PDIA4 INHIBITORS AND THEIR USE TO INHIBIT BETA-CELL PATHOGENESIS AND TREAT DIABETES - Patent application

Abstract

Disulfide isomerase A4 (PDIA4) inhibitors and their use for inhibiting pancreatic beta cell pathogenic mechanisms and treating diabetes are disclosed. Drug candidates that inhibit PDIA4 and have IC50 values ​​of 4 μM to 300 nM are identified. These compounds have high activity on insulin secretion in pancreatic beta cells. Representative compound 8 (4,5-dimethoxy-2-propiolamidobenzoic acid), alone or in combination with metformin, can effectively preserve pancreatic beta cell function, treat diabetes, and / or reverse and restore blood glucose concentrations to normal levels in diabetic patients.

Description

[Technical field]

[0001] The present invention relates to PDIA4 inhibitors. [Background technology]

[0002] Type 2 diabetes (T2D) is characterized by peripheral insulin resistance, insufficient insulin secretion, and progressive loss of β-cells. Decline in β-cell function and quality is a major hallmark of T2D. Data suggest that maintaining functional β-cell quality at early stages could slow and reverse T2D. Therefore, identification of key players in β-cell dysfunction and death may lead to a better understanding of β-cell pathogenesis and T2D development, as well as new strategies for T2D treatment.

[0003] The protein disulfide isomerase (PDI) family (21 members) plays multiple roles in cellular functions and is reported to be involved in infection, fertilization, coagulation, immunity, tumor metastasis, or cell viability / proliferation. The role of the protein disulfide isomerase (PDI) family in health and disease is not well studied. Protein disulfide isomerase A3 (PDIA3) and protein disulfide isomerase A4 (PDIA4) are two members of this family. Their functions may not be redundant, as mice lacking Pdia3 but not PDIA4 were shown to be embryonic lethal. So far, no PDI-based drugs have been developed.

[0004] There is a strong need for methods that can reverse type 2 diabetes by preserving β-cell function and quality at the early stages of diabetes. Summary of the Invention

[0005] In one aspect, the present invention relates to a compound of formula (I) or a pharma- ceutically acceptable salt thereof. JPEG0007620629000001.jpg2937 In formula (1), R 1 -C(=O)OR 6 and R 2 is H, a (C1-C6) alkyl group, a benzyl group, or -LR 11 where L is a bond or a (C1-C6) alkylene group, and R 11 is a phenyl group, a halo group, a dioxoisoindoline, -NR 7 R 8 or -NR 7 -CO-R 8 and R 3 is H, a (C1-C6) alkyl group or a benzyl group, R 4 is H or a (C1-C6)alkoxy group, R 5 is H, (C1-C6) amine, nitro, -NR 7 R 8 or -NR 7 -CO-R 8 and X 1 and X 2 each independently represents -O-, -S- or a covalent bond; R 6 is H, halogen, (C1-C6) alkyl group, (C1-C6) aryl group, benzyl group, halo(C1-C6) alkyl group, -(CH2) n -Si(CH3)3 or a carbonyl(C1-C6)alkyne group, where n is 1 to 6; R 7 and R 8 are each independently H, a (C1-C6)alkoxy group, a phenyl group, a benzoyl group, a halobenzoyl group, a benzyl group, a carbonyl(C1-C6)alkyne group, a (C1-C6)alkyne group, an oxazole group, a thiazole group, or an imidazole group, wherein the (C1-C6)alkoxy group, the phenyl group, the benzoyl group, and / or the benzyl group are each independently optionally substituted with one or more substituents selected from F, Cl, Br, I, a (C1-C6)alkoxy group, a (C1-C6)alkyl group, or a halo(C1-C6)alkyl group; 7 and R8 forms benzimidic acid together with the N atom.

[0006] In one embodiment of the present invention, R 6 is H or a (C1-C6) alkyl group, a benzyl group, a halo(C1-C6) alkyl group, -(CH2) n -Si(CH3)3 or a carbonyl (C1-C6) alkyne group.

[0007] In another embodiment of the present invention, R 2 is a (C1-C6) alkyl group or -LR 11 where L is a (C1-C6) alkylene group and R 11 is dioxoisoindoline, -NR 7 R 8 or -NR 7 -CO-R 8 where R 7 and R 8 are each independently H, a phenyl group, a benzoyl group, a halobenzoyl group, a benzyl group, a carbonyl (C1-C6) alkyne group, or an oxazole group.

[0008] In another embodiment, L is a (C1-C6) alkylene group and R 11 -NR 7 -CO-R 8 It is.

[0009] In another embodiment, L is a (C1-C6) alkylene group and R 11 -NR 7 -CO-R 8 where R 7 is a carbonyl (C1-C6) alkyne group, and R 8 is an oxazole group, a thiazole group or an imidazole group.

[0010] In another embodiment, R 11 is 1,3-dioxoisoindoline.

[0011] In other embodiments of the invention, the phenyl, benzoyl and / or benzyl groups are each independently substituted with one or more substituents selected from F, Cl, Br, I or a halo(C1-C6)alkyl group.

[0012] In another embodiment of the present invention, R 5 -NR 7 R 8 or -NR 7 -CO-R 8 where R 7 and R 8 are each independently H or a carbonyl (C1-C6) alkyne group.

[0013] In another embodiment of the present invention, R 5 is a nitro group.

[0014] In another embodiment of the present invention, R 6 is a carbonyl (C1-C6) alkyne group.

[0015] In another embodiment of the present invention, R 2 Ha-LR 11 where L is a (C1-C6) alkylene group and R 11 -NR 7 R 8 where R 7 and R 8 forms a benzimidic acid with the N atom.

[0016] In another embodiment of the present invention, R 7 and R 8 forms a benzimidic acid with the N atom.

[0017] In another embodiment of the present invention, R 2 Ha-LR 11 and R 11 -NR 7 R 8 or -NR 7 -CO-R 8 and R 7 is a carbonyl (C1-C6) alkyne group, and R8 is a benzoyl group.

[0018] In another embodiment of the present invention, R 2 is a (C1-C6) alkyl group, and R 3 is a (C1-C6) alkyl group, and R 4 is H and R 5 -NR 7 -CO-R 8 where R 7 is H and R 8 is a (C2) alkyne group.

[0019] In another embodiment of the present invention, R 5 -NR 7 R 8 or -NR 7 -CO-R 8 where R 7 and R 8 are each independently H, a carbonyl (C1-C6) alkyne group, or a (C1-C6) alkyne group.

[0020] In another embodiment of the present invention, R 2 Ha-LR 11 where L is a (C1-C6) alkylene group and R 11 is dioxoisoindoline or -NR 7 R 8 where R 7 and R 8 are each independently a benzoyl group or a carbonyl (C1-C6) alkyne group.

[0021] In another embodiment, the compounds of the present invention, and pharma- ceutically acceptable salts thereof, are selected from the compounds shown in Table 6.

[0022] In another aspect, the present invention relates to a pharmaceutical composition comprising a compound of the present invention or a pharma- ceutically acceptable salt thereof and a pharma- ceutically acceptable carrier or excipient, said pharmaceutical composition further comprising metformin.

[0023] In another aspect, the present invention relates to a pharmaceutical composition comprising: (a) a compound of the present invention, or a pharma- ceutically acceptable salt thereof; and (b) metformin.

[0024] In another aspect, the invention relates to the use of a compound or a pharma- ceutically acceptable salt or pharmaceutical composition of the invention in the manufacture of a medicament for enhancing insulin secretion in pancreatic β cells, treating diabetes, and / or reversing and restoring blood glucose concentrations to normal levels.

[0025] The present invention also relates to compounds or pharma- ceutically acceptable salts or pharmaceutical compositions of the present invention for enhancing insulin secretion in pancreatic β cells, treating diabetes, and / or reversing and restoring blood glucose concentrations to normal levels.

[0026] In one embodiment, the compounds or pharma- ceutically acceptable salts or pharmaceutical compositions of the invention are used to inhibit PDIA4 activity, inhibit pancreatic beta cell pathogenesis, treat diabetes, and / or reverse and restore blood glucose concentrations to normal levels.

[0027] The present invention also relates to a method of inhibiting pancreatic β-cell pathogenic mechanisms, enhancing insulin secretion in pancreatic β-cells, treating diabetes, and / or reversing and restoring blood glucose concentrations to normal levels, comprising administering to a subject a therapeutically effective amount of a compound of the present invention or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0028] In another aspect, the invention relates to a method of inhibiting PDIA4 activity, the method comprising contacting PDIA4 with a compound of the invention, or a pharma- ceutically acceptable salt thereof, thereby inhibiting PDIA4 activity.

[0029] These and other aspects will become apparent from the following description of the preferred embodiments taken in conjunction with the following drawings, in which variations and modifications may be effected without departing from the spirit and scope of the novel concepts of the present disclosure.

[0030] The accompanying drawings illustrate one or more embodiments of the invention and, together with the description, serve to explain the principles of the invention. Wherever possible, the same reference numerals are used in all the drawings to refer to the same or similar elements of an embodiment. [Brief description of the drawings]

[0031] [Figure 1] A flow chart showing the screening of PDIA4 inhibitors from hit compounds to lead compounds and drug candidates. Figure 1A shows a scheme illustrating the screening strategy of PDIA4 inhibitors from hit compounds to lead compounds and drug candidates by combining molecular docking, PDIA4 bioassay and total synthesis. Figure 1B shows a Min6 cell-based assay for investigating glucose-induced insulin secretion (GSIS) and cell viability. Figure 1C shows the use of PDIA4 inhibitors on diabetic db / db mice.

[0032] [Diagram 2] Molecular docking showing the interaction of selected PDIA4 inhibitors with the active motif (AM) of PDIA4 is shown. In FIG. 2A, AM1, 2, and 3 represent the first, second, and third CGHC domains, respectively. Sulfur, nitrogen, and oxygen atoms are shown in yellow, blue, and red, respectively. Hydrogen bonds and hydrophobic interactions between CPD P1 and amino acid residues of the PDIA4 model are shown, respectively. FIG. 2B shows molecular docking showing the mutual activity of selected CPD8 and the active motif (AM) of PDIA4, similar to FIG. 2A.

[0033] [Diagram 3]The insulin release effect and cytotoxicity of drug candidates are shown. Figure 3A shows the effect of drug candidates on insulin secretion in β cells. Min6 cells were incubated in serum-free oxygen-saturated Krebs-Ringer bicarbonate (KRB) buffer containing high glucose (16.7 mM), CCF642 and drug candidates for 30 minutes. The supernatant was collected and used for insulin ELISA assay. In Figure 3B, Min6 cells were incubated with PBS, CCF642 and drug candidates for 24 hours, respectively, then washed with PBS, and WST-1 was added to each well and incubated for another 20 minutes.

[0034] [Figure 4] We show that CPD8 alone and in combination with metformin can treat and reverse diabetes in Leprdb / db mice. In Figure 4A, B, newly onset diabetic Leprdb / db mice were grouped and treated with PBS vehicle (CTR), sitagliptin (STG, 30 mg / kg), CPD8 (2.5 mg / kg), and a combination of CPD8 (2.5 mg / kg) and metformin 960 mg / kg (CPD8+Met) for the indicated times. Fasting blood glucose (FBG (A)) and postprandial blood glucose (PBG (B)) of the mice were monitored. Values ​​(n) are the number of mice shown in brackets. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] definition Representative acyl groups include acetyl, propionyl, butanoyl and benzoyl groups.

[0036] A dash ("-") that is not between two letters or symbols indicates the point of attachment of the moiety or substituent. For example, the moiety -CONH2 is attached via a carbon atom.

[0037] As used herein, the term "ester" refers to compounds containing substituted carboxylic acids (eg, --COO-alkyl).

[0038] As used herein, the term "sulfonyl" refers to a group having the structure of formula -S(O)2-.

[0039] The term "alkylene" refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of 1 to 18 carbon atoms and having two monovalent radical centers obtained by the removal of two hydrogen atoms from the same or different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to, methylene (-CH2-), 1,2-ethylene (-CH2CH2-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and the like.

[0040] "(C m -C n The term "m, n are integers, n>m" means that all integer amounts in the range from m to n are specifically disclosed as part of the invention. Thus, (C m -C n ) is C m , C m+1 , C m+2 , , C n-2 , C n-1 , C n , (C m -C m+1 ), (C m -C m+2 ), (C m -C m+3 ), ..., (C m -C n-2 ), (C m -C n-1 ), (C m -C n );(C m+1 -C m+2 ), (C m+1 -C m+3 ), (C m+1 -C m+4 ), ..., (C m+1 -C n-2 ), (C m+1 -C n-1 ), (C m+1 -C n ), ..., (C n-2 -C n-1 ), (Cn-2 -C n ) and (C n-1 -C n ) are meant to be included as embodiments of the present invention.

[0041] By "(C1-C6)" it is meant that all integer unit amounts in the range of 1 to 6 are specifically disclosed as part of the present invention. Thus, C1, C2, C3, C4, C5, C6; (C1-C2), (C1-C3), (C1-C4), (C1-C5), (C1-C6); (C2-C3), (C2-C4), (C2-C5), (C2-C6); (C3-C4), (C3-C5), (C3-C6); (C4-C5), (C4-C6); and (C5-C6) unit amounts are included as embodiments of the present invention.

[0042] The term "treatment" or "treating" refers to curing, alleviating, ameliorating, or preventing a disease or a symptom or tendency toward a disease by administering a therapeutically effective amount of a therapeutic agent to a subject having the disease or a symptom or tendency toward the disease.

[0043] The "Guidance for Industry and Reviewers for Estimating Safe Starting Dose for Clinical Trials of Therapeutic Products in Healthy Adult Volunteers" published by the U.S. Department of Health and Human Services Food and Drug Administration discloses that the "human equivalent dose" can be calculated using the following formula. HED = Animal dose (mg / kg) x (animal weight (kg) / human weight (kg)) 0.33

[0044] Abbreviation PDIA4: Protein disulfide isomerase family A member 4 CCTB: Carbon / carbon triple bond I C 50 :50% inhibitory concentration

[0045] The present invention relates to the discovery of PDIA4 inhibitors for the treatment of type 2 diabetes. A combination of molecular docking and PDIA4 bioassays is used to identify PDIA4-based hit compounds. Lead optimization is used to identify the IC of drug candidates.50 Improve its biological activity until values ​​reach the nM range.

[0046] Working Example Materials and Methods Virtual Screening To obtain a reliable model of PDIA4 action, multiple sequence alignment was performed against the A-chains of 3IDV (catalytic domain of human PDIA4 (residues 53-284)), 3EC3 (non-catalytic domain of rat PDIA4 (residues 283-523)) and 3F8U (residues 25-501 of human PDIA3) from the Protein Data Bank. The sequences of these two proteins have 100%, 88% and 42% identity with human PDIA4, respectively. A homology model of PDIA4 was built by Discovery Studio v.4.1 based on the crystallographic structures of the A-chains of 3IDV, 3EC3 and 3F8U of PDIA4 used as structural templates. 261 compounds from an in-house phytochemical library were converted to 3D coordinates using the CHARMM force field and the compounds were minimized by the Prepare Ligand module of Discovery Studio. The protonation states of the residues were adjusted to the predominant ionic form at pH 7.5. The residues surrounding the second CGHC motif of PDIA4, which is composed of the binding packet between the first and second active domains, were used as the docking packet. Molecular docking between the compounds and the second active domain of PDIA4 was performed using Goldscore in the GOLDv.5.1 program (CCDC Software Limited, Cambridge, UK). Finally, among the 100 docking conformations of the compounds, the best candidates (with higher GOLD fitness scores) were selected to explore the "inhibitor-bound" conformation of the second active domain of PDIA4.

[0047] chemical methods NMR spectrum ( 1 H and 13C NMR (C NMR) were acquired using Bruker Fourier300 and AVIII500 spectrometers with the standard plus program. Chemical shifts are given in parts per million (ppm, δ) using TMS as the internal standard. MS data were measured on a Finnigan Mat TSQ-7000 mass spectrometer (ESIMS and HRESIMS). Melting points were recorded on a Fisher-Johns apparatus (uncorrected). HPLC was performed on a C18 column (150 mm × 4.6 mm) with an L-2130 pump (Hitachi, Ibaraki, Japan). Column chromatography was performed on silica gel (70-230 mesh, Merck, Darmstadt, Germany). TLC analysis was performed on silica gel plates (KG60-F254, Merck). Unless otherwise stated, all chemicals and materials were used as received from commercial suppliers without purification. Anhydrous dichloromethane was distilled under N2 from calcium hydride.

[0048] Scheme 1 [ka]

[0049] Methyl 4,5-dimethoxy-2-nitrobenzoate (2) [ka] To a mixture of 1 (5.00 g, 22.00 mmol) and K2CO3 (12.17 g, 88.04 mmol) in DMF (50 mL) was added CH3I (5.48 mL, 88.04 mmol) dropwise. The resulting solution was heated to 100 °C under N2 and maintained for 15 h. The reaction mixture was suspended in H2O (150 mL) and then extracted with EtOAc (3 x 150 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo to give a solid. The solid was washed with MeOH to give 2 (5.22 g, 98%). 1H NMR(500MHz,CDCl3)δ7.43(s,1H),7.06(s,1H),3.96(s,3H),3.95(s,3H),3.89(s,3H)

[0050] Methyl 2-amino-4,5-dimethoxybenzoate (3) [ka] To a solution of 2 (5.22 g, 21.66 mmol) in EtOAc (10 mL) was added a catalytic amount of 10% Pd / C (1 g). The resulting mixture was stirred at RT under H2 atmosphere for 8 h. The reaction mixture was filtered through diatomaceous earth and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc / n-hexane=1:9) to give 3 (3.00 g, 66%) as a yellow solid. 1 H NMR (500MHz, CDCl3) δ7.27(s,1H),6.12(s,1H),5.55(s,2H),3.84(s,3H),3.83(s,3H),3.80(s,3H).

[0051] Methyl 4,5-dimethoxy-2-propiolamidobenzoate (4) [ka] A solution of 3 (50 mg, 0.24 mmol) in anhydrous CHCl (10 mL) was added dropwise to a solution of propiolic acid (0.022 mL, 0.36 mmol) and N,N'-dicyclohexylcarbodiimide (73 mg, 0.36 mmol) in anhydrous CHCl (5 mL) in an ice bath. The resulting solution was stirred under N for 2 h. The reaction mixture was suspended in H0 (50 mL) and extracted with CHCl (3 x 50 mL). The organic layer was extracted, dried over MgSO, filtered and concentrated in vacuo. The mixture was filtered and washed with EtOAc / n-hexane = 1:1 (10 mL). The residue was purified by silica gel chromatography (EtOAc / n-hexane = 1:5) to give 4 (57 mg, 91%). 1H NMR(500MHz,CDCl3)δ11.56(s,1H),8.31(s,1H),7.44(s,1H),3.93(s,3H),3.90(s,3H),3.87(s,3H),2.92(s,1H).

[0052] Scheme 2 [ka]

[0053] 2-(trimethylsilyl)ethyl 4,5-dimethoxy-2-nitrobenzoate (5) [ka] Following the procedure described for compound 4, a reaction mixture of 1 (100 mg, 0.44 mmol), N,N'-dicyclohexylcarbodiimide (109 mg, 0.528 mmol), and DMAP (5 mg, 0.04 mmol) in anhydrous THF (10 mL) was treated with 2-(trimethylsilyl)ethanol (0.094 mL, 0.66 mmol) dropwise to give 5 (59 mg, 41%). 1 H NMR(300MHz,CDCl3)δ7.43(s,1H),7.05(s,1H),4.38(m,2H),3.06(s,3H),3.95(s,3H),1.08(m,2H),0.04(s,9H).

[0054] 2-Amino-4,5-dimethoxybenzoic acid 2-(trimethylsilyl)ethyl ester (6) [ka] Following the procedure described for compound 3, a catalytic amount of 10% Pd / C (10 Mg) was added to a reaction of 5 (100 mg, 0.31 mmol) in EtOAc (10 mL) to give 6 (41 mg, 45%). 1H NMR(300MHz,CDCl3)δ7.29(s,1H),6.12(s,1H),4.34(m,2H),3.84(s,3H),3.80(s,3H),1.10(m,2H),0.07(s,9H).

[0055] 4,5-Dimethoxy-2-propiolamidobenzoic acid 2-(trimethylsilyl)ethyl ester (7) [ka] Following the procedure described for compound 4, a reaction system of 6 (100 mg, 0.34 mmol) in anhydrous CHCl (10 mL) in an ice bath was treated dropwise with a solution of propiolic acid (0.031 mL, 0.50 mmol) and N,N'-dicyclohexylcarbodiimide (104 mg, 0.50 mmol) in anhydrous CHCl (5 mL) to give 7 (108 mg, 92%). 1 H NMR(300MHz,CDCl3)δ11.65(s,1H),8.32(s,1H),7.45(s,1H),4.41(m,2H),3.94(s,3H),3.87(s,3H),1.14(m,2H),0.08(s,9H).

[0056] 4,5-Dimethoxy-2-propiolamidobenzoic acid (8) [ka] To a solution of 7 (50 mg, 0.14 mmol) in anhydrous THF (4 mL) at room temperature, TBAF (1 M in THF, 0.43 mL, 0.43 mmol) was added dropwise. The resulting solution was stirred under N2 for 24 h. The reaction mixture was diluted with 1N HCl. (aq) (50 mL) and EtOAc (3×50 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (MeOH / CH2Cl2=3:97) to give 8 (30 mg, 84%). 1H NMR(500MHz,DMSO)δ11.87(s,1H),8.06(s,1H),7.45(s,1H),4.45(s,1H),3.80(s,3H),3.77(s,3H).

[0057] Scheme 3 [ka]

[0058] 2-Amino-4,5-dimethoxybenzoic acid (9) [ka] To a solution of 1 (5.00 g, 22.00 mmol) in MeOH (20 mL) was added a catalytic amount of 10% Pd / C (1.5 g) and the mixture was stirred at room temperature under an atmosphere of H for 24 h. The reaction mixture was filtered through diatomaceous earth and concentrated in vacuo to give 9 (4.00 g, 92%). 1 H NMR(500MHz,DMSO)δ7.14(s,1H),6.33(s,1H),3.73(s,3H),3.63(s,3H).

[0059] 2-Amino-4,5-dimethoxybenzoic acid propiolic anhydride (10) [ka] Following the procedure described for compound 4, a reaction of 9 (500 mg, 2.54 mmol) in CHCl (10 mL) in an ice bath was treated with anhydrous CHCl (5 mL) containing propiolic acid (0.234 mL, 3.81 mmol) and N,N'-dicyclohexylcarbodiimide (466 mg, 2.26 mmol) dropwise to give 10 (15 mg, 2%). 1 H NMR(300MHz,CDCl3)δ7.50(s,1H),7.02(s,1H),3.97(s,3H),3.96(s,3H).

[0060] Scheme 4 [ka]

[0061] Benzyl 5-(benzyloxy)-4-methoxy-2-nitrobenzoate (12) [ka] Following the procedure described for compound 2, benzyl bromide (0.61 mL, 5.15 mmol) was added to a reaction of 11 (439 mg, 2.00 mmol) and K2CO3 (710 mg, 5.15 mmol) in DMF (20 mL). The resulting solution was heated to 110 °C under N2 to give 12 (807 mg, 99%). 1 H NMR (500MHz, CDCl3) δ7.43(s,1H),7.34(m,10H),7.13(s,1H),5.30(s,2H),5.18(s,2H),3.96(s,3H).

[0062] 2-Amino-5-(benzyloxy)-4-methoxybenzoate benzyl (13) [ka] To a solution of 12 (1.00 g, 2.54 mmol) in HOAc (20 mL) was added Zn (1.66 g, 25.44 mmol) and the mixture was heated to 50° C. under N2 and kept for 24 h. The reaction mixture was filtered through diatomaceous earth. The residue was dissolved in EtOAc (50 mL) and then washed with NaHCO3 (3×50 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc / n-hexane=1:3) to give 13 (330 mg, 36%). 1 H NMR (500MHz, CDCl3) δ7.37(m,11H),6.15(s,1H),5.64(s,2H),5.28(s,2H),5.04(s,2H),3.87(s,3H).

[0063] 5-(Benzyloxy)-4-methoxy-2-propiolamidobenzoate benzyl (14) [ka] To a solution of 13 (500 mg, 1.37 mmol) in anhydrous CHCl (30 mL) was added propiolic acid chloride (1.00 g, 11.36 mmol) and KCO (475 mg, 3.44 mmol) dropwise in an ice bath. The resulting solution was stirred at 0 °C under N for 0.5 h. The reaction mixture was suspended in H0 (50 mL) and extracted with CHCl (3 × 50 mL). The combined organic layers were dried over MgSO, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc / n-hexane = 1:4) to give 14 (76 mg, 13%). 1 H NMR(500MHz,CDCl3)δ11.52(s,1H),8.36(s,1H),7.56(s,1H),7.37(m,10H),5.33(s,2H),5.13(s,2H),4.71(s,1H),3.97(s,3H).

[0064] Scheme 5 [ka]

[0065] 2-Amino-5-(benzyloxy)-4-methoxybenzoic acid (15) [ka] A solution of 13 (150 mg, 0.41 mmol) and LiOH (100 mg, 2.30 mmol) in MeOH (10 mL) was heated to 70 °C under N2 for 18 h. The resulting solution was acidified to pH 2 and extracted with 1 N HCl (50 mL) and EtOAc (3 x 50 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was recrystallized to give 15 (100 mg, 89%). 1 H NMR(500MHz,CDCl3)δ7.43(s,1H),7.41(s,2H),7.35(t,J=7.4Hz,2H),7.28(t,J=7.3Hz,1H),6.12(s,1H),5.02(s,2H),3.85(s,3H).

[0066] 2-Amino-5-(benzyloxy)-4-methoxybenzoic acid propiolic anhydride (16) [ka] Following the procedure described for compound 4, a reaction system of 15 (250 mg, 0.92 mmol) in anhydrous CHCl (10 mL) in an ice bath was treated with the dropwise addition of propiolic acid (0.084 mL, 0.92 mmol) and N,N'-dicyclohexylcarbodiimide (246 mg, 1.19 mmol) in anhydrous CHCl (5 mL) to give 16 (36 mg, 12%). 1 H NMR(500MHz,CDCl3)δ7.57(s,1H),7.44(d,J=7.3Hz,2H),7.37(t,J=7.5Hz,2H ),7.32(t,J=7.3Hz,1H),7.04(s,1H),5.21(s,2H),3.97(s,3H),3.20(s,1H).

[0067] Scheme 6 [ka]

[0068] Methyl 3,4,5-trimethoxy-2-nitrobenzoate (18) [ka] A solution of 17 (500 mg, 2.21 mmol) in HOAc (4 mL) was added with HNO3 (2 mL) and heated to 50 °C under N2 for 1.5 h. The reaction mixture was suspended in H2O (50 mL) and then extracted with EtOAc (3 x 50 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc / n-hexane = 1:4) to give 18 (231 mg, 38%). 1 H NMR(500MHz,CDCl3)δ7.25(s,1H),3.94(s,3H),3.93(s,3H),3.92(s,3H),3.85(s,3H).

[0069] Methyl 2-amino-3,4,5-trimethoxybenzoate (19) [ka] Following the procedure described for compound 13, Zn (2.41 g, 36.90 mmol) was added to a reaction of 18 (1.00 g, 3.69 mmol) in HOAc (20 mL) and the mixture was heated to 50° C. under N2 for 24 h. The reaction mixture was filtered through diatomaceous earth. The residue was extracted with EtOAc (50 mL) and NaHCO3 (3×50 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc / n-hexane=1:4) to give 19 (590 mg, 66%). 1 H NMR (500MHz, CDCl3) δ7.13(s,3H),3.93(s,3H),3.84(s,6H),3.79(s,3H).

[0070] Methyl 3,4,5-trimethoxy-2-propiolamidobenzoate (20) [ka] Following the procedure described for compound 14, a reaction mixture of 19 (1.36 g, 5.68 mmol) in anhydrous CH2Cl2 (30 mL) in an ice bath was treated with dropwise addition of propiolic acid chloride (1.00 g, 11.36 mmol) and K2CO3 (1.57 g, 3.11.36 mmol) to give 20 (148 mg, 9%). 1 H NMR(500MHz,CDCl3)δ7.20(s,1H),3.92(s,3H),3.91(s,3H),3.88(s,3H),3.87(s,3H),2.88(s,1H).

[0071] Scheme 7 [ka]

[0072] 2-Amino-3,4,5-trimethoxybenzoic acid (21) [ka] Following the procedure described for compound 15, the reaction of 19 (560 mg, 2.32 mmol) and LiOH (240 mg, 5.72 mmol) in MeOH (10 mL) was heated to 70 °C under N for 18 h to give 21 (380 mg, 73%). 1 H NMR (500MHz, CDCl3) δ7.19(s,1H),3.95(s,3H),3.86(s,3H),3.80(s,3H).

[0073] 2-Amino-3,4,5-trimethoxybenzoic acid propiolic anhydride (22) [ka] Following the procedure described for compound 4, a reaction system of 21 (320 mg, 1.41 mmol) in anhydrous CHCl (10 mL) in an ice bath was treated with the dropwise addition of propiolic acid (0.130 mL, 2.11 mmol) and N,N'-dicyclohexylcarbodiimide (435 mg, 2.11 mmol) in anhydrous CHCl (5 mL) to give 22 (6 mg, 2%). 1 H NMR(500MHz,CDCl3)δ7.39(s,1H),4.05(s,3H),4.01(s,3H),3.95(s,3H),3.19(s,1H).

[0074] Scheme 8 [ka]

[0075] 2-Amino-5-hydroxy-4-methoxybenzoic acid (23) [ka] A catalytic amount of 10% Pd / C (200 Mg) was added to a solution of 11 (500 mg, 2.35 mmol) in MeOH (30 mL), and the mixture was stirred at room temperature under H2 atmosphere for 9 h. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (MeOH / CH2Cl2=1:9) to give 23 (350 mg, 82%). 1 H NMR(300MHz,CDCl3)δ8.30(s,1H),7.18(s,1H),6.38(s,1H),3.83(s,3H),3.27(s,1H).

[0076] Scheme 9 [ka]

[0077] Propyl 3,4-dipropoxybenzoate (25) [ka] Following the procedure described for compound 2, 1-bromopropane (13.2 mL, 145 mmol) was added to a reaction of 24 (5 g, 32.47 mmol) and K2CO3 (22.36 g, 162 mmol) in DMF (50 mL). The resulting solution was heated to 110 °C under N2 to give 25 (8.5 g, 89%). 1 H NMR(300MHz,CDCl3)δ7.62(dd,J=8.4, 2.0Hz,1H),7.53(d,J=2.0Hz,1H),6.85(d,J=8.4Hz,1H),4.22(t,J=6.7Hz,2H),3.99(t,J=6.6Hz,4H),1.80(m,6H),1.02(m,9H).

[0078] 2-Nitro-4,5-dipropoxybenzoic acid propyl ester (26) [ka] Following the procedure described for compound 18, a reaction of 25 (8.10 g, 28.93 mmol) in HOAc (20 mL) was added with HNO3 (5 mL) and heated to 50 °C under N2 to give 26 (8.50 g, 90%). 1 H NMR (300MHz, CDCl3 )δ7.40(s,1H),7.04(s,1H),4.24(t,J=6.7Hz,2H),4.02(m,4H),1.86(sxt,J=7.1Hz,4H),1.72(m,2H),1.04(td,J=7.4, 1.5Hz,6H),0.95(t,J=7.4Hz,3H).

[0079] 2-Amino-4,5-dipropoxybenzoic acid propyl ester (27) [ka] To a solution of 26 (8.50 g, 26.15 mmol) in HOAc (20 mL) was added Zn (17.00 g, 261.54 mmol) and the mixture was stirred at room temperature under N2 for 2 h. The reaction mixture was filtered through diatomaceous earth. The residue was extracted with EtOAc (150 mL) and NaHCO3 (3 x 150 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc / n-hexane = 1:7) to give 27 (4.90 g, 64%). 1 H NMR (300MHz, CDCl3) δ7.36(s,1H),6.10(s,1H),4.18(t,J=6.7Hz,2H),3.88(m,4H),1.79(m,6H),1.01(m,9H).

[0080] Methyl 2-amino-4,5-dipropoxybenzoate (28) [ka] A solution of 27 (1 g, 3.86 mmol) and LiOH (810 mg, 19.30 mmol) in MeOH (20 mL) was heated to 70 °C under N2 for 24 h. The resulting solution was acidified to pH 2 and extracted with 1 N HCl (50 mL) and EtOAc (3 x 50 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was recrystallized to give 28 (450 mg, 46%). 1 H NMR(300MHz,CDCl3)δ7.32(s,1H),6.10(s,1H),3.89(m,2H),3.81(s,3H),1.80(m,4H),1.01(m,6H).

[0081] 2-Propiolamido-4,5-dipropoxybenzoic acid methyl ester (29) [ka] Following the procedure described for compound 4, a reaction system of 28 (440 mg, 1.74 mmol) in water (10 mL) in an ice bath was treated with dropwise addition of propiolic acid (0.183 mL, 2.61 mmol) and N,N'-dicyclohexylcarbodiimide (466 mg, 2.26 mmol) in anhydrous CHCl (5 mL) to give 29 (66 mg, 12%). 1 H NMR(300MHz,CDCl3)δ11.52(s,1H),8.27(s,1H),7.46(s,1H),4.03(t,J=6.6Hz,2H), 3.94(t,J=6.6Hz,2H),3.89(s,3H),2.91(s,1H),1.83(m,4H),1.02(t,J=7.4Hz,6H).

[0082] Scheme 10 [ka]

[0083] 2-Propiolamido-4,5-dipropoxybenzoic acid propyl ester (30) [ka] Following the procedure described for compound 4, a reaction system of 27 (590 mg, 2.00 mmol) in anhydrous CHCl (10 mL) in an ice bath was treated with dropwise addition of propiolic acid (0.184 mL, 3.00 mmol) and N,N'-dicyclohexylcarbodiimide (453 mg, 2.19 mmol) in anhydrous CHCl (5 mL) to give 30 (342 mg, 49%). 1 H NMR(300MHz,CDCl3)δ11.56(s,1H),8.27(s,1H),7.48(s,1H),4.25(t,J=6.7Hz,2H), 4.03(t,J=6.6Hz,2H),3.94(t,J=6.6Hz,2H),2.90(s,1H),1.82(m,6H),1.02(m,9H).

[0084] Scheme 11 [ka]

[0085] Benzyl 3,4-bis(benzyloxy)benzoate (31) [ka] Following the procedure described for compound 2, benzyl bromide (27 mL, 227.3 mmol) was added to a reaction of 24 (10.00 g, 64.94 mmol) and K2CO3 (44.00 g, 318.23 mmol) in DMF (100 mL). The resulting solution was heated to 110 °C under N2 to give 31 (27.10 g, 98%). 1 H NMR (300MHz, CDCl3) δ7.53(s,1H),7.35(m,15H),7.18(s,1H),5.33(s,2H),5.23(s,4H).

[0086] Benzyl 4,5-bis(benzyloxy)-2-nitrobenzoate (32) [ka] Following the procedure described for compound 18, a reaction of 31 (16.00 g, 37.69 mmol) in HOAc (30 mL) was added to HNO3 (15 mL) and stirred at room temperature under N2 to give 32 (16.44 g, 95%). 1 H NMR (300MHz, CDCl3) δ7.50(s,1H),7.36(m,15H),7.15(s,1H),5.30(s,2H),5.20(s,4H).

[0087] Scheme 12 [ka]

[0088] 2-(trimethylsilyl)ethyl 5-hydroxy-4-methoxy-2-nitrobenzoate (33) [ka] To a solution of 11 (5000 mg, 23.46 mmol) and triphenylphosphine (12.31 g, 46.92 mmol) in anhydrous THF (150 mL) was added 2-(trimethylsilyl)ethanol (5.04 mL, 35.19 mmol) and diisopropyl azodicarboxylate (6.93 mL, 35.19 mmol) dropwise in an ice bath. The resulting solution was stirred at room temperature under N for 7 h. The reaction mixture was suspended in H2O (150 mL) and extracted with EtOAc (3 x 150 mL). The organic layer was collected, dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc / n-hexane = 1:9) to give 33 (2.03 g, 29%). 1 H NMR (300MHz, CDCl3) δ7.49(s,1H),7.11(s,1H),4.38(m,2H),3.99(s,3H),1.07(m,2H),0.03(s,9H).

[0089] 2-(Trimethylsilyl)ethyl 5-(3-chloropropoxy)-4-methoxy-2-nitrobenzoate (34) [ka] Following the procedure described for compound 2, 1-bromo-3-chloropropane (0.921 mL, 9.32 mmol) was added to a reaction of 33 (2.43 g, 7.76 mmol) and K2CO3 (2.15 g, 15.53 mmol) in DMF (150 mL). The resulting solution was heated to 100 °C under N2 to give 34 (2.88 g, 95%). 1 H NMR(300MHz,CDCl3)δ7.42(s,1H),7.08(s,1H),4.38(m,2H),4.24(m,2H),3.67(m,2H),2.34(m,2H),1.08(m,2H),0.04(s,9H).

[0090] 2-(trimethylsilyl)ethyl 5-(3-(1,3-dioxoisoindolin-2-yl)propoxy)-4-methoxy-2-nitrobenzoate (35) [ka] Following the procedure described for compound 2, potassium phthalimide salt (1.64 g, 8.85 mmol) was added to a reaction of 34 (2.88 g, 7.37 mmol) and K2CO3 (2.04 g, 14.75 mmol) in DMF (100 mL). The resulting solution was heated to 100 °C under N2 to give 35 (3.42 g, 93%). 1 H NMR(300MHz,CDCl3)δ7.81(m,2H),7.70(m,2H),7.32(s,1H),6.99(s,1H),4.36(m,2H),4.14 (q,J=5.5Hz,2H),3.90(t,J=6.4Hz,2H),3.66(s,3H),2.25(m,2H),1.06(m,2H),0.03(s,9H).

[0091] 2-(trimethylsilyl)ethyl 5-(3-aminopropoxy)-4-methoxy-2-nitrobenzoate (36) [ka] To a solution of 35 (1.50 g, 3.00 mmol) in MeOH (150 mL) was added hydrazine monohydrate (1.45 mL, 29.97 mmol) dropwise. The resulting solution was stirred at reflux under N for 24 h. The reaction mixture was concentrated in vacuo, suspended in H2O (150 mL), and extracted with EtOAc (3 x 150 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (MeOH / CH2Cl2 = 1:99) to give 36 (140 mg, 13%). 1H NMR(300MHz,CDCl3)δ7.45(s,1H),6.52(s,1H),5.38(s,2H),4.39(m,2H),3.91(s ,3H),3.79(t,J=5.8Hz,2H),3.37(m,2H),1.90(m,2H),1.08(m,2H),0.03(s,9H).

[0092] 2-(trimethylsilyl)ethyl 5-(3-benzamidopropoxy)-4-methoxy-2-nitrobenzoate (37) [ka] To a solution of 36 (140 mg, 0.38 mmol) in anhydrous CHCl (10 mL) was added pyridine (0.122 mL, 1.51 mmol) and benzoyl chloride (0.131 mL, 1.13 mmol) dropwise. The resulting solution was stirred at room temperature under N for 12 h. The reaction mixture was suspended in H0 (50 mL) and then extracted with CHCl (3×50 mL). The combined organic layers were dried over MgSO, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc / n-hexane=1:4) to give 37 (177 mg, 99%). 1 H NMR(300MHz,CDCl3)δ8.02(d,J=7.1Hz,2H),7.57(tt,J=7.4, 1.2Hz,1H),7.45(m,3H),6.54(s,1H),5.24(s,1H),4.45(t,J=5.9Hz,2H),4.38(m ,2H),3.85(s,3H),3.42(q,J=6.3Hz,2H),2.13(m,2H),1.08(m,2H),0.03(s,9H).

[0093] 2-Amino-5-(3-benzamidopropoxy)-4-methoxybenzoate 2-(trimethylsilyl)ethyl ester (38) [ka] A solution of 37 (180 mg, 0.38 mmol) in HOAc / THF=1:4 (20 mL) was added to Zn (248 mg, 3.79 mmol) and the mixture was stirred at room temperature under N2 for 24 h. The reaction mixture was filtered through diatomaceous earth. The residue was extracted with EtOAc (50 mL) and NaHCO3 (3 x 50 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc / n-hexane=1:3) to give 38 (158 mg, 94%). 1 H NMR(300MHz,CDCl3)δ8.04(m,2H),7.55(m,1H),7.42(m,2H),7.06(s,1H),6.08(s,1H), 4.45(t,J=6.2Hz,2H),4.32(m,2H),3.77(s,3H),3.26(t,J=6.8Hz,2H),2.11(tt,J=6.2, 6.8Hz,2H),1.07(m,2H),0.05(s,9H).

[0094] 2-(trimethylsilyl)ethyl 4-methoxy-2-propiolamido-5-(3-(N-propioloylbenzamido)propoxy)benzoate (39) [ka] Following the procedure described for compound 4, a reaction system of 38 (125 mg, 0.28 mmol) in anhydrous CHCl (10 mL) in an ice bath was treated with dropwise addition of propiolic acid (0.037 mL, 0.56 mmol) and N,N'-dicyclohexylcarbodiimide (116 mg, 0.56 mmol) in anhydrous CHCl (5 mL) to give 39 (149 mg, 97%). 1H NMR(300MHz,CDCl3)δ11.82(s,1H),8.38(s,1H),7.94(dd,J=8.4,1.4Hz,2H),7.87(s,1H),7.52(m,1H),7.40(m ,2H),4.37(m,2H),3.93(m,4H),3.72(m,1H),2.97(s,1H),2.68(s,1H),1.99(m,2H),1.12(m,2H),0.06(s,9H).

[0095] 4-Methoxy-2-propiolamido-5-(3-(N-propioloylbenzamido)propoxy)benzoic acid (40) [ka] To a solution of 39 (75 mg, 0.14 mmol) in anhydrous THF (3 mL) was added TBAF (1 M in THF, 0.75 mL, 0.75 mmol) dropwise at room temperature. The resulting solution was stirred under N2 for 5 h. The reaction mixture was diluted with 1N HCl (aq) (50 mL) and extracted with EtOAc (3×50 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (MeOH / CH2Cl2=3:97) to give 40 (46 mg, 75%). 1 H NMR(300MHz,DMSO)δ11.57(s,1H),8.40(s,1H),7.94(m,2H),7.52(m,1H),7.40(m,2H), 4.35(t,J=6.3Hz,2H),3.95(m,4H),3.72(m,1H),2.99(s,1H),2.71(s,1H),2.00(m,2H).

[0096] Scheme 13 [ka]

[0097] 4-Methoxy-2-nitro-5-propoxybenzoic acid propyl ester (41) [ka] Following the procedure described for compound 2, 1-bromopropane (3.63 mL, 40.00 mmol) was added to 11 (2.13 g, 10.00 mmol) and K2CO3 (5.52 g, 39.92 mmol) in DMF (50 mL). The resulting solution was heated to 100 °C under N2 to give 41 (2.80 g, 94%). 1 H NMR(500MHz,CDCl3)δ7.41(s,1H),7.04(s,1H),4.24(t,J=6.7Hz,2H),4.04(t,J=6.7Hz,2H),4.92(s,3H),1.87(dt,J=6.7, 7.4Hz,2H),1.71(dt,J=6.7, 7.4Hz,2H),1.03(t,J=7.4Hz,3H),0.95(t,J=7.4Hz,3H).

[0098] 2-Amino-4-methoxy-5-propoxybenzoic acid propyl ester (42) [ka] To a solution of 41 (2.70 g, 9.08 mmol) in HOAc (20 mL) was added Zn (5.94 g, 90.81 mmol) and the mixture was stirred at room temperature under N2 for 48 h. The reaction mixture was filtered through diatomaceous earth. The residue was extracted with EtOAc (150 mL) and NaHCO3 (3 x 150 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc / n-hexane = 1:7) to give 27 (1.90 g, 78%). 1 H NMR(500MHz,CDCl3)δ7.33(s,1H),6.11(s,1H),4.19(t,J=6.7Hz,2H),3.87(t,J=6.7Hz,2H),3.82(s,3H),1.77(m,4H),1.00(m,6H).

[0099] Propyl 4-methoxy-2-propiolamido-5-propoxybenzoate (43) [ka] Following the procedure described for compound 4, a reaction system of 41 (440 mg, 1.74 mmol) in anhydrous CHCl (10 mL) in an ice bath was treated with dropwise addition of propiolic acid (0.183 mL, 2.61 mmol) and N,N'-dicyclohexylcarbodiimide (466 mg, 2.26 mmol) in anhydrous CHCl (5 mL) to give 43 (202 mg, 38%). 1 H NMR(300MHz,CDCl3)δ11.56(s,1H),8.30(s,1H),7.46(s,1H),4.26(t,J=6.7Hz,2H),3.95(t,J=6.8Hz,2H),3.91(s,3H),1.81(m,4H),1.02(m,6H).

[0100] Scheme 14 [ka]

[0101] 2-Amino-4-methoxy-5-propoxybenzoic acid (44) [ka] Following the procedure described for compound 15, the reaction of 42 (500 mg, 1.87 mmol) and LiOH (790 mg, 18.70 mmol) in MeOH (20 mL) was heated to 70 °C under N to give 44 (310 mg, 75%). 1 H NMR(500MHz,CDCl3)δ7.35(s,1H),6.11(s,1H),3.89(t,J=6.8Hz,2H),3.84(s,3H),1.81(m,2H),1.01(t,J=7.5Hz,3H).

[0102] 2-Amino-4-methoxy-5-propoxybenzoic acid propiolic anhydride (45) [ka] Following the procedure described for compound 4, a reaction system of 44 (225 mg, 1.00 mmol) in anhydrous CHCl (10 mL) in an ice bath was treated with dropwise addition of propiolic acid (0.092 mL, 1.50 mmol) and N,N'-dicyclohexylcarbodiimide (267 mg, 1.29 mmol) in anhydrous CHCl (5 mL) to give 45 (5 mg, 2%). 1 H NMR(300MHz,CDCl3)δ7.49(s,1H),7.02(s,1H),4.06(t,J=6.7Hz,2H),3.96(s,3H),3.20(s,1H),1.89(m,2H),1.05(t,J=7.4Hz,3H).

[0103] Scheme 15 [ka]

[0104] Methyl 5-hydroxy-4-methoxy-2-nitrobenzoate (46) [ka] A solution of 11 (20.00 g, 93.83 mmol) in MeOH (300 mL) was diluted with 12 N HCl (aq) (30 mL) was added dropwise. The resulting solution was refluxed under N2 for 72 h. The reaction mixture was concentrated in vacuo. The residue was washed with H2O (100 mL) and filtered to give 46 (19.05 g, 89%) as a white solid. 1 H NMR (300MHz, CDCl3) δ7.50(s,1H),7.12(s,1H),6.17(s,1H),3.99(s,3H),3.88(s,3H).

[0105] Methyl 5-(2-chloroethoxy)-4-methoxy-2-nitrobenzoate (47a) [ka] Following the procedure described for compound 2, 1-bromo-2-chloroethane (0.44 mL, 5.28 mmol) was added to a reaction of 46 (1.00 g, 4.40 mmol) and K2CO3 (1.21 g, 8.80 mmol) in DMF (50 mL). The resulting solution was heated to 100 °C under N2 to give 47a (414 mg, 32%). 1 H NMR(300MHz,CDCl3)δ7.24(s,1H),7.09(s,1H),4.34(t,J=5.9Hz,2H),3.95(s,3H),3.88(s,3H),3.86(t,J=5.9Hz,2H).

[0106] Methyl 5-(3-chloropropoxy)-4-methoxy-2-nitrobenzoate (47b) [ka] Following the procedure described for compound 2, the reaction of 11 (19.05 g, 83.86 mmol), K2CO3 (23.18 g, 167.71 mmol) and 1-bromo-3-chloropropane (12.44 mL, 125.79 mmol) in DMF (350 mL) was heated to 100 °C under N2 to give 47b (21.45 g, 84%). 1 H NMR(300MHz,CDCl3)δ7.43(1, sH),7.09(s,1H),4.24(t,J=5.9Hz,2H),3.93(s,3H),3.89(s,3H),3.74(t,J=6.1Hz,2H),2.30(tt,J=5.9, 6.1Hz,2H).

[0107] Methyl 5-(4-chlorobutoxy)-4-methoxy-2-nitrobenzoate (47c) [ka] Following the procedure described for compound 2, the reaction of 11 (1.00 g, 83.86 mmol), K2CO3 (1.21 g, 8.80 mmol) and 1-bromo-4-chlorobutane (0.61 mL, 5.28 mmol) in DMF (50 mL) was heated to 100 °C under N2 to give 47c (1.09 g, 78%). 1 H NMR(300MHz,CDCl3)δ7.43(s,1H),7.04(s,1H),4.12(t,J=5.9Hz,2H),3.93(s,3H),3.88(s,3H),3.61(t,J=6.2Hz,3H),1.99(m,4H).

[0108] Methyl 5-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)-4-methoxy-2-nitrobenzoate (48a) [ka] Following the procedure described for compound 2, the reaction of 47a (390 mg, 1.35 mmol), K2CO3 (372 mg, 2.69 mmol) and potassium phthalimide (299 mg, 1.61 mmol) in DMF (50 mL) was heated to 100 °C under N2 to give 48a (463 mg, 86%) as a white solid. 1 H NMR(300MHz,CDCl3)δ7.85(m,2H),7.72(m,2H),7.36(s,1H),7.10(s,1H),4.35(t,J=5.8Hz,2H),4.14(t,J=5.9Hz,2H),3.86(s,3H),3.85(s,3H).

[0109] Methyl 5-(3-(1,3-dioxoisoindolin-2-yl)propoxy)-4-methoxy-2-nitrobenzoate (48b) [ka] Following the procedure described for compound 2, the reaction of 47b (21.45 g, 70.63 mmol), K2CO3 (19.52 g, 141.26 mmol) and potassium phthalimide salt (19.62 g, 105.95 mmol) in DMF (300 mL) was heated to 100 °C under N2 to give 48b (29.00 g, 99%) as a white solid. 1 H NMR(300MHz,CDCl3)δ7.84(m,2H),7.72(m,2H),7.33(s,1H),7.00(s,1H),4.15(t ,J=5.9Hz,2H),3.90(t,J=6.4Hz,2H),3.87(s,3H),3.67(s,3H),2.26(tt,J=5.9, 6.4Hz,2H).

[0110] 5-(4-(1,3-dioxoisoindolin-2-yl)butoxy)-4-methoxy-2-nitrobenzoic acid methyl ester (48c) [ka] Following the procedure described for compound 2, the reaction of 47c (950 mg, 2.99 mmol), K2CO3 (826 mg, 5.98 mmol) and potassium phthalimide salt (665 mg, 3.59 mmol) in DMF (50 mL) was heated to 100 °C under N2 to give 48c (1064 mg, 83%) as a white solid. 1 H NMR(300MHz,CDCl3)δ7.82(m,2H),7.70(m,2H),7.40(s,1H),7.03(s,1H),4.11 (t,J=5.9Hz,2H),3.91(s,3H),3.88(s,3H),3.76(t,J=6.2Hz,3H),1.89(m,4H).

[0111] 2-Amino-5-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)-4-methoxybenzoic acid (49a) methyl [ka] Following the procedure described for compound 3, 48a (100 mg, 0.25 mmol) was reacted with 10% Pd / C (10 Mg) in EtOH / EtOAc = 1:1 (10 mL) under H2 to give 49a (74 mg, 80%). 1 H NMR(300MHz,CDCl3)δ7.84(m,2H),7.70(m,2H),7.35(s,1H),6.03(s,1H),5.55 (s,2H),4.16(t,J=5.8Hz,2H),4.06(t,J=5.9Hz,2H),3.79(s,3H),3.67(s,3H).

[0112] Methyl 2-amino-5-(3-(1,3-dioxoisoindolin-2-yl)propoxy)-4-methoxybenzoate (49b) [ka] Following the procedure described for compound 3, 48b (100 mg, 0.24 mmol) was reacted with 10% Pd / C (10 Mg) in EtOH / EtOAc = 1:1 (10 mL) under H2 to give 49b (76 mg, 82%). 1 H NMR(300MHz,CDCl3)δ7.81(m,2H),7.68(m,2H),7.30(s,1H),6.05(s,1H),5.54(s,2H), 3.99(t,J=6.2Hz,2H),3.88(t,J=6.9Hz,2H),3.80(s,3H),3.68(s,3H),2.16(tt,J=6.2, 6.9Hz,2H).

[0113] Methyl 2-amino-5-(4-(1,3-dioxoisoindolin-2-yl)butoxy)-4-methoxybenzoate (49c) [ka] Following the procedure described for compound 3, 48c (100 mg, 0.23 mmol) was reacted with 10% Pd / C (10 Mg) in EtOH / EtOAc = 1:1 (10 mL) under H2 to give 49c (74 mg, 80%). 1 H NMR(300MHz,CDCl3)δ7.80(m,2H),7.68(m,2H),7.28(s,1H),6.08(s,1H),5.55(s,2H) ,3.93(t,J=6.0Hz,2H),3.80(s,3H),3.79(s,3H),3.74(t,J=6.7Hz,3H),1.83(m,4H).

[0114] Methyl 5-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)-4-methoxy-2-propiolamidobenzoate (50a) [ka] Following the procedure described for compound 4, 49a (50 mg, 0.14 mmol), propiolic acid (0.013 mL, 0.20 mmol) and N,N'-dicyclohexylcarbodiimide (42 mg, 0.20 mmol) were reacted in CH2Cl2 (15 mL) under N2 to give 50a (57 mg, 99%). 1 H NMR(500MHz,CDCl3)δ11.52(s,1H),8.25(s,1H),7.84(dd,J=5.5, 3.1Hz,2H),7.71(dd,J=5.5, 3.1Hz,2H),7.51(s,1H),4.25(t,J=5.9Hz,2H),4.10(t,J=5.9Hz,2H),3.88(s,3H),3.79(s,3H),2.90(s,2H).

[0115] Methyl 5-(3-(1,3-dioxoisoindolin-2-yl)propoxy)-4-methoxy-2-propiolamidobenzoate (50b) [ka] Following the procedure described for compound 4, 49b (50 mg, 0.13 mmol), propiolic acid (0.012 mL, 0.20 mmol) and N,N'-dicyclohexylcarbodiimide (40 mg, 0.20 mmol) were reacted in CH2Cl2 (15 mL) under N2 to give 50b (34 mg, 60%). 1 H NMR(500MHz,CDCl3)δ11.53(s,1H),8.22(s,1H),7.80(dd,J=5.4, 3.1Hz,2H),7.69(dd,J=5.4, 3.0Hz,2H),7.43(s,1H),4.08(t,J=6.0Hz,2H),3.90(m,5H),3.68(s,3H),2.91(s,1H),2.21(tt,J=6.0,6.6Hz,2H).

[0116] Methyl 5-(4-(1,3-dioxoisoindolin-2-yl)butoxy)-4-methoxy-2-propiolamidobenzoate (50c) [ka] Following the procedure described for compound 4, 49c (50 mg, 0.13 mmol), propiolic acid (0.012 mL, 0.19 mmol) and N,N'-dicyclohexylcarbodiimide (39 mg, 0.19 mmol) were reacted in CH2Cl2 (15 mL) under N2 to give 50c (56 mg, 99%). 1 H NMR(500MHz,CDCl3)δ11.52(s,1H),8.28(s,1H),7.81(dd,J=5.3, 3.0Hz,2H),7.69(dd,J=5.5, 3.0Hz,2H),7.43(s,1H),4.02(t,J=6.0Hz,2H),3.89(m,6H),3.75(t,J=6.6Hz,2H),2.91(s,1H),1.87(m,4H).

[0117] Scheme 16 [ka]

[0118] Methyl 5-(2-aminoethoxy)-4-methoxy-2-nitrobenzoate (51) [ka] To a solution of 48a (250 mg, 0.62 mmol) in MeOH (25 mL) was added hydrazine monohydrate (0.303 mL, 6.24 mmol) dropwise. The resulting solution was stirred at reflux under N for 4 h. The reaction mixture was concentrated in vacuo, suspended in H2O (150 mL), and extracted with EtOAc (3 x 150 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (MeOH / CH2Cl2 = 1:99) to give 51 (121 mg, 72%) as a brown oil. 1 H NMR (300MHz, CDCl3) δ7.47(s,1H),6.55(s,1H),5.36(s,2H),3.93(2,3H),3.89(m,5H),3.39(m,2H).

[0119] Methyl 5-(2-benzamidoethoxy)-4-methoxy-2-nitrobenzoate (52) [ka] To a solution of 51 (50 mg, 0.19 mmol) in anhydrous CHCl (10 mL) was added pyridine (0.060 mL, 0.74 mmol) and benzoyl chloride (0.065 mL, 0.56 mmol) dropwise. The resulting solution was stirred at room temperature under N for 2.5 h. The reaction mixture was suspended in H0 (50 mL) and then extracted with CHCl (3×50 mL). The combined organic layers were dried over MgSO, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc / n-hexane=1:5) to give 52 (65 mg, 94%). 1H NMR(300MHz,CDCl3)δ8.00(d,J=7.2Hz,2H),7.56(m,1H),7.44(m,3H),6.66(s,1H),5.34(s, 1H),4.54(t,J=5.4Hz,2H),4.10(q,J=7.2Hz,2H),3.93(s,3H),3.89(s,3H),3.3.65(m,2H).

[0120] 2-Amino-5-(2-benzamidoethoxy)-4-methoxybenzoate methyl (53) [ka] Following the procedure described for compound 38, Zn (253 mg, 3.86 mmol) was added to a reaction mixture of 52 (63 mg, 0.17 mmol) in HOAc / THF=1:4 (10 mL), and the mixture was stirred at room temperature under N2 to give 53 (45 mg, 78%). 1 H NMR(300MHz,CDCl3)δ8.03(dd,J=8.3, 1.4Hz,2H),7.53(tt,J=3.7, 1.6Hz,1H),7.42(t,J=7.5Hz,2H),7.12(s,1H),6.09(s,1H),5.38(s,2H) ,4.52(t,J=5.2Hz,2H),3.81(s,3H),3.80(s,3H),3.50(t,J=5.4Hz,2H).

[0121] 4-Methoxy-2-propiolamido-5-(2-(N-propioloylbenzamido)ethoxy)benzoate methyl (54) [ka] Following the procedure described for compound 4, a reaction mixture of 53 (40 mg, 0.12 mmol) in anhydrous CHCl (10 mL) in an ice bath was treated with dropwise addition of propiolic acid (0.011 mL, 0.17 mmol) and N,N'-dicyclohexylcarbodiimide (36 mg, 0.17 mmol) in anhydrous CHCl (5 mL) to give 54 (49 mg, 94%). 1 H NMR(300MHz,CDCl3)δ11.70(s,1H),8.34(s,1H),7.90(s,1H),7.83(m,2H),7.51(tt,J=7.4, 1.6Hz,2H),7.35(m,2H),4.44(m,2H),4.25(m,1H),3.89(m,1H),3.83(s,3H),3.76(s,3H),2.98(s,1H),2.71(s,1H).

[0122] Scheme 17 [ka]

[0123] Methyl 5-(3-aminopropoxy)-4-methoxy-2-nitrobenzoate (55) [ka] To a solution of 48b (29.00 g, 69.99 mmol) in MeOH (500 mL) was added hydrazine monohydrate (33.95 mL, 699.86 mmol) dropwise. The resulting solution was stirred at reflux under N2 for 24 h. The reaction mixture was concentrated in vacuo, suspended in H2O (150 mL), and extracted with EtOAc (3 x 150 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (MeOH / CH2Cl2 = 1:99) to give 55 (3.25 g, 16%) as a brown oil. 1 H NMR(300MHz,CDCl3)δ7.46(s,1H),6.53(s,1H),5.38(s,2H),3.92(2, 3H),3.89(s,3H),3.80(t,J=5.7Hz,2H),3.38(dt,J=5.6, 6.6Hz,2H),1.91(tt,J=5.7, 6.5Hz,2H).

[0124] Methyl 5-(3-benzamidopropoxy)-4-methoxy-2-nitrobenzoate (56a) [ka] To a solution of 55 (100 mg, 0.35 mmol) in anhydrous CHCl (10 mL) was added pyridine (0.113 mL, 1.41 mmol) and benzoyl chloride (0.113 mL, 1.41 mmol). The resulting solution was stirred at room temperature under N for 2.5 h. The reaction mixture was suspended in H0 (50 mL) and then extracted with CHCl (3×50 mL). The combined organic layers were dried over MgSO, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc / n-hexane=1:5) to give 56a (124 mg, 91%). 1 H NMR(300MHz,CDCl3)δ8.02(d,J=7.1Hz,2H),7.57(tt,J=7.4, 1.2Hz,1H),7.45(m,3H),6.54(s,1H),5.26(s,1H),4.45(t,J=5.9Hz,2H),3.87(s,3H),3.85(s,3H),3.42(t,J=5.6Hz,2H),2.14(tt,J=5.9, 5.6Hz,2H).

[0125] Methyl 5-(3-(2-fluorobenzamido)propoxy)-4-methoxy-2-nitrobenzoate (56b) [ka] Following the procedure described for compound 56a, 55 (90 mg, 0.32 mmol), pyridine (0.102 mL, 1.27 mmol) and 2-fluorobenzoyl chloride (0.113 mL, 0.95 mmol) were reacted in CH2Cl2 (10 mL) under N2 to give 56b (116 mg, 90%). 1H NMR(300MHz,CDCl3)δ7.93(td,J=7.5, 1.8Hz,1H),7.53(m,1H),7.45(s,1H),7.23(m,1H),7.16(dd,J=11.1, 2.8Hz,1H),6.55(s,1H),5.24(t,J=5.7Hz,1H),4.45(t,J=5.9Hz,2H),3.87(s,6H),3.44(dt,J=6.0,6.5Hz,2H),2.12(tt,J=5.9,6.5Hz,2H).

[0126] Methyl 5-(3-(3-fluorobenzamido)propoxy)-4-methoxy-2-nitrobenzoate (56c) [ka] A mixture of 55 (100 g, 0.35 mmol), 4-(dimethylamino)pyridine (43 mg, 0.35 mmol) and 3-fluorobenzoic acid (148 mg, 1.05 mmol) in anhydrous CHCl (10 mL) was added dropwise to anhydrous CHCl (5 mL) containing N,N'-dicyclohexylcarbodiimide (363 mg, 1.76 mmol) in an ice bath. The resulting solution was stirred at room temperature under N for 12 h. The reaction mixture was suspended in H0 (50 mL) and then extracted with CHCl (3 x 50 mL). The combined organic layers were dried over MgSO, filtered and concentrated in vacuo. The mixture was filtered and washed with EtOAc / n-hexane = 1:1 (10 mL). The residue was purified by silica gel chromatography (EtOAc / n-hexane = 1:5) to give 56c (129 mg, 90%). 1 H NMR(300MHz,CDCl3)δ7.82(dt,J=7.6, 1.3Hz,1H),7.69(m,1H),7.46(s,H),7.42(m,1H),7.28(m,1H),6.54(s,1H),5.22(s, 1H),4.45(t,J=5.9Hz,2H),3.88(s,6H),3.42(dt,J=5.0,6.2Hz,2H),2.14(tt,J=6.2, 5.9Hz,2H).

[0127] Methyl 5-(3-(4-fluorobenzamido)propoxy)-4-methoxy-2-nitrobenzoate (56d) [ka] Following the procedure described for compound 56a, 55 (150 mg, 0.53 mmol), pyridine (0.171 mL, 2.11 mmol) and 4-fluorobenzoyl chloride (0.187 mL, 1.58 mmol) were reacted in CH2Cl2 (10 mL) under N2 to give 56d (191 mg, 89%). 1 H NMR(300MHz,CDCl3)δ8.04(m,2H),7.46(s,1H),7.11(m,2H),6.54(s,1H),5.21(s,1H),4.43(t,J=5.9Hz,2H),3.88(s,6H),3.41(dt,J=5.9, 6.7Hz,2H),2.13(tt,J=6.7, 5.9Hz,3H).

[0128] Methyl 5-(3-(2,6-difluorobenzamido)propoxy)-4-methoxy-2-nitrobenzoate (56e) [ka] Following the procedure described for compound 56c, 55 (100 mg, 0.35 mmol), 2,6-difluorobenzoic acid (167 mg, 1.05 mmol), 4-(dimethylamino)pyridine (43 mg, 0.35 mmol) and N,N'-dicyclohexylcarbodiimide (363 mg, 1.76 mmol) in CHCl (15 mL) under N gave 56e (147 mg, 98%). 1 H NMR(300MHz,CDCl3)δ7.46(s,1H),7.38(m,1H),6.96(m,2H),6.54(s,1H),5.20(s,1H), 4.47(t,J=5.9Hz,2H),3.88(s,3H),3.88(s,3H),3.42(t,J=6.8Hz,2H),2.10(tt,J=5.9, 6.8Hz,3H).

[0129] Methyl 5-(3-(2,6-dimethoxybenzamido)propoxy)-4-methoxy-2-nitrobenzoate (56f) [ka] Following the procedure described for compound 56a, 55 (120 mg, 0.42 mmol), pyridine (0.136 mL, 1.69 mmol) and 2,6-dimethoxybenzoyl chloride (254 mg, 1.27 mmol) in CH2Cl2 (10 mL) were reacted under N2 to give 56f (189 mg, 99%). 1 H NMR(300MHz,CDCl3)δ7.45(s,1H),7.29(t,J=8.5Hz,1H),6.56(d,J=8.5Hz,2H),6.52(s,1H) ,5.37(s,1H),4.45(t,J=5.8Hz,2H),3.86(s,3H),3.83(s,3H),3.78(s,6H),3.42(dt,J=6.1, 6.4Hz,2H),2.07(tt,J=5.8, 6.4Hz,3H).

[0130] Methyl 4-methoxy-5-(3-(3-methoxybenzamido)propoxy)-2-nitrobenzoate (56g) [ka] Following the procedure described for compound 56a, 55 (150 mg, 0.53 mmol), pyridine (0.171 mL, 2.11 mmol) and 3-methoxybenzoyl chloride (222 mL, 1.58 mmol) were reacted in CH2Cl2 (10 mL) under N2 to give 56g (218 mg, 99%). 1H NMR(300MHz,CDCl3)δ7.78(dt,J=7.8, 1.2Hz,1H),7.69(m,1H),7.61(s,1H),7.51(t,J=7.9Hz,1H),7.27(ddd,J=8.3, 2.7, 0.9Hz,1H),6.69(s,1H),5.41(s,1H),4.60(t,J=5.9Hz,2H),4.04(s,3H) ,4.02(s,3H),4.00(s,3H),3.58(dt,J=6.0,6.5Hz,2H),2.11(tt,J=5.9, 6.5Hz,2H).

[0131] 4-Methoxy-5-(3-(4-methoxybenzamido)propoxy)-2-nitrobenzoate methyl (56h) [ka] Following the procedure described for compound 56a, 55 (90 mg, 0.32 mmol), pyridine (0.102 mL, 1.27 mmol) and 4-methoxybenzoyl chloride (0.128 mL, 0.95 mmol) were reacted in CH2Cl2 (10 mL) under N2 to give 56h (131 mg, 99%). 1 H NMR(300MHz,CDCl3)δ7.97(d,J=9.0Hz,2H),7.45(s,1H),6.91(d,J=9.0Hz,2H),6.54(s,1H),5.26(s,1H), 4.41(t,J=5.9Hz,2H),3.88(s,3H),3.87(s,3H),3.85(s,3H),3.41(dt,J=6.0,6.5Hz,2H),2.11(tt,J=5.9, 6.0Hz,2H).

[0132] Methyl 4-methoxy-2-nitro-5-(3-(2-phenylacetamido)propoxy)benzoate (56i) [ka] Following the procedure described for compound 56c, 55 (50 mg, 0.18 mmol), phenylacetic acid (48 mg, 0.35 mmol), 4-(dimethylamino)pyridine (21 mg, 0.18 mmol) and N,N'-dicyclohexylcarbodiimide (73 mg, 0.35 mmol) in CHCl (15 mL) under N afforded 56i (68 mg, 96%). 1 H NMR(300MHz,CDCl3)δ7.47(s,1H),7.29(m,5H),6.47(s,1H),5.11(s,1H),4.20(t,J=6.0Hz,2H), 3.92(s,3H),3.90(s,3H),3.63(s,2H),3.24(dt,J=6.0,6.5Hz,2H),1.96(tt,J=6.0,6.9Hz,2H).

[0133] Methyl 5-(3-(2-(2-fluorophenyl)acetamido)propoxy)-4-methoxy-2-nitrobenzoate (56j) [ka] Following the procedure described for compound 56c, 55 (120 mg, 0.42 mmol), 2-fluorophenylacetic acid (195 mg, 1.27 mmol), 4-(dimethylamino)pyridine (51 mg, 0.42 mmol) and N,N'-dicyclohexylcarbodiimide (435 mg, 2.11 mmol) in CHCl (15 mL) under N afforded 56j (176 mg, 99%). 1 H NMR(300MHz,CDCl3)δ7.47(s,1H),7.24(m,2H),7.10(m,1H),7.06(m,1H),6.48(m,1H),5.14(s ,1H),4.22(t,J=6.0Hz,2H),3.92(s,3H),3.90(s,3H),3.68(d,J=1.1Hz,2H),3.27(dt,J=5.9, 6.7Hz,2H),1.97(tt,J=6.0,6.7Hz,2H).

[0134] Methyl 5-(3-(2-(3-fluorophenyl)acetamido)propoxy)-4-methoxy-2-nitrobenzoate (56k) [ka] Following the procedure described for compound 56a, 55 (150 mg, 0.53 mmol), pyridine (0.170 mL, 2.11 mmol) and 3-fluorophenyl chloride (109 mg, 0.63 mmol) were reacted in CH2Cl2 (10 mL) under N2 to give 56k (210 mg, 95%). 1 H NMR(300MHz,CDCl3)δ7.47(s,1H),7.27(m,1H),7.03(d,J=8.5Hz,1H),6.96(m,2H),6.48(s,1H ),5.10(s,1H),4.21(t,J=6.1Hz,2H),3.92(s,3H),3.90(s,3H),3.62(s,2H),3.26(dt,J=5.9, 6.6Hz,2H),1.98(tt,J=6.1, 6.6Hz,2H).

[0135] Methyl 5-(3-(2-(4-fluorophenyl)acetamido)propoxy)-4-methoxy-2-nitrobenzoate (56l) [ka] Following the procedure described for compound 56c, 55 (150 mg, 0.53 mmol), 4-fluorophenylacetic acid (244 mg, 1.58 mmol), 4-(dimethylamino)pyridine (64 mg, 0.53 mmol) and N,N'-dicyclohexylcarbodiimide (544 mg, 2.64 mmol) were reacted in CHCl (15 mL) under N to give 56l (219 mg, 99%). 1H NMR(300MHz,CDCl3)δ7.47(s,1H),7.22(m,2H),6.99(m,2H),6.49(s,1H),5.11(s, 1H),4.20(t,J=6.0Hz,2H),3.92(s,3H),3.90(s,3H),3.60(s,2H),3.26(dt,J=5.9, 6.6Hz,2H),1.97(tt,J=6.0,6.6Hz,2H).

[0136] 4-Methoxy-5-(3-(2-(2-methoxyphenyl)acetamido)propoxy)-2-nitrobenzoate methyl (56m) [ka] Following the procedure described for compound 56c, 55 (120 mg, 0.42 mmol), 2-methoxyphenylacetic acid (210 mg, 1.27 mmol), 4-(dimethylamino)pyridine (51 mg, 0.42 mmol) and N,N'-dicyclohexylcarbodiimide (435 mg, 2.11 mmol) in CHCl (15 mL) under N afforded 56m (118 mg, 65%). 1 H NMR(300MHz,CDCl3)δ7.46(s,1H),7.25(td,J=7.8, 1.7Hz,1H),7.16(dd,J=7.4, 1.7Hz,1H),6.89(td,J=7.4, 1.1Hz,1H),6.86(d,J=8.2Hz,1H),6.47(s,1H),5.19(s,1H),4.21(t,J=6.0H z,2H),3.91(s,3H),3.90(s,3H),3.77(s,3H),3.63(s,2H),3.26(dt,J=5.9, 6.6Hz,2H),1.96(tt,J=6.0,6.6Hz,2H).

[0137] 4-Methoxy-5-(3-(2-(3-methoxyphenyl)acetamido)propoxy)-2-nitrobenzoate methyl (56n) [ka] Following the procedure described for compound 56a, 55 (150 mg, 0.53 mmol), pyridine (0.128 mL, 1.58 mmol) and 3-methoxyphenyl chloride (0.089 mL, 0.63 mmol) were reacted in CH2Cl2 (10 mL) under N2 to give 56n (206 mg, 90%). 1 H NMR(300MHz,CDCl3)δ7.46(s,1H),7.25(td,J=7.5, 1.2Hz,1H),6.81(m,3H),6.48(s,1H),5.13(s,1H),4.20(t,J=6.0Hz,2H ),3.92(s,3H),3.90(s,3H),3.77(s,3H),3.60(s,2H),3.25(dt,J=5.9, 6.6Hz,2H),1.97(tt,J=6.0,6.6Hz,2H).

[0138] 4-Methoxy-5-(3-(2-(4-methoxyphenyl)acetamido)propoxy)-2-nitrobenzoate methyl (56o) [ka] Following the procedure described for compound 56c, 55 (130 mg, 0.46 mmol), 4-methoxyphenylacetic acid (228 mg, 1.37 mmol), 4-(dimethylamino)pyridine (51 mg, 0.46 mmol) and N,N'-dicyclohexylcarbodiimide (472 mg, 2.29 mmol) in CHCl (15 mL) under N afforded 56o (172 mg, 87%). 1 H NMR(300MHz,CDCl3)δ7.47(s,1H),7.17(m,2H),6.84(m,2H),6.49(s,1H),5.48(t,J=5.5Hz,1H),4.19(t,J=6.0Hz, 2H),3.92(s,3H),3.90(s,3H),3.77(s,3H),3.56(s,2H),3.26(dt,J=6.0,6.6Hz,2H),1.96(tt,J=6.0,6.6Hz,2H).

[0139] 4-Methoxy-5-(3-(2-(3,4,5-trimethoxyphenyl)acetamido)propoxy)-2-nitrobenzoate methyl (56p) [ka] Following the procedure described for compound 56c, 55 (120 mg, 0.42 mmol), 3,4,5-trimethoxyphenylacetic acid (286 mg, 1.27 mmol), 4-(dimethylamino)pyridine (52 mg, 0.42 mmol) and N,N'-dicyclohexylcarbodiimide (435 mg, 2.11 mmol) in CHCl (15 mL) under N afforded 56p (168 mg, 81%). 1 H NMR(300MHz,CDCl3)δ7.47(s,1H),6.52(s,1H),6.48(s,2H),5.14(s,1H),4.21(t,J=6.0H z,2H),3.92(s,3H),3.90(s,3H),3.82(s,6H),3.80(s,3H),3.56(s,2H),3.30(dt,J=5.9, 6.7Hz,2H),1.99(tt,J=6.0,6.7Hz,2H).

[0140] Methyl 5-(3-(2-(2-chlorophenyl)acetamido)propoxy)-4-methoxy-2-nitrobenzoate (56q) [ka] Following the procedure described for compound 56a, 55 (150 mg, 0.53 mmol), pyridine (0.128 mL, 1.58 mmol) and 2-chlorophenylacetyl chloride (0.092 mL, 0.63 mmol) were reacted in CH2Cl2 (10 mL) under N2 to give 56q (207 mg, 90%). 1H NMR(300MHz,CDCl3)δ7.47(s,1H),7.37(m,1H),7.24(m,3H),6.47(s,1H),5.13(s,1H),4.23(t,J=6.0H z,2H),3.92(s,3H),3.90(s,3H),3.78(s,2H),3.26(dt,J=6.0,6.6Hz,2H),1.97(tt,J=6.0,6.6Hz,2H).

[0141] Methyl 5-(3-(2-(3-chlorophenyl)acetamido)propoxy)-4-methoxy-2-nitrobenzoate (56r) [ka] Following the procedure described for compound 56c, 55 (120 mg, 0.42 mmol), 3-chlorophenylacetic acid (216 mg, 1.27 mmol), 4-(dimethylamino)pyridine (51 mg, 0.42 mmol) and N,N'-dicyclohexylcarbodiimide (435 mg, 2.11 mmol) in CHCl (15 mL) under N afforded 56r (182 mg, 99%). 1 H NMR(300MHz,CDCl3)δ7.47(s,1H),7.25(m,3H),7.15(m,1H),6.49(s,1H),5.10(t,J=5. 3Hz,1H),4.21(t,J=6.1Hz,2H),3.92(s,3H),3.90(s,3H),3.60(s,2H),3.27(dt,J=5.9, 6.7Hz,2H),1.98(tt,J=6.1, 6.7Hz,2H).

[0142] 4-Methoxy-5-(3-(2-(2-(trifluoromethyl)phenyl)acetamido)propoxy)-2-nitrobenzoate methyl (56s) [ka] Following the procedure described for compound 56c, 55 (150 mg, 0.53 mmol), 2-(trifluoromethyl)phenylacetic acid (323 mg, 1.58 mmol), 4-(dimethylamino)pyridine (64 mg, 0.53 mmol) and N,N'-dicyclohexylcarbodiimide (544 mg, 2.64 mmol) in CHCl (15 mL) under N afforded 56s (230 mg, 93%). 1 H NMR(300MHz,CDCl3)δ7.65(d,J=7.9Hz,1H),7.51(t,J=7.4Hz,1H),7.46(s,1H),7.38(t,J=6.1Hz,2H),6.47(s,1H),5.12(s,1H), 4.21(t,J=6.0Hz,2H),3.92(s,3H),3.90(s,3H),3.84(d,J=1.1Hz,2H),3.26(dt,J=6.0,6.7Hz,2H),1.96(tt,J=6.0,6.7Hz,2H).

[0143] 4-Methoxy-5-(3-(2-(3-(trifluoromethyl)phenyl)acetamido)propoxy)-2-nitrobenzoate methyl (56t) [ka] Following the procedure described for compound 56c, 55 (150 mg, 0.53 mmol), 3-(trifluoromethyl)phenylacetic acid (323 mg, 1.58 mmol), 4-(dimethylamino)pyridine (64 mg, 0.53 mmol) and N,N'-dicyclohexylcarbodiimide (544 mg, 2.64 mmol) were reacted in CHCl (15 mL) under N to give 56t (240 mg, 97%). 1H NMR(300MHz,CDCl3)δ7.53(m,2H),7.45(m,3H),6.49(s,1H),5.10(t,J=5.4Hz,1H ),4.22(t,J=6.0Hz,2H),3.92(s,3H),3.90(s,3H),3.69(s,2H),3.27(dt,J=5.9, 6.7Hz,2H),1.98(tt,J=6.0,6.7Hz,2H).

[0144] 4-Methoxy-5-(3-(2-(4-(trifluoromethyl)phenyl)acetamido)propoxy)-2-nitrobenzoate methyl (56u) [ka] Following the procedure described for compound 56a, 55 (150 mg, 0.53 mmol), pyridine (0.171 mL, 2.11 mmol) and 4-(trifluoromethyl)phenylacetyl chloride (141 mg, 0.63 mmol) in CH2Cl2 (10 mL) were reacted under N2 to give 56u (223 mg, 90%). 1 H NMR(300MHz,CDCl3)δ7.58(d,J=8.1Hz,2H),7.47(s,1H),7.39(d,J=8.1Hz,2H),6.51(s,1H),5.11(s,1H),4.21( t,J=6.1Hz,2H),3.92(s,3H),3.90(s,3H),3.69(s,2H),3.29(dt,J=5.9,6.5Hz,2H),1.98(tt,J=6.1,6.5Hz,2H).

[0145] 4-Methoxy-5-(3-(oxazole-5-carboxamido)propoxy)-2-nitrobenzoate methyl (56v) [ka] Following the procedure described for compound 56c, 55 (150 mg, 0.53 mmol), oxazole-5-carboxylic acid (179 mg, 1.58 mmol), 4-(dimethylamino)pyridine (64 mg, 0.53 mmol) and N,N'-dicyclohexylcarbodiimide (544 mg, 2.64 mmol) in CHCl (15 mL) under N afforded 56v (185 mg, 92%). 1 H NMR(300MHz,CDCl3)δ8.02(s,1H),7.79(s,1H),7.46(s,1H),6.54(s,1H),5.18(t,J=5.6Hz,1H),4.45 (t,J=6.0Hz,2H),3.92(s,3H),3.88(s,3H),3.40(dt,J=6.0,6.6Hz,2H),2.11(tt,J=6.0,6.6Hz,2H).

[0146] 2-Amino-5-(3-benzamidopropoxy)-4-methoxybenzoate methyl ester (57a) [ka] Following the procedure described for compound 38, Zn (253 mg, 3.86 mmol) was added to the reaction mixture of 56a (150 mg, 0.39 mmol) in HOAc / THF=1:4 (10 mL), and the mixture was stirred at room temperature under N2 to give 57a (113 mg, 82%). 1 H NMR(300MHz,CDCl3)δ8.05(m,2H),7.54(tt,J=7.4, 1.6Hz,1H),7.42(m,2H),7.04(s,1H),6.08(s,1H),4.44(t,J=6.1Hz,2H),3.79(s,3H),3.77(s,3H),3.27(t,J=5.8Hz,2H),2.11(tt,J=6.1, 5.8Hz,2H).

[0147] Methyl 2-amino-5-(3-(2-fluorobenzamido)propoxy)-4-methoxybenzoate (57b) [ka] Following the procedure described for compound 38, 56b (100 mg, 0.25 mmol) and Zn (161 mg, 2.46 mmol) were reacted in HOAc / THF=1:4 (10 mL) under N2 to give 57b (89 mg, 96%). 1 H NMR(300MHz,CDCl3)δ7.93(td,J=7.5, 1.9Hz,1H),7.51(m,1H),7.18(tt,J=7.6, 1.1Hz,1H),7.12(ddd,J=10.8, 8.3, 1.0Hz,1H),7.04(s,1H),6.08(s,1H),4.45(t,J=6.1Hz,2H),3.79(s,3H),3.78(s,3H),3.28(t,J=6.7Hz,2H),2.10(tt,J=6.1, 6.7Hz,2H).

[0148] Methyl 2-amino-5-(3-(3-fluorobenzamido)propoxy)-4-methoxybenzoate (57c) [ka] Following the procedure described for compound 38, 56c (200 mg, 0.49 mmol) and Zn (321 mg, 4.92 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57c (120 mg, 65%). 1 H NMR(300MHz,CDCl3)δ7.82(d,J=7.7Hz,1H),7.72(m,1H),7.41(m,1H),7.25(m,1H),7.03(s,1H),6.09(s, 1H),5.35(s,2H),4.45(t,J=6.1Hz,2H),3.80(s,3H),3.79(s,3H),3.27(t,J=6.7Hz,2H),2.11(tt,J=6.1, 6.7Hz,2H).

[0149] 2-Amino-5-(3-(4-fluorobenzamido)propoxy)-4-methoxybenzoate methyl (57d) [ka] Following the procedure described for compound 38, 56d (180 mg, 0.44 mmol) and Zn (289 mg, 4.43 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57d (125 mg, 75%). 1 H NMR(300MHz,CDCl3)δ8.06(m,2H),7.10(m,2H),7.03(s,1H),6.09(s,1H),5.02(s,2H), 4.43(t,J=6.2Hz,2H),3.80(s,3H),3.78(s,3H),3.26(t,J=6.7Hz,2H),2.10(tt,J=6.2, 6.7Hz,3H).

[0150] 2-Amino-5-(3-(2,6-difluorobenzamido)propoxy)-4-methoxybenzoate methyl (57e) [ka] Following the procedure described for compound 38, 56e (200 mg, 0.47 mmol) and Zn (308 mg, 4.71 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57e (149 mg, 80%). 1 H NMR(300MHz,CDCl3)δ7.40(m,1H),7.03(s,1H),6.94(t,J=8.2Hz,2H),6.08(s,1H),4. 48(t,J=6.2Hz,2H),3.80(s,3H),3.79(s,3H),3.26(t,J=6.8Hz,2H),2.08(tt,J=6.2, 6.8Hz,3H).

[0151] 2-Amino-5-(3-(2,6-dimethoxybenzamido)propoxy)-4-methoxybenzoate methyl (57f) [ka] Following the procedure described for compound 38, 56f (150 mg, 0.34 mmol) and Zn (218 mg, 3.35 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57f (70 mg, 50%). 1 H NMR(300MHz,CDCl3)δ7.27(t,J=8.5Hz,1H),7.03(s,1H),6.54(d,J=8.3Hz,2H),6.08(s,1H),4.4 4(t,J=6.0Hz,2H),3.78(s,3H),3.77(s,9H),3.25(t,J=6.9Hz,2H),2.05(tt,J=6.0,6.9Hz,3H).

[0152] 2-Amino-4-methoxy-5-(3-(3-methoxybenzamido)propoxy)benzoic acid methyl ester (57g) [ka] Following the procedure described for compound 38, 56g (200 mg, 0.48 mmol) and Zn (313 mg, 4.79 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57g (166 mg, 89%). 1 H NMR(300MHz,CDCl3)δ7.64(dt,J=7.6, 1.2Hz,1H),7.56(dd,J=2.6, 1.5Hz,1H),7.33(t,J=8.0Hz,1H),7.09(ddd,J=8.2, 2.7, 1.0Hz,1H),7.04(s,1H),6.08(s,1H),4.44(t,J=6.1Hz,2H),4.83(s,3H),3.79(s,3H),3.78(s,3H),3.27(t,J=6.7Hz,2H),2.10(tt,J=6.1, 6.7Hz,2H).

[0153] 2-Amino-4-methoxy-5-(3-(4-methoxybenzamido)propoxy)benzoic acid methyl ester (57h) [ka] Following the procedure described for compound 38, 56h (115 mg, 0.27 mmol) and Zn (180 mg, 2.75 mmol) were reacted in HOAc / THF=1:4 (10 mL) under N2 to give 57h (85 mg, 80%). 1 H NMR(300MHz,CDCl3)δ8.00(m,2H),7.04(s,1H),6.90(m,2H),6.08(s,1H),4.41(t,J=6. 1Hz,2H),3.84(s,3H),3.80(s,3H),3.78(s,3H),3.26(t,J=6.8Hz,2H),2.09(tt,J=6.1, 6.8Hz,2H).

[0154] Methyl 2-amino-4-methoxy-5-(3-(2-phenylacetamido)propoxy)benzoate (57i) [ka] Following the procedure described for compound 38, 56i (81 mg, 0.20 mmol) and Zn (132 mg, 2.01 mmol) were reacted in HOAc / THF=1:4 (10 mL) under N2 to give 57i (69 mg, 92%). 1 H NMR(300MHz,CDCl3)δ7.28(m,5H),7.00(s,1H),6.09(s,1H),4.21(t,J=6.2Hz,2H ),3.83(s,3H),3.81(s,3H),3.62(s,2H),3.13(t,J=6.8Hz,2H),1.94(tt,J=6.2, 6.8Hz,2H).

[0155] 2-Amino-5-(3-(2-(2-fluorophenyl)acetamido)propoxy)-4-methoxybenzoate methyl (57j) [ka] Following the procedure described for compound 38, 56j (150 mg, 0.36 mmol) and Zn (233 mg, 3.57 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57j (74 mg, 53%). 1 H NMR(300MHz,CDCl3)δ7.24(m,2H),7.05(m,2H),7.00(s,1H),6.09(s,1H),4.23(t,J=6. 2Hz,1H),3.83(s,3H),3.81(s,3H),3.68(s,2H),3.13(t,J=6.8Hz,2H),1.95(tt,J=6.2, 6.8Hz,2H).

[0156] 2-Amino-5-(3-(2-(3-fluorophenyl)acetamido)propoxy)-4-methoxybenzoate methyl (57k) [ka] Following the procedure described for compound 38, 56k (150 mg, 0.36 mmol) and Zn (233 mg, 3.57 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57k (110 mg, 79%). 1 H NMR(300MHz,CDCl3)δ7.26(m,1H),6.98(m,4H),6.09(s,1H),4.22(t,J=6.3Hz,2H ),3.83(s,3H),3.81(s,3H),3.61(s,2H),3.13(t,J=6.8Hz,2H),1.95(tt,J=6.3,6 .8Hz,2H).

[0157] 2-Amino-5-(3-(2-(4-fluorophenyl)acetamido)propoxy)-4-methoxybenzoate methyl (57l) [ka] Following the procedure described for compound 38, 56l (175 mg, 0.42 mmol) and Zn (272 mg, 4.16 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57l (120 mg, 74%). 1 H NMR(300MHz,CDCl3)δ7.23(m,2H),6.98(m,3H),6.09(s,1H),4.21(t,J=6.2Hz,2H ),3.83(s,3H),3.81(s,3H),3.59(s,2H),3.13(t,J=6.8Hz,2H),1.95(tt,J=6.2, 6.8Hz,2H).

[0158] 2-Amino-4-methoxy-5-(3-(2-(2-methoxyphenyl)acetamido)propoxy)benzoic acid methyl ester (57m) [ka] Following the procedure described for compound 38, 56m (100 mg, 0.23 mmol) and Zn (151 mg, 2.31 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57m (73 mg, 78%). 1 H NMR(300MHz,CDCl3)δ7.25(td,J=7.9, 1.7Hz,1H),7.16(dd,J=7.5, 1.7Hz,1H),7.00(s,1H),6.89(td,J=7.4, 1.0Hz,1H),6.84(d,J=8.2Hz,1H),6.09(s,1H),4.21(t,J=6.2Hz,2H),3.83(s,3H) ),3.80(s,3H),3.77(s,3H),3.63(s,2H),3.12(t,J=6.8Hz,2H),1.94(tt,J=6.2, 6.8Hz,2H).

[0159] 2-Amino-4-methoxy-5-(3-(2-(3-methoxyphenyl)acetamido)propoxy)benzoic acid methyl ester (57n) [ka] Following the procedure described for compound 38, 56n (150 mg, 0.35 mmol) and Zn (227 mg, 3.47 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57n (97 mg, 69%). 1 H NMR(300MHz,CDCl3)δ7.21(t,J=7.8Hz,1H),6.99(s,1H),6.85(m,1H),6.79(m,2H),6.09(s,1H),4.21(t,J =6.3Hz,2H),3.82(s,3H),3.81(s,3H),3.76(s,3H),3.59(s,2H),3.13(t,J=6.8Hz,2H),1.95(tt,J=6.3,6 .8Hz,2H).

[0160] 2-Amino-4-methoxy-5-(3-(2-(4-methoxyphenyl)acetamido)propoxy)benzoic acid methyl ester (57o) [ka] Following the procedure described for compound 38, 56o (150 mg, 0.35 mmol) and Zn (227 mg, 3.47 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57o (120 mg, 86%). 1 H NMR(300MHz,CDCl3)δ7.18(m,2H),7.00(s,1H),6.83(m,2H),6.09(s,1H),4.20(t,J=6.3Hz,2H),3.8 3(s,3H),3.81(s,3H),3.76(s,3H),3.56(s,2H),3.13(t,J=6.8Hz,2H),1.94(tt,J=6.3,6.8Hz,2H).

[0161] 2-Amino-4-methoxy-5-(3-(2-(3,4,5-trimethoxyphenyl)acetamido)propoxy)benzoic acid methyl ester (57p) [ka] Following the procedure described for compound 38, 56p (130 mg, 0.26 mmol) and Zn (173 mg, 2.64 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57p (99 mg, 81%). 1 H NMR(300MHz,CDCl3)δ7.00(s,1H),6.48(s,2H),6.09(s,1H),4.23(t,J=6.2Hz,2H),3.82(s,3 H),3.81(s,9H),3.80(s,3H),3.55(s,2H),3.16(t,J=6.7Hz,2H),1.97(tt,J=6.2,6.7Hz,2H).

[0162] 2-Amino-5-(3-(2-(2-chlorophenyl)acetamido)propoxy)-4-methoxybenzoate methyl (57q) [ka] Following the procedure described for compound 38, 56q (150 mg, 0.34 mmol) and Zn (224 mg, 3.43 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57q (118 mg, 84%). 1 H NMR(300MHz,CDCl3)δ7.36(m,1H),7.23(m,3H),7.00(s,1H),6.09(s,1H),4.24(t,J=6. 2Hz,2H),3.83(s,3H),3.81(s,3H),3.78(s,2H),3.13(t,J=6.8Hz,2H),1.95(tt,J=6.2, 6.8Hz,2H).

[0163] 2-Amino-5-(3-(2-(3-chlorophenyl)acetamido)propoxy)-4-methoxybenzoate methyl (57r) [ka] Following the procedure described for compound 38, 56r (150 mg, 0.34 mmol) and Zn (224 mg, 3.43 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57r (100 mg, 72%). 1 H NMR(300MHz,CDCl3)δ7.27(s,1H),7.22(m,2H),7.15(m,1H),7.00(s,1H),6.09(s,1H),4.22(t ,J=6.2Hz,2H),3.83(s,3H),3.81(s,3H),3.59(s,2H),3.14(t,J=6.8Hz,2H),1.95(tt,J=6.2, 6.8Hz,2H).

[0164] 2-Amino-4-methoxy-5-(3-(2-(2-(trifluoromethyl)phenyl)acetamido)propoxy)benzoic acid methyl ester (57s) [ka] Following the procedure described for compound 38, 56s (175 mg, 0.37 mmol) and Zn (243 mg, 3.72 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57s (151 mg, 92%). 1 H NMR(300MHz,CDCl3)δ7.64(d,J=7.4Hz,1H),7.49(t,J=7.2Hz,1H),7.36(m,2H),6.99(s,1H),6.0 9(s,1H),4.23(t,J=6.2Hz,2H),3.83(s,6H),3.80(s,2H),3.12(t,J=6.8Hz,2H),1.94(tt,J=6.2, 6.8Hz,2H).

[0165] 2-Amino-4-methoxy-5-(3-(2-(3-(trifluoromethyl)phenyl)acetamido)propoxy)benzoate methyl (57t) [ka] Following the procedure described for compound 38, 56t (200 mg, 0.43 mmol) and Zn (278 mg, 4.25 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57t (112 mg, 60%). 1 H NMR(300MHz,CDCl3)δ7.46(m,4H),7.00(s,1H),6.09(s,1H),4.23(t,J=6.3Hz,2H ),3.82(s,3H),3.81(s,3H),3.68(s,2H),3.14(t,J=6.8Hz,2H),1.96(tt,J=6.3,6 .8Hz,2H).

[0166] 2-Amino-4-methoxy-5-(3-(2-(4-(trifluoromethyl)phenyl)acetamido)propoxy)benzoic acid methyl ester (57u) [ka] Following the procedure described for compound 38, 56u (200 mg, 0.43 mmol) and Zn (278 mg, 4.25 mmol) were reacted in HOAc / THF=1:4 (20 mL) under N2 to give 57u (136 mg, 73%). 1 H NMR(300MHz,CDCl3)δ7.56(d,J=8.2Hz,2H),7.39(d,J=8.2Hz,2H),7.01(s,1H),6.09(s,1H),4.23 (t,J=6.3Hz,2H),3.82(s,3H),3.81(s,3H),3.68(s,2H),3.14(dt,J=6.8Hz,2H),1.96(tt,J=6.3,6 .8Hz,2H).

[0167] 2-Amino-4-methoxy-5-(3-(oxazole-5-carboxamido)propoxy)benzoic acid methyl ester (57v) [ka] Following the procedure described for compound 38, 56v (150 mg, 0.40 mmol) was reacted with Zn (259 mg, 3.95 mmol) in HOAc / THF=1:4 (20 mL) under N2 to give 57v (132 mg, 96%). 1 H NMR(300MHz,CDCl3)δ8.00(s,1H),7.78(s,1H),7.02(s,1H),6.09(s,1H),4.46(t ,J=6.2Hz,2H),3.81(s,3H),3.81(s,3H),3.24(t,J=6.7Hz,2H),2.09(tt,J=6.2, 6.7Hz,2H).

[0168] Methyl 4-methoxy-2-propiolamido-5-(3-(N-propioloylbenzamido)propoxy)benzoate (58a) [ka] Following the procedure described for compound 4, 57a (50 mg, 0.14 mmol), propiolic acid (0.018 mL, 0.28 mmol) and N,N'-dicyclohexylcarbodiimide (58 mg, 0.28 mmol) were reacted in CHCl (15 mL) under N to give 58a (61 mg, 94%). 1 H NMR(500MHz,CDCl3)δ11.71(s,1H),8.38(s,1H),7.95(d,J=7.9Hz,2H),7.88(s,1H),7.52(t,J=7.4Hz,1H),7.39(t,J=7.7Hz ,2H),4.34(t,J=6.4Hz,2H),3.95(m,1H),3.90(s,3H),3.89(s,3H),3.71(m,1H),2.98(s,1H),2.69(s,1H),2.00(tt,J=6.4, 6.7Hz,2H).

[0169] 5-(3-(2-fluoro-N-propioloylbenzamido)propoxy)-4-methoxy-2-propiolamidobenzoate methyl ester (58b) [ka] Following the procedure described for compound 4, 57b (50 mg, 0.13 mmol), propiolic acid (0.016 mL, 0.27 mmol) and N,N'-dicyclohexylcarbodiimide (55 mg, 0.27 mmol) were reacted in CHCl (15 mL) under N to give 58b (57 mg, 89%). 1 H NMR(500MHz,CDCl3)δ11.71(s,1H),8.38(s,1H),7.88(s,1H),7.86(td,J=7.6, 1.8Hz,1H),7.48(m,1H),7.16(t,J=7.2Hz,1H),7.07(dd,J=10.5, 8.4Hz,1H),4.35(t,J=6.5Hz,2H),3.94(m,1H),3.91(s,3H),3.90(s,3H),3.72(m,1H),2.98(s,1H),2.68(s,1H),2.00(tt,J=6.5, 7.0Hz,2H).

[0170] 5-(3-(3-fluoro-N-propioloylbenzamido)propoxy)-4-methoxy-2-propiolamidobenzoate methyl ester (58c) [ka] Following the procedure described for compound 4, 57c (90 mg, 0.24 mmol), propiolic acid (0.029 mL, 0.48 mmol) and N,N′-dicyclohexylcarbodiimide (99 mg, 0.47 mmol) were reacted in CHCl (15 mL) under N to give 58c (113 mg, 98%). 1H NMR(500MHz,CDCl3)δ11.71(s,1H),8.39(s,1H),7.75(d,J=7.8Hz,1H),7.61(ddd,J=9.3, 2.4, 1.5Hz,1H),7.37(m,1H),7.22(m,1H),4.35(t,J=6.3Hz,2H),3.93(m,1H),3.91 (s,3H),3.90(s,3H),3.73(m,1H),2.98(s,1H),2.69(s,1H),1.99(tt,J=6.3,6 .7Hz,2H).

[0171] 5-(3-(4-fluoro-N-propioloylbenzamido)propoxy)-4-methoxy-2-propiolamidobenzoate methyl ester (58d) [ka] Following the procedure described for compound 4, 57d (70 mg, 0.19 mmol), propiolic acid (0.023 mL, 0.37 mmol) and N,N'-dicyclohexylcarbodiimide (77 mg, 0.37 mmol) were reacted in CHCl (15 mL) under N to give 58d (68 mg, 76%). 1 H NMR(500MHz,CDCl3)δ11.71(s,1H),8.39(s,1H),7.98(m,2H),7.87(s,1H),7.06(m,1H),4.33(td,J=6.3, 1.6Hz,2H),3.93(m,1H),3.91(s,3H),3.90(s,3H),3.72(m,1H),2.99(s,1H),2.69(s,1H),1.98(tt,J=6.3,6.9Hz,2H).

[0172] 5-(3-(2,6-difluoro-N-propioloylbenzamido)propoxy)-4-methoxy-2-propiolamidobenzoate methyl ester (58e) [ka] Following the procedure described for compound 4, 57e (100 mg, 0.25 mmol), propiolic acid (0.031 mL, 0.51 mmol) and N,N'-dicyclohexylcarbodiimide (105 mg, 0.51 mmol) were reacted in CHCl (15 mL) under N to give 58e (113 mg, 89%). 1 H NMR(500MHz,CDCl3)δ11.73(s,1H),8.39(s,1H),7.89(s,1H),7.37(tt,J=8.5, 6.2Hz,1H),6.90(t,J=8.2Hz,2H),4.38(t,J=6.3Hz,2H),3.91(s,6H),3.86(m,1H),3.70(m,1H),2.98(s,1H),2.69(s,1H),1.99(tt,J=6.3,6 .9Hz,2H).

[0173] 5-(3-(2,6-dimethoxy-N-propioloylbenzamido)propoxy)-4-methoxy-2-propiolamidobenzoic acid methyl ester (58f) [ka] Following the procedure described for compound 4, 57f (50 mg, 0.12 mmol), propiolic acid (0.015 mL, 0.24 mmol) and N,N'-dicyclohexylcarbodiimide (49 mg, 0.24 mmol) were reacted in CH2Cl2 (15 mL) under N2 to give 58f (61 mg, 98%). 1 H NMR(500MHz,CDCl3)δ11.74(s,1H),8.39(s,1H),7.88(s,1H),7.24(m,1H),6.50(d,J=8.4Hz,2H),4.34(t,J =6.3Hz,2H),3.90(s,6H),3.88(m,1H),3.74(s,6H),3.68(m,1H),2.98(s,1H),2.67(s,1H),1.96(tt,J=6.9, 7.2Hz,2H).

[0174] 4-Methoxy-5-(3-(3-methoxy-N-propioloylbenzamido)propoxy)-2-propiolamidobenzoic acid methyl ester (58g) [ka] Following the procedure described for compound 4, 57g (130 mg, 0.33 mmol), propiolic acid (0.041 mL, 0.67 mmol) and N,N'-dicyclohexylcarbodiimide (138 mg, 0.67 mmol) were reacted in CHCl (15 mL) under N to give 58g (127 mg, 77%). 1 H NMR(500MHz,CDCl3)δ11.70(s,1H),8.37(s,1H),7.88(s,1H),7.53(d,J=7.6Hz,1H),7.45(dd,J=2.5, 1.6Hz,1H),7.29(t,J=8.0Hz,1H),7.06(dd,J=8.6, 2.2Hz,1H),4.33(t,J=6.4Hz,2H),3.94(m,1H),3.90(s,3H),3.89(s,3H ),3.81(s,3H),3.72(m,1H),2.98(s,1H),2.69(s,1H),2.00(tt,J=6.4, 6.8Hz,2H).

[0175] 4-Methoxy-5-(3-(4-methoxy-N-propioloylbenzamido)propoxy)-2-propiolamidobenzoic acid methyl ester (58h) [ka] Following the procedure described for compound 4, 57h (60 mg, 0.15 mmol), propiolic acid (0.019 mL, 0.31 mmol) and N,N'-dicyclohexylcarbodiimide (64 mg, 0.31 mmol) were reacted in CH2Cl2 (15 mL) under N2 to give 58h (72 mg, 95%). 1H NMR(500MHz,CDCl3)δ11.71(s,1H),8.37(s,1H),7.89(m,3H),6.86(d,J=8.8Hz,2H),4.30(t,J=6.0Hz,2H),3.93( m,1H),3.90(s,3H),3.89(s,3H),3.83(s,3H),3.70(m,1H),2.98(s,1H),2.69(s,1H),1.98(tt,J=6.0,6.8Hz,2H).

[0176] 4-Methoxy-5-(3-(N-(2-phenylacetyl)propiolamido)propoxy)-2-propiolamidobenzoate methyl ester (58i) [ka] Following the procedure described for compound 4, 57i (50 mg, 0.13 mmol), propiolic acid (0.017 mL, 0.27 mmol) and N,N'-dicyclohexylcarbodiimide (55 mg, 0.27 mmol) were reacted in CH2Cl2 (15 mL) under N2 to give 58i (63 mg, 98%). 1 H NMR(500MHz,CDCl3)δ11.75(s,1H),8.39(s,1H),7.85(s,1H),7.26(m,5H),4.11(t,J=6.3Hz,2H),3.91( s,3H),3.88(s,3H),3.78(m,1H),3.56(s,2H),3.55(m,1H),3.00(s,1H),2.70(s,1H),1.83(tt,J=6.3,6 .8Hz,2H).

[0177] 5-(3-(N-(2-(2-fluorophenyl)acetyl)propiolamido)propoxy)-4-methoxy-2-propiolamidobenzoate methyl ester (58j) [ka] Following the procedure described for compound 4, 57j (50 mg, 0.13 mmol), propiolic acid (0.018 mL, 0.26 mmol) and N,N′-dicyclohexylcarbodiimide (53 mg, 0.26 mmol) were reacted in CH2Cl2 (15 mL) under N2 to give 58j (58 mg, 92%). 1 H NMR(500MHz,CDCl3)δ11.75(s,1H),8.40(s,1H),7.86(s,1H),7.21(m,2H),7.05(m,1H),6.98(d,J=9.1Hz,1H),4.13(t,J=6.3, 2.2Hz,2H),3.91(s,3H),3.89(s,3H),3.79(m,1H),3.61(s,2H),3.56(m,1H),2.99(s,1H),2.68(s,1H),1.84(tt,J=6.3,6.8Hz,2H).

[0178] 5-(3-(N-(2-(3-fluorophenyl)acetyl)propiolamido)propoxy)-4-methoxy-2-propiolamidobenzoate methyl ester (58k) [ka] Following the procedure described for compound 4, 57k (65 mg, 0.17 mmol), propiolic acid (0.020 mL, 0.33 mmol) and N,N'-dicyclohexylcarbodiimide (69 mg, 0.33 mmol) were reacted in CH2Cl2 (15 mL) under N2 to give 58k (79 mg, 96%). 1 H NMR(500MHz,CDCl3)δ11.74(s,1H),8.39(s,1H),7.85(s,1H),7.23(m,1H),6.99(d,J=7.5Hz,1H),6.92(m,2H),4.12(t,J =6.0Hz,2H),3.91(s,3H),3.89(s,3H),3.78(m,1H),3.56(m,3H),2.99(s,1H),2.68(s,1H),1.84(tt,J=6.0,6.9Hz,2H).

[0179] 5-(3-(N-(2-(4-fluorophenyl)acetyl)propiolamido)propoxy)-4-methoxy-2-propiolamidobenzoate methyl ester (58l) [ka] Following the procedure described for compound 4, 57l (90 mg, 0.23 mmol), propiolic acid (0.028 mL, 0.46 mmol) and N,N'-dicyclohexylcarbodiimide (95 mg, 0.46 mmol) were reacted in CH2Cl2 (15 mL) under N2 to give 58l (112 mg, 98%). 1 H NMR(500MHz,CDCl3)δ11.74(s,1H),8.40(s,1H),7.84(s,1H),7.18(dd,J=8.5, 5.4Hz,2H),6.95(t,J=8.7Hz,2H),4.11(t,J=6.3Hz,2H),3.91(s,3H),3.89(s,3H), 3.77(m,1H),3.57(m,1H),3.54(s,2H),2.99(s,1H),2.68(s,1H),1.83(tt,J=6.3,6 .8Hz,2H).

[0180] 4-Methoxy-5-(3-(N-(2-(2-methoxyphenyl)acetyl)propiolamido)propoxy)-2-propiolamidobenzoic acid methyl ester (58m) [ka] Following the procedure described for compound 4, 57m (50 mg, 0.12 mmol), propiolic acid (0.015 mL, 0.25 mmol) and N,N'-dicyclohexylcarbodiimide (51 mg, 0.25 mmol) were reacted in CHCl (15 mL) under N to give 58m (49 mg, 78%). 1H NMR(500MHz,CDCl3)δ11.75(s,1H),8.39(s,1H),7.86(s,1H),7.20(td,J=3.9, 1.5Hz,1H),7.11(dd,J=7.4, 1.4Hz,1H),6.85(m,1H),6.81(d,J=8.3Hz,1H),4.11(td,J=6.3, 2.1Hz,2H),3.90(s,3H),3.89(s,3H),3.80(m,1H),3.75(s,3H),3.56(s,2H),3.59(m,1H),2.99(s,1H),2.68(s,1H),1.83(tt,J=6.3,6 .8Hz,2H).

[0181] 4-Methoxy-5-(3-(N-(2-(3-methoxyphenyl)acetyl)propiolamido)propoxy)-2-propiolamidobenzoic acid methyl ester (58n) [ka] Following the procedure described for compound 4, 57n (65 mg, 0.16 mmol), propiolic acid (0.020 mL, 0.32 mmol) and N,N'-dicyclohexylcarbodiimide (67 mg, 0.32 mmol) were reacted in CHCl (15 mL) under N to give 58n (70 mg, 86%). 1 H NMR(500MHz,CDCl3)δ11.74(s,1H),8.39(s,1H),7.84(s,1H),7.17(dd,J=8.9, 7.7Hz,1H),6.78(m,3H),4.11(t,J=6.3Hz,2H),3.91(s,3H),3.89(s,3H),3.79(m,1H) ),3.76(s,3H),3.57(m,1H),3.53(s,2H),2.99(s,1H),2.68(s,1H),1.84(tt,J=6.3,6 .8Hz,2H).

[0182] 4-Methoxy-5-(3-(N-(2-(4-methoxyphenyl)acetyl)propiolamido)propoxy)-2-propiolamidobenzoic acid methyl ester (58o) [ka] Following the procedure described for compound 4, 57o (90 mg, 0.22 mmol), propiolic acid (0.028 mL, 0.45 mmol) and N,N'-dicyclohexylcarbodiimide (92 mg, 0.45 mmol) were reacted in CHCl (15 mL) under N to give 58o (80 mg, 71%). 1 H NMR(500MHz,CDCl3)δ11.74(s,1H),8.39(s,1H),7.85(s,1H),7.12(d,J=8.6Hz,2H),6.79(m,2H),4.10(t,J=6.3Hz,2H),3 .91(s,3H),3.89(s,3H),3.80(m,1H),3.76(s,3H),3.56(m,1H),3.49(s,2H),2.99(s,1H),2.68(s,1H),1.83(tt,J=6.3,6 .7Hz,2H).

[0183] 4-Methoxy-2-propiolamido-5-(3-(N-(2-(3,4,5-trimethoxyphenyl)acetyl)propiolamido)propoxy)benzoic acid methyl ester (58p) [ka] Following the procedure described for compound 4, 57p (75 mg, 0.16 mmol), propiolic acid (0.020 mL, 0.32 mmol) and N,N'-dicyclohexylcarbodiimide (67 mg, 0.32 mmol) were reacted in CH2Cl2 (15 mL) under N2 to give 58p (80 mg, 87%). 1H NMR(500MHz,CDCl3)δ11.73(s,1H),8.40(s,1H),7.86(s,1H),6.47(s,2H),4.12(t,J=6.3Hz,2H),3.91(s,3H) ,3.90(s,3H),3.82(m,7H),3.80(s,3H),3.60(m,1H),3.51(s,2H),2.99(s,1H),2.68(s,1H),1.84(tt,J=6.3,6 .7Hz,2H).

[0184] 5-(3-(N-(2-(2-chlorophenyl)acetyl)propiolamido)propoxy)-4-methoxy-2-propiolamidobenzoate methyl ester (58q) [ka] Following the procedure described for compound 4, 57q (80 mg, 0.20 mmol), propiolic acid (0.024 mL, 0.39 mmol) and N,N'-dicyclohexylcarbodiimide (81 mg, 0.39 mmol) were reacted in CHCl (15 mL) under N to give 58q (100 mg, 99%). 1 H NMR(500MHz,CDCl3)δ11.75(s,1H),8.40(s,1H),7.85(s,1H),7.30(dd,J=5.6, 3.6Hz,1H),7.22(m,1H),7.18(m,2H),4.13(t,J=6.1Hz,2H),3.91(s,3H),3.89(s,3H) ),3.78(m,1H),3.70(s,2H),3.56(m,1H),2.99(s,1H),2.68(s,1H),1.85(tt,J=6.1, 6.7Hz,2H).

[0185] 5-(3-(N-(2-(3-chlorophenyl)acetyl)propiolamido)propoxy)-4-methoxy-2-propiolamidobenzoate methyl ester (58r) [ka] Following the procedure described for compound 4, 57r (75 mg, 0.18 mmol), propiolic acid (0.023 mL, 0.37 mmol) and N,N'-dicyclohexylcarbodiimide (76 mg, 0.37 mmol) were reacted in CHCl (15 mL) under N to give 58r (84 mg, 89%). 1 H NMR(500MHz,CDCl3)δ11.74(s,1H),8.39(s,1H),7.85(s,1H),7.21(s,1H),7.19(m,2H),7.10(m,1H),4.12(t,J=6.4 Hz,2H),3.91(s,3H),3.89(s,3H),3.78(m,1H),3.57(m,1H),3.54(s,2H),2.99(s,1H),2.68(s,1H),1.84(tt,J=6.4, 6.8Hz,2H).

[0186] 4-Methoxy-2-propiolamido-5-(3-(N-(2-(2-(trifluoromethyl)phenyl)acetyl)propiolamido)propoxy)benzoic acid methyl ester (58s) [ka] Following the procedure described for compound 4, 57s (100 mg, 0.23 mmol), propiolic acid (0.028 mL, 0.45 mmol) and N,N'-dicyclohexylcarbodiimide (94 mg, 0.45 mmol) were reacted in CHCl (15 mL) under N to give 58s (116 mg, 94%). 1 H NMR(500MHz,CDCl3)δ11.75(s,1H),8.39(s,1H),7.85(s,1H),7.59(d,J=7.8Hz,1H),7.47(t,J=7.7Hz,1H),7.34(t,J=7.4Hz,2H), 4.12(t,J=6.0Hz,2H),3.91(s,3H),3.89(s,3H),3.76(m,3H),3.55(m,1H),2.99(s,1H),2.68(s,1H),1.83(tt,J=6.0,6.8Hz,2H).

[0187] 4-Methoxy-2-propiolamido-5-(3-(N-(2-(3-(trifluoromethyl)phenyl)acetyl)propiolamido)propoxy)benzoate methyl (58t) [ka] Following the procedure described for compound 4, 57t (75 mg, 0.17 mmol), propiolic acid (0.021 mL, 0.34 mmol) and N,N'-dicyclohexylcarbodiimide (70 mg, 0.34 mmol) were reacted in CH2Cl2 (15 mL) under N2 to give 58t (92 mg, 99%). 1 H NMR(500MHz,CDCl3)δ11.74(s,1H),8.40(s,1H),7.85(s,1H),7.49(m,2H),7.41(m,2H),7.34(t,J=7.4Hz,2H),4.13(t,J= 6.3Hz,2H),3.91(s,3H),3.89(s,3H),3.78(m,1H),3.64(s,2H),3.59(m,1H),2.99(s,1H),2.68(s,1H),1.84(tt,J=6.3,6 .8Hz,2H).

[0188] 4-Methoxy-2-propiolamido-5-(3-(N-(2-(4-(trifluoromethyl)phenyl)acetyl)propiolamido)propoxy)benzoate methyl (58u) [ka] Following the procedure described for compound 4, 57u (102 mg, 0.23 mmol), propiolic acid (0.029 mL, 0.46 mmol) and N,N'-dicyclohexylcarbodiimide (96 mg, 0.46 mmol) were reacted in CHCl (15 mL) under N to give 58u (115 mg, 91%). 1H NMR(500MHz,CDCl3)δ11.74(s,1H),8.41(s,1H),7.86(s,1H),7.54(d,J=8.2Hz,2H),7.35(d,J=8.0Hz,2H),4.13(t,J=6 .3Hz,2H),3.91(s,3H),3.90(s,3H),3.79(m,1H),3.64(d,2H),3.60(m,1H),2.99(s,1H),2.69(s,1H),1.83(tt,J=6.3,6 .8Hz,2H).

[0189] 4-Methoxy-2-propiolamido-5-(3-(N-propioloyloxazole-5-carboxamido)propoxy)benzoic acid methyl ester (58v) JPEG0007620629000143.jpg2342 Following the procedure described for compound 4, 57v (100 mg, 0.29 mmol), propiolic acid (0.035 mL, 0.57 mmol) and N,N'-dicyclohexylcarbodiimide (118 mg, 0.57 mmol) were reacted in CHCl (15 mL) under N to give 58v (118 mg, 91%). 1 H NMR(500MHz,CDCl3)δ11.72(s,1H),8.40(s,1H),7.97(s,1H),7.89(s,1H),7.72(s,1H),4.37(t, J=6.4Hz,2H),3.91(s,6H),3.87(m,1H),3.70(m,1H),2.99(s,1H),2.69(s,1H),1.97(tt,J=6.3,6 .8Hz,2H).

[0190] Scheme 18 [ka]

[0191] Methyl 5-(3-((tert-butoxycarbonyl)amino)propoxy)-4-methoxy-2-nitrobenzoate (59) [ka] To a solution of 46 (300 mg, 1.32 mmol) and triphenylphosphine (692 mg, 2.64 mmol) in anhydrous THF (30 mL) was added 3-(boc-amino)-1-propanol (0.270 mL, 1.58 mmol) and diisopropyl azodicarboxylate (6.93 mL, 35.19 mmol) dropwise in an ice bath. The resulting solution was stirred at room temperature under N for 6 h. The reaction mixture was suspended in H2O (150 mL) and then extracted with EtOAc (3 x 150 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc / n-hexane = 1:3) to give 59 (500 mg, 99%). 1 H NMR(300MHz,CDCl3)δ7.43(s,1H),7.04(s,1H),5.18(s,1H),4.16(t,J=5. 9Hz,2H),3.95(s,3H),3.88(s,3H),3.34(m,2H),2.04(m,2H),1.43(m,9H).

[0192] 2-Amino-5-(3-((tert-butoxycarbonyl)amino)propoxy)-4-methoxybenzoate methyl (60) [ka] Following the procedure described for compound 38, Zn (85 mg, 1.30 mmol) was added to a reaction mixture of 59 (50 mg, 0.13 mmol) in HOAc / THF=1:4 (10 mL), and the mixture was stirred at room temperature under N2 to give 60 (33 mg, 72%). 1 H NMR(300MHz,CDCl3)δ7.29(s,1H),7.11(s,1H),5.58(s,2H),5.45(s,1H),3.99(t ,J=5.8Hz,2H),3.84(s,3H),3.82(s,3H),3.33(m,2H),1.94(m,2H),1.43(s,9H).

[0193] 5-(3-((tert-butoxycarbonyl)amino)propoxy)-4-methoxy-2-propiolamidobenzoate methyl ester (61) [ka] Following the procedure described for compound 4, a reaction system of 60 (33 mg, 0.09 mmol) in anhydrous CHCl (10 mL) in an ice bath was treated with dropwise addition of propiolic acid (0.009 mL, 0.14 mmol) and N,N'-dicyclohexylcarbodiimide (29 mg, 0.14 mmol) in anhydrous CHCl (5 mL) to give 61 (37 mg, 98%). 1 H NMR(300MHz,CDCl3)δ11.56(s,1H),8.32(s,1H),7.44(s,1H),4.07(t,J=5. 6Hz,2H),3.93(s,3H),3.90(s,3H),3.35(m,2H),1.99(m,2H),1.43(s,9H).

[0194] Scheme 19 [ka]

[0195] (Z)-N-(3-chloropropyl)benzimidic acid (63) [ka] Benzoic acid (140 mg, 1.12 mmol) and N,N'-dicyclohexylcarbodiimide (238 mg, 1.15 mmol) in anhydrous CHCl (5 mL) were added dropwise to a solution of 62 (100 mg, 0.77 mmol) in anhydrous CHCl (10 mL) in an ice bath. The resulting solution was stirred at room temperature under N for 12 h. The reaction mixture was suspended in H0 (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic layers were dried over MgSO, filtered and concentrated in vacuo. The mixture was filtered and washed with EtOAc / n-hexane = 1:1 (10 mL). The residue was purified by silica gel chromatography (EtOAc / n-hexane = 1:3) to give 63 (142 mg, 93%). 1 H NMR(300MHz,CDCl3)δ7.74(m,2H),7.45(m,3H),6.38(s,1H),3.62(m,4H),2.11(m,2H).

[0196] (Z)-N-(3-(2-methoxy-5-(methoxycarbonyl)-4-nitrophenoxy)propyl)benzimidic acid (64) [ka] Following the procedure described for compound 2, 63 (1.50 g, 7.59 mmol) was added to a reaction mixture of 46 (1.72 g, 7.59 mmol) and K2CO3 (2.10 g, 15.17 mmol) in DMF (150 mL). The resulting solution was heated to 100 °C under N2 to give 64 (359 mg, 12%). 1 H NMR(300MHz,CDCl3)δ7.78(m,2H),7.45(m,4H),7.08(s,1H),6.91(s,1H), 4.25(t,J=5.7Hz,2H),3.88(s,3H),3.76(s,3H),3.70(m,2H),2.19(m,2H).

[0197] (Z)-N-(3-(4-amino-2-methoxy-5-(methoxycarbonyl)phenoxy)propyl)benzimidic acid (65) [ka] Following the procedure described for compound 38, Zn (134 mg, 2.06 mmol) was added to a reaction mixture of 64 (80 mg, 0.21 mmol) in HOAc / THF=1:4 (10 mL), and the mixture was stirred at room temperature under N2 to give 65 (69 mg, 93%). 1 H NMR(300MHz,CDCl3)δ7.80(m,2H),7.42(m,3H),7.33(s,1H),7.29(s,1H),6.10(s,1H), 5.60(s,2H),4.10(t,J=5.5Hz,2H),3.82(s,3H),3.69(m,2H),3.62(s,3H),2.09(m,2H).

[0198] (Z)-N-(3-(2-methoxy-5-(methoxycarbonyl)-4-propiolamidophenoxy)propyl)benzimidic acid (66) [ka] Following the procedure described for compound 4, a reaction system of 65 (35 mg, 0.10 mmol) in anhydrous CHCl (10 mL) in an ice bath was treated with dropwise addition of propiolic acid (0.010 mL, 0.15 mmol) and N,N'-dicyclohexylcarbodiimide (30 mg, 0.15 mmol) in anhydrous CHCl (5 mL) to give 66 (26 mg, 65%). 1 H NMR(300MHz,CDCl3)δ11.54(s,1H),8.30(s,1H),7.79(m,2H),7.44(m,4H),7.16( s,1H),4.17(t,J=5.5Hz,2H),3.90(s,3H),3.70(m,5H),2.92(s,1H),2.14(m,2H).

[0199] Scheme 20 [ka]

[0200] (Z)-5-(3-((hydroxy(phenyl)methylene)amino)propoxy)-4-methoxy-2-nitrobenzoic acid (67) [ka] To a solution of 64 (229 mg, 0.59 mmol) in MeOH (20 mL) and THF (20 mL) was added 5N NaOH (2.5 mL) in an ice bath. The reaction mixture was stirred at room temperature under N2 for 8 h. The resulting solution was acidified to pH 2 and extracted with 1N HCl (50 mL) and EtOAc (3 x 50 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was recrystallized to give 67 (220 mg, 99%). 1 H NMR(300MHz,MeOD)δ7.84(m,2H),7.52(m,4H),7.31(s,1H),4.28(t,J=6.0Hz,2H),3.88(s,3H),3.64(t,J=6.6Hz,2H),2.19(m,2H).

[0201] (Z)-N-(3-(2-methoxy-4-nitro-5-((2-(trimethylsilyl)ethoxy)carbonyl)phenoxy)propyl)benzimidic acid (68) [ka] To a solution of 67 (210 mg, 0.56 mmol) and triphenylphosphine (294 mg, 1.12 mmol) in anhydrous THF (30 mL) was added 2-(trimethylsilyl)ethanol (0.097 mL, 0.67 mmol) and diisopropyl azodicarboxylate (0.133 mL, 0.67 mmol) dropwise in an ice bath. The resulting solution was stirred under N for 3 h. The reaction mixture was suspended in H2O (100 mL) and then extracted with EtOAc (3 x 100 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc / n-hexane = 1:2) to give 68 (221 mg, 83%). 1 H NMR(300MHz,CDCl3)δ7.78(m,2H),7.47(m,3H),7.39(s,1H),7.07(s,1H),6.91(s,1H),4.38(m ,2H),4.25(t,J=5.7Hz,2H),3.75(s,3H),3.71(m,2H),2.19(m,2H),1.07(m,2H),0.04(s,9H).

[0202] (Z)-N-(3-(4-amino-2-methoxy-5-((2-(trimethylsilyl)ethoxy)carbonyl)phenoxy)propyl)benzimidic acid (69) [ka] Following the procedure described for compound 38, Zn (69 mg, 1.05 mmol) was added to a reaction mixture of 68 (50 mg, 0.11 mmol) in HOAc / THF=1:4 (10 mL), and the mixture was stirred at room temperature under N2 to give 69 (40 mg, 85%). 1 H NMR(300MHz,CDCl3)δ7.80(m,2H),7.42(m,3H),7.34(s,1H),7.31(s,1H),6.09(s,1H),5.60(s,2H), 4.33(m,2H),4.09(t,J=7.1Hz,2H),3.69(m,2H),3.61(s,3H),2.09(m,2H),1.09(m,2H),0.06(m,9H).

[0203] (Z)-N-(3-(2-methoxy-4-propiolamido-5-((2-(trimethylsilyl)ethoxy)carbonyl)phenoxy)propyl)benzimidic acid (70) [ka] Following the procedure described for compound 4, a reaction of 69 (170 mg, 0.38 mmol) in anhydrous CHCl (10 mL) in an ice bath was treated with dropwise addition of propiolic acid (0.035 mL, 0.15 mmol) and N,N'-dicyclohexylcarbodiimide (118 mg, 0.57 mmol) in anhydrous CHCl (5 mL) to give 70 (180 mg, 95%). 1 H NMR(300MHz,CDCl3)δ11.64(s,1H),8.31(s,1H),7.80(m,2H),7.43(m,4H),7.17(s,1H),4.40( m,2H),4.17(t,J=5.5Hz,2H),3.71(m,5H),2.91(s,1H),2.14(m,2H),1.13(m,2H),0.07(s,9H).

[0204] (Z)-5-(3-((hydroxy(phenyl)methylene)amino)propoxy)-4-methoxy-2-propiolamidobenzoic acid (71) [ka] To a solution of 70 (50 mg, 0.10 mmol) in anhydrous THF (2 mL) was added TBAF (1 M in THF, 0.50 mL, 0.50 mmol) dropwise at room temperature. The resulting solution was stirred under N2 for 12 h. The reaction mixture was diluted with 1N HCl. (aq) (50 mL) and EtOAc (3×50 mL). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (MeOH / CH2Cl2=3:97) to give 71 (19 mg, 48%). 1 H NMR(300MHz,DMSO)δ11.87(s,1H),8.52(t,J=5.5Hz,1H),8.08(s,1H),7.83(m,2H),7. 48(m,3H),4.47(s,1H),4.04(t,J=6.2Hz,2H),3.80(s,3H),3.02(m,2H),1.98(m,2H).

[0205] PDIA4 assay Recombinant PDI protein PDIA4 (23 pM) was incubated with insulin (0.2 mM) and DTT (0.1 mM) in the presence of solvent and a given dose of compound for 30 min at 25°C. The resulting mixture was measured at 595 nm using a SpectraMax i3x reader according to the manufacturer's instructions (Molecular device, CA, USA). Published formula: 100% × (OD of solvent) 595 -CP OD 595 ) / (OD of solvent 595 ) The inhibition of PDIA4 enzyme activity was calculated by:

[0206] Glucose-stimulated insulin secretion Unless otherwise stated, Min6 cells were grown in complete Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 100 units / ml penicillin, and 100 g / ml streptomycin in the presence of 3.3 mM glucose with 5% CO2 incubation. Min6 cells were preincubated in serum-free, oxygen-saturated Krebs-Ringer bicarbonate (KRB) buffer containing 3.3 mM glucose at 37°C for 30 min. The cells (7.5 × 10 4 ) was further incubated with KRB buffer containing high glucose (16.7 mM) or low glucose (3.3 mM) for 30 min. The supernatant was collected and used for insulin ELISA assay.

[0207] Cell viability test Min6 cells were cultured at 37°C in complete medium. WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate) was used to determine cytotoxicity according to the manufacturer's guidelines. Briefly, cells were grown in the presence of a given compound in 96-well plates containing complete medium for 24 h. After washing with PBS, WST-1 was added to each well and incubated for an additional 20 min. After removing the medium, cells in the plates were measured under 450 nm using a SpectraMax i3x.

[0208] statistics Data obtained from three or more independent experiments are expressed as the mean ± standard deviation (SD). Comparisons between multiple groups were performed by ANOVA unless otherwise stated. P<0.05 (*), <0.01 (**), or <0.001 (***) were considered statistically significant between the control and treatment groups.

[0209] Table 1-5 shows the IC of PDIA4 inhibitors. 50 Indicates the value.

[0210] [Table 1] JPEG0007620629000160.jpg86133

[0211] [Table 2]

[0212] [Table 3]

[0213] [Table 4] JPEG0007620629000164.jpg6157

[0214] [Table 5]

[0215] result Screening of PDIA4 inhibitors from hit compounds to lead compounds

[0216] As shown in Figure 1, virtual screening and PDIA4 were used to search for hit compounds, lead compounds and / or drug candidates. Details of virtual screening are shown in the Experimental Procedure (Figure 1). From 261 kinds of phytochemicals, cytopiloyne (CPD P1, Table 1) and cytopiloyne aglycone (CPD P2, Table 1) were identified as two optimal hit compounds. Molecular docking of CPD P1 into PDIA4 with three active motifs is shown in Figure 2. The PDIA4 assay platform was constructed to determine the median inhibitory concentration (IC) of CPD P2. 50 ) was measured (167.9 μM, Table 1).

[0217] Characterization of PDIA4-based drug candidates

[0218] Next, based on the pharmacophore features of CP / CPA and the binding pocket of PDIA4, 10 compounds with one to three carbon-carbon triple bonds (CCTBs) were selected from the ZINC database by virtual screening, and their IC 50The values ​​were measured and the core structures of the lead compounds were determined (Table 1). These compounds are (E)-7-(hexa-2,4-diyn-1-ylidene)-1,6-dioxaspiro[4.4]nona-2,8-dien-4-yl acetate (C1), (Z)-5-(hexa-2,4-diyn-1-ylidene)furan-2(5H)-one (C2), 1-phenylhexa-2,4-diyne-1,6-diol (C3), N-phenylpropiolamido (C4), (2-(N-(2,4-dimethoxyphenyl)propiolamido)-2-(thiophen-2-yl)acetyl)glycinic acid. Ethyl (C5), (E)-7-(hexa-2,4-diyn-1-ylidene)-1,6-dioxaspiro[4.4]nona-2,8-dien-4-yl-3-methylbut-2-enoate (C6), 1-phenylbut-2-yn-1,4-diol (C7), N-(4-(but-2-yn-1-ylamino)phenyl)acetamide (C8), hexa-2,4-diyn-1,6-diyl-bis(2-phenylacetate) and hexa-2,4-diyn-1 (C9), and dibenzoate-6-diyl (C10). N-phenylpropiolamido (C4) (IC 50 = 182.7 μM, Table 1) was selected as the active core structure, and a partial structure search for lead compounds was performed. Therefore, (4-propiolamidobenzoyl)glycine (C11), 2-(4-propiolamidophenyl)acetic acid (C12), 4,5-dimethoxy-2-propiolamidobenzoic acid (8), 4-propiolamidobenzoic acid (C13), and 2-hydroxy-5-propiolamidobenzoic acid (C14), which contain the core structure of N-phenylpropiolamido (C4), were selected to obtain IC 50 As a result, 4,5-dimethoxy-2-propiolamidobenzoic acid (8) was selected as a potential lead compound, with an IC 50 The activity was 4 μM, 53-fold higher than that of the hit compound CPD P1 (Table 2). 4,5-Dimethoxy-2-propiolamidobenzoic acid (8) was synthesized from 4,5-dimethoxy-2-nitrobenzoic acid (1) according to Scheme 2.

[0219] To optimize 4,5-dimethoxy-2-propiolamidobenzoic acid (8), 4,5-dimethoxy-2-nitrobenzoic acid (1) was chemically modified to give compounds 2-8 based on Schemes 1 and 2. As a result, 4,5-dimethoxy-2-propiolamidobenzoic acid (8) was selected as a potential lead compound, and its IC 50 The IC value was 4 μM, 40-fold higher than that of the hit compound CPA (Tables 1 and 2). Furthermore, chemical modification of 4,5-dimethoxy-2-propiolamidobenzoic acid (8) was carried out according to Scheme 3-20. Compounds 9-58v were synthesized and tested for inhibition against PDIA4 (Table 1). Their IC 50 The values ​​are shown in Tables 3 and 4. The details of synthesis and structure description of CPD1-58v are described in the chemistry section of Materials and Methods. Therefore, some compounds 8, 58v, 58s and 58t can be used as drug candidates due to their low IC50 values.

[0220] Pharmacological effects of selected PDIA4 inhibitors on insulin secretion and cell viability in β-cells

[0221] Next, the effects of the selected PDIA4 inhibitors CCF642 (a commercially available PDIA4 inhibitor) and CPD P1, 8, 58v, 58s, 58t on the insulin release activity of Min6 (mouse β cells) were examined. The EC50 values ​​of CCF642 and CPD P1, 8, 58v, 58s, 58t are 0.25, 1.55, 0.06, 0.005, 0.03, and 0.02 μM, respectively (Figure 3 and Table 2). They have little effect on insulin release in Min6 cells. The CC50 values ​​of CCF642 and CPD P1, 8, 58v, 58s, and 58t are 23.9, >100, >100, >100, >100, and 64.9, respectively (Figure 3 and Tables 1-4), indicating that, like CCF642, CPD P1, 8, 58v, 58s, and 58t can enhance insulin secretion in β cells. Molecular docking of CPD8 into PDIA4, which has three activity motifs, is shown in Figure 2. The data show that CPD8 fits better with PDIA4 than CPD P1 (Figure 2).

[0222] Pharmacological effects of selected PDIA4 inhibitors on diabetes in db / db mice

[0223] Furthermore, we investigated the antidiabetic effect of CPD8 (a drug candidate for Pdia4 inhibition) in vivo using db / db mice (a mouse model of type 2 diabetes). As expected, 30 mg / kg sitagliptin (STG) (positive control) significantly reduced fasting blood glucose (FBG) in db / db mice (Figure 4A). CPD8 significantly reduced FBG in db / db mice at a dose of 2.5 mg / kg (Figure 3A). Moreover, the combination of CPD8 (2.5 mg / kg) and metformin (60 mg / kg) further reduced FBG in db / db mice than CPD8 alone (Figure 4A). 30 mg / kg sitagliptin (positive control) significantly reduced postprandial blood glucose (PBG) in db / db mice (Figure 4B). CPD8 significantly reduced PBG in db / db mice at a dose of 2.5 mg / kg (Figure 4B). Moreover, the combination of CPD8 (2.5 mg / kg) and metformin (60 mg / kg) further reduced FBG in db / db mice than CPD8 alone (Figure 4B). Overall the data indicate that CPD8 (a PDIA4-based drug candidate) by itself and in combination can reverse type 2 diabetes in db / db mice.

[0224] In summary, pancreatic β-cell damage is a hallmark of type 2 diabetes (T2D). Strategies that can preserve β-cell function / quality at the early stage of diabetes and reverse T2D are highly sought after. The present invention relates to screening PDIA4-based inhibitors from hit compounds to lead compounds by a combination of molecular docking and PDIA4 bioassay. N-phenylpropiolamido (C4) was identified as the active core structure of PDIA4 inhibitors. 4,5-dimethoxy-2-propiolamidobenzoic acid (8) was identified as a lead compound by a combination of chemical synthesis, molecular docking and PDIA4 bioassay. Based on the structure-activity relationship of the compounds to PDIA4, 48 compounds (Table 6) were synthesized and characterized. As a result, the IC 50Several drug candidates (CPD P1, 8, 58v, 58s and 58t) with potency values ​​ranging from 4 μM to 300 nM were identified. These compounds were tested for beta cell function and diabetes treatment. Similar to CCF642 (a commercial PDIA4 inhibitor), CPD P1, 8, 58v, 58s and 58t had higher activity on beta cell insulin secretion. Thus, CPD8 itself and its combination can treat and reverse T2D in db / db mice (diabetic mouse model). Overall data indicates that PDIA4 is a new therapeutic target for type 2 diabetes (T2D). Table 6 shows the compounds of the present invention.

[0225] JPEG0007620629000166.jpg3338JPEG0007620629000167.jpg203140JPEG0007620629000168.jp g193140JPEG0007620629000169.jpg203140JPEG0007620629000170.jpg188140JPEG00076206290 00171.jpg203140JPEG0007620629000172.jpg203140JPEG0007620629000173.jpg203140JPEG000 7620629000174.jpg187140JPEG0007620629000175.jpg198140JPEG0007620629000176.jpg38140

[0226] All references cited and discussed in this specification are incorporated herein by reference in their entirety to the same extent as if each reference was individually incorporated by reference.

Claims

【Request 1】 【Chemistry 1】 【change】 【change】 and pharma- ceutically acceptable salts thereof. A protein disulfide isomerase A4 (PDIA4) inhibitor, an antidiabetic drug which is a PDIA4 inhibitor, or an insulin secretion enhancer which is a PDIA4 inhibitor, selected from the group consisting of:

2. A pharmaceutical composition comprising a PDIA4 inhibitor, The PDIA4 inhibitor is 【Chemistry 2】 【change】 【change】 and pharma- ceutically acceptable salts thereof.

3. The PDIA4 inhibitor is 【Chemistry 3】 3. A pharmaceutical composition comprising the PDIA4 inhibitor of claim 2, selected from the group consisting of: and pharma- ceutically acceptable salts thereof.

4. A pharmaceutical composition comprising the PDIA4 inhibitor of claim 2 or 3, further comprising a pharma- ceutically acceptable carrier or excipient.

5. A pharmaceutical composition comprising the PDIA4 inhibitor of claim 2 or 3, further comprising metformin.

6. The PDIA4 inhibitor is 【Chemistry 4】 A pharmaceutical composition comprising a PDIA4 inhibitor according to any one of claims 2 to 5, selected from the group consisting of: and pharma- ceutically acceptable salts thereof.

7. 1. A protein disulfide isomerase A4 (PDIA4) inhibitor for enhancing insulin secretion from pancreatic beta cells, treating diabetes, and / or reversing and restoring blood glucose concentrations to normal levels in a subject, comprising: Chemical structure: 【Chemistry 5】 or a pharma- ceutically acceptable salt thereof.

8. 1. Use of a protein disulfide isomerase A4 (PDIA4) inhibitor in the manufacture of a medicament for enhancing insulin secretion from pancreatic beta cells, treating diabetes, and / or reversing and restoring blood glucose concentrations to normal levels in a subject, comprising: The PDIA4 inhibitor has the chemical structure: 【Chemistry 6】 or a pharma- ceutically acceptable salt thereof.

9. 1. A protein disulfide isomerase A4 (PDIA4) inhibitor for enhancing insulin secretion from pancreatic beta cells, treating diabetes, and / or reversing and restoring blood glucose concentrations to normal levels in a subject, comprising: 【Chemistry 7】 【change】 【change】 and pharma- ceutically acceptable salts thereof.

10. 1. Use of a protein disulfide isomerase A4 (PDIA4) inhibitor in the manufacture of a medicament for enhancing insulin secretion from pancreatic beta cells, treating diabetes, and / or reversing and restoring blood glucose concentrations to normal levels in a subject, comprising: The PDIA4 inhibitor 【Chemistry 8】 【change】 【change】 and pharma- ceutically acceptable salts thereof.

11. A pharmaceutical composition comprising the PDIA4 inhibitor described in claim 2 or 3 for enhancing insulin secretion from pancreatic beta cells in a subject, treating diabetes, and / or reversing and restoring blood glucose concentrations to normal levels.

12. Use of a pharmaceutical composition comprising a PDIA4 inhibitor according to claim 2 or 3 in the manufacture of a medicament for enhancing insulin secretion from pancreatic beta cells, treating diabetes, and / or reversing and restoring blood glucose concentrations to normal levels in a subject.

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