6), heteroalkyl, alkoxy, alkylamino, aryl amino, alkyl (C1-C12) ester, (hetero)aryl ester, alkyl phosphonate, (hetero)aryl phosphonate, tetrazole;
R2 and R3 are either same or different and selected from the group consisting of H, COOR4, cyano, tetrazole, phosphonate, alkoxybenzyl, hydroxy, nitro, halogen, amino, alkyl (C1-C12), aryl, heteroaryl, cycloalkyl (C1-C7), heteroalkyl, alkoxy, alkylamino, aryl amino, (hetero)aryl ester, alkyl phosphonate, (hetero)aryl phosphonate or tetrazole;
wherein each of these groups [R1, R2 and R3] may further substituted with one or more substituents selected from the group consisting of hydrogen, hydroxy, halogen, cyano, alkyl amino, aryl amino, alkoxy, amino, nitro, aldehyde, carboxylic acid, ester or phosphoester, (hetero)aryl;
or 5-7 membered hetrocyclic ring wherein heteroatom is O, N or S,
wherein R4 is selected from the group consisting of alkyl (C1-C12), allyl, methoxyethyl and benzyl;
R5, R6, R7 are either same or different and selected from the group consisting of H, OMe, CH3, t-butyl, halogen, OCF3, CN, OBu, vinyl, dimethylamino and
wherein the halogen is selected from the group consisting of chlorine (Cl), bromine (Br), fluorine (F) and iodine (I);
wherein R5 and R6 is optionally joined to form a 5 to 6 membered hetrocyclic ring.
In an embodiment of the present invention, the compound of formula I is selected from the group consisting of:
In yet another embodiment, the present invention provides a pharmaceutical composition comprising the compound of formula I along with pharmaceutically acceptable excipients and pharmaceutically acceptable salts thereof.
In yet another embodiment of present invention, the excipients are selected from the group consisting of Carboxy methyl cellulose, tween 20, polyethylene glycol (PEG 400), dimethyl formamide (DMF) and olive oil.
In yet another embodiment of present invention, the salts are selected from salts of basic or acidic groups present in compounds of the invention and basic salts are selected from the group consisting of aluminum, calcium, lithium, magnesium, potassium or sodium and acidic salts are selected from the group consisting of hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate or p-toluenesulfonate.
In yet another embodiment, present invention provides a process for the preparation of Azirine containing compound of formula I comprising the steps of:
In yet another embodiment of present invention, the amine base is selected from the group consisting of pyridine, triethylamine, pyrrolidine or imidazole.
In yet another embodiment of present invention, the organic solvent is selected from the group consisting of toluene, tetrahydrofuran, dioxin, dichloromethane, chloroform, hexane, pentane, heptane or acetonitrile and alcoholic solvent is selected from the group consisting of methanol, ethanol, propanol, butanol or isopropanol.
In yet another embodiment, present invention provides a method of treating angiogenesis comprises administering the compound of formula I or the pharmaceutical composition comprising the compound of formula I, to a subject in need thereof.
In yet another embodiment of present invention, said compounds are useful as anti-angiogenesis.
In yet another embodiment of present invention, said compounds are useful as anti-angiogenesis for the treatment of eye disorders, macular degeneration and reduce intraocular pressure.
In yet another embodiment, present invention provides use of compound of formula I for the treatment of abnormal angiogenesis.
Olive Oil used in the present invention was purchased from Sigma Aldrich Ltd (St Louis, USA).
Present invention provides an azirine containing compound of formula I, which have been synthesized by diverse functional modifications with azirine as the basic core. Accordingly, the present invention affords a new class of azirine containing compounds of formula I,
wherein
R1 is selected from alkyl (C1-C12); alkenyl (C1-C12), hydroxy, nitro, halogen, amino, cyano, aryl, heteroaryl, cycloalkyl (C1-C7), cyclo ether (C1-C6), heteroalkyl, alkoxy, alkylamino, aryl amino, alkyl (C1-C12) ester, (hetero)aryl ester, alkyl phosphonate, (hetero)aryl phosphonate, tetrazole;
R2 and R3 are either same or different and selected from the group consisting of H, COOR4, cyano, tetrazole, phosphonate, alkoxybenzyl, hydroxy, nitro, halogen, amino, alkyl (C1-C12), aryl, heteroaryl, cycloalkyl (C1-C7), heteroalkyl, alkoxy, alkylamino, aryl amino, (hetero)aryl ester, alkyl phosphonate, (hetero)aryl phosphonate or tetrazole;
wherein each of these groups [R1, R2 and R3] may further substituted with one or more substituents selected from the group consisting of hydrogen, hydroxy, halogen, cyano, alkyl amino, aryl amino, alkoxy, amino, nitro, aldehyde, carboxylic acid, ester or phosphoester, (hetero)aryl;
or 5-7 membered hetrocyclic ring wherein heteroatom is O, N or S.
R4 is selected from the group consisting of alkyl (C1-C12), allyl, Methoxyethyl; benzyl; R5 R6 and R7 are either same or different and selected from the group consisting of H, OMe; H, CH3, t-butyl; Halogen (Cl, Br, F, I), OCF3, CN, OBu, vinyl; dimethylamino or
R5 and R6 may be joined to form a 5 to 6 membered hetrocyclic ring.
The compounds of the present invention are with asymmetric centers; thus, they are mixture of enantiomers and mixture of diastereomers in some cases. The present invention includes the individual enantiomers and diastereomeric forms of the compound formula I besides the mixtures thereof.
The present invention also provides a process for the preparation of azirine containing compound of formula I.
A large number of various azirine containing compounds possessing diversely substituted architecture were found to exhibit several biological properties. These functionalities are prominent structural motifs of new medicines from different pharmacological groups. The development of new structural scaffolds of azirine containing architecture is very important for the drug discovery process. In this connection a large number of azirine containing compounds were developed as depicted in the formula I. The processes for the synthesis of these azirine containing compounds involve operationally simple and highly efficient improved synthetic protocol giving rise to the desired products in high yields.
A process for the preparation of azirine containing compound of formula I wherein the said process comprising the steps of:
Optionally, the process steps (b) and (c) can also be performed in one-pot operation using p-toluenesulfonyl chloride and an amine base in organic solvent at temperature in the range of −5 to 35° C. for period in the range of 6-12 h to obtain azirine containing compound of formula I.
The amine base used is selected from the group consisting of pyridine, triethylamine, pyrrolidine or imidazole.
The alcoholic solvents used is selected from the group consisting of methanol, ethanol, propanol, butanol or isopropanol either alone or combination thereof.
The organic solvent used is selected from the group consisting of toluene, tetrahydrofuran, dioxin, dichloromethane, chloroform, hexane, pentane, heptane or acetonitrile either alone or combination thereof.
All the products are purified by column chromatography.
Azirine containing compounds are efficient structural motifs capable of showing diverse biological activities. The azirine containing compounds prepared are tested for anti-angiogenesis activity and in this study, compound 1 possessed an effective and potent anti-angiogenic activity on primary tubulogenesis screening (75 nM) and 3D angiogenesis screening (250 nm) activity. This compound showed its anti-angiogenic activity in different models such as matrigel implantation assay, CAM and ear angiogenesis assay. The compound 1 inhibits the tumor growth in the in vivo xenograft mouse model both in the intramuscular and intra peritoneal injection 25 mg/kg compound. Further, the compound 1 partially inhibits the expression of VEGFR2 and SRC kinase expression. The compound 1 also inhibited angiogenesis through its binding of the ligand endothelin. In total, the compound 1 is a potent inhibitor angiogenesis with a new target.
Following examples are given by way of illustration and therefore should not be construed to limit the scope of the invention.
To a solution of compound II (1 g, 4.00 mmol) in methanol (7.9 mL) was added hydroxylamine hydrochloride (0.568 g, 12.00 mmol) at 0° C. Then, pyridine (0.3 mL, 4.40 mmol) was added and stirred until completion of the reaction. Upon completion, the crude mixture was quenched with cold water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo and the crude material III was used in the next step.
To a solution of E/Z-isomers of ketoxime III (1.2 g, 4.528 mmol) in dichloromethane (9 mL) was added p-toluenesulfonic anhydride (1.77 g, 5.434 mmol) at 0° C. Then, pyridine (0.4 mL, 4.981 mmol) was added and stirred the reaction mixture at room temperature. Upon completion, the reaction mixture was quenched with cold water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo and purified by using silica gel column chromatography (10% EtOAc/hexane) to yield IV (0.85 g, 47%) (Rf=0.6, 20% EtOAc/hexane). Conversion of the hydroxyl group to a leaving group is also achieved from other reagents such as acetic anhydride, acyl chloride, 2,4,6-triisopropylbenzenesulfonyl chloride, 2,4,6-triisopropylbenzenesulfonyl anhydride, and tosyl chloride.
To a solution of E/Z isomers of compound IV (0.85 g, 2.028 mmol) in dichloromethane (4 mL) was added triethylamine (1.12 mL, 8.145 mmol) and stirred at 0° C. Upon completion, the reaction mixture was quenched with cold water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo and purified by using silica gel column chromatography (7% EtOAc/hexane) to give I (0.24 g, 48%) Rf=0.5, 20% EtOAc/hexane).
General Procedure for the Synthesis of Compound of Formula I from Compound of Formula III
To a solution of E/Z-isomers of ketoxime III (1.2 g, 4.528 mmol) in dichloromethane (9 mL) was added p-toluenesulfonyl chloride (1 g, 5.433 mmol) at 0° C., followed by triethylamine (2.5 mL, 18.113 mmol) at 0° C. Then, the reaction mixture was stirred, and upon completion, the crude reaction mixture was quenched with cold water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo and purified by using silica gel column chromatography (7% EtOAc/hexane) to give the compound I (0.642 g, 58%) Rf=0.5, 20% EtOAc/hexane).
Ethyl 3-(4-methoxyphenethyl)-2H-azirine-2-carboxylate (1) Compound 1 was synthesized from the corresponding keto-compound following the general procedure. Yield: 48% as pale yellow color oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.15 (d, J=8.6 Hz, 2H), 6.85 (d, J=8.6 Hz, 2H), 4.24-4.08 (m, 2H), 3.79 (s, 3H), 3.16-3.07 (m, 2H), 3.07-2.96 (m, 2H), 2.42 (s, 1H), 1.26 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 171.9, 161.7, 158.3, 131.2, 129.1, 114.0, 61.0, 55.1, 29.4, 28.9, 28.6, 14.1; IR (Neat): νmax 2982, 2936, 2836, 1790, 1721, 1611, 1512, 1464, 1444, 1369, 1333, 1245, 1176, 1107, 1030, 972, 822, 789, 701 cm−1; Mass (m/z): 248.15 (M+H)+ (calculated for C14H17NO3: M 247.1208); HPLC: 99% (Purity).
Ethyl 3-(pent-4-en-1-yl)-2H-azirine-2-carboxylate (2) Yield: 32% Rf=0.5 (10% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 5.8 (m, 1H), 5.1-5.0 (m, 2H), 4.3-4.12 (m, 2H), 2.85 (m, 2H), 2.42 (s, 1H), 2.25 (m, 2H), 1.9-1.8 (m, 2H), 1.24 (t, J=7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 172.1, 161.9, 136.8, 116.1, 61.0, 32.7, 28.7, 25.9, 23.3, 14.2; IR (Neat): νmax 3447, 3078, 2923, 2853, 1782, 1728, 1370, 1334, 1285, 1193, 1035, 994, 916, 772 cm−1; Mass (m/z): 182.20 (M+H)+ (calculated for C10H15NO2: M 181.1102).
Yield: 28% Rf=0.5 (10% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 4.25-4.05 (m, 2H), 2.8 (m, 2H), 2.42 (s, 1H), 1.8-1.6 (m, 2H), 1.5-1.0 (m, 21H), 0.9 (t, J=7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 172.1, 162.0, 61.0, 31.8, 29.5, 29.5, 29.3, 29.2, 29.0, 28.7, 26.7, 24.2, 22.6, 14.1; IR (Neat): νmax 3446, 2925, 2854, 1791, 1731, 1463, 1333, 1283, 1188, 1036, 974, 770 cm−1; Mass (m/z): 282.25 (M+H)+ (calculated for C17H31NO2: M 281.2354).
Yield: 31% Rf=0.5 (10% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 5.15 (m, 1H), 4.3-4.1 (m, 2H), 2.8 (m, 2H), 2.45 (m, 2H), 2.42 (s, 1H), 1.7 (s, 3H), 1.6 (s, 3H), 1.25 (t, J=7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 172.1, 161.9, 134.2, 121.3, 61.0, 28.8, 27.0, 25.6, 22.9, 17.7, 14.2; IR (Neat): νmax 3447, 2925, 2859, 1727, 1635, 1455, 1374, 1333, 1263, 1193, 1036, 759, 573 cm−1; Mass (m/z): 196.15 (M+H)+ (calculated for C11H17NO2: M 195.1259).
Ethyl (E)-3-(4, 8-dimethylnona-3, 7-dien-1-yl)-2H-azirine-2-carboxylate (5) Yield: 30% Rf=0.5 (10% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 5.15 (m, 1H), 5.05 (m, 1H), 4.25-4.20 (m, 2H), 2.8 (m, 2H), 2.45 (m, 2H), 2.42 (s, 1H), 2.1-2.0 (m, 2H), 2.0-1.9 (m, 2H), 1.69 (s, 3H), 1.62 (s, 3H), 1.6 (s, 3H), 1.26 (t, J=7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 171.8, 161.6, 137.4, 131.1, 123.7, 121.0, 60.7, 39.3, 28.6, 26.7, 26.2, 25.4, 22.6, 17.4, 15.8, 13.9; IR (Neat): νmax 3444, 2975, 2925, 1791, 1728, 1662, 1372, 1333, 1285, 1192, 1100, 1036, 973, 768 cm−1; Mass (m/z): 264.25 (M+H)+ (calculated for C16H25NO2: M 263.1885).
Yield: 49% as colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.31 (t, J=7.9 Hz, 2H), 7.25-7.19 (m, 3H), 4.22-4.09 (m, 2H), 3.19-3.11 (m, 2H), 3.11-3.03 (m, 2H), 2.42 (s, 1H), 1.25 (t, J=7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ (ppm) 171.9, 161.7, 139.1, 128.6, 128.1, 126.6, 61.0, 30.2, 29.0, 28.4, 14.1; IR (Neat): νmax 3441, 3028, 2982, 2931, 1790, 1726, 1334, 1266, 1196, 1034, 971, 747, 700 cm−1; Mass (m/z): 218.00 (M+H)+ (calculated for C13H15NO2: M 217.1102); HPLC: 99% (Purity).
Yield: 49% as a colorless oil Rf=0.4 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.12 (s, 4H), 4.23-4.09 (m, 2H), 3.16-3.08 (m, 2H), 3.08-3.01 (m, 2H), 2.42 (s, 1H), 2.32 (s, 3H), 1.25 (t, J=7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 171.9, 161.8, 136.2, 136.1, 129.3, 128.0, 61.0, 29.8, 29.0, 28.5, 20.9, 14.1; IR (Neat): νmax 3447, 2923, 2854, 1791, 1726, 1370, 1333, 1266, 1193, 1035, 808, 761, 668 cm−1; Mass (m/z): 232.00 (M+H)+ (calculated for C14H17NO2: M 231.1259); HPLC: 97% (Purity).
Yield: 45% as colorless oil Rf=0.4 (10% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.29 (d, J=8.4 Hz, 2H), 7.18 (d, J=8.4 Hz, 2H), 4.25-4.08 (m, 2H), 3.20-3.01 (m, 4H), 2.43 (s, 1H), 1.26 (t, J=7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 171.8, 161.6, 137.6, 132.4, 129.6, 128.7, 61.1, 29.6, 29.0, 28.2, 14.1; IR (Neat): νmax 3440, 2983, 2933, 1790, 1726, 1492, 1370, 1334, 1267, 1197, 1093, 1035, 813, 669 cm−1; Mass (m/z): 252.00 (M+H)+ (calculated for C13H14ClNO2: M 251.0713); HPLC: 96% Purity).
Yield: 35% as colorless oil Rf=0.6 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.3 (d, J=8.3 Hz, 2H), 7.1 (d, J=8.3 Hz, 2H), 4.2-4.0 (m, 2H), 3.18-3.08 (m, 2H), 3.08-2.97 (m, 2H), 2.42 (s, 1H), 1.30 (s, 9H), 1.23 (t, J=7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 171.6, 161.5, 149.1, 135.9, 127.6, 125.2, 60.6, 34.0, 31.0, 29.4, 28.7, 28.1, 13.9; IR (Neat): νmax 3441, 2961, 2869, 1791, 1727, 1367, 1334, 1267, 1193, 1110, 1036, 821, 761, 666 cm−1; Mass (m/z): 274.00 (M+H)+ (calculated for C17H23NO2: M 273.1728); HPLC: 95% (Purity).
Yield: 48% as colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (300 MHz, CDCl3): δ (ppm) 7.21 (td, J=5.2, 1.8, Hz, 2H), 7.0 (t, J=8.6 Hz, 2H), 4.2-4.0 (m, 2H), 3.19-3.08 (m, 4H), 2.43 (s, 1H), 1.26 (t, J=6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 171.9, 161.6, 129.7, 129.6, 115.5, 115.3, 61.1, 29.5, 29.0, 28.5, 14.1; IR (Neat): νmax 3440, 2984, 2932, 1790, 1726, 1511, 1370, 1334, 1222, 1196, 1035, 829, 786, 705 cm−1; Mass (m/z): 236.00 (M+H)+ (calculated for C13H14FNO2: M 235.1008); HPLC: 91% (Purity).
Yield: 35% as colorless oil Rf=0.4 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.38-7.18 (m, 5H), 6.48 (d, J=15.8 Hz, 1H), 6.23 (m, 1H), 4.21-4.07 (m, 2H), 3.0 (t, J=7.3 Hz, 2H), 2.68 (qd, 14.7, 7.3 Hz, 2H), 2.46 (s, 1H), 1.23 (t, J=7.09 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 171.9, 161.7, 136.8, 131.9, 128.4, 127.3, 126.8, 126.0, 61.0, 28.8, 27.6, 26.6, 14.1; IR (Neat): νmax 3441, 3026, 2982, 2929, 1791, 1726, 1369, 1333, 1266, 1196, 1033, 966, 771, 745, 694 cm−1; Mass (m/z): 266.00 (M+Na)+244.00 (M+H)+(calculated for C15H17NO2: M 243.1259); HPLC: 90% (Purity).
Yield: 40% as a colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm), 6.45 (s, 2H), 4.23-4.07 (m, 2H), 3.84 (s, 6H), 3.81 (s, 3H), 3.12 (t, J=7.2 Hz, 2H), 3.02 (t, J=7.2 Hz, 2H), 2.4 (s, 1H), 1.24 (t, J=7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 171.9, 161.7, 153.2, 136.6, 134.9, 105.1, 61.1, 60.7, 56.0, 30.6, 29.0, 28.6, 14.1; IR (Neat): νmax 3444, 2937, 2837, 1724, 1590, 1507, 1459, 1240, 1193, 1125, 1009, 824, 774 cm−1; Mass (m/z): 308.15 (M+H)+ (calculated for C16H21NO5: M 307.1419); HPLC: 88% (Purity).
Yield: 30% as a colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.53 (d, J=8.8 Hz, 2H), 7.29 (d, J=15.7 Hz, 1H), 7.0 (d, J=15.7 Hz, 1H), 6.9 (d, J=8.8 Hz, 2H), 4.28-4.16 (m, 2H), 3.86 (s, 3H), 2.67 (s, 1H), 1.28 (t, J=7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 171.9, 162.0, 156.7, 149.0, 130.2, 126.7, 114.5, 106.7, 61.1, 55.4, 28.7, 14.2; IR (Neat): νmax 2979, 2842, 1757, 1724, 1601, 1511, 1257, 1176, 1033, 971, 822, 770, 565 cm−1; Mass (m/z): 268.10 (M+Na)+ (calculated for C14H15NO3: M 245.1051); HPLC: 96% (Purity).
Yield: 48% as a colorless oil Rf=0.6 (20% EtOAc/hexane); 1H NMR (500 MHz, CDCl3): δ (ppm) 7.15 (d, J=8.6 Hz, 2H), 6.85 (d, J=8.6 Hz, 2H), 3.79 (s, 3H), 3.70 (s, 3H), 3.14-3.07 (m, 2H), 3.06-2.98 (m, 2H), 2.43 (s, 1H); 13C NMR (100 MHz, CDCl3): δ (ppm) 172.3, 161.7, 158.2, 131.1, 129.1, 113.9, 55.1, 52.0, 29.3, 28.7, 28.6; IR (Neat): νmax 3446, 3001, 2952, 2839, 1790, 1729, 1611, 1513, 1439, 1341, 1248, 1203, 1182, 1032, 823, 770, 709 cm−1; Mass (m/z): 256.10 (M+Na)+ (calculated for C13H15NO3: M 233.1051); HPLC: 90% (Purity).
Yield: 35% as a colorless oil; Rf=0.6 (20% EtOAc/hexane); 1H NMR (500 MHz, CDCl3): δ (ppm) 7.47-7.37 (m, 2H), 7.11 (d, J=6.8 Hz, 2H), 4.21-4.07 (m, 2H), 3.17-3.08 (m, 2H), 3.08-2.99 (m, 2H), 2.43 (d, J=2.2 Hz, 1H), 1.23 (td, J=7.0, 2.2 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 171.8, 161.5, 138.1, 131.6, 129.9, 120.5, 61.1, 29.6, 29.0, 28.2, 14.1; IR (Neat): νmax 3445, 2930, 1792, 1724, 1629, 1486, 1265, 1195, 1034, 810, 766 cm−1; Mass (m/z): 318.00 (M+Na)+ (calculated for C13H14BrNO2: M 295.0207); HPLC: 98% (Purity).
Yield: 39% as a colorless oil; Rf=0.6 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.63 (d, J=8.3 Hz, 2H), 7.0 (d, J=8.3 Hz, 2H), 4.2-4.0 (m, 2H), 3.2-3.0 (m, 2H), 3.0-3.0 (m, 2H), 2.43 (s, 1H), 1.26 (t, J=7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 171.8, 161.6, 138.8, 137.7, 130.3, 91.9, 61.1, 29.8, 29.1, 28.1, 14.1; IR (Neat): νmax 3447, 2925, 2854, 1791, 1724, 1266, 1195, 1034, 1008, 807, 765, 669 cm−1; Mass (m/z): 366.00 (M+Na)+ (calculated for C13H14NO2: M 343.0069); HPLC: 97% (Purity).
Yield: 49% as a colorless oil Rf=0.6 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.15 (d, J=8.6 Hz, 2H), 6.84 (d, J=8.6 Hz, 2H), 3.78 (s, 3H), 3.13-3.04 (m, 2H), 3.04-2.96 (m, 2H), 2.43 (s, 1H), 1.44 (s, 9H); 13C NMR (100 MHz, CDCl3): δ (ppm) 171.1, 162.0, 158.2, 131.3, 129.1, 114.0, 81.3, 55.1, 29.8, 29.4, 28.7, 27.9; IR (Neat): νmax 3443, 2929, 1720, 1615, 1513, 1343, 1248, 1156, 1033, 974, 826, 763 cm−1; Mass (m/z): 298.00 (M+Na)+ (calculated for C16H21NO3: M 275.1521); HPLC: 95% (Purity).
Yield: 35% as a colorless oil Rf=0.6 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.37-7.26 (m, 5H), 7.01 (d, J=8.5 Hz, 2H), 6.78 (d, J=8.5 Hz, 2H), 5.12 (q, J=12.3 Hz, 2H), 3.76 (s, 3H), 3.52 (d, J=15.1 Hz, 1H), 2.99 (d, J=15.1 Hz, 1H), 2.19 (s, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 172.6, 162.8, 158.1, 135.6, 130.5, 129.1, 128.4, 128.1, 127.8, 113.7, 66.8, 55.1, 38.9, 35.8, 12.2; IR (Neat): νmax 3450, 2925, 2855, 1725, 1196, 1100, 1034, 765, 553 cm−1; Mass (m/z): 332.00 (M+Na)+, 310.00 (M+H)+ (calculated for C19H19NO3: M 309.364); HPLC: 93% (Purity).
Yield: 45% as a colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (500 MHz, CDCl3): δ (ppm) 7.15 (d, J=8.6 Hz, 2H), 6.85 (d, J=8.6 Hz, 2H), 4.12-4.0 (m, 2H), 3.79 (s, 3H), 3.4-3.06 (m, 2H), 3.06-2.96 (m, 2H), 2.43 (s, 1H), 1.64 (m, 2H), 0.93 (t, J=7.4 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 172.0, 161.7, 158.2, 131.2, 129.1, 114.0, 66.4, 55.1, 29.4, 28.9, 28.7, 21.8, 10.2; IR (Neat): νmax 3447, 2923, 1798, 1651, 1514, 1342, 1247, 1156, 1032, 973, 827, 763 cm−1; Mass (m/z): 294.25 (M-OMe)+, 262.20 (M+H)+ (calculated for C15H19NO3: M 261.1364); HPLC: 97% (Purity).
Yield: 39% as a colorless oil Rf=0.6 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.16 (d, J=8.6 Hz, 2H), 6.85 (d, J=8.6 Hz, 2H), 5.03 (m, 1H), 3.79 (s, 3H), 3.14-3.06 (m, 2H), 3.05-2.98 (m, 2H), 2.40 (s, 1H), 1.24 (d, J=6.2 Hz, 6H); 13C NMR (75 MHz, CDCl3): δ (ppm) 171.5, 161.8, 158.3, 131.3, 129.1, 114.0, 68.6, 55.1, 29.4, 29.2, 28.7, 21.7, 21.7; IR (Neat): νmax 2981, 2932, 1788, 1721, 1612, 1513, 1248, 1202, 1107, 1033, 981, 822, 768 cm−1; Mass (m/z): 262.20 (M+H)+ (calculated for C15H19NO3: M 261.1364); HPLC: 93% (Purity).
Yield: 45% as a colorless oil Rf=0.6 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.15 (d, J=8.6 Hz, 2H), 6.85 (d, J=8.6 Hz, 2H), 3.94-3.84 (m, 2H), 3.79 (s, 3H), 3.14-3.09 (m, 2H), 3.06-2.99 (m, 2H), 2.4 (s, 1H), 1.92 (m, 1H), 0.92 (d, J=6.7 Hz, 6H); 13C NMR (100 MHz, CDCl3): δ (ppm) 172.0, 161.7, 158.3, 131.2, 129.2, 114.0, 71.1, 55.1, 29.4, 28.9, 28.7, 27.6, 18.9; IR (Neat): νmax 3418, 2961, 1788, 1725, 1613, 1513, 1463, 1249, 1188, 1028, 821, 698 cm−1; Mass (m/z): 308.25 (M-OMe)+, 276.20 (M+H)+ (calculated for C16H21NO3: M 275.1521); HPLC: 97% (Purity).
Yield: 50% as a colorless oil; Rf=0.6 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.15 (d, J=8.6 Hz, 2H), 6.85 (d, J=8.6 Hz, 2H), 4.87 (m, 1H), 3.79 (s, 3H), 3.14-3.06 (m, 2H), 3.06-2.99 (m, 2H), 2.4 (s, 1H), 1.65-1.48 (m, 2H), 1.20 (dd, J=6.2, 0.9 Hz, 3H), 0.89 (t, J=7.4 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 171.6, 161.8, 158.3, 131.3, 129.1, 114.0, 73.1, 55.1, 29.4, 29.1, 28.7, 28.6, 19.3, 9.5; IR (Neat): νmax 2928, 1788, 1721, 1614, 1513, 1249, 1199, 1116, 1030, 824, 764 cm−1; Mass (m/z): 276.20 (M+H)+ (calculated for C16H21NO3: M 275.1521); HPLC: 97% (Purity).
Yield: 33% as a colorless oil Rf=0.6 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.15 (d, J=8.5 Hz, 2H), 6.84 (d, J=8.5 Hz, 2H), 4.12-4.0 (m, 2H), 3.78 (s, 3H), 3.15-3.06 (m, 2H), 3.06-2.96 (m, 2H), 2.43 (s, 1H), 1.71-1.56 (m, 2H), 1.39-1.24 (m, 4H), 0.93 (t, J=6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 172.0, 161.7, 158.3, 131.2, 129.1, 114.0, 65.2, 55.1, 29.4, 28.9, 28.7, 28.2, 27.9, 22.2, 13.8; IR (Neat): νmax 2956, 2864, 1790, 1725, 1612, 1512, 1461, 1336, 1248, 1187, 1035, 822, 549 cm−1; Mass (m/z): 322.25 (M-OMe)+, 290.30 (M+H)+ (calculated for C17H23NO3: M 289.1677); HPLC: 95% (Purity).
Isopentyl 3-(4-methoxyphenethyl)-2H-azirine-2-carboxylate (24): Yield: 45% as colorless oil Rf=0.6 (20% EtOAc/hexane); 1H NMR (500 MHz, CDCl3): δ (ppm) 7.15 (d, J=8.6 Hz, 2H), 6.84 (d, J=8.6 Hz, 2H), 4.19-4.06 (m, 2H), 3.78 (s, 3H), 3.14-3.06 (m, 2H), 3.06-2.98 (m, 2H), 2.4 (s, 1H), 1.71-1.60 (m, 1H), 1.51 (q, J=6.8 Hz, 2H), 0.91 (d, J=6.6 Hz, 6H); 13C NMR (100 MHz, CDCl3): δ (ppm) 172.0, 161.7, 158.3, 131.2, 129.2, 114.0, 63.8, 55.1, 37.2, 29.4, 29.0, 28.7, 24.9, 22.4, 22.3; IR (Neat): νmax 3782, 3446, 2957, 2926, 1791, 1726, 1613, 1512, 1248, 1188, 1035, 823, 770, 671, 535 cm−1; Mass (m/z): 290.20 (M+H)+(calculated for C17H23NO3: M 289.1677); HPLC: 96% (Purity).
2-Methoxyethyl 3-(4-methoxyphenethyl)-2H-azirine-2-carboxylate (25): Yield: 31% as a colorless oil Rf=0.5 (30% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.14 (d, J=8.6 Hz, 2H), 6.84 (d, J=8.6 Hz, 2H), 4.32-4.19 (m, 2H), 3.78 (s, 3H), 3.63-3.54 (m, 2H), 3.38 (m, 3H), 3.14-3.06 (m, 2H), 3.05-2.95 (m, 2H), 2.4 (s, 1H); 13C NMR (100 MHz, CDCl3): δ (ppm) 171.9, 161.5, 158.2, 131.2, 129.1, 113.9, 70.1, 64.0, 58.8, 55.1, 29.3, 28.7, 28.6; IR (Neat): νmax 2930, 1791, 1725, 1610, 1511, 1246, 1184, 1128, 1034, 824, 533 cm−1; Mass (m/z): 278.20 (M+H)+ (calculated for C15H19NO4: M 277.1314); HPLC: 91% (Purity).
Allyl 3-(4-methoxyphenethyl)-2H-azirine-2-carboxylate (26): Yield: 21% as a colorless oil Rf=0.6 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.15 (d, J=8.5 Hz, 2H), 6.85 (d, J=8.5 Hz, 2H), 5.95-5.85 (m, 1H), 5.31 (dd, J=17.2, 1.3 Hz, 1H), 5.24 (dd, J=10.3, 0.9 Hz, 1H), 4.60 (m, 2H), 3.79 (s, 3H), 3.17-3.07 (m, 2H), 3.07-2.98 (m, 2H), 2.4 (s, 1H); 13C NMR (75 MHz, CDCl3): δ (ppm) 171.6, 161.6, 158.3, 131.7, 131.2, 129.2, 118.5, 114.0, 65.7, 55.2, 29.4, 28.9, 28.7; IR (Neat): νmax 2979, 2842, 1757, 1724, 1601, 1511, 1257, 1176, 1033, 972, 823, 771, 566 cm−1; Mass (m/z): 260.20 (M+H)+ (calculated for C15H17NO3: M 259.1208); HPLC: 97% (Purity).
Ethyl 3-(2-(naphthalen-2-yl) ethyl)-2H-azirine-2-carboxylate (27): Yield: 45% as a colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.84-7.73 (m, 3H), 7.67 (s, 1H), 7.49-7.41 (m, 2H), 7.35 (dd, J=8.3, 1.7 Hz, 1H), 4.18-4.04 (m, 2H), 3.29-3.16 (m, 4H), 2.4 (s, 1H), 1.20 (t, J=7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 171.8, 161.7, 136.6, 133.4, 132.2, 128.3, 127.5, 127.4, 126.6, 126.5, 126.1, 125.5, 61.0, 30.3, 29.0, 28.2, 14.0; IR (Neat): νmax 3019, 2981, 1721, 1369, 1332, 1266, 1191, 1033, 971, 855, 816, 746, 667 cm−1; Mass (m/z): 268.00 (M+H)+ (calculated for C17H17NO2: M 267.1259); HPLC: 95% (Purity).
Ethyl 3-(4-(trifluoromethoxy) phenethyl)-2H-azirine-2-carboxylate (28): Yield: 21% as a colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.28 (d, J=8.6 Hz, 2H), 7.17 (d, J=8.6 Hz, 2H), 4.22-4.09 (m, 2H), 3.19-3.06 (m, 4H), 2.4 (s, 1H), 1.25 (t, J=7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 171.8, 161.6, 147.9, 137.9, 129.6, 121.2, 61.1, 29.6, 29.1, 28.3, 14.1; IR (Neat): νmax 2925, 2853, 1725, 1509, 1254, 1192, 1154, 1020, 973, 920, 846, 809, 788, 672 cm−1; Mass (m/z): 302.00 (M+H)+(calculated for C14H14F3NO3: M 301.0925); HPLC: 97% (Purity).
Octyl 3-(4-methoxyphenethyl)-2H-azirine-2-carboxylate (29): Yield: 50% as a colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.15 (d, J=8.6 Hz, 2H), 6.84 (d, J=8.6 Hz, 2H), 4.15-4.0 (m, 2H), 3.78 (s, 3H), 3.13-3.06 (m, 2H), 3.05-2.97 (m, 2H), 2.42 (s, 1H), 1.69-1.52 (m, 2H), 1.38-1.19 (m, 10H), 0.88 (t, J=7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 172.0, 161.7, 158.3, 131.2, 129.2, 114.0, 65.2, 55.1, 31.7, 29.4, 29.1, 29.0, 28.7, 28.5, 25.7, 22.5, 14.0; IR (Neat): νmax 2926, 2855, 1790, 1724, 1612, 1512, 1464, 1246, 1177, 1107, 1034, 976, 810, 723 cm−1; Mass (m/z): 332.00 (M+H)+(calculated for C20H29NO3: M 331.2147); HPLC: 98% (Purity).
Ethyl 3-(2-(tetrahydrofuran-2-yl) ethyl)-2H-azirine-2-carboxylate (30): Yield: 21% as a colorless oil Rf=0.4 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 4.24-4.09 (m, 2H), 3.99-3.79 (m, 2H), 3.73 (m, 1H), 3.02-2.85 (m, 2H), 2.45 (s, 1H), 2.09-1.82 (m, 4H), 1.57-1.46 (m, 2H), 1.26 (t, J=7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 172.1, 162.1, 67.7, 61.0, 31.0, 30.0, 29.0, 25.7, 23.8, 23.9, 14.2; IR (Neat): νmax 2925, 2855, 1791, 1725, 1445, 1369, 1332, 1263, 1186, 1068, 1032, 972, 790 cm−1; Mass (m/z): 212.00 (M+H)+ (calculated for C11H17NO3: M 211.1208); HPLC: 93% (Purity).
Ethyl 3-(4-cyanophenethyl)-2H-azirine-2-carboxylate (31): Yield: 39% as a colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (500 MHz, CDCl3): δ (ppm) 7.61 (d, J=8.4 Hz, 2H), 7.37 (d, J=8.4 Hz, 2H), 4.21-4.07 (m, 2H), 3.16 (m, 4H), 2.4 (s, 1H), 1.24 (t, J=7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 171.6, 161.4, 144.6, 132.4, 129.1, 118.5, 110.7, 61.1, 30.2, 29.2, 27.8, 14.1; IR (Neat): νmax 2982, 2934, 2227, 1789, 1721, 1193, 1033, 908, 823, 754, 729, 667 cm−1; Mass (m/z): 243.45 (M+H)+ (calculated for C14H14N2O2: M 242.1055); HPLC: 93% (Purity).
Ethyl 3-(4-butoxyphenethyl)-2H-azirine-2-carboxylate (32): Yield: 55% as a colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.13 (d, J=8.6 Hz, 2H), 6.83 (d, J=8.6 Hz, 2H), 4.22-4.09 (m, 2H), 3.93 (t, J=6.6 Hz, 2H), 3.17-3.06 (m, 2H), 3.06-2.97 (m, 2H), 2.42 (s, 1H), 1.80-1.70 (m, 2H), 1.54-1.42 (m, 2H), 1.25 (t, J=7.0 Hz, 3H), 0.97 (t, J=7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 171.9, 161.8, 157.9, 131.0, 129.1, 114.6, 67.6, 61.0, 31.2, 29.4, 28.9, 28.7, 19.2, 14.1, 13.8; IR (Neat): νmax 2958, 2933, 1726, 1612, 1511, 1244, 1193, 1034, 906, 752, 730, 668 cm−1; Mass (m/z): 290.25 (M+H)+ (calculated for C17H23NO3: M 289.1677); HPLC: 99% (Purity).
Ethyl 3-(2-(anthracen-9-yl)ethyl)-2H-azirine-2-carboxylate (33): Yield: 32% as a colorless oil Rf=0.4 (20% EtOAc/hexane); 1H NMR (500 MHz, CDCl3): δ (ppm) 8.4 (s, 1H), 8.23 (d, J=8.8 Hz, 2H), 8.04 (d, J=8.3 Hz, 2H), 7.56 (t, J=8.8 Hz, 2H), 7.49 (t, J=8.3 Hz, 2H), 4.29-4.15 (m, 2H), 4.1 (t, J=8.0 Hz, 2H), 3.35-3.23 (td, J=7.6, 3.0 Hz, 2H), 2.5 (s, 1H), 1.30 (t, J=7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 171.9, 161.9, 131.5, 130.9, 129.4, 129.4, 126.9, 126.2, 125.0, 123.4, 61.2, 29.5, 27.8, 22.4, 14.2; IR (Neat): νmax 2980, 1789, 1722, 1332, 1262, 1194, 1095, 1031, 884, 839, 787, 731, 665 cm−1; Mass (m/z): 318.20 (M+H)+ (calculated for C21H19NO2: M 317.1415); HPLC: 98% (Purity).
Ethyl 3-(3,4-dimethoxyphenethyl)-2H-azirine-2-carboxylate (34): Yield: 46% solid, M.P=55-57° C. Rf=0.5 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 6.82-6.73 (m, 3H), 4.21-4.08 (m, 2H), 3.86 (s, 3H), 3.84 (s, 3H), 3.14-3.07 (m, 2H), 3.06-2.98 (m, 2H), 2.43 (s, 1H), 1.24 (t, J=7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 171.8, 161.6, 148.8, 147.6, 131.7, 120.0, 111.5, 111.2, 61.0, 55.7, 55.7, 29.8, 28.9, 28.6, 14.0; IR (Neat): νmax 2936, 2835, 1721, 1514, 1418, 1259, 1236, 1188, 1025, 850, 806, 763 cm−1; Mass (m/z): 278.20 (M+H)+ (calculated for C15H19NO4: M 277.1314); HPLC: 96% (Purity).
Diethyl 3-(4-methoxyphenethyl)-2H-azirine-2,2-dicarboxylate (35): Yield: 11% as a colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.17 (d, J=8.6 Hz, 2H), 6.84 (d, J=8.6 Hz, 2H), 4.27 (q, J=7.0 Hz, 2H), 3.7 (s, 3H), 3.6 (q, J=7.0 Hz, 2H), 3.05-2.98 (m, 2H), 2.96-2.87 (m, 2H), 1.33 (t, J=7.0 Hz, 3H), 1.24 (t, J=7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 170.0, 164.6, 162.7, 157.8, 133.6, 129.2, 113.8, 86.7, 59.9, 55.2, 44.8, 33.1, 29.6, 14.4, 13.4; IR (Neat): νmax 2975, 2932, 1696, 1582, 1511, 1443, 1363, 1301, 1244, 1190, 1165, 1081, 1035, 822, 785; Mass (m/z): 274.00 (M-OEt)+ (calculated for C17H21NO5: M 319.1419); HPLC: 90% (Purity).
0.5 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.36 (d, J=8.1 Hz, 2H), 7.20 (d, J=8.1 Hz, 2H), 6.69 (m, 1H), 5.72 (dd, J=17.6, 0.8 Hz, 1H), 5.23 (dd, J=10.8, 0.8 Hz, 1H), 4.22-4.09 (m, 2H), 3.17-3.11 (m, 2H), 3.11-3.03 (m, 2H), 2.43 (s, 1H), 1.25 (t, J=7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 171.9, 161.7, 138.8, 136.3, 136.1, 128.4, 126.4, 113.6, 61.1, 30.0, 29.0, 28.3, 14.1; IR (Neat): νmax 2980, 2925, 2853, 1722, 1512, 1369, 1333, 1264, 1189, 1032, 990, 908, 839, 828, 708 cm−1; Mass (m/z): 244.00 (M+H)+ (calculated for C15H17NO2: M 243.1259); HPLC: 90% (Purity).
Ethyl 3-(3-(2, 3-dihydrobenzofuran-5-yl)propyl)-2H-azirine-2-carboxylate (37): Yield: 24% as a colorless oil Rf=0.4 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.01 (s, 1H), 6.90 (d, J=8.0 Hz, 1H), 6.70 (d, J=8.0 Hz, 1H), 4.54 (t, J=8.6 Hz, 2H), 4.24-4.10 (m, 2H), 3.17 (t, J=8.6 Hz, 2H), 2.80 (t, J=7.2 Hz, 2H), 2.68 (t, J=7.3 Hz, 2H), 2.41 (s, 1H), 2.09-1.98 (m, 2H), 1.26 (t, J=7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 172.0, 161.9, 158.5, 132.4, 127.8, 127.1, 124.9, 109.0, 71.1, 61.0, 34.2, 29.7, 28.6, 26.2, 25.8, 14.1; IR (Neat): νmax 2935, 1791, 1723, 1615, 1491, 1369, 1191, 1102, 982, 944, 818, 750 cm−1; Mass (m/z): 274.20 (M+H)+ (calculated for C16H19NO3: M 273.1364); HPLC: 96% (Purity).
tert-Butyl 3-(2-(2-(ethoxycarbonyl)-2H-azirin-3-yl)ethyl)-1H-indole-1-carboxylate (38): Yield: 38% as colorless oil Rf=0.4 (30% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 8.3 (s, 1H), 7.52 (d, J=7.8 Hz, 1H), 7.45 (s, 1H), 7.37-7.31 (td, J=7.2, 1.2 Hz, 1H), 7.28-7.23 (dt, J=7.4, 0.8 Hz, 1H), 4.23-4.09 (m, 2H), 3.27-3.21 (m, 2H), 3.21-3.15 (m, 2H), 2.48 (s, 1H), 1.67 (s, 6H), 1.56 (s, 3H), 1.25 (t, J=7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3): δ (ppm) 171.9, 161.9, 125.0, 124.6, 124.1, 122.9, 122.5, 118.5, 118.2, 115.4, 83.7, 81.6, 61.1, 29.1, 28.1, 26.7, 19.9, 14.1; IR (Neat); νmax 2980, 2932, 1724, 1608, 1453, 1370, 1308, 1192, 1084, 1034, 856, 747, 667 cm−1; Mass (m/z): 257.15 (M-Boc)+ (calculated for C20H24N2O4: M 356.1736); HPLC: 94% (Purity).
Ethyl 3-(2-(2, 3-dihydrobenzofuran-5-yl)ethyl)-2H-azirine-2-carboxylate (39): Yield: 48% as colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (500 MHz, CDCl3): δ (ppm) 7.06 (s, 1H), 6.95 (d, J=8.0 Hz, 1H), 6.71 (d, J=8.0 Hz, 1H), 4.55 (t, J=8.6 Hz, 2H), 4.24-4.10 (m, 2H), 3.18 (t, J=8.6 Hz, 2H), 3.13-3.05 (m, 2H), 3.04-2.95 (m, 2H), 2.42 (s, 1H), 1.26 (t, J=7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ (ppm) 171.9, 161.8, 158.8, 131.2, 127.7, 127.4, 124.8, 109.2, 71.1, 61.0, 29.7, 29.6, 29.0, 14.1; IR (Neat): νmax 2980, 2930, 1790, 1721, 1492, 1187, 1102, 1033, 981, 942, 814, 789, 773, 711 cm−1; Mass (m/z): 260.15 (M+H)+ (calculated for C15H17NO3: M 259.1208); HPLC: 98% (Purity).
Ethyl 3-(3-(benzofuran-5-yl)propyl)-2H-azirine-2-carboxylate (40): Yield: 49% as colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.61 (d, J=2.0 Hz, 1H), 7.43 (d, J=8.4 Hz, 1H), 7.40 (s, 1H), 7.11 (dd, J=8.4, 1.3 Hz, 1H), 6.72 (d, J=2.0 Hz, 1H), 4.26-4.11 (m, 2H), 2.92-2.76 (m, 4H), 2.43 (s, 1H), 2.20-2.04 (m, 2H), 1.27 (t, J=7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 172.1, 161.9, 153.7, 145.2, 134.9, 127.6, 124.8, 120.6, 111.2, 106.3, 61.1, 34.6, 28.7, 26.3, 25.9, 14.2; IR (Neat): νmax 2981, 2934, 2865, 1791, 1721, 1537, 1261, 1189, 1028, 973, 883, 814, 770, 736, 640 cm−1; Mass (m/z): 271.25 (M+H)+ (calculated for C16H17NO3: M 271.1208); HPLC: 100% (Purity).
Ethyl 3-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-2H-azirine-2-carboxylate (41): Yield: 29% as a colorless oil Rf=0.4 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 6.75 (d, J=7.8 Hz, 1H), 6.72 (d, J=1.5 Hz, 1H), 6.68 (dd, J=7.8, 1.7 Hz, 1H), 5.94 (s, 2H), 4.24-4.10 (m, 2H), 3.13-3.05 (m, 2H), 3.04-2.94 (m, 2H), 2.43 (s, 1H), 1.27 (t, J=7.2 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ (ppm) 171.9, 161.7, 147.7, 146.2, 132.9, 121.1, 108.6, 108.3, 100.9, 61.1, 30.0, 29.0, 28.7, 14.1; IR (Neat): νmax 2982, 2904, 1790, 1723, 1608, 1503, 1245, 1191, 1037, 975, 931, 858, 810, 772 cm−1; Mass (m/z): 262.15 (M+H)+ (calculated for C14H15NO4: M 261.1001); HPLC: 99% (Purity).
Ethyl 3-(4-(2, 3-dihydrobenzofuran-5-yl)butyl)-2H-azirine-2-carboxylate (42): Yield: 39% as a colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 6.99 (s, 1H), 6.88 (d, J=8.0 Hz, 1H), 6.68 (d, J=8.0 Hz, 1H), 4.54 (t, J=8.5 Hz, 2H), 4.24-4.10 (m, 2H), 3.16 (t, J=8.5 Hz, 2H), 2.82 (t, J=6.7 Hz, 2H), 2.58 (t, J=6.7 Hz, 2H), 2.41 (s, 1H), 1.85-1.65 (m, 4H), 1.25 (t, J=7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 172.0, 161.8, 158.2, 133.5, 127.6, 126.9, 124.7, 108.8, 71.1, 61.0, 34.6, 31.1, 29.7, 28.7, 26.5, 23.6, 14.1; IR (Neat): νmax 2978, 2934, 2859, 1791, 1723, 1491, 1188, 1031, 982, 944, 813, 714 cm−1; Mass (m/z): 310.00 (M+Na)+ (calculated for C17H21NO3: M 287.1521); HPLC: 97% (Purity).
Ethyl 3-(3-(benzo[d][1,3]dioxol-5-yl)propyl)-2H-azirine-2-carboxylate (60): Yield: 48% as colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 6.73 (d, J=7.9 Hz, 1H), 6.67 (d, J=1.5 Hz, 1H), 6.63 (dd, J=7.9, 1.5 Hz, 1H), 5.93 (s, 2H), 4.25-4.07 (m, 2H), 2.81 (t, J=7.2 Hz, 2H) 2.69 (t, J=7.4 Hz, 2H), 2.43 (s, 1H), 2.10-1.93 (m, 2H), 1.27 (t, J=7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 172.0, 161.8, 147.6, 145.9, 134.2, 121.2, 108.8, 108.2, 100.8, 61.0, 34.4, 28.7, 26.0, 25.8, 14.1; IR (Neat): νmax 2981, 2926, 2872, 1791, 1722, 1503, 1488, 1369, 1333, 1244, 1188, 1035, 973, 927, 861, 809, 633 cm−1; Mass (m/z): 298.00 (M+Na)+, 276.00 (M+H)+ (calculated for C15H17NO4: M 275.1157); HPLC: 97% (Purity).
Ethyl 3-(3-(dibenzo[b,d]furan-2-yl)propyl)-2H-azirine-2-carboxylate (44): Yield: 49% as a colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.94 (d, J=8.3 Hz, 1H), 7.70 (d, J=1.4 Hz, 1H), 7.5 (d, J=8.3 Hz, 1H), 7.49 (d, J=8.3 Hz, 1H), 7.47-7.42 (td, J=7.3, 1.3 Hz, 1H), 7.37-7.30 (td, J=7.3, 0.8 Hz, 1H), 7.27 (dd, J=8.3, 1.7 Hz, 1H), 4.27-4.13 (m, 2H), 2.93 (t, J=7.4 Hz, 2H), 2.85 (t, J=7.2 Hz, 2H), 2.4 (s, 1H), 2.24-2.09 (m, 2H), 1.27 (t, J=7.09 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 172.0, 161.8, 156.3, 154.7, 134.9, 127.4, 127.0, 124.2, 123.9, 122.5, 120.4, 120.1, 111.5, 111.3, 61.0, 34.5, 28.6, 26.2, 25.8, 14.1; IR (Neat): νmax 2981, 2934, 2866, 1728, 1479, 1447, 1332, 1187, 1028, 800, 767, 748, 725, 624, 615 cm−1; Mass (m/z): 322.00 (M+H)+ (calculated for C20H19NO3: M 321.1364); HPLC: 99% (Purity).
Ethyl 3-(3-(chroman-6-yl)propyl)-2H-azirine-2-carboxylate (45): Yield: 49.5% as a colorless oil Rf=0.5 (20% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 6.88 (dd, J=8.1, 2.0 Hz, 1H), 6.84 (s, 1H), 6.71 (d, J=8.1 Hz, 1H), 4.24-4.11 (m, 4H), 2.81 (t, J=7.21 Hz, 2H), 2.75 (t, J=6.4 Hz, 2H), 2.65 (t, J=7.4 Hz, 2H), 2.41 (s, 1H), 2.09-1.95 (m, 4H), 1.27 (t, J=7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 172.1, 161.9, 153.2, 131.9, 129.6, 127.1, 122.0, 116.6, 66.3, 61.0, 33.9, 28.6, 26.0, 25.9, 24.8, 22.3, 14.1; IR (Neat): νmax 2930, 2857, 1724, 1499, 1369, 1333, 1266, 1188, 1126, 1061, 1033, 1007, 820 cm−1; Mass (m/z): 288.10 (M+H)+ (calculated for C17H21NO3: M 287.1521); HPLC: 98% (Purity).
Ethyl (E)-3-(4-(dimethylamino)styryl)-2H-azirine-2-carboxylate (46): Yield: 49% yellow color solid, M.P=119-121° C. Rf=0.5 (30% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.46 (d, J=8.8 Hz, 2H), 7.24 (d, J=15.5 Hz, 1H), 6.86 (d, J=15.5 Hz, 1H), 6.68 (d, J=8.8 Hz, 2H), 4.27-4.14 (m, 2H), 3.05 (s, 6H), 2.63 (s, 1H), 1.28 (t, J=7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ (ppm) 172.3, 156.4, 152.1, 149.9, 130.2, 121.7, 111.7, 103.2, 61.0, 40.0, 28.6, 14.2; IR (Neat): νmax 2981, 2905, 1751, 1725, 1590, 1524, 1367, 1330, 1170, 1037, 968, 919, 810, 728, 567 cm−1; Mass (m/z): 259.15 (M+H)+ (calculated for C15H18N2O2: M 258.1368); HPLC: 83% (Purity).
Ethyl 3-(4-(dimethylamino)phenethyl)-2H-azirine-2-carboxylate (47): Yield: 47% as a pale color oil Rf=0.5 (30% EtOAc/hexane); 1H NMR (400 MHz, CDCl3): δ (ppm) 7.10 (d, J=8.8 Hz, 2H), 6.70 (d, J=8.8 Hz, 2H), 4.24-4.10 (m, 2H), 3.15-3.06 (m, 2H), 3.03-2.95 (m, 2H), 2.92 (s, 6H), 2.42 (s, 1H), 1.26 (t, J=7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) 172.0, 161.8, 149.4, 128.8, 127.1, 112.8, 61.0, 40.6, 29.3, 28.9, 28.8, 14.1; IR (Neat): νmax max 2981, 2928, 2801, 1789, 1724, 1614, 1333, 1190, 1164, 1034, 946, 810 cm−1; Mass (m/z): 261.15 (M+H)+ (calculated for C15H20N2O2: M 260.1524); HPLC: 95% (Purity).
3-(4-methoxyphenethyl)-2H-azirine-2-carbonitrile (48): Compound 48 was achieved from the 5-(4-methoxyphenyl)-3-oxo-pentanenitrile following the general procedure described for the compound 1. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.1 (d, 2H), 6.8 (d, 2H), 3.7 (s, 3H), 3.2 (m, 2H), 3.1 (m, 2H), 2.1 (s, 0.9H), 1.9 (d, 0.4H).
Diethyl (3-(4-methoxyphenethyl)-2H-azirin-2-yl)phosphonate (49): The synthesis of compound 49 was achieved from the diethyl [4-(4-methoxyphenyl)-2-oxobutyl]phosphonate following the general procedure described for compound 1. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.1 (d, 2H), 6.8 (d, 2H), 4.1 (m, 4H), 3.7 (s, 3H), 3.1 (m, 2H), 3.0 (m, 2H), 1.7-1.6 (d, 1H), 1.3 (q, 6H); Mass (m/z): 312 (M+H)+ (calculated for: C15H22NO4P: M 311.31).
Ethyl 3-(4-((tetrahydrofuran-3-yl)oxy)phenethyl)-2H-azirine-2-carboxylate (50): The synthesis of compound 50 was achieved from p-ketoester following the general procedure described for the compound 1. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.1 (d, 2H), 6.8 (d, 2H), 4.8 (m, 1H), 4.1 (m, 2H), 3.9 (m, 3H), 3.8 (m, 1H), 3.1 (m, 4H), 2.4 (s, 1H), 2.1 (m, 2H), 1.25 (t, 3H); Mass (m/z): 304.2 (M+H)+ (calculated for C17H21NO4: M 303.3).
5-(3-(4-Methoxyphenethyl)-2H-azirin-2-yl)-1H-tetrazole (51): To a stirred mixture of 3-[2-(4-methoxyphenyl)ethyl]-2H-azirene-2-carbonitrile (600 mg, 1 equiv), ammonium chloride (1.1 g, 7 equiv) and sodium azide (390 mg, 2 equiv) was added IPA (2.5 mL) and water (2.5 mL) at room temperature and stirred for 10 min. The reaction mixture was evaporated under reduced pressure to dryness. Crude was taken into diethyl ether (20 mL), stirred for 10 min then ether layer was separated, dried over sodium sulfate and filtered. Dioxane HCl (1 mL) was added to ether layer, compound was precipitated out which was filtered, washed with DCM and dried to obtain 5-{3-[2-(4-methoxyphenyl)ethyl]-2H-aziren-2-yl}-1H-tetrazole.hydrochloride (103 mg, 14.12%) as an off white solid. 1H NMR (400 MHz, D2O): δ (ppm) 7.2 (d, 2H), 6.9 (d, 2H), 3.8 (s, 3H), 3.3 (s, 1H), 2.9 (m, 2H), 2.6 (m, 2H); 1H NMR (400 MHz, CD3OD): δ (ppm) 7.1 (d, 2H), 6.8 (d, 2H), 3.7 (s, 3H), 3.3 (s, 1H), 2.8 (m, 2H), 2.6 (m, 2H); 1H NMR (400 MHz, DMSO): δ (ppm) 9.3 (s, 2H), 7.3 (d, 1H), 7.1 (m, 4H), 6.8 (d, 2H), 3.7 (s, 3H), 2.8 (m, 2H).
Compound 1 possessed a potent anti-angiogenic activity on primary tubulogenesis screening (75 nM) and 3D angiogenesis screening (250 nm) activity. The compound showed its anti-angiogenic activity in different models such as matrigel implantation assay, CAM and ear angiogenesis assay. The compound 1 inhibits the tumor growth in the in vivo xenograft mouse model both in the intramuscular and intra peritoneal injection 25 mg/kg compound. The compound 1 partially inhibits the expression of VEGFR2 and SRC kinase expression. The compound 1 inhibited angiogenesis it through its binding of the ligand endothelin. In total, the compound 1 is a potent inhibitor angiogenesis with a new target.
To examine the effect of Compound 1 on angiogenesis, capillary tube formation assay was performed on regular or reduced growth factor Matrigel, taking VEGF (50 ng/mL) treated cells as a positive control and sunitinib (50 nM) as a negative control (
Detailed Screening data of Compound 1
Compound 1 exhibited no post endothelial mediated tubule disruption activity Next, we have checked the vascular disrupting activity of preformed tubules. Compound 1 could not be able to disrupt the preformed tubules, suggesting that this molecule could inhibit only the newly forming angiogenesis (
As per previous reports, some anti-angiogenic compounds also show anti-migratory effect by modulating the expression of proteins responsible for blood vessels formation. A Scratch was made to the confluent endothelial cells containing VEGF (50 ng/mL) and sunitinib (50 nM) as a positive and negative control respectively and Compound 1 at 250 nM and 125 nM as a test compound. 0%, 75% and 60% wound closure was observed at 0 nM, 125 nM and 250 nM respectively observed at 0 h and 12 h and complete migration was observed at 24 h of Compound 1 treated cells. 75% wound closure at 125 nM and 60% wound closure at 250 nM showed similar effect like sunitinib at 50 nM whereas control and positive control (VEGF at 50 ng/mL) have resulted 80% and 90% of wound closure effect. This result demonstrates that compound 1 have got a potent anti-migratory effect at a very low dose concentration till 12 h (
Anti-angiogenic effect of Compound 1 was further validated by 3D angiogenesis assay (
The anti-angiogenic effect of compound 1 was further confirmed by CAM assay that inhibit the formation of new blood vessels onto the CAM membrane that provides the nourishment to the developing chick embryo (
Compound 1 prevented the in vivo blood vessel formation Apart from in vitro, Compound 1 also exhibited its anti-angiogenic effect in vivo through matrigel plug assay. Matrigel injected along with Compound 1 at 750 nM concentration into the dorsal skin of the mice exhibited significant reduction in blood vessels formation through the implanted Matrigel (
As VEGF induced vessel permeability is one of the crucial parts of angiogenesis, so the anti-angiogenic effect of Compound 1 on VEGF induced micro-vessels formation in Balb/c mice through ear angiogenesis assay was performed (
Isomer (1-1) means chiral isomer 1 of racemic compound 1;
Isomer (2-1) means chiral isomer 2 of racemic compound 1.
As previously, the anti-angiogenic activity of Compound 1 has already been confirmed by various approaches, it becomes mandatory to check the anti-angiogenic effect of the chiral isomers (1-1 and 2-1) of Compound 1 (
Synergistic Effect of Chiral Isomers (1-1) and (2-1) and Sunitinib at Lower Doses have Better Antiangiogenic Effect
As it has been clearly demonstrated that chiral isomers (1-1) and (2-1) of compound 1 in combination have better anti-angiogenic effect at 500 nM in comparison to individual effect (
Reduced Expression of p-Src and p-VEGFR2 Might Contribute to Anti-Angiogenic Effect of Compound 1
VEGFR2, a receptor tyrosine kinase mediates angiogenesis by binding to its ligand, VEGF that phosphorylates VEGFR2 and induces further downstream signaling responsible for angiogenesis. VEGFR2 expression as well as the expression of Src was checked in HUVECs as reported earlier both the proteins are very important in regulating the angiogenesis (
A nude mice model bearing orthotopic HCT116 colon tumor or MDA-MB 468 was used to evaluate the anti-tumor angiogenesis effect of Compound 1 in vivo (
Modulation in the number of genes and their expression was observed in compound 1 treated HUVECs by mRNA sequence analysis. Heat map graph clearly demonstrates that the genes that are commonly up-regulated and the ones that are down-regulated with Compound 1 treatment at all concentrations (10 μM, 1 μM, 500 nM and 250 nM) with respect to control samples of HUVECs have summarized in Table 2. The gene expression that was observed to be up-regulated and down regulated genes in the tumor samples treated with Compound 1 at 25 mg/kg for 15 days with respect to control samples are summarized in Table 3. Amongst all the genes those were modulated by the treatment of Compound 1, Thrombospondin gene is one of them whose up-regulation is well reported to inhibit angiogenesis. Therefore, this data implies that Thrombospondin 1 could be one of the crucial molecular targets that is being regulated by Compound 1 molecule and exhibiting its anti-angiogenic effect.
Intact protein band was observed in Compound 1 10 mM lane whereas no band in control lane at the same position was observed. This could be due to the binding of drug to the protein lysate that has protected them against the protease digestion whereas control sample with same volume of vehicle has undergone complete protease digestion that leads to absence of any band in the control lane. LC/MS-MS analysis for the excised band from Compound 1 sample has revealed several proteins that were bounded by the drug. According to the PLGS score the proteins involved in angiogenesis are summarized in Table 4 given below. Amongst these obtained proteins Thrombospondin 1, Endothelin, Angiopoietin well known to be involved in the regulation of angiogenesis. Hence, DART analysis once again confirmed the mRNA sequence analysis data that thrombospondin1, endothelin and angiopoetin are the protein target that are being regulated by Compound 1 and imparting it an anti-angiogenic effect.
To test for the selectivity of the putative Endothelin-1 receptor, we applied endothelin-1 or Compound 1 in a concentration dependent manner to the aortic ring to observe its contraction inhibition activity (
Pharmacokinetic Assessment of Compound 1 and its Chiral Isomers 1 and 2 after Intramuscular Administration
In vivo pharmacokinetic study was performed in male Sprague Dawley (SD) rats (n=4). Intramuscular (IM) formulation of compound 1 Chiral 1 and 2 were prepared by individually dissolving accurately weighed quantity of compound 1, chiral isomers 1 and 2 (20 mg) in 1 mL of olive oil followed by vortexing for 2 min and sterilize through syringe filter. Blood samples were collected from the retro orbital plexus of rats under light ether anesthesia into microfuge tubes containing heparin as an anti-coagulant at 0.25, 0.5, 1, 3, 5, 7, 9, 24, 30, 48 and 72 hours post-dosing after intramuscular dose. Plasma sample was harvested by centrifuging the blood at 13000 rpm for 10 min on Sigma 1-15 K (Frankfurt, Germany) and stored frozen at −70±10° C. until bioanalysis. Each plasma sample (100 μl) was processed using protein precipitation method using 200 μl acetonitrile containing medicarpine as internal standard (I.S.) as protein precipitant, and 10 μl of the supernatant was injected for LC-MS/MS.
The mean plasma concentration-time profiles of compound 1 are shown in the table 5 and
Pharmacokinetic Assessment of Bulk Compound 1, Chiral Isomer-1 of Compound 1 and Chiral Isomer-2 of Compound 1 after Intramuscular Administration
In vivo pharmacokinetic study was performed in male Sprague Dawley (SD) rats (n=4). Intramuscular (IM) formulation of bulk of compound 1 as well as chiral isomer-1 and chiral isomer-2 was prepared by dissolving accurately weighed quantity of compound 1 (20 mg) in 1 mL of olive oil followed by vortexing for 2 min and sterilize through syringe filter. Blood samples were collected from the retro orbital plexus of rats under light ether anesthesia into microfuge tubes containing heparin as an anti-coagulant at 0.25, 0.5, 1, 3, 5, 7, 9, 24, 30, 48 and 72 hours post-dosing after intramuscular dose. Plasma sample was harvested by centrifuging the blood at 13000 rpm for 10 min on Sigma 1-15 K (Frankfurt, Germany) and stored frozen at −70±10° C. until bioanalysis. Each plasma sample (100 μl) was processed using protein precipitation method using 200 μl acetonitrile containing medicarpine as internal standard (I.S.) as protein precipitant, and 10 μl of the supernatant was injected for LC-MS/MS.
The mean plasma concentration-time profiles of compound 1 are shown in the table 7 and
DMSO was purchased from Sigma (D2650); Mouse-Fibroblast Growth Factor-basic (FGF) was from Sigma (St. Louis, Mo.), human-VEGF was purchased from (Sigma; V7259), mouse-VEGF was purchased from (Sigma; V4512), hemoglobin (H7379). Phospho-VEGFR2 (Tyr1175) CST; Raf Family Antibody Sampler kit (#2330 CST); anti-CD31 (sc-1506) Santa Cruz Biotechnology. Alexa 488-conjugated goat anti-rabbit IgG secondary antibodies were obtained from (Invitrogen; A27034). Matrigel was purchased from (CORNING; 354230) and hemoglobin from (Sigma; H7379); Sunitinib Maleate, used as a positive control was purchased from (Sigma; 341031-54-7); all other procured chemicals were of the highest grade commercially available.
The human endothelial cell line, HUVEC was purchased from (Himedia, India) at passage 2 and was cultured in complete Dulbecco's Modified Eagle Medium (DMVEM, Himedia, India) supplemented with 10% fetal bovine serum (FBS, Himedia, India) and 1% Penicillin/Streptomycin (Himedia, India) at 37° C. in 5% CO2 and was allowed to attain a confluency rate of 80%-90%. The cultured cells were then used for different experiments.
HUVECs (Human Umbilical Vein Endothelial Cells) were seeded onto the pre-coated matrigel (CORNING; 354230) in 96 wells plate at the rate of 11000 cells/well in incomplete ECM basal medium. Control group received vehicle, VEGF at 50 ng/mL as a positive and sunitinib at 50 nM as a negative control were used. Compound 1 at different dose concentrations (10 μM, 5 μM, 1 μM, 800 nM, 500 nM, 250 nM, 100 nM, 75 nM, 50 nM and 25 nM) were employed on seeded HUVECs in incomplete medium. Post 6 h of incubation Calcein-AM (Invitrogen) was added to all the respective groups and then the tubule pattern was observed and photographs were taken using an inverted fluorescent microscope (DM 6000, Leica Microsystems, USA). Relative tubule lengths were measured using Leica Q-Win software version 3.5.1. (Leica Microsystems, Switzerland).
Endothelial cells were grown inside 6-well plate at a confluency rate of 90%. And then with the help of 200 μL tip a straight line scratch was made in the middle of the grown ECs. After giving a gentle wash with 1×PBS, control group received vehicle in 2.5% FBS media. VEGF at 50 ng/mL as a positive control and sunitinib at 50 nM concentration was used as a negative control. Compound 1 at a dose concentration of 250 nM and 125 nM were used onto the wounded cells to observe its anti-migratory effect. After 0 h, 12 h and 24 h of post treatment the wound closure area was observed and micrographed using an inverted microscope (Olympus, Centre Valley, Pa., USA) and were analysed by Image J software. Control well at 100% was used to express inhibition percentage and the assay was performed for three-five times independently.
Endothelial cells (ECs) were trypsinized and then coated on cytodex beads at the rate of 1500 cells per bead in 5% DMEM medium at 37° C. for 4 h by rotating it gently on IP rotor. Fibrinogen (2 mg/mL) and Aprotonin (40 U/μL) were added to the ECs coated beads and this was subsequently added to the 96 well plate pre-coated with pro-thrombin (10 U/μL) and incubated for 15 min. Vehicle control in 2.5% DMEM media with different doses of Compound 1 (500 nM, 250 nM, 125 nM and 75 nM) were used to observe the anti-angiogenic effect of Compound 1 on 3D angiogenesis pattern. Post 3 days of incubation the results were observed and photographs were taken under inverted microscope (Olympus, Centre Valley, Pa., USA) and relative tubule lengths were measured using Leica Q-Win software version 3.5.1. (Leica Microsystems, Switzerland).
All experiments were conducted according to international ethical standards with prior approval from the Institutional Animal Ethics Committee (IAEC) [IAEC/2015/129] of CSIR-Central Drug Research Institute (CSIR-CDRI) and Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Male Balb/c mice (20-25 g) of aged 8-10 weeks were procured from the National Laboratory Animal Centre (NLAC) of CSIR-CDRI, Lucknow, India. CrTac:NCr-Foxn1nu (athymic nude mice, female, 8-10 weeks) were procured from (Taconic Biosciences, U.S.). Animals were housed under standard environmental conditions of 12 h/12 h light-dark cycle, 23±2° C., ambient temperature and humidity 60-65%, in polypropylene cages. Food, in the form of dry pellets ad libitum, and water were available to the mice.
Compound 1 ability to modulate neovascularization was evaluated by matrigel plug assay, using mouse-VEGF and bFGF (R&D systems, Minneapolis) as stimulus. 300 μL of matrigel (Corning Inc., Corning, N.Y., USA) were allowed to liquefy at 4° C., supplemented with either VEGF (50 ng/μl), bFGF (50 ng/μl) and heparin (10 mg/mL) as a control or with sunitinib (2 M) as a positive control. All groups and test groups having similar condition along with Compound 1 (750 nM) were carefully mixed with pre-chilled pipette tips to prevent aeration and gelatinization. 4-5 weeks old male Balb/c mice anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) i.p. were injected subcutaneously (s.c.) with matrigel mixtures (0.300 ml) in two locations into the dorsal part. After ten days, mice were euthanized, the dorsal skin retaining the Matrigel implants were dissected. Matrigel plugs were excised after photographs were taken and angiogenic response was evaluated by spectrophotometric measurement of hemoglobin content using the Drabkin's method according to the manufacturer's protocol (Cat no. D5941-6VL; Sigma, St. Louis, Mo.). Absorbance was measured at 540 nm and hemoglobin concentration was calculated.
Sterile 1×PBS having mouse VEGF (50 ng/mL), bFGF (50 ng/mL), Heparin (10 mg/mL) and sunitinib (2 μM) as a positive control along with compound 1 (750 nM) as a test compound were injected subcutaneously into the ear of 8- to 12-week old male Balb/c mice. Equal volume of 1×PBS with all components except drug was used as a control and was injected in contra lateral ear. After 6 days, mice were sacrificed and the ears were excised and embedded in OCT for cryo-sectioning. Frozen sections of 5 m sizes were immunostained with endothelial cell markers anti CD31 and anti PECAM 1 antibody. This was quantified by total number of blood vessels count.
The eggs were incubated horizontally for 3 days at 37° C. in a 60% humidified atmosphere, using a hybridization incubator (combi-SV 12) with one hour scheduled rotation. Post 3 days of incubation, 2-3 mL of albumin was aspirated from the acute pole of the egg to create false air sac directly over the CAM. Post fertilization (Day 8), a fine blade was used to make an approximate 0.5 cc square incision on the eggshell under sterile conditions and the eggshell window fragment was removed to access the CAM beneath. A piece of gelatin sponge embedded in Compound 1 (500 nM and 250 nM) and sunitinib (2 μM) individually were placed on the CAM of the chicken embryo and incubated for 12 days. Sterile parafilm tape was used to seal back the window and the eggs were further incubated without the rotation setting. After 12 days of incubation the CAM integration was assessed by visual inspection and photographed with a digital camera (Canon EOS); the CAM membrane along with gelatin sponge were harvested and its hemoglobin (Hb.) estimation was done by Drabkin's method according to the manufacturer's protocol (Sigma, St. Louis, Mo.). Absorbance was measured at 540 nm and hemoglobin concentration was calculated.
Whole cell lysates of endothelial cells at 6 h of treatment with Compound 1 (250 nM) and sunitinib (50 nM) were prepared using the RIPA-lysis buffer. Proteins were fractionated and resolved on 8% SDS-PAGE gels and transferred onto PVDF membranes (Millipore). After incubation in blocking buffer (5% BSA in 1×TBST) for 1 h the membranes were incubated with primary antibodies specific for p-Akt and p-Src. GAPDH was used as housekeeping and loading control. The blots were then incubated with specific HRP-conjugated secondary antibodies and bands were visualized using ECL (Millipore) on Image Quant (LAS4010) chemiluminescence detection system. The band intensities were quantified using My Image Analysis software.
HCT 116 (Human colon cancer cell line) cells were trypsinised and the pellet was dissolved in ice chilled 1×PBS. HCT 116 cells suspension to a concentration of (1*106) cells in 50 μL PBS solution, were left on ice with occasional agitation. Mice were anaesthetized with ketamine: xylazine (100:10 mg/kg), abdominal side area was shaved and sterilized using 70% ethanol. 50 μL of cell suspension was injected slowly subcutaneously to all the mice. After 2 days, booster dose of tumor cells was injected to all mice as performed previously. The growth of the tumor was measured by using vernier caliper on day 2, 4, 6, 8, 10, 12, 14, 16 18, 20, 23 and presented in the form of tumor volume (mm3)=[4/3*π*(long axis/2)*(short axis/2)]. After attaining a volume of 100 mm3 of tumor size, all mice were distributed equally (n=6−8) in 3 groups of controls received vehicle (Olive oil). Sutent (25 mg/kg) as a positive control was given orally and Compound 1 (25 mg/kg) dissolved in olive oil was injected intra-muscularly for 15 days and the tumor size regression was measured on every alternate day as described previously. Post 15 days of treatment, mice from all groups were sacrificed and tumor was excised out and processed further for immunohistochemistry and molecular analysis.
mRNA Sequence Analysis
High quality intact RNA was isolated using RNeasy Minikit (QIAGEN) from the tumor samples of control and Compound 1 at (25 mg/kg) treated groups and also from the HUVECs treated with compound 1 at different dose concentrations (10 μM, 1 μM, 500 nM, 250 nM) for 6 h>3-4 μg of isolated RNA was sent for mRNA sequence analysis. Heat map graph for the down-regulated and up-regulated genes were generated for each of the respective samples.
HUVECs with confluency of 80-85% in 100 mm dish were trypsinised and the media was removed and washed twice with 1×PBS. These cells were lysed with lysis buffer at 4° C. Control and treatment group with 100 ag of protein were incubated with vehicle and Compound 1 at a concentration of 10 mM for 15-30 mins at RT with gentle shaking. Then different proteases like thermolysin (10 μg/15 μL) and pronase at (10 μg/L) were added to both the control and treatment groups for 5-10 mins at RT. Digestion was stopped by adding protease inhibitor (20×) at specific interval and allowed to incubate at 4° C. for 10 mins. 6×SDS-PAGE loading dye was added to all the respective samples and heated at 70° C. for 10 mins. SDS-PAGE was performed for the given control and treatment group samples and then the gel was stained with 0.05% Coomassie Brilliant Blue (CBB-R250). After destaining the gel, bands of different molecular weight of treatment and control groups were excised off from the gel and further employed for the protein sequence analysis through LC/MS-MS (Liquid chromatography/Mass spectroscopy).
The results from at least three independent experiments are expressed as mean±SEM. Statistical significance were analyzed using two-tailed Student' T-test or ANOVA was used to test the variability amongst groups. Using graph pad prism software version 5.00.28 (GraphPad Software, Inc., San Diego, Calif., USA) all the statistical test was performed. A Probability (P) value less than 0.05 were considered statistically significant.
HUVEC, Human Umbilical Vein Endothelial cell; VEGF, Vascular Endothelial Growth Factor; bFGF, basic Fibroblast Growth Factor; PBS, Phosphate Buffer Saline; BSA, Bovine Serum Albumin; CD31, cluster of differentiation 31; PECAM1, Platelet endothelial cell adhesion molecule; RT, room temperature; LC/MS-MS, Liquid Chromatography/Mass-Mass Spectroscopy.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202011023422 | Jun 2020 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IN2021/050544 | 6/4/2021 | WO |
