Selective BCRP / ABCG2 transporter inhibitor as an agent for eliminating resistance to anticancer drugs

Abstract

The present invention relates to a compound of formula (I) [Formula 1] JPEG2022541334000102.jpg3960 or enantiomers, pharmaceutically acceptable salts or solvates of this compound, or mixtures thereof, wherein the nucleus A, the substituents Z, Y and R1 are as defined herein.

Description

Technical Field

[0001] The present invention relates to novel compounds, methods for their production, and their use as BCRP / ABCG2 inhibitors.

Background Art

[0002] These new organic compounds belong to the same chemical family derived from chromone, all of which are natural compounds.

[0003] Its main purpose is to selectively inhibit the BCRP protein (breast cancer resistance protein, also known as ABCG2) involved in the phenomenon of multidrug resistance of tumors to anticancer agents. BCRP acts as an efflux pump and is overexpressed in the tumor cell membrane. These compounds act as selective and non-cytotoxic inhibitors against BCRP / ABCG2.

[0004] Finally, it has been found that these compounds have higher inhibitory activity and selectivity and lower cytotoxicity than the reference compound Ko143, while the organic synthesis is very simple. Clinically, by using these compounds in combination with anticancer agents, the effect of the anticancer agents can be enhanced, and they can be used as adjuvants to counter the resistance of tumors to treatments aimed at eradicating tumors.

[0005] According to the WHO, cancer is the second leading cause of death in the world, with approximately 9.6 million people dying annually. The treatment cost of this disease is estimated to be 1160 billion yen annually in 2010. Currently, there are three main strategies for the treatment of cancer: 1) surgery, 2) radiotherapy, and 3) chemotherapy, depending on the stage and type. It should be noted that these strategies can be used in addition to each other to complement the overall effect of the treatment.

[0006] However, in chemotherapy, by repeatedly using the same anticancer agent, tumor cells develop a defensive response against the anticancer agent. Unfortunately, this reduces the sensitivity of tumors to various anticancer agents, renders chemotherapy ineffective, and increases harm to patients. One of the defensive strategies of tumor cells is the overexpression of transmembrane proteins called ATP-binding cassette (ABC) transporters in the cell membrane. (Borst, P.; Elferink, R. O. Mammalian ABC Transporters in Health and Disease. Annu. Rev. Biochem. 2002, 71(1), 537-592. Linton, K. J. Struture and Function of ABC Transporters. Physiology 2007, 22(2), 122-130. Sharom, F. J. ABC Multidrug Transporters: Structure, Function and Role in Chemoresistance. Pharmacogenomics 2 - 008, 9(1), 105-127).

[0007] Among the 48 transmembrane proteins of this superfamily, three have been clearly shown to play a major role in chemotherapy failure: P-gp / ABCB1, MRP1 / ABCC1, BCRP / ABCG2 (Leslie, E.M.; Deeley, R.G.; Cole, S.P.C. Multidrug Resistance Proteins: Role of P-Glycoprotein, MRP1, MRP2, and BCRP(ABCG2) in Tissue Defense. Toxicol. Appl. Pharmacol. 2005, 204(3), 216-237. Eckford, P.D.W.; Sharom, F.J. ABC Efflux Pump-Based Resistance to Chemotherapy Drugs. Chem. Rev. 2009, 109(7), 2989-3011.).

[0008] These membranes enable the transport of various types of chemicals and are naturally present in most cell membranes and physiological barriers in our bodies. Therefore, their physiological role in healthy individuals is to defend and protect organs and tissues from foreign and / or xenobiotic substances. However, it has been shown that these ABC transporters can dramatically alter the absorption, distribution, metabolism, and excretion of active ingredients. (Sharom, F.J. ABC Multidrug Transporters: Structure, Function and Role in Chemoresistance. Pharmacogenomics 2008, 9(1), 105-127).

[0009] Due to these functions, these three transporters have become effective, novel, and major therapeutic targets in research as an effective therapeutic solution for suppressing the problem of chemotherapy resistance. (Bugde, P.; Biswas, R.; Merien, F.; Lu, J.; Liu, D.-X.; Chen, M.; Zhou, S.; Li, Y. The Therapeutic Potential of Targeting ABC Transporters to Combat Multi-Drug Resistance. (The potential of treating by targeting ABC transporters to overcome multi-drug resistance) Expert Opin. Ther. Targets 2017, 21(5), 511-530.)

[0010] Ko143 is a polycyclic organic molecule containing three asymmetric centers and is currently used in research as a reference inhibitor of BCRP. However, its optimized five-step synthesis has a cumbersome overall yield of 5%, and the control of the three asymmetric centers remains a limiting parameter (Li, Y.; Hayman, E.; Plesescu, M.; Prakash, S. R. Synthesis of Potent BCRP Inhibitor - Ko143. (Synthesis of a potent BCRP inhibitor - Ko143) Tetrahedron Lett. 2008, 49(9), 1480-1483.)

[0011] Despite having good activity (50% inhibitory concentration, IC50 = 0.09 μM ± 0.01), this compound has relatively low solubility, which affects its bioavailability. Ko143 has been shown in clinical trials to be rapidly metabolized (60 minutes) by hydrolysis of the tertiary butyl ester to produce an inactive metabolite. (Liu, K; Zhu, J; Huang, Y; Li, C.; Lu, J; Sachar, M; Li, S.; Ma, X. Metabolism of KO143, an ABCG2 inhibitor. (ABCG2 inhibitor KO143's metabolism) Drug Metab. Pharmacokinet. 2017, 32(4), 193 - 200.) As a result, the clinical trial was terminated. This compound, initially reported to be selective for BCRP, was ultimately found to be non - selective for ABCG2 (Weidner, L.D.; Zoghbi, S.S.; Lu, S.; Shukla, S; Ambudkar, S.V.; Pike, V.W.; Mulder, J.; Gottesman, M.M.; Innis, R.B.; Hall, M.D. The Inhibitor Ko143 Is Not Specific for ABCG2. (Inhibitor Ko143 is not specific for ABCG2) J. Pharmacol. Exp. Ther. 2015, 354(3), 384 - 393. Allen, J.D.; van Loevezijn, A.; Lakhai, J.M.; van der Valk, M.; van Tellingen, O.; Reid, G.; Schellens, J.H.M.; Koomen, G.-J.; Schinkel, A.H. Potent and Specific Inhibition of the Breast Cancer Resistance Protein Multidrug Transporter in Vitro and in Mouse Intestine by a Novel Analogue of Fumitremorgin C. (Fumitremorgin C's novel analogue's in vitro and in mouse intestine's potent and specific inhibition of the breast cancer resistance protein multidrug transporter.) Mol. Cancer Ther. 2002, 1(6), 417.)

[0012] The applicant has previously developed an inhibitor called MBL-II-141, which is selective, non-toxic, and has good activity in preclinical models. (Honorat,M;Guitton,J;Gauthier,C;Bouard,C;Lecerf-Schmidt,F;Peres,B;Terreux,R;Gervot,H;Rioufol,C;Boumendjel,A;et al.MBL-II-141,a Chromone Derivative,Enhances Irinotecan(CPT-11)Anticancer Efficiency in ABCG2-Positive Xenografts.(Chromone derivative MBL-II-141 enhances the anticancer efficiency of irinotecan (CPT-11) in ABCG2-positive xenografts.)Oncotarget 2014,5(23),11957-11970.Henin,E;Honorat,M;Guitton,J;Di Pietro,A;Payen,L;Tod,M.Pharmacokinetic Interactions in Mice between Irinotecan and MBL-II-141,an ABCG2 Inhibitor:Irinotecan MBLI-II-141 Interaction.(Pharmacokinetic interactions between irinotecan and the ABCG2 inhibitor MBL-II-141 in mice: Irinotecan MBLI-II-141 interaction.)Biopharm.Drug Dispos.2017.Valdameri,G.;Genoux-Bastide,E.;Peres,B.;Gauthier,C.;Guitton,J.;Terreux,R.;Winnischofer,S.M.B.;Rocha,M.E.M.;Boumendjel,A.;Di Pietro,A. Substituted Chromones as Highly Potent Nontoxic Inhibitors, Specific for the Breast Cancer Resistance Protein.(Substituted chromones as highly potent non-toxic inhibitors specific for the breast cancer resistance protein.))J.Med.Chem.2012,55(2),966-970.Lecerf-Schmidt,F.;Peres,B.;Valdameri,G.;Gauthier,C.;Winter,E.;Payen,L.;Di Pietro,A.;Boumendjel,A.ABCG2: Recent Discovery of Potent and Highly Selective Inhibitors(ABCG2: Recent Discovery of Potent and Highly Selective Inhibitors.)Future Med.Chem.2013,5(9),1037-1045.Winter,E.;Lecerf-Schmidt,F.;Gozzi,G.;Peres,B.;Lightbody,M.;Gauthier,C.;Ozvegy-Laczka,C.;Szakacs,G.;Sarkadi,B.;Creczynski-Pasa,T.B.;et al.Structure-Activity Relationships of Chromone Derivatives toward the Mechanism of Interaction with and Inhibition of Breast Cancer Resistance Protein ABCG2.(Structure-Activity Relationships of Chromone Derivatives toward the Mechanism of Interaction with and Inhibition of Breast Cancer Resistance Protein ABCG2.)J.Med.Chem.2013,56(24),9849-9860.Pires,A.do R.A.;Lecerf-Schmidt,F.;Guragossian,N.;Pazinato,J.;Gozzi,G.J.;Winter,E.;Valdameri,G.;Veale,A.;Boumendjel,A;Di Pietro,A.;et al.New,Highly Potent and Non-Toxic, Chromone Inhibitors of the Human Breast Cancer Resistance Protein ABCG2.(New, Highly Potent and Non-Toxic, Chromone Inhibitors of the Human Breast Cancer Resistance Protein ABCG2.)Eur.J.Med.Chem.2016,122,291-301.)。In this latest publication, it has been found that the compound used as a synthetic intermediate is an inhibitor of the breast cancer multidrug resistance protein (breast cancer resistance protein BCRP / ABCG2).

[0013] Since the renewal of marketed anticancer drugs is not carried out promptly, the problem of chemotherapy resistance is increasing. To counter the shortage of new anticancer drugs, it is a realistic and economical solution to target the root cause of the resistance problem. In fact, by suppressing the self - defense ability of tumor cells, the effectiveness of current anticancer drugs can be maintained and the effective dose can be limited. As a result, side effects can be reduced and the total cost of chemotherapy for patients and society can be suppressed. Furthermore, it would be interesting to find new compounds that are even more effective than MBL - II - 141 and Ko143.

Summary of the Invention

[0014] The present invention relates to a compound of formula (I):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Mode for Carrying Out the Invention

[0015] In particular, ring A may be substituted with one or two Br at positions 2, 3, 4, and 5, and Y = -OH; -OMe; -NH-(CH2)2-(3-indolyl); -NH(CH2)2-3-((5-hydroxy)indolyl); -NH-CH(R3)-COR2, where R1, R2, and R3 are as defined above.

[0016] In certain embodiments, Y is -NH-(CH2)2-(3-indolyl) or -NH(CH2)2-3-((5-hydroxy)indolyl), provided that

Chemical formula

Chemical formula

Chemical formula

[0017] In particular, the compounds according to the invention can be selected from the following. Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucinate; Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-leucinate; Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-valinate; Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-phenylalaninate; Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-phenylalaninate; Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-tryptophanate; Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-tryptophanate; Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-D-tryptophanate; (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucine; (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-phenylalanine; (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-phenylalanine; (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-D-tryptophan; Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucyl-L-valinate; Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucyl-L-leucinate; Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucyl-L-valinate; Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucyl-L-leucinate; (S)-5-((2-bromobenzyl)oxy)-N-(1-((2-(5-hydroxy-1H-indol-3-yl)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)-4-oxo-4H-chromene-2-carboxamide; (S)-N-(1-((2-(1H-Indol-3-yl)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)-5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxamide; and (R)-N-(1-((2-(1H-Indol-3-yl)ethyl)amino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)-5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxamide.

[0018] The present invention also relates to a method for obtaining a compound according to the present invention, characterized by comprising the following steps: (a) An alkylated compound of the formula [Chemical formula] , where ring A is as defined in claim 1 and X is a halogen selected from F, Cl, Br, and I, is reacted with 2,6-dihydroxyacetophenone of the formula at the reflux temperature of acetone in acetone to obtain an intermediate of the formula [Chemical formula] ; [Chemical formula] ; (b) The intermediate obtained in step (a) is reacted with diethyl oxalate of the formula [Chemical formula] at a temperature of 0 °C to 50 °C in a mixture of tetrahydrofuran (THF) / ethanol (1:1) to obtain a compound of the formula [Chemical formula] to obtain the intermediate; (c) Reacting the intermediate obtained in step (b) at a temperature of 50 °C in an acidic or basic medium in a THF / ethanol / water solvent (3:1:1.5) by a hydrolysis reaction of the ester functional group to obtain an intermediate of the formula [Chemical formula] to obtain the intermediate; (d) Reacting the intermediate obtained in step (c) with a coupling compound of the formula [Chemical formula] wherein R1, Z and Y are as defined in claim 1 at room temperature in anhydrous DMF to form an amide bond to obtain the compound of formula (I).

[0019] The present invention also relates to a compound of formula (I) for use in inhibiting the breast cancer multidrug resistance protein (breast cancer resistance protein BCRP / ABCG2): [Chemical formula] or a pharmaceutically acceptable enantiomer, salt or solvate thereof, or a mixture thereof. In the formula · Ring A is unsubstituted or substituted by one or two of F; Cl; Br; I; OR at the 2, 3, 4, 5 positions, where R = Me, Et, Pr, i-Pr, n-Bu; O-CH2-(O-CH2CH2) n -O-CH3, n = 3, 4, 5, 6 · Z is [Chemical formula] or -CH2- · Y = -OH; -OMe; -OEt; -OPr; -NH2; -NHMe; -N(Me)2; -N(Me)OCH3; -NH-(CH2)2-(3-indolyl); -NH(CH2)2-3-((5-hydroxy)indolyl); -NH-CH(R3)-COR2, where R2 is selected from the following: -OH; -OMe; -OEt; -OPr; -NH2; -NHMe; -N(Me)2; -N(Me)OCH3; 3-(5-methoxy)indolyl; -NH-(CH2)2-(3-indolyl); -NH(CH2)2-3-((5-hydroxy)indolyl); -NH(CH2)2-3-((5-methoxy)indolyl), · In formula (I)

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0020] Inhibition of the breast cancer multidrug resistance protein (breast cancer resistance protein BCRP / ABCG2) may, in addition to its role in breast cancer treatment, enable re-sensitization of tumor cells and improvement of the pharmacokinetics and effects of drugs that need to cross physiological membranes or barriers such as the blood-brain barrier and the gastrointestinal barrier to exert their therapeutic effects.

[0021] In particular, ring A may be substituted with one or two Br at the 2, 3, 4, and 5 positions, and Y = -OH; -OMe; -NH-(CH2)2-(3-indolyl); -NH(CH2)2-3-((5-hydroxy)indolyl); -NH-CH(R3)-COR2, where R1, R2, and R3 are as defined above.

[0022] In certain embodiments, Y is -NH-(CH2)2-(3-indolyl) or -NH(CH2)2-3-((5-hydroxy)indolyl), provided that

Chemical formula

Chemical formula

Chemical formula

[0023] Compounds for use in inhibiting the breast cancer multidrug resistance protein (breast cancer resistance protein BCRP / ABCG2) according to the present invention can be selected from the following: Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucinate; Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucinate; Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-leucinate Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-valinate; Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-phenylalaninate; Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-phenylalaninate; Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-tryptophanate; Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-tryptophanate; Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-D-tryptophanate; (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucine; (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-phenylalanine; (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-phenylalanine; (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-D-tryptophan; Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucyl-L-valinate; Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucyl-L-leucinate; Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucyl-L-valinate. Methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucyl-L-leucinate; (S)-5-((2-Bromobenzyl)oxy)-N-(1-((2-(5-Hydroxy-1H-indol-3-yl)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)-4-oxo-4H-chromene-2-carboxamide; (S)-N-(1-((2-(1H-Indol-3-yl)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)-5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxamide, and (R)-N-(1-((2-(1H-Indol-3-yl)ethyl)amino)-3-(1H-Indol-3-yl)-1-oxopropan-2-yl)-5-(((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxamide.

[0024] Also, the present invention relates to at least one pharmaceutically acceptable active agent; and at least one compound as defined above and relates to a pharmaceutical composition comprising the same.

[0025] In particular, the pharmaceutically acceptable active agent may be selected from an anticancer agent, an intestinal anti-inflammatory agent, a cholesterol-lowering agent, an antihyperlipidemic agent, and a kinase inhibitor.

[0026] For example, as the intestinal anti-inflammatory agent, sulfasalazine can be used, as the cholesterol-lowering agent, atorvastatin can be used, as the antihyperlipidemic agent, rosuvastatin can be used, and as the anticancer agent or kinase inhibitor, imatinib mesylate can be used.

[0027] These compounds, distinguishable by the fact that a natural amino acid or an enantiomer of a natural amino acid is bound to the chromone unit, can be synthesized in four steps from commercially available 2,6-dihydroxyacetophenone 1 (Scheme 1), with an overall yield of 13 - 41%. Intermediate 2 can be accessed in three standard steps: alkylation, Kostanecki reaction, and saponification, with an overall yield of 45%. The final step is carried out by a coupling reaction, for example, a peptide coupling that binds a natural amino acid or an enantiomer of a natural amino acid to the chromone unit, and is assisted by a coupling agent that accelerates the reaction and limits the number of equivalents of the reactants.

[0028] The reagents are inexpensive and commonly used in laboratories. Furthermore, this reaction consumes little energy, and since the product is purified by precipitation or recrystallization, the amount of organic solvent used and, consequently, the amount of waste are limited. Finally, the compound is isolated in pure form, and its structure is established and verified by various analytical techniques (NMR, mass spectrometry, X-ray diffraction). Note that X-ray diffraction confirmed the absence of racemization of the asymmetric centers of the commercially available amino acids.

[0029] Scheme 1: Synthetic route of the BCRP inhibitor of the present invention.

Chemical formula

[0030] The following examples illustrate the present invention without limiting its scope.

[0031] In these examples: NMR spectra were recorded on a Bruker Avance-400 400 MHz instrument (400 MHz) or a Bruker Avance-500 500 MHz instrument (500 MHz).

[0032] Chemical shifts (δ) were reported in ppm relative to Me4Si used as an internal standard.

[0033] The electrospray ionization (ESI) mass spectrum was obtained by the Analytical Department of the University of Grenoble using a Waters Xevo G2-S Q TOF instrument with a nano spray input. The exact mass was indicated in m / z.

[0034] HPLC analysis was performed using an Agilent 1100 series system with a diode array detector and a C18 reverse-phase column (Nucleosil C18, Macherey-Nagel, particle size 5 mm, 125 mm × 3 mm) at 45 °C with a mobile phase consisting of A: water and 0.1% trifluoroacetic acid (TFA) and B: methanol (MeOH) and 0.1% TFA. The A:B gradient was changed from 85:15 to 0:100 over 14 minutes at a flow rate of 1 mL / min, with a 10 μL injection and detection at 254 nm.

[0035] The melting point (m.p.) expressed in degrees Celsius (°C) was obtained using a Buchi B540 melting point apparatus.

[0036] Thin layer chromatography (TLC) was performed using Merck F-254 silica gel plates (0.25 mm thick).

[0037] Unless otherwise specified, the reagents were obtained from commercial sources (Alpha Aesar, Sigma-Aldrich, and TCI) and used without further purification.

Example

[0038] Examples 1 - 20 Series 1

Chemical Formula

[0039] Note) In the following protocol, "Molecular number + a" refers to the case where bromine is at the 2-position of the aromatic ring, and "Molecular number + b" refers to the case where bromine is at the 4-position of the aromatic ring.

[0040] General Procedure A: To a solution of 2,6-dihydroxyacetophenone 1 (1 equiv) in acetone (6 mL / mmol) were simultaneously added K2CO3 (3 equiv) and tetra-n-butylammonium bromide (0.4 equiv) which had been pre-homogenized together.

[0041] After refluxing this solution for 30 - 60 minutes, the corresponding bromobenzyl bromide (1 equiv) in acetone (15 mL / mmol) was added dropwise.

[0042] Thereafter, the solution was refluxed for 4 - 5 hours and monitored by TLC (ethyl acetate / cyclohexane 3:7). The solution was poured into water and extracted with ethyl acetate. The organic phase was collected, washed with water and brine, dried over magnesium sulfate, filtered, and evaporated under vacuum.

[0043] General procedure B: To a solution of 2 (1 equiv) in anhydrous THF (10 mL / mmol) was added a solution of sodium ethoxide generated in situ from sodium (6 equiv) in absolute ethanol (15 mL / mmol) at 0 °C under an inert atmosphere.

[0044] This solution was stirred at room temperature for 30 minutes, and diethyl oxalate (4 equiv) was added dropwise to the solution. Thereafter, the solution was heated to 50 °C until precipitation occurred and refluxed for 2 hours. The reaction was monitored by TLC (cyclohexane / ethyl acetate 1:1).

[0045] Thereafter, a few drops of concentrated hydrochloric acid (37%) were added to the solution until the formed precipitate became white.

[0046] This solution was refluxed for 1 hour and then cooled to room temperature. After concentration under vacuum, the solution was poured into water and extracted with ethyl acetate. The organic phase was collected, washed with water and brine, dried over magnesium sulfate, filtered, and evaporated under vacuum.

[0047] General operating procedure C: A solution of 3 (1 equiv) in THF (25 mL / mmol) and ethanol (8 mL / mmol) was added to a solution of K2CO3 (1.3 equiv) in water (12 mL / mmol).

[0048] The solution was heated at 50 °C for 4 h and monitored by TLC (ethyl acetate / cyclohexane 1:1). The solution was concentrated under vacuum, then poured into basic water (20% K2CO3) and washed with ethyl acetate. The basic aqueous phase was acidified with concentrated hydrochloric acid (37%) and extracted with ethyl acetate.

[0049] Subsequently, the organic phases were collected, washed with water and brine, dried over magnesium sulfate, filtered, and evaporated.

[0050] General procedure D: To a solution of carboxylic acid derivative 4 (1 equiv) in anhydrous dimethylformamide (DMF) (20 mL / mmol) was added 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) (2 equiv). The solution was stirred at room temperature for 30 min.

[0051] Next, a solution of amino acid derivative (2 equiv) in DMF (10 mL / mmol) was carefully added thereto in the presence of N,N-diisopropylethylamine (DIEA) (4 equiv). The reaction was stirred at room temperature for 12 h or 24 h and monitored by TLC (ethyl acetate / cyclohexane 1:1). The solution was concentrated under vacuum, then poured into acidified water (1 M HCl) and extracted with ethyl acetate.

[0052] The organic phases were collected, washed with NaHCO3 solution (20%), water, and brine, dried over magnesium sulfate, filtered, and concentrated under vacuum.

[0053] General procedure E: A solution of ester derivative 5 (1 equiv) in THF (25 mL / mmol) and methanol (10 mL / mmol) was added dropwise to a solution of LiOH (1.5 equiv) in H2O (10 mL / mmol). The reaction was stirred at room temperature for 2 h and monitored by TLC (ethyl acetate / cyclohexane 1:1).

[0054] This solution was poured into basic water (20% NaHCO3) and washed with ethyl acetate. The aqueous phase was acidified with concentrated hydrochloric acid (37%) and extracted with ethyl acetate.

[0055] Next, the organic phases were combined, washed with brine, dried over magnesium sulfate, filtered, and concentrated in vacuo.

[0056] Example 1 Preparation of 1-(2-((2-bromobenzyl)oxy)-6-hydroxyphenyl)ethan-1-one (2a)

Chemical formula

[0057] Starting from 1 (1.500 g, 9.86 mmol), the crude product was prepared according to the general procedure A and purified by recrystallization from methanol or ethyl acetate to give 2a (2.583 g, 82%) C 15 H 13 BrO3 was obtained.

[0058] 1 1H NMR (400 MHz, DMSO) δ 11.69 (s, 1H), 7.71 (dd, J = 8.0, 1.1 Hz, 1H), 7.61 (dd, J = 7.6, 1.6 Hz, 1H), 7.46 (td, J = 7.5, 1.2 Hz, 1H), 7.39 - 7.29 (m, 2H), 6.67 (dd, J = 8.4, 0.5 Hz, 1H), 6.56 (dd, J = 8.3, 0.7 Hz, 1H), 5.19 (s, 2H), 2.47 (s, 3H).

[0059] 1313C NMR (101 MHz, DMSO) δ 203.38, 159.60, 157.95, 135.31, 133.90, 132.72, 130.64, 130.41, 128.00, 123.04, 114.62, 109.83, 103.19, 70.02, 32.86.

[0060] m.p.: 75.5 - 77.4 °C.

[0061] MS (ESI) m / z 321 ( 79 Br), 323 ( 81 Br) [M+H] + , 319 ( 79 Br), 321 ( 81 Br) [M-H] + .

[0062] Example 2 Preparation of 1-(2-((4-bromobenzyl)oxy)-6-hydroxyphenyl)ethan-1-one (2b)

Chem.

[0063] Starting from 1 (1.500 g, 9.86 mmol), the crude product was prepared according to the general procedure A and purified by recrystallization from ethyl acetate to give 2b (2.321, 73%) C 15 H 13 BrO3.

[0064] 1 1H NMR (400 MHz, DMSO) δ 11.64 (s, 1H), 7.62 - 7.58 (m, 2H), 7.45 - 7.41 (m, 2H), 7.30 (t, J = 8.3 Hz, 1H), 6.62 (dd, J = 8.4, 0.5 Hz, 1H), 6.52 (dd, J = 8.3, 0.7 Hz, 1H), 5.14 (s, 2H), 2.48 (s, 3H).

[0065] 1313C NMR (101 MHz, DMSO) δ 203.43, 159.52, 157.94, 135.96, 133.77, 131.41, 129.98, 121.15, 114.75, 109.63, 103.41, 69.30, 33.04.

[0066] m.p.: 114.8 - 116.8 °C.

[0067] MS (ESI) m / z 320 ( 79 Br), 322 ( 81 Br) [M] + .

[0068] Example 3 Preparation of ethyl 5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxylate (3a)

Chem.

[0069] Starting from 2a (2.583 g, 8.04 mmol), the crude product was prepared according to the general procedure B and purified by recrystallization from methanol or ethyl acetate to give 3a (2.478, 76%) C 19 H 15 BrO5.

[0070] 1 1H NMR (400 MHz, DMSO) δ 8.11 (dd, J = 7.7, 1.4 Hz, 1H), 7.79 (t, J = 8.4 Hz, 1H), 7.68 (dd, J = 8.0, 1.0 Hz, 1H), 7.51 (td, J = 7.6, 1.1 Hz, 1H), 7.33 (td, J = 7.8, 1.7 Hz, 1H), 7.30 - 7.25 (m, 1H), 7.18 - 7.13 (m, 1H), 6.79 (s, 1H), 5.23 (s, 2H), 4.40 (q, J = 7.1 Hz, 2H), 1.36 (t, J = 7.1 Hz, 3H).

[0071] 13 13C NMR (101 MHz, DMSO) δ 176.40, 172.49, 160.01, 158.25, 157.43, 150.24, 135.70, 135.44, 132.17, 129.57, 129.12, 127.85, 121.05, 115.43, 114.62, 110.78, 108.85, 69.72, 62.61, 13.87.

[0072] Melting point: 155.3 - 157.1 °C.

[0073] MS (ESI) m / z 403 ( 79 Br), 405 ( 81 Br) [M-H] + .

[0074] Example 4 Preparation of ethyl 5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxylate (3b)

Chem.

[0075] Starting from 2b (1.718 g, 5.35 mmol), the crude product was prepared according to the general procedure B and purified by recrystallization from methanol or ethyl acetate to obtain 3b (1.516, 70%) C 19 H 15 BrO5.

[0076] 1 1H NMR (400 MHz, DMSO) δ 7.74 (t, J = 8.4 Hz, 1H), 7.64 - 7.56 (m, 4H), 7.25 - 7.21 (m, 1H), 7.13 - 7.08 (m, 1H), 6.77 (s, 1H), 5.24 (s, 2H), 4.38 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H).

[0077] 1313C NMR (101 MHz, DMSO) δ 176.39, 160.01, 157.70, 157.23, 150.19, 136.23, 135.29, 131.21, 128.90, 120.61, 115.43, 114.68, 110.54, 109.02, 69.22, 62.59, 13.87.

[0078] m.p.: 148.0 - 149.4 °C.

[0079] MS (ESI) m / z 402 ( 79 Br), 404 ( 81 Br) [M] + .

[0080] Example 5 Preparation of 5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxylic acid (4a)

Chem.

[0081] Starting from 3a (1.000 g, 2.48 mmol), the crude product was prepared according to the general procedure C, triturated with methanol for purification, and washed with diethyl ether to obtain 4a (0.777 g, 84%). Also, the desired product can be obtained as crystals by recrystallization from methanol. C 17 H 11 BrO 5。

[0082] 11H NMR (400 MHz, DMSO) δ 8.14 (dd, J = 7.7, 1.1 Hz, 1H), 7.77 (t, J = 8.4 Hz, 1H), 7.68 (dd, J = 8.0, 0.9 Hz, 1H), 7.51 (td, J = 7.6, 0.9 Hz, 1H), 7.32 (td, J = 7.8, 1.5 Hz, 1H), 7.26 (d, J = 8.1 Hz, 1H), 7.13 (d, J = 8.3 Hz, 1H), 6.74 (s, 1H), 5.23 (s, 2H).

[0083] 13 13C NMR (101 MHz, DMSO) δ 176.70, 161.44, 157.42, 157.36, 151.31, 135.75, 135.24, 132.13, 129.52, 129.13, 127.82, 121.01, 115.17, 114.63, 110.80, 108.71, 69.71.

[0084] m.p.: 244.3 °C.

[0085] MS (ESI) m / z 373 ( 79 Br), 375 ( 81 Br) [M-H] - .

[0086] Example 6 Preparation of 5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxylic acid (4b)

Chem.

[0087] Starting from 3b (1.586 g, 3.93 mmol), the crude product was prepared according to the general procedure C, triturated with methanol for purification, and washed with diethyl ether to obtain 4b (1.259 g, 85%). The target product can be crystallized from methanol to obtain white crystals. C 17 H 11 BrO 5。

[0088] 1 1H NMR (400 MHz, DMSO) δ 7.76 (t, J = 8.4 Hz, 1H), 7.66 - 7.59 (m, 4H), 7.24 (dd, J = 8.4, 0.7 Hz, 1H), 7.14 - 7.10 (m, 1H), 6.76 (s, 1H), 5.27 (s, 2H).

[0089] 13 13C NMR (101 MHz, DMSO) δ 176.69, 161.44, 157.69, 157.35, 151.15, 136.27, 135.14, 131.20, 128.89, 120.58, 115.21, 114.69, 110.58, 108.93, 69.23.

[0090] m.p.: 204.6 - 205.3 °C.

[0091] MS (ESI) m / z 374 ( 79 Br), 376 ( 81 Br) [M] + .

[0092] Example 7 Preparation of methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucinate (5a)

Chem.

[0093] Starting from 4a (511 mg, 1.36 mmol) and isoleucine methyl ester hydrochloride (0.485 g, 2.72 mmol), the crude product was prepared according to the general procedure D and purified by recrystallization from methanol to give 5a (0.352 g, 51%) C 24 H 24 BrNO6.

[0094] 11H NMR (400 MHz, DMSO) δ 9.23 (d, J = 7.7 Hz, 1H), 8.14 (d, J = 7.4 Hz, 1H), 7.82 (t, J = 8.4 Hz, 1H), 7.69 (d, J = 7.9 Hz, 1H), 7.52 (t, J = 7.4 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.33 (t, J = 7.3 Hz, 1H), 7.17 (d, J = 8.3 Hz, 1H), 6.74 (s, 1H), 5.25 (s, 2H), 4.39 (t, J = 7.7 Hz, 1H), 3.70 (s, 3H), 2.10 - 1.99 (m, 1H), 1.60 - 1.48 (m, J = 11.6, 5.8 Hz, 1H), 1.33 - 1.23 (m, 1H), 0.97 - 0.86 (m, 6H).

[0095] 13 13C NMR (101 MHz, DMSO) δ 176.44, 171.41, 159.61, 157.38, 157.05, 152.97, 135.76, 135.11, 132.17, 129.56, 129.14, 127.85, 121.04, 114.49, 112.75, 111.09, 108.81, 69.70, 57.26, 51.90, 35.48, 25.09, 15.37, 10.77.

[0096] m.p.: 123.1 - 125.7 °C.

[0097] HRMS (ESI / QTOF): Calculated for C 24 H 25 BrNO6 (M + H + ): 502.0865, Found 502.0860.

[0098] Purity (HPLC) > 95%.

[0099] Example 8 Preparation of methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucinate (5b)

Chem.

[0100] Starting from 4b (300 mg, 0.80 mmol) and L-isoleucine methyl ester hydrochloride (290 mg, 1.60 mmol), the crude product was prepared according to the general procedure D and purified by recrystallization from methanol to give 5b (318 mg, 79%) C 24 H 24 BrNO6 was obtained.

[0101] 1 H NMR (400 MHz, DMSO) δ 9.17 (d, J = 7.9 Hz, 1H), 7.77 (t, J = 8.4 Hz, 1H), 7.64 - 7.56 (m, 4H), 7.34 (dd, J = 8.5, 0.7 Hz, 1H), 7.12 (d, J = 8.1 Hz, 1H), 6.70 (s, 1H), 5.25 (s, 2H), 4.37 (t, J = 7.7 Hz, 1H), 3.68 (s, 3H), 2.07 - 1.96 (m, 1H), 1.58 - 1.45 (m, 1H), 1.32 - 1.19 (m, 1H), 0.95 - 0.84 (m, 6H).

[0102] 13 C NMR (101 MHz, DMSO) δ 176.41, 171.40, 159.62, 157.63, 157.04, 152.91, 136.29, 134.95, 131.21, 128.92, 120.59, 114.54, 112.73, 110.86, 108.99, 69.19, 57.24, 51.88, 35.48, 25.09, 15.37, 10.77.

[0103] Example 9 Preparation of methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-leucinate (5c)

Chem.

[0104] Starting from 4a (500 mg, 1.33 mmol) and L-leucine methyl ester hydrochloride (0.483 g, 2.66 mmol), the crude product was prepared according to General Procedure D and purified by recrystallization in methanol to give 5c (234 mg, 35%) C 24 H 24 BrNO6 was obtained.

[0105] 1 H NMR (400 MHz, CDCl3) δ 8.13 (d, J = 7.4 Hz, 1H), 7.57 (t, J = 8.3 Hz, 1H), 7.48 (d, J = 7.9 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 7.18 - 7.05 (m, 3H), 6.98 (s, 1H), 6.90 (d, J = 8.3 Hz, 1H), 5.18 (s, 2H), 4.82 - 4.73 (m, 1H), 3.74 (s, 3H), 1.80 - 1.60 (m, 3H), 0.99 - 0.87 (m, 6H).

[0106] 13 C NMR (101 MHz, CDCl3) δ 177.52, 172.93, 159.05, 158.46, 157.33, 152.34, 135.55, 134.74, 132.10, 129.07, 128.79, 128.12, 120.82, 115.42, 114.21, 110.63, 108.66, 70.38, 52.73, 51.14, 41.81, 25.02, 22.82, 22.05.

[0107] m.p.: 60.3 - 60.4 °C.

[0108] HRMS (ESI / QTOF): Calculated for C 24 H 25 BrNO6 (M + H +) : 502,0865, Measured value: 502,0865.

[0109] Purity (HPLC) > 98%.

[0110] Example 10 Preparation of methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-valinate (5d)

Chem.

[0111] Starting from 4a (0.200 g, 0.53 mmol) and L-valine methyl ester hydrochloride (0.179 g, 1.07 mmol), the crude product was prepared according to the general procedure D and purified by recrystallization in methanol to give 5d (0.126 g, 48%) C 23 H 22 BrNO6 was obtained.

[0112] 1 H NMR (400 MHz, DMSO) δ 9.20 (d, J = 7.8 Hz, 1H), 8.12 (d, J = 7.4 Hz, 1H), 7.82 (t, J = 8.4 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.51 (t, J = 7.5 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.33 (t, J = 7.4 Hz, 1H), 7.16 (d, J = 8.3 Hz, 1H), 6.74 (s, 1H), 5.24 (s, 2H), 4.33 (t, J = 7.7 Hz, 1H), 3.70 (s, 3H), 2.32 - 2.19 (m, 1H), 1.06 - 0.92 (m, 6H).

[0113] 1313C NMR (101 MHz, DMSO) δ 176.42, 171.34, 159.69, 157.41, 157.07, 153.03, 135.76, 135.09, 132.17, 129.57, 129.18, 127.84, 121.07, 114.53, 112.74, 111.10, 108.88, 69.76, 58.56, 51.90, 29.46, 19.04, 18.95.

[0114] m.p.: 127.0 - 128.2 °C.

[0115] HRMS (ESI / QTOF): Calculated for C 23 H 23 BrNO6 (M + H + ): 488.0709, Found 488.0719.

[0116] Purity (HPLC) > 98%.

[0117] Example 11 Preparation of methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-phenylalaninate (5e)

Chem.

[0118] Starting from 4a (0.194 g, 0.52 mmol) and L-phenylalanine methyl ester hydrochloride (0.223 g, 1.04 mmol), the crude product was prepared according to the general procedure D and purified by precipitation with diethyl ether and cyclohexane to give 5e (0.187 g, 70%) C 27 H 22 BrNO6.

[0119] 11H NMR (400 MHz, DMSO) δ 9.47 (s, 1H), 8.13 (dd, J = 7.7, 1.5 Hz, 1H), 7.82 (t, J = 8.4 Hz, 1H), 7.68 (dd, J = 8.0, 1.1 Hz, 1H), 7.51 (td, J = 7.6, 1.1 Hz, 1H), 7.35 - 7.27 (m, 6H), 7.25 - 7.19 (m, 1H), 7.16 (d, J = 8.0 Hz, 1H), 6.64 (s, 1H), 5.24 (s, 2H), 4.77 - 4.71 (m, 1H), 3.69 (s, 3H), 3.26 (dd, J = 13.8, 5.4 Hz, 1H), 3.21 - 3.13 (m, 1H).

[0120] 13 13C NMR (101 MHz, DMSO) δ 176.33, 171.26, 159.21, 157.42, 156.95, 152.77, 137.22, 135.74, 135.24, 132.15, 129.54, 129.11, 129.06, 128.31, 127.84, 126.62, 121.01, 114.43, 112.57, 110.82, 108.85, 69.69, 54.13, 52.20, 35.93, 30.67.

[0121] m.p.: 145.5 - 147.5 °C.

[0122] HRMS (ESI / QTOF): Calculated for C 27 H 23 BrNO6 (M+H + ): 536.0709, Found 536.0711.

[0123] Purity (HPLC) > 97%.

[0124] Example 12 Preparation of methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-phenylalaninate (5f) [Chemistry]

[0125] Starting from 4b (0.472 g, 1.26 mmol) and L-phenylalanine methyl ester hydrochloride (0.453 g, 2.52 mmol), a crude product was prepared according to the general procedure D and purified by recrystallization from methanol to give 5f (0.551 g, 90%) C 27 H 22 BrNO6 was obtained.

[0126] 1 H NMR (400 MHz, DMSO) δ 9.43 (d, J = 7.9 Hz, 1H), 7.79 (t, J = 8.4 Hz, 1H), 7.68 - 7.54 (m, 4H), 7.30 (d, J = 12.6 Hz, 5H), 7.23 (d, J = 6.1 Hz, 1H), 7.13 (d, J = 8.3 Hz, 1H), 6.63 (s, 1H), 5.26 (s, 2H), 4.73 (dd, J = 13.5, 9.1 Hz, 1H), 3.68 (s, 3H), 3.26 (dd, J = 13.8, 5.2 Hz, 1H), 3.16 (dd, J = 13.6, 10.3 Hz, 1H).

[0127] 13 C NMR (101 MHz, DMSO) δ 176.31, 171.22, 159.22, 157.68, 156.94, 152.71, 137.20, 136.26, 135.08, 131.20, 129.05, 128.91, 128.31, 126.62, 120.59, 114.50, 112.56, 110.60, 109.06, 69.21, 54.10, 52.20, 35.93.

[0128] Decomposition point: 153.1 °C.

[0129] HRMS (ESI / QTOF): Calculated C27 H 23 BrNO6(M+H + ) : 536,0709, Measured 536,0704.

[0130] Purity (HPLC) > 96%.

[0131] Example 13 Preparation of methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-tryptophanate (5g) [Chemical formula]

[0132] Starting from 4a (0.100 g, 0.27 mmol) and L-tryptophan methyl ester hydrochloride (0.136 g, 0.53 mmol), a crude product was prepared according to the general procedure D and purified by recrystallization in methanol to give 5g (0.043 g, 28%) C 29 H 23 BrN2O6 was obtained.

[0133] 1 H NMR (400 MHz, DMSO) δ 10.89 (s, 2H), 9.36 (d, J = 7.8 Hz, 2H), 8.12 (d, J = 7.7 Hz, 2H), 7.83 (t, J = 8.4 Hz, 2H), 7.69 (dd, J = 8.0, 0.9 Hz, 2H), 7.62 (d, J = 7.8 Hz, 2H), 7.51 (td, J = 7.5, 0.9 Hz, 2H), 7.39 - 7.28 (m, 6H), 7.25 (d, J = 2.2 Hz, 2H), 7.17 (d, J = 8.3 Hz, 2H), 7.07 (td, J = 7.7, 0.9 Hz, 2H), 6.98 (t, J = 7.4 Hz, 2H), 6.65 (s, 2H), 5.24 (s, 4H), 4.80 - 4.70 (m, 2H), 3.69 (s, 7H).

[0134] 1313C NMR (101 MHz, DMSO) δ 176.36, 171.52, 159.18, 157.43, 156.95, 152.81, 136.09, 135.74, 135.21, 132.17, 129.57, 129.15, 127.85, 127.03, 123.79, 121.05, 121.02, 118.44, 118.04, 114.43, 112.55, 111.48, 110.83, 109.47, 108.89, 69.73, 53.80, 52.19, 26.38. Two proton signals under the peak of water.

[0135] m.p.: 250 - 252.8 °C.

[0136] HRMS (ESI / QTOF): Calculated for C 29 H 24 BrN2O6 (M + H + ) : 575.0818, Found 575.0821.

[0137] Purity (HPLC) > 99%.

[0138] Example 14 Preparation of methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-tryptophanate (5h)

Chem.

[0139] Starting from 4b (0.100 g, 0.27 mmol) and L-tryptophan methyl ester hydrochloride (0.136 g, 0.533 mmol), the crude product was prepared according to the general procedure D and purified by recrystallization from methanol to give 5h (0.126 g, 82%) C 29 H 23 BrN2O6.

[0140] 11H NMR (400 MHz, DMSO) δ 10.90 (s, 1H), 9.34 (d, J = 7.8 Hz, 1H), 7.79 (t, J = 8.4 Hz, 1H), 7.69 - 7.52 (m, 5H), 7.35 (d, J = 8.1 Hz, 1H), 7.29 (d, J = 8.1 Hz, 1H), 7.25 (d, J = 2.2 Hz, 1H), 7.12 (d, J = 8.3 Hz, 1H), 7.10 - 7.03 (m, 1H), 7.02 - 6.94 (m, 1H), 6.63 (s, 1H), 5.25 (s, 2H), 4.80 - 4.69 (m, 1H), 3.68 (s, 3H).

[0141] 13 13C NMR (101 MHz, DMSO) δ 176.34, 171.50, 159.19, 157.67, 156.92, 152.76, 136.25, 136.10, 135.04, 131.20, 128.92, 127.03, 123.78, 121.02, 120.59, 118.44, 118.04, 114.48, 112.54, 111.48, 110.61, 109.47, 109.06, 69.23, 53.79, 52.18, 26.39. Two proton signals under the peak of water.

[0142] m.p.: 235.3 - 236.2 °C.

[0143] HRMS (ESI / QTOF): Calculated for C 29 H 24 BrN2O6 (M + H + ): 575.0818, Found 575.0807.

[0144] Purity (HPLC) > 99%.

[0145] Example 15 Preparation of methyl (5-((4-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-D-tryptophanate (5i)

Chem.

[0146] Starting from 4b (0.300 g, 0.80 mmol) and D-tryptophan methyl ester hydrochloride (0.408 g, 1.60 mmol), the crude product was prepared according to General Procedure D and purified by precipitation with ethyl acetate and cyclohexane to give 5i (0.375 g, 81%) C 29 H 23 BrN2O6 was obtained.

[0147] 1 H NMR (400 MHz, DMSO) δ 10.90 (s, 1H), 9.34 (d, J = 7.8 Hz, 1H), 7.79 (t, J = 8.4 Hz, 1H), 7.66 - 7.57 (m, 5H), 7.35 (d, J = 8.1 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 7.25 (d, J = 2.1 Hz, 1H), 7.12 (d, J = 8.3 Hz, 1H), 7.07 (t, J = 7.2 Hz, 1H), 6.98 (t, J = 7.4 Hz, 1H), 6.63 (s, 1H), 5.25 (s, 2H), 4.79 - 4.70 (m, 1H), 3.69 (s, 3H), 3.43 - 3.38 (m, 1H), 3.33 - 3.27 (m, 1H).

[0148] 13 C NMR (101 MHz, DMSO) δ 176.33, 171.51, 159.18, 157.67, 156.93, 152.75, 136.26, 136.10, 135.04, 131.20, 128.91, 127.03, 123.78, 121.01, 120.59, 118.43, 118.04, 114.48, 112.54, 111.48, 110.60, 109.47, 109.04, 69.21, 53.79, 52.18, 26.38.

[0149] m.p.: 236.2 - 237.9 °C.

[0150] HRMS (ESI / QTOF): Calculated for C 29 H 24 BrN2O6 (M + H + ): 575.0818, Found 575.0822.

[0151] Purity (HPLC) > 99%.

[0152] Example 16 Preparation of 5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucine (6a)

Chem.

[0153] Starting from 5a (0.256 g, 0.51 mmol), the crude product was prepared according to the general procedure E and purified by recrystallization in methanol to give 6a (0.114 g, 46%) C 23 H 22 BrNO6 was obtained.

[0154] 11H NMR (400 MHz, DMSO) δ 8.99 (d, J = 8.1 Hz, 1H), 8.14 (d, J = 7.5 Hz, 1H), 7.82 (t, J = 8.4 Hz, 1H), 7.69 (dd, J = 7.9, 0.8 Hz, 1H), 7.52 (td, J = 7.6, 0.8 Hz, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.33 (td, J = 7.8, 1.5 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 6.73 (s, 1H), 5.25 (s, 2H), 4.39 - 4.31 (m, 1H), 2.08 - 1.98 (m, 1H), 1.61 - 1.48 (m, 1H), 1.35 - 1.23 (m, 1H), 0.97 (d, J = 6.8 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H).

[0155] 13 13C NMR (101 MHz, DMSO) δ 176.46, 172.23, 159.43, 157.39, 157.06, 153.18, 135.76, 135.07, 132.16, 129.57, 129.16, 127.84, 121.06, 114.49, 112.64, 111.12, 108.84, 69.73, 57.21, 35.62, 25.04, 15.52, 10.96.

[0156] m.p.: 242.8 - 243.7 °C.

[0157] HRMS (ESI / QTOF): Calculated for C 23 H 23 BrNO6 (M + H + ): 488.0709, Found 488.0712.

[0158] Purity (HPLC) > 98%.

[0159] Example 17 Preparation of 5-((4-Bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonyl)-L-alloisoleucine (6b)

Chem.

[0160] Starting from 5b (671 mg, 0.51 mmol), the crude product was prepared according to the general procedure E and purified by recrystallization in methanol to give 6b (419 mg, 64%) C 23 H 22 BrNO6 was obtained.

[0161] 1 H NMR (400 MHz, DMSO) δ 12.91 (s, 1H), 8.98 (d, J = 8.1 Hz, 1H), 7.78 (t, J = 8.4 Hz, 1H), 7.67 - 7.57 (m, 4H), 7.37 (dd, J = 8.5, 0.6 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H), 6.72 (s, 1H), 5.26 (s, 2H), 4.40 - 4.29 (m, 1H), 2.07 - 1.96 (m, 1H), 1.61 - 1.45 (m, 1H), 1.38 - 1.20 (m, 1H), 0.96 (d, J = 6.8 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H). (ERO1-94)

[0162] 13 C NMR (101 MHz, DMSO) δ 176.44, 172.23, 159.44, 157.63, 157.04, 153.12, 136.28, 134.91, 131.20, 128.93, 120.59, 114.54, 112.63, 110.89, 108.99, 69.21, 57.19, 35.63, 25.04, 15.52, 10.96.

[0163] m.p.: 230.1 - 230.4 °C.

[0164] HRMS (ESI / QTOF): Calculated C 23 H 23 BrNO6(M+H + ): 488.0709, Found 488.0715.

[0165] Purity (HPLC) > 98%.

[0166] Example 18 Preparation of 5-((2-Bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-phenylalanine (6c)

Chem.

[0167] Starting from 5e (0.320 g, 0.60 mmol), the crude product was prepared according to the general procedure E and purified by recrystallization from methanol to give 6c (0.210 g, 67%) C 26 H 20 BrNO6.

[0168] 1 H NMR (400 MHz, DMSO) δ 9.29 (d, J = 8.2 Hz, 2H), 8.11 (dd, J = 7.6, 1.0 Hz, 2H), 7.82 (t, J = 8.4 Hz, 2H), 7.68 (dd, J = 7.9, 0.8 Hz, 2H), 7.50 (td, J = 7.6, 0.9 Hz, 2H), 7.37 - 7.25 (m, 11H), 7.24 - 7.10 (m, 4H), 6.63 (s, 2H), 5.23 (s, 4H), 4.72 - 4.58 (m, 2H), 3.14 (dd, J = 13.8, 10.3 Hz, 5H). One peak under the signal of water.

[0169] 1313C NMR (101 MHz, DMSO) δ 176.37, 172.20, 159.04, 157.44, 156.96, 153.00, 137.72, 135.73, 135.20, 132.16, 129.57, 129.17, 129.02, 128.25, 127.83, 126.47, 121.06, 114.45, 112.43, 110.85, 108.91, 69.75, 54.18, 36.01.

[0170] m.p.: 240.4 - 241.7 °C.

[0171] HRMS (ESI / QTOF): Calculated for C 26 H 21 BrNO6 (M + H + ): 522.0552, Found 522.0558

[0172] Example 19 Preparation of 5-((4-Bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-phenylalanine (6d)

Chem.

[0173] Starting from 5f (0.551 g, 1.03 mmol), the crude product was prepared according to the general procedure E and purified by recrystallization in methanol to give 6d (0.446 g, 83%) of C 26 H 20 BrNO6.

[0174] 11H NMR (400 MHz, DMSO) δ 8.51 (d, J = 6.8 Hz, 1H), 7.72 (t, J = 8.4 Hz, 1H), 7.66 - 7.57 (m, 4H), 7.24 - 7.16 (m, 5H), 7.16 - 7.07 (m, 2H), 6.62 (s, 1H), 5.24 (s, 2H), 4.24 (dd, J = 11.0, 5.9 Hz, 1H), 3.26 (dd, J = 13.5, 4.6 Hz, 1H), 3.11 (dd, J = 13.5, 6.8 Hz, 1H). (ERO1-71)

[0175] 13 13C NMR (101 MHz, DMSO) δ 176.35, 172.19, 159.11, 157.67, 156.94, 152.89, 137.66, 136.26, 135.06, 131.20, 129.02, 128.91, 128.27, 126.50, 120.58, 114.47, 112.44, 110.62, 109.04, 69.21, 54.08, 35.94.

[0176] Decomposition point: 231.5 °C.

[0177] HRMS (ESI / QTOF): Calculated for C 26 H 21 BrNO6(M+H + ): 522.0552, Found 522.0559.

[0178] Purity (HPLC) > 95%.

[0179] Example 20 Preparation of 5-((4-Bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-D-tryptophan (6e)

Chem.

[0180] Starting from 5i (0.250 g, 0.27 mmol), the crude product was prepared according to the general procedure E and purified by precipitation with ethyl acetate and cyclohexane to give 6e (0.158 g, 66%) C 28 H 21 BrN2O6 was obtained.

[0181] 1 H NMR (400 MHz, DMSO) δ 10.87 (d, J = 1.4 Hz, 1H), 9.18 (d, J = 8.1 Hz, 1H), 7.79 (t, J = 8.4 Hz, 1H), 7.69 - 7.56 (m, 5H), 7.34 (d, J = 8.1 Hz, 1H), 7.29 (d, J = 8.2 Hz, 1H), 7.24 (d, J = 2.2 Hz, 1H), 7.13 (d, J = 8.3 Hz, 1H), 7.10 - 7.04 (m, 1H), 7.01 - 6.95 (m, 1H), 6.63 (s, 1H), 5.25 (s, 2H), 4.73 - 4.65 (m, 1H). Two signals under the peak of water.

[0182] 13 C NMR (101 MHz, DMSO) δ 176.37, 172.52, 159.06, 157.66, 156.92, 152.92, 136.26, 136.08, 135.04, 131.20, 128.91, 127.09, 123.66, 120.98, 120.58, 118.39, 118.16, 114.44, 112.42, 111.43, 110.59, 109.93, 109.00, 69.18, 53.73, 26.37. Two proton signals under the peak of water.

[0183] m.p.: 185.5 - 186.7 °C.

[0184] HRMS (ESI / QTOF): Calculated C 28 H 22 BrN2O6 (M + H +) : 561,0661, Measured 561,0672.

[0185] Purity (HPLC) > 99%.

[0186] Examples 21 - 27 Series 2

Chem.

[0187] Note) In the following protocol, "Molecular number + a" refers to the case where bromine is at the 2 - position of the aromatic ring, and "Molecular number + b" refers to the case where bromine is at the 4 - position of the aromatic ring.

[0188] Note: Up to step e, the synthetic scheme is the same as that of Series 1. Only step f, which enables access to compound 7, is different.

[0189] General operating procedure F: To a solution of carboxylic acid derivative 6 (1 equivalent) in anhydrous DMF (20 mL / mmol), TBTU (1.5 equivalents or 2 equivalents) was added. This solution was stirred at room temperature for 30 minutes. Then, in the presence of DIEA (5 equivalents), a solution of amino acid derivative (1.5 or 2 equivalents) dissolved in DMF (10 mL / mmol) was carefully added to the above - mentioned solution. The reaction mixture was stirred at room temperature for 24 hours and monitored by TLC (cyclohexane / ethyl acetate 3:2). This solution was poured into acidic water (1M HCl) and extracted with ethyl acetate. The organic phase was collected, washed with 20% NaHCO3 solution and brine, dried over magnesium sulfate, filtered, and evaporated under vacuum.

[0190] Example 21 Preparation of methyl (5-((2-Bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucyl-L-valinate (7a)

Chem.

[0191] Starting from 6a (0.091 g, 0.19 mmol) and L-valine methyl ester (0.064 g, 0.38 mmol), a crude product was prepared according to General Procedure F, purified by precipitation in ethyl acetate and a minimal amount of cyclohexane, and washed with diethyl ether to give 7a (0.042 g, 37%) C 29 H 33 BrN2O7 was obtained.

[0192] 1 H NMR (400 MHz, DMSO) δ 8.79 (d, J = 8.4 Hz, 0.5H), 8.60 - 8.50 (m, 1H), 8.45 (d, J = 7.2 Hz, 0.5H), 8.13 (d, J = 7.5 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.52 (t, J = 7.3 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.33 (t, J = 7.3 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 6.72 (d, J = 5.0 Hz, 1H), 5.25 (s, 2H), 4.73 - 4.65 (m, 0.5H), 4.54 - 4.45 (m, 0.5H), 4.27 - 4.21 (m, 0.5H), 4.21 - 4.14 (m, 0.5H), 3.65 (d, J = 4.7 Hz, 3H), 2.13 - 1.92 (m, 2H), 1.64 - 1.47 (m, 1H), 1.28 - 1.14 (m, 1H), 1.04 - 0.70 (m, 12H).

[0193] 1313C NMR (101 MHz, DMSO) δ 172.31, 171.91, 171.88, 157.84, 157.83, 157.82, 157.51, 157.49, 157.46, 157.43, 157.42, 153.69, 153.67, 153.65, 135.92, 132.79, 130.34, 129.78, 128.33, 121.94, 114.74, 114.72, 114.70, 114.66, 112.89, 112.87, 112.69, 111.52, 111.46, 109.48, 109.46, 109.37, 109.35, 70.33, 70.31, 58.31, 58.30, 58.21, 58.05, 58.02, 57.96, 52.33, 52.26, 52.23, 52.21, 36.32, 30.00, 25.02, 19.31, 19.17, 19.13, 18.66, 18.59, 15.29, 15.28, 10.85.

[0194] HRMS (ESI / QTOF): Calculated for C 29 H 34 BrN2O7 (M+H + ): 601.1549, Found 601.1533.

[0195] Example 22 Preparation of methyl (5-((2-Bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucyl-L-leucinate (7b)

Chem.

[0196] Starting from 6a (0.082 mg, 0.16 mmol) and L-leucine methyl ester (0.061 g, 0.32 mmol), the crude product was prepared according to the general procedure F, purified by precipitation in ethyl acetate and a minimal amount of cyclohexane, and washed with diethyl ether to give 7b (0.030 g, 31%) C 30 H 35 BrN2O7 was obtained.

[0197] 1 1H NMR (400 MHz, DMSO) δ 8.79 (d, J = 8.8 Hz, 0.5H), 8.64 (d, J = 7.6 Hz, 1H), 8.56 (d, J = 7.7 Hz, 0.5H), 8.13 (d, J = 7.6 Hz, 1H), 7.81 (t, J = 8.2 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.40 (dd, J = 8.3, 4.4 Hz, 1H), 7.33 (t, J = 7.5 Hz, 1H), 7.16 (d, J = 8.1 Hz, 1H), 6.73 (d, J = 7.3 Hz, 1H), 5.25 (s, 2H), 4.64 - 4.56 (m, 0.5H), 4.41 (t, J = 8.7 Hz, 1H), 4.37 - 4.27 (m, 0.5H), 3.63 (s, 1H), 2.05 - 1.92 (m, 1H), 1.74 - 1.43 (m, 4H), 1.27 - 1.13 (m, 1H), 0.98 - 0.81 (m, 12H).

[0198] 13 13C NMR (126 MHz, DMSO) δ 177.64, 174.72, 173.46, 173.29, 171.55, 171.48, 159.81, 159.64, 157.82, 157.41, 153.65, 153.63, 135.89, 135.84, 132.77, 130.31, 129.75, 128.32, 121.89, 114.67, 112.93, 112.88, 111.50, 109.43, 70.31, 58.05, 57.49, 52.53, 52.42, 52.32, 51.52, 51.02, 50.90, 41.57, 40.89, 37.32, 36.39, 25.92, 24.96, 24.70, 24.64, 23.10, 23.01, 22.99, 21.82, 21.57, 21.44, 21.23, 15.31, 14.95, 11.64, 10.85.

[0199] HRMS (ESI / QTOF): Calculated C 30 H 36 BrN2O7(M+H + ) : 615,1690, Found 615,1684.

[0200] Example 23 Preparation of methyl (5-((4-Bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucyl-L-valinate (7c) [Chemical formula]

[0201] Starting from 6a (0.123 g, 0.25 mmol) and L-valine methyl ester (0.084 g, 0.50 mmol), a crude product was prepared according to the general procedure F and purified by trituration in diethyl ether to give 7c (0.061 g, 41%) C 29 H 33 BrN2O7 was obtained.

[0202] 11H NMR (400 MHz, DMSO) δ 8.76 (d, J = 8.6 Hz, 0.5H), 8.54 (d, J = 9.4 Hz, 1H), 8.43 (d, J = 7.5 Hz, 0.5H), 7.76 (t, J = 8.3 Hz, 1H), 7.60 (q, J = 8.6 Hz, 4H), 7.35 (d, J = 8.2 Hz, 1H), 7.11 (d, J = 8.3 Hz, 1H), 6.69 (d, J = 5.4 Hz, 1H), 5.25 (s, 2H), 4.67 (dd, J = 8.8, 6.9 Hz, 0.5H), 4.48 (t, J = 8.8 Hz, 0.5H), 4.22 (dd, J = 7.9, 6.9 Hz, 0.5H), 4.15 (t, J = 7.0 Hz, 0.5H), 3.63 (d, J = 4.9 Hz, 3H), 2.12 - 1.90 (m, 2H), 1.59 - 1.36 (m, 1H), 1.25 - 1.11 (m, 1H), 0.99 - 0.81 (m, 12H).

[0203] 13 13C NMR (101 MHz, DMSO) δ 176.43, 171.93, 171.64, 170.88, 170.67, 159.10, 158.94, 157.64, 157.01, 153.21, 136.29, 134.90, 131.19, 128.91, 120.58, 114.52, 112.61, 112.54, 110.83, 109.00, 69.21, 57.66, 57.42, 57.24, 56.72, 51.72, 51.53, 37.09, 35.99, 29.85, 29.58, 25.65, 24.62, 19.02, 18.84, 18.35, 18.20, 14.95, 14.58, 11.37, 10.55.

[0204] HRMS (ESI / QTOF): Calculated for C 29 H 34 BrN2O7(M+H + ) : 601.1549, Actual measurement: 601,1545.

[0205] Example 24 Preparation of methyl (5-((4-Bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucyl-L-leucinate (7d) [Chemical formula]

[0206] Starting from 6b (0.123 g, 0.25 mmol) and L-leucine methyl ester (0.091 mg, 0.50 mmol), a crude product was prepared according to the general procedure F and purified by trituration in diethyl ether to give 7d (0.052 g, 34%) C 30 H 35 BrN2O7 was obtained.

[0207] 1 H NMR (400 MHz, DMSO) δ 8.76 (d, J = 8.7 Hz, 0.5H), 8.62 (d, J = 7.8 Hz, 0.5H), 8.54 (dd, J = 8.0, 4.5 Hz, 1H), 7.76 (t, J = 8.2 Hz, 1H), 7.66 - 7.56 (m, 4H), 7.35 (dd, J = 8.4, 4.2 Hz, 1H), 7.11 (d, J = 8.2 Hz, 1H), 6.70 (d, J = 7.5 Hz, 1H), 5.25 (s, 2H), 4.58 (dd, J = 8.6, 6.5 Hz, 0.5H), 4.39 (t, J = 8.6 Hz, 0.5H), 4.36 - 4.24 (m, 1H), 3.62 (d, J = 2.3 Hz, 3H), 2.03 - 1.89 (m, 1H), 1.70 - 1.55 (m, 2H), 1.55 - 1.45 (m, 2H), 1.24 - 1.10 (m, 1H), 0.96 - 0.79 (m, 12H).

[0208] 1313C NMR 176.43, 172.81, 172.60, 170.56, 170.42, 159.14, 158.95, 157.63, 157.01, 153.19, 136.28, 134.90, 131.19, 128.91, 120.57, 114.51, 112.62, 112.54, 110.84, 109.00, 69.20, 57.29, 56.64, 51.86, 51.71, 50.39, 50.23, 36.98, 36.00, 25.59, 24.56, 24.23, 24.18, 22.78, 22.66, 21.28, 20.90, 14.99, 14.62, 10.54.

[0209] HRMS (ESI / QTOF): Calculated for C 30 H 36 BrN2O7(M+H + ) : 615.1690, Found 615.1690.

[0210] Example 25 Preparation of (S)-5-((2-Bromobenzyl)oxy)-N-(1-((2-(5-Hydroxy-1H-indol-3-yl)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)-4-oxo-4H-chromene-2-carboxamide (7e)

Chem.

[0211] Starting from 6c (0.127 mg, 0.24 mmol) and 5-hydroxytryptamine hydrochloride (0.103 mg, 0.48 mmol), the crude product was prepared according to the general procedure F, purified by trituration with methanol, and subsequently washed with diethyl ether to give 7e (0.063 g, 38%) C 36 H 30 BrN3O6 was obtained.

[0212] 11H NMR (400 MHz, DMSO) δ 10.49 (d, J = 1.6 Hz, 1H), 9.11 (d, J = 8.4 Hz, 1H), 8.61 (s, 1H), 8.31 (t, J = 5.5 Hz, 1H), 8.10 (dd, J = 7.7, 0.8 Hz, 1H), 7.79 (t, J = 8.4 Hz, 1H), 7.65 (dd, J = 8.0, 0.7 Hz, 1H), 7.48 (td, J = 7.6, 0.7 Hz, 1H), 7.37 - 7.23 (m, 6H), 7.18 (t, J = 7.2 Hz, 1H), 7.12 (d, J = 8.6 Hz, 2H), 7.04 (d, J = 2.1 Hz, 1H), 6.86 (d, J = 2.1 Hz, 1H), 6.63 (s, 1H), 6.60 (dd, J = 8.6, 2.2 Hz, 1H), 5.21 (s, 2H), 4.73 - 4.65 (m, 1H), 3.34 - 3.26 (m, 1H), 3.17 (dd, J = 13.7, 4.5 Hz, 1H), 3.05 (dd, J = 13.6, 10.2 Hz, 1H), 2.73 (t, J = 7.5 Hz, 2H).

[0213] 13 13C NMR (101 MHz, DMSO) δ 176.46, 170.03, 158.99, 157.41, 156.99, 153.19, 150.16, 137.88, 135.74, 135.14, 132.15, 130.81, 129.55, 129.15, 128.14, 127.83, 126.38, 123.13, 121.06, 114.44, 112.36, 111.65, 111.27, 110.93, 110.64, 108.83, 102.20, 69.74, 54.91, 37.11, 25.14. 31C + 2EQ 3C disappeared.

[0214] HRMS (ESI / QTOF): Calculated for C 36 H 31BrN3O6(M+H + ) : 680,1396, Measured 680,1392.

[0215] Example 26 Preparation of (S)-N-(1-((2-(1H-Indol-3-yl)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)-5-((4-Bromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxamide (7f)

Chem.

[0216] Starting from 6d (0.090 g, 0.17 mmol) and tryptamine hydrochloride (0.067 g, 0.34 mmol), the crude product was prepared according to the general procedure F, triturated in ethyl acetate and a minimum amount of cyclohexane, and washed with diethyl ether to give 7f (0.014 g, 12%) C 36 H 30 BrN3O5 was obtained.

[0217] 1 H NMR (400 MHz, CDCl3) δ 8.20 (s, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.57 (t, J = 8.4 Hz, 1H), 7.54 - 7.44 (m, 5H), 7.32 - 7.15 (m, 6H), 7.12 - 7.06 (m, 2H), 7.06 - 7.01 (m, 1H), 6.89 (s, 1H), 6.85 (d, J = 8.2 Hz, 1H), 6.80 (s, 1H), 5.99 - 5.91 (m, 1H), 5.19 (s, 2H), 4.72 (dd, J = 13.5, 7.7 Hz, 1H), 3.53 (dd, J = 11.8, 5.9 Hz, 2H), 3.20 (dd, J = 13.4, 5.6 Hz, 1H), 3.06 (dd, J = 13.3, 8.4 Hz, 1H), 2.88 (dt, J = 13.1, 6.4 Hz, 1H), 2.84 - 2.73 (m, 1H).

[0218] 1313C NMR (101 MHz, CDCl3) δ 177.67, 170.09, 159.03, 158.55, 157.39, 152.34, 136.50, 136.16, 135.38, 134.72, 131.87, 129.44, 128.91, 128.45, 127.44, 127.17, 122.32, 122.19, 121.84, 119.59, 118.58, 115.56, 113.88, 112.34, 111.42, 110.84, 109.06, 70.35, 60.54, 55.24, 39.94, 38.96, 29.82, 25.00, 21.18, 14.33. 36C instead of 32.

[0219] HRMS (ESI / QTOF): Calculated for C 36 H 31 BrN3O5(M+H + ) : 664.1447, Found 664.1432.

[0220] Example 27 Preparation of (R)-N-(1-((2-(1H-Indol-3-yl)ethyl)amino)-3-(1H-Indol-3-yl)-1-oxopropan-2-yl)-5-((4-Bromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxamide (7g)

Chem.

[0221] Starting from 6e (0.069 g, 0.13 mmol) and tryptamine hydrochloride (0.048 g, 0.25 mmol), the crude product was prepared according to the general procedure F and purified by trituration in diethyl ether to give 7 g (0.051 mg, 59%) of C 38 H 31 BrN4O5.

[0222] 11H NMR (400 MHz, DMSO) δ 10.83 (s, 2H), 8.98 (d, J = 8.2 Hz, 1H), 8.34 (t, J = 5.3 Hz, 1H), 7.78 (t, J = 8.4 Hz, 1H), 7.70 (d, J = 7.7 Hz, 1H), 7.60 (q, J = 8.6 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.36 - 7.26 (m, 3H), 7.18 (dd, J = 31.1, 0.9 Hz, 2H), 7.11 (d, J = 8.4 Hz, 1H), 7.08 - 7.01 (m, 2H), 7.00 - 6.92 (m, 2H), 6.61 (s, 1H), 5.24 (s, 2H), 4.72 - 4.63 (m, 1H), 3.30 - 3.14 (m, 2H), 2.82 (t, J = 7.3 Hz, 2H). Two signals under the peak of water.

[0223] 13 13C NMR (101 MHz, DMSO) δ 176.46, 170.44, 158.90, 157.64, 156.94, 153.16, 136.27, 136.19, 136.04, 134.99, 131.20, 128.90, 127.24, 127.15, 123.76, 122.67, 120.89, 120.88, 120.58, 118.53, 118.21, 114.43, 112.31, 111.66, 111.33, 110.67, 110.08, 108.95, 69.17, 54.44, 27.38, 25.02. 32C + 2 EQ 4C disappeared.

[0224] HRMS (ESI / QTOF): Calculated for C 38 H 31 BrN4O5(M+H + ): 703.1556, Found 703.1553.

[0225] Examples 28 - 36 Series 3 [Chemistry]

[0226] Note) In the following protocol, "Molecular number + a" refers to the case where bromine is at the 2- and 4-positions of the aromatic ring, and "Molecular number + b" refers to the case where bromine is at the 3- and 5-positions of the aromatic ring.

[0227] General operation mode A: 2,5-Dihydroxyacetophenone 3 (1 equivalent) was dissolved in acetone (11 mL / mmol). Next, K2CO3 (3 equivalents) and tetra-n-butylammonium bromide (TBAB) (1.5 equivalents) were mixed, weighed, and added to the solution. The resulting suspension was refluxed for 30 minutes, and a solution of dibromo-1-(bromomethyl)benzene (1 equivalent) in acetone (4 mL / mmol) was added. After refluxing this suspension for 30 minutes, it was concentrated under vacuum. The reaction was monitored by TLC (cyclohexane / ethyl acetate 7:3).

[0228] The reaction mixture was poured into ethyl acetate and acidic water (1 M HCl). The aqueous layer was extracted with ethyl acetate (3 times), and then the combined organic layers were washed with acidic water (1 M HCl) and brine (1 time). The combined organic layers were dried over MgSO4, filtered, and evaporated under vacuum. Finally, this crude product was dried under high vacuum.

[0229] General procedure B: Sodium (6 equivalents) was dissolved in cold anhydrous ethanol (3 mL / mmol) to obtain a fresh solution of sodium ethoxide. This solution was added dropwise to a cold (0 °C) solution of 4 (1 equivalent) in dry THF (the same amount as ethanol). Next, diethyl oxalate (4 equivalents) was added to the solution, and it was stirred at room temperature for 30 minutes. The resulting solution was warmed to 50 °C and monitored by TLC (cyclohexane / ethyl acetate 3:2). A precipitate of the reaction intermediate occurred during the reaction.

[0230] After 4 hours, 37% hydrochloric acid was added dropwise to the solution until the precipitate became white. After the color of the reaction solution changed, it was refluxed for 1.5 hours. Then, the reaction mixture was evaporated and poured into ethyl acetate and acidic water (1M HCl). The aqueous layer was extracted with ethyl acetate until the color changed (3 times). The combined organic layers were washed with acidic water (1M HCl) and brine (1 time), dried over MgSO4, and then evaporated.

[0231] General operating procedure C: A solution of K2CO3 (1.3 equivalents) in water (15 mL / mmol) was added to a solution of 5 (1 equivalent) in THF (30 mL / mmol) and ethanol (EtOH) (10 mL / mmol). The resulting solution was heated to 50 °C and stirred for 1.5 hours. The reaction was monitored by TLC (cyclohexane / ethyl acetate 7:3). The reaction mixture was concentrated and poured into dichloromethane and acidic water (1M HCl).

[0232] To increase the solubility of the target product in the organic layer, a few drops of methanol were added. The aqueous layer was extracted with dichloromethane (3 times), and the combined organic layers were washed with acidic water (1M HCl) and brine (1 time). Then, the combined organic layers were dried over MgSO4, filtered, and evaporated.

[0233] Example 28 Preparation of 2,4-Dibromo-1-(bromomethyl)benzene (2)

Chemical formula

[0234] 2,4-Dibromotoluene 1 (1000 g, 4.00 mmol) and freshly purified N-bromosuccinimide (NBS) (0.925 g, 5.20 mmol) were dissolved in 12 mL of 1,2-dichloroethane under an inert atmosphere. The solution was refluxed for 10 minutes, and azobisisobutyronitrile (AIBN) (0.328 g, 2.00 mmol) was added. The resulting suspension was stirred and refluxed for 6 hours. The reaction was monitored by TLC (100% cyclohexane). Thereafter, the reaction mixture was evaporated, and a cold 1:1 cyclohexane / dichloromethane solution was added to precipitate the by-products (white solid). After filtration and evaporation, the crude product 2 (1.476 g, 4.49 mmol) was used without purification. C7H5Br3

[0235] Example 29 Preparation of 1-(2-((2,4-dibromobenzyl)oxy)-6-hydroxyphenyl)ethan-1-one (4a) [Chemical formula]

[0236] Starting from 2 (1.315 g, 4.00 mmol) and 3 (1.000 g, 4.00 mmol), the crude product was prepared according to the general procedure A. The crude product was precipitated with a 1:1 cyclohexane / dichloromethane solution and then recrystallized from isopropanol to give 4a (0.793 g, 50%) C 15 H 12 Br2O3 was obtained.

[0237] 1 1H NMR (400 MHz, DMSO) δ 11.64 (s, 1H), 7.95 (d, J = 1.8 Hz, 1H), 7.66 (dd, J = 8.2, 1.8 Hz, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.32 (t, J = 8.3 Hz, 1H), 6.62 (d, J = 8.3 Hz, 1H), 6.55 (d, J = 8.2 Hz, 1H), 5.13 (s, 2H), 2.45 (s, 3H).

[0238] 13 C NMR (101 MHz, DMSO) δ 203.27, 159.54, 157.71, 134.92, 134.60, 133.83, 132.06, 131.01, 123.92, 122.19, 114.68, 109.93, 103.16, 69.38, 32.88.

[0239] HRMS (ESI / LTQ Orbitrap): Calculated for C 15 H 11 O3Br2 (M - H + ): 396.9080, Found 396.9082.

[0240] Example 30 Preparation of 1-(2-((3,5-dibromobenzyl)oxy)-6-hydroxyphenyl)ethan-1-one (4b)

Chemistry

[0241] The crude product was prepared from 1,3 - dibromo - 5 - (bromomethyl)benzene (1.315 g, 4.00 mmol) and 3 (1.000 g, 4.00 mmol) according to the general procedure A. It was purified by trituration with diethyl ether to give 4b (1.103 g, 70%) C 15 H 12 O3Br2.

[0242] 1 H NMR (400 MHz, DMSO) δ 11.53 (s, 1H), 7.79 (s, 1H), 7.69 (d, J = 1.5 Hz, 2H), 7.29 (t, J = 8.3 Hz, 1H), 6.58 (d, J = 8.3 Hz, 1H), 6.54 (d, J = 8.2 Hz, 1H), 5.17 (s, 2H), 2.53 - 2.46 (m, 5H).

[0243] 1313C NMR (101 MHz, DMSO) δ 203.16, 158.98, 157.30, 141.43, 133.41, 132.81, 129.45, 122.49, 115.30, 109.78, 103.40, 68.24, 32.91.

[0244] HRMS (ESI / LTQ Orbitrap): Calculated for C 15 H 11 O3Br2 (M - H + ): 396.9080, Found 396.9078.

[0245] Example 31 Preparation of ethyl 5-((2,4-dibromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxylate (5a)

Chem.

[0246] Starting from 4a (0.435 g, 1.09 mmol) and diethyl oxalate (0.636 g, 4.35 mmol), the crude product was prepared according to General Procedure B. The resulting oil was solidified under high vacuum and heated with the addition of isopropanol.

[0247] The resulting suspension was filtered, the pasty product was dissolved in dichloromethane and evaporated to give a white solid. The desired product 5a (0.144 g, 27%). C 19 H 14 O5Br2

[0248] 11H NMR (400 MHz, DMSO) δ 8.08 (d, J = 8.3 Hz, 1H), 7.94 (d, J = 1.9 Hz, 1H), 7.80 (t, J = 8.4 Hz, 1H), 7.74 (dd, J = 8.3, 1.9 Hz, 1H), 7.29 (d, J = 8.5 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H), 6.81 (s, 1H), 5.19 (s, 2H), 4.40 (q, J = 7.1 Hz, 2H), 1.36 (t, J = 7.1 Hz, 3H).

[0249] 13 13C NMR (101 MHz, DMSO) δ 176.44, 159.99, 157.23, 157.22, 150.30, 135.46, 135.43, 134.04, 130.85, 130.60, 121.82, 121.29, 115.42, 114.59, 110.90, 108.90, 69.29, 62.62, 13.87.

[0250] HRMS (ESI / LTQ Orbitrap): Calculated for C 19 H 15 O5Br2(M+H + ) : 480.9281, Found 480.9271.

[0251] Example 32 Preparation of ethyl 5-((3,5-dibromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxylate (5b)

Chemical formula

[0252] Starting from 4b (1.103 g, 2.76 mmol) and diethyl oxalate (1.612 g, 14.03 mmol), the crude product was prepared according to General Procedure B. It was purified by trituration with diethyl ether to give 5b (0.785 g, 59%) C 19 H 14 Br2O5 was obtained.

[0253] 1 1H NMR (400 MHz, DMSO) δ 7.91 (d, J = 1.6 Hz, 2H), 7.81 - 7.74 (m, 2H), 7.26 (d, J = 8.1 Hz, 1H), 7.08 (d, J = 8.3 Hz, 1H), 6.83 (s, 1H), 5.27 (s, 2H), 4.38 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H).

[0254] 13 13C NMR (101 MHz, DMSO) δ 176.55, 159.99, 157.32, 157.22, 150.28, 141.68, 135.40, 132.26, 128.38, 122.44, 115.46, 114.62, 110.82, 108.83, 68.21, 62.60, 13.86.

[0255] HRMS (ESI / LTQ Orbitrap): Calculated for C 19 H 15 Br2O5(M+H + ): 480.9281, Found 480.9274.

[0256] Example 33 Preparation of 5-((2,4-dibromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxylic acid (6a)

Chem.

[0257] Starting from 5a (0.144 g, 0.30 mmol), the crude product was prepared according to the general procedure C and purified by trituration with 1:1 diethyl ether / dichloromethane to give 6a (0.074 g, 55%) C 17 H 10 Br2O5 was obtained.

[0258] 1 1H NMR (400 MHz, DMSO) δ 8.07 (d, J = 8.3 Hz, 1H), 7.90 (d, J = 1.9 Hz, 1H), 7.76 (t, J = 8.4 Hz, 1H), 7.70 (dd, J = 8.3, 1.9 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.75 (s, 1H), 5.15 (s, 2H).

[0259] 13 13C NMR (101 MHz, DMSO) δ 176.72, 161.42, 157.30, 157.17, 151.21, 135.46, 135.28, 133.97, 130.81, 130.55, 121.71, 121.23, 115.20, 114.54, 110.90, 108.69, 69.23, 64.89.

[0260] HRMS (ESI / LTQ Orbitrap): Calculated for C 17 H 11 Br2O5(M+H + ) : 454.8948, Found 454.8937.

[0261] Example 34 Preparation of 5-((3,5-dibromobenzyl)oxy)-4-oxo-4H-chromene-2-carboxylic acid (6b)

Chem.

[0262] Starting from 5b (0.785 g, 1.63 mmol), the crude product was prepared according to General Procedure C and purified by trituration with 1:1 diethyl ether / dichloromethane to give 6b (0.493 g, 67%) C 17 H 10 Br2O5 was obtained.

[0263] 11H NMR (400 MHz, DMSO) δ 7.91 (s, 2H), 7.82 - 7.72 (m, 2H), 7.24 (d, J = 8.4 Hz, 1H), 7.06 (d, J = 8.3 Hz, 1H), 6.79 (s, 1H), 5.27 (s, 2H).

[0264] 13 13C NMR (101 MHz, DMSO) δ 176.82, 161.44, 157.31, 157.29, 151.21, 141.70, 135.23, 132.22, 128.34, 122.42, 115.23, 114.58, 110.83, 108.67, 68.19.

[0265] HRMS (ESI / LTQ Orbitrap): Calculated for C 17 H 11 Br2O5(M+H + ) : 454.8948, Found 454.8941.

[0266] Example 35 Preparation of 5-((2,4-dibromobenzyl)oxy)-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-4-oxo-4H-chromene-2-carboxamide (7a)

Chem.

[0267] 6a (0.074 g, 0.16 mmol) was dissolved in 2 mL of anhydrous DMF. Then, DIEA (0.084 g, 0.65 mmol) and TBTU (0.105 g, 0.33 mmol) were sequentially added to the solution. After complete dissolution, 5-methoxytryptamine (0.074 g, 0.33 mmol) was added, and the resulting solution was stirred at room temperature for 24 h. The reaction was monitored by TLC (cyclohexane / ethyl acetate 1:4).

[0268] The reaction mixture was poured into acidic water (1 M HCl) and extracted with dichloromethane (3 times). The organic phases were collected, washed with acidic water (1 M HCl), basic water (10% NaOH), and brine (1 time), dried over MgSO4, filtered, and evaporated. The resulting oil was precipitated with a few drops of diethyl ether.

[0269] After filtration, the crude product was purified using a 12 g silica column with eluents ranging from cyclohexane / dichloromethane 4:1 to dichloromethane 100%. After the first product was eluted, the desired product was obtained with 9:1 dichloromethane / methanol. The desired product 7a (0.006 g, 5.6%) is a solid. C 28 H 22 Br2N2O5

[0270] 1 1H NMR (500 MHz, CDCl3) δ 8.12 (d, J = 8.4 Hz, 1H), 8.00 (s, 1H), 7.72 (d, J = 1.9 Hz, 1H), 7.60 - 7.55 (m, 2H), 7.31 (d, J = 8.8 Hz, 1H), 7.10 (dd, J = 9.8, 2.3 Hz, 2H), 7.05 (s, 1H), 6.95 - 6.89 (m, 3H), 6.85 (dd, J = 8.4, 0.5 Hz, 1H), 5.16 (s, 2H), 3.84 - 3.78 (m, 5H), 3.11 (t, J = 6.6 Hz, 2H).

[0271] 13 13C NMR (126 MHz, CDCl3) δ 177.59, 159.14, 158.11, 157.22, 154.32, 152.90, 134.78, 134.48, 134.36, 131.54, 131.28, 130.13, 127.80, 122.96, 121.80, 121.15, 115.29, 113.68, 112.63, 112.36, 112.17, 110.59, 108.41, 100.40, 69.86, 55.84, 40.52, 24.94.

[0272] HRMS (ESI / LTQ Orbitrap): Calculated C 28 H 23 Br2N2O5(M + H + ): 626,9948, Found 626,9929.

[0273] Example 36 Preparation of ((3,5-dibromobenzyl)oxy)-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-4-oxo-4H-chromene-2-carboxamide (7b)

Chem.

[0274] 6b (0.100 g, 0.22 mmol) was dissolved in anhydrous DMF (10 mL) and stirred until completely dissolved. Next, (1H-benzotriazol-1-yloxy)(tri-1-pyrrolidinyl)phosphonium hexafluorophosphate (PyBOP) coupling agent (0.195 g, 0.44 mmol) was added to the solution, followed by the addition of DIEA (0.114 g, 0.88 mmol). The solution was stirred at room temperature for 1 hour. A color change occurred.

[0275] Next, 5-methoxytryptamine (0.100 g, 0.44 mmol) was added and the solution was stirred at room temperature for 2 days. When there was no change in cyclohexane / ethyl acetate 1:1 TLC, bis(2-oxo-1,3-oxazolidin-3-yl)phosphinic acid chloride (BOP-Cl) (0.112 g, 0.44 mmol) was added to the solution. After 2 days, the formation of the product was confirmed by 1:1 cyclohexane / ethyl acetate TLC.

[0276] The reaction mixture was evaporated and poured into ethyl acetate. The organic phase was washed with basic water (saturated K2CO3), then acidic water (1 M HCl) and brine (3 times). The organic layer was dried over MgSO4 and evaporated to give a brown solid.

[0277] This solid was purified by silica column chromatography using a dried sample followed by an eluent such as 100% dichloromethane and then 2.4:0.1 dichloromethane / methanol. 5 mL fractions were left standing overnight in a fume hood to precipitate the desired product. After filtration, the desired product 7b, (0.007 g, 6.6%) was obtained. C 28 H 22 O5N2Br2

[0278] 1 H NMR (400 MHz, DMSO) δ 10.71 (s, 1H), 9.23 (t, J = 5.8 Hz, 1H), 7.94 (d, J = 1.3 Hz, 2H), 7.85 - 7.79 (m, 2H), 7.30 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 8.7 Hz, 1H), 7.19 (d, J = 2.1 Hz, 1H), 7.12 - 7.06 (m, 2H), 6.76 - 6.70 (m, 2H), 5.30 (s, 2H), 3.75 (s, 3H), 3.58 (dd, J = 14.2, 6.6 Hz, 2H), 2.97 (t, J = 7.4 Hz, 2H).

[0279] 13 C NMR (126 MHz, DMSO) δ 177.14, 159.37, 157.83, 157.49, 154.21, 153.51, 142.26, 135.54, 132.78, 131.88, 128.94, 128.03, 123.91, 122.94, 114.95, 112.57, 112.53, 111.72, 111.56, 111.36, 109.32, 100.65, 68.76, 55.81, 25.35.

[0280] HRMS (ESI / LTQ Orbitrap): Calculated for C 28 H 23 O5N2Br2(M+H + ) : 626.9948, Found 626.9937.

[0281] Example 37 Biological evaluation Materials High glucose DMEM (Dulbecco / Vogt modified Eagle’s minimal medium) supplemented with GlutaMAX™ (Gibco) and fetal bovine serum (FBS, GE Healthcare Hyclone) were purchased from Fisher Scientific. Penicillin / streptomycin (10,000 U / 10 mg per 1 ml), G418, trypsin, and Dulbecco's phosphate buffered saline (DPBS) were purchased from Sigma Aldrich (France). Mitoxantrone (MX), rhodamine 123 (R123), calcein-AM (cAM), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were also the same. All commercially available products were used at the highest purity available.

[0282] Compound All chromone derivatives were dissolved in dimethyl sulfoxide (DMSO) and then diluted with high glucose DMEM. Stock solutions were stored at -20 °C and warmed to 25 °C immediately before use.

[0283] Cell lines and culture ABCB1-transfected NIH / 3T3, ABCC1-transfected Flp-In 293, and ABCG2-transfected HEK293 cells and their empty plasmid counterparts were generated as previously described (Borst, P.; Elferink, R.O. Mammalian ABC Transporters in Health and Disease. Ann. Rev. Biochem. 2002, 71(1), 537-592).

[0284] Specifically, an ABCG2-transfected HEK293 monoclonal cell line was selected after fluorescence-activated cell sorting (FACS) using a phycoerythrin-conjugated 5D3 antibody (Santa Cruz Biotech) as an endogenous expression reporter.

[0285] Cells were grown and maintained in high-glucose DMEM with GlutaMAX™ supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin / streptomycin in a humidified atmosphere of 37 °C and 5% CO2. Furthermore, 200 μg / mL hygromycin B, 90 ng / mL colchicine, or 750 μg / mL G418 was added to the growth medium as a selection agent for NIH / 3T3, Flp-In 293, or HEK293 transfected cells, respectively.

[0286] Cytotoxicity assay The cytotoxicity of the compounds was determined using a colorimetric MTT assay as reported in the literature (Linton, K. J. Structure and Function of ABC Transporters. (ABC transporters' structure and function.) Physiology 2007, 22(2), 122 - 130. Sharom, F. J. ABC Multidrug Transporters: Structure, Function and Role in Chemoresistance (ABC multidrug transporters: structure, function and role in chemoresistance). Pharmacogenomics 2008, 9(1), 105 - 127).

[0287] Briefly, cells were seeded at 1 × 10 in a 96-well plate 5Cells were seeded at a density of cells / well, with a total growth medium volume of 100 μL, and incubated overnight. Next, 100 μL of fresh medium with increasing concentrations of the compound to be tested (dissolved in DMSO in a concentration range of 0, 2, 20 μM) was added to each well, while the DMSO control was fixed at 0.5% (v / v). After incubation for 72 hours, 22 μL of MTT dye (5 mg / mL) in PBS was added to each well, and the plate was incubated at 37 °C for an additional 4 hours. After removing and drying the medium, the formazan dye crystals were solubilized with 200 μL of DMSO / ethanol (1:1, v / v). Absorbance was measured using a spectrophotometer with 570 nm and 690 nm as reference wavelengths. The effect of each compound on cell viability in all cell lines was calculated as the difference in absorbance between the test medium wells and the control medium wells.

[0288] The cytotoxicity results of the compounds according to the present invention are shown in Table 1.

[0289]

Table 1

[0290] Therefore, the compounds according to the present invention exhibit very low cytotoxicity or no cytotoxicity.

[0291] MDR-related drug efflux inhibition test These cells were seeded in a 96-well plate at 5×10 4Cells / wells were seeded in 200 μL of medium at a density and incubated overnight. Thereafter, the growth medium was changed to fresh medium containing the compound, and the final concentration of DMSO was 0.5% (v / v) in the presence of 4 μM MX as a fluorescent probe for efflux via BCRP. After incubation at 37 °C for 30 minutes, the medium was removed, the cells were washed with 100 μL of Dulbecco's phosphate-buffered saline (DPBS), and then the cells were dissociated at 37 °C for 5 minutes via 25 μL of trypsin. Finally, the trypsin was neutralized with 175 μL of ice-cold DPBS containing 2% bovine serum albumin (BSA), and the cells were carefully resuspended. As a selectivity assay, the same experiment was performed for efflux via P-gp and MRP1 using 0.5 μM R123 or 0.2 μM cAM as the respective fluorescent substrates instead of MX.

[0292] Intracellular fluorescence was measured using a MacsQUANT VRB Analyzer flow cytometer (Miltenyi Biotec) by recording at least 5000 events. MX was excited at 635 nm and fluorescence emission was recorded in the window of 655 - 730 nm, while R123 and cAM were excited at 488 nm and recorded with a 525 / 50 nm filter. The compound inhibition rate was estimated by the following formula.

Equation

[0293] Activity of chromones as ABCG2 inhibitors [Table 2]

[0294] From the above table, it can be seen that the compounds show good IC50 values. In particular, compound 5d of series 1, compounds 7a and 7b of series 2, and compound 7a of series III show better results than the MBL-II-141 inhibitor and the reference inhibitor Ko143.

[0295] Selectivity of chromones for BCRP vs. P-gp and MRP1 [Table 3]

Claims

1. A compound selected from the following, or a pharmaceutically acceptable enantiomer, salt or solvate thereof, or a mixture thereof: Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-valinate 【Chemical 1】 Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucyl-L-valinate [Chemical Formula 2] Or Methyl (5-((2-bromobenzyl)oxy)-4-oxo-4H-chromene-2-carbonylamino)-L-alloisoleucyl-L-leucinate [Chemical Formula 3]

2. A method for obtaining the compound according to Claim 1, characterized by comprising the following steps: (a) An alkylated compound of the formula 【Chemical Formula 4】 where ring A is substituted by Br at the 2-position and X is a halogen selected from F, Cl, Br and I, is reacted with 2,6-dihydroxyacetophenone of the formula in acetone at the reflux temperature of acetone to obtain an intermediate of the formula 【Chemical Formula 5】 ; [Chemical Formula 6] (b) The intermediate obtained in step (a) is reacted with diethyl oxalate of the formula at a temperature of 0 °C to 50 °C in a mixture of tetrahydrofuran (THF) / ethanol (1:1) to obtain an intermediate of the formula 【Chemical Formula 7】 ; 【Chemical 8】 (c) The intermediate obtained in step (b) is reacted at a temperature of 50 °C in an acidic or basic medium in THF / ethanol / water solvent (3:1:1.5) by hydrolysis of the ester functional group to obtain an intermediate of the formula ; 【Chemical Formula 9】 (d) The intermediate obtained in step (c) is reacted with a coupling compound of the formula (wherein 【Chemical Formula 10】 Z = R １ = CH(CH₃)₂ or CH(CH₃)CH₂CH₃, Y = -OMe or -NH-CH(R3)-COR2, 【Chemical Formula 11】 、 in anhydrous DMF at room temperature to form an amide bond to obtain the compound according to Claim 1. R ２ = -OMe, R ３ is independently selected from the following: 【Chemical Formula 12】

3. A pharmaceutical composition comprising the compound according to Claim 1 for use in inhibiting the multidrug resistance protein of breast cancer (breast cancer resistance protein BCRP / ABCG2).

4. A pharmaceutical composition comprising at least one pharmaceutically acceptable active agent; and at least one compound according to Claim 1 .

5. The pharmaceutical composition according to Claim 4, characterized in that the pharmaceutically acceptable active agent is selected from anticancer agents, enteral anti-inflammatory agents, cholesterol-lowering agents, anti-dyslipidemia agents and kinase inhibitors. ​

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