STAT3 inhibitor

Abstract

To provide a naphthoquinone compound exhibiting enhanced STAT3 inhibitory activity over conventional compounds.SOLUTION: A STAT3 phosphorylation inhibitor comprising, for example, a compound represented by formula (21S) as an active ingredient exhibits stronger STAT3 phosphorylation inhibitory activity than conventional STAT3 inhibitors having a furanonaphthoquinone skeleton.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a STAT3 inhibitor, and more particularly to a novel naphthoquinone compound and a pharmaceutical composition containing the same as an active ingredient. [Background technology]

[0002] Signal Transducer and Activator of Transcription 3 (STAT3) is a member of the STAT family that regulates cell cycle progression and anti-apoptosis, and is therefore involved in the progression and poor prognosis of various human cancers (Non-Patent Document 1). When cells are stimulated by growth factors or cytokines such as interleukin 6 (IL-6), associated upstream kinases (growth factor receptors, tyrosine kinases, Janus kinases (JAKs), Src family kinases, etc.) are activated. Upon activation of the upstream kinases, STAT3 monomers are phosphorylated at Tyr705 or Ser727, which are the transactivation domains of the STAT3 protein. Phosphorylated STAT3 monomers interact with each other symmetrically to form transcriptionally active homodimers. These homodimers then translocate to the nucleus, bind to specific DNA sequences, and regulate the transcription of target genes.

[0003] STAT3 signaling is tightly regulated to maintain a transiently active state under normal physiological conditions. However, constitutive STAT3 activation has been detected in various solid tumors and hematological malignancies. Therefore, constitutive STAT3 activation may induce tumor angiogenesis and suppress antitumor immune responses. Furthermore, constitutive STAT3 activation correlates with chemotherapy resistance and poor cancer prognosis (Non-Patent Document 2).

[0004] Therefore, STAT3 is an ideal target for cancer therapy, and various small molecule inhibitors of STAT3, such as STA-21, LLL-12, Stattic, and niclosamide, have been discovered and are progressing through early stages of the drug development pipeline (Figure 6) (Non-patent Document 3).

[0005] Regarding the upstream kinases of STAT3, certain JAK inhibitors, such as ruxolitinib, tofacitinib, oclacitinib, and baricitinib, have achieved clinical success and are approved by the US Food and Drug Administration. In contrast, no STAT3-targeting drugs have been approved by the US Food and Drug Administration to date. Therefore, the development of novel, potent STAT3 inhibitors is expected to bring significant challenges and opportunities to the field of cancer treatment.

[0006] A wide variety of 1,4-naphthoquinones are found in several natural and artificial substances and are known to exhibit a wide range of biological activities, including antitumor activity, cancer chemoprevention activity (Non-Patent Document 4), antibacterial activity (Non-Patent Document 5), antimalarial activity (Non-Patent Document 6), trypanocidal activity (Non-Patent Document 7), and antituberculosis activity (Non-Patent Document 8). Therefore, 1,4-naphthoquinones are expected to be extremely useful in the development of therapeutic agents for treating diseases such as cancer and infectious diseases (Non-Patent Document 9).

[0007] Recently, various quinone derivatives that selectively inhibit STAT3 have also been widely studied (Non-Patent Document 1(b)). Among all reported STAT3 inhibitors, only napabucasin (2, BBI608) has been shown to inhibit cancer stem cell pathway activity and has reached phase III clinical trials for the treatment of various cancers.

[0008] Based on the structures of napabucasin and β-lapachone (formula 3: Figure 7), Muller et al. synthesized isonapabucasin (formula 4: Figure 7) and demonstrated that its STAT3 inhibitory activity was twice as strong as that of napabucasin using a time-resolved fluorescence resonance energy transfer assay.

[0009] In contrast to most studies that focused on the 2-acetyl group of the napabucasin furan ring (Non-Patent Document 10), Tian et al. modified the napabucasin structure by cleaving the benzene ring and introducing various functional groups to access the pY-X site. The aim was to improve STAT3 binding affinity based on the predicted binding mode in which the 2-acetylfuran group is located at the pY705 site, leaving the critical pY-X site empty (Non-Patent Document 11). [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] (a)Lai,PS;Rosa,DA;MagdyAli,A.;Gomez-Biagi,RF;Ball,DP;Shouksmith,AE;Gunning,PTA STAT inhibitor patent review:progress since 2011.Expert Opin.Ther.Pat. 2015,25,1397-1421,DOI:10.1517 / 13543776.2015.1086749.(b)Dong J,Cheng XD,Zhang WD,Qin JJ Degradation.J Med Chem. 2021 Jul 8;64(13):8884-8915.doi:10.1021 / acs.jmedchem.1c00629.(c)Huang Q,Zhong Y,Dong H,Zheng Q,Shi S,Zhu K,Qu X,Hu W,Zhang X,Wang Y.Revisiting signal transducer and activator of transcription 3(STAT3) as an anticancer target and its inhibitor discovery:Where are we and where should we go? Eur J Med Chem. 2020 Feb 1;187:111922.doi:10.1016 / j.ejmech.2019.111922.

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[0011] The inventors have previously evaluated representative naphthoquinones such as NQ801 (formula (1): Figure 7) isolated from Tabebuia avellanedae, and found that a furano-naphthoquinone structure is required for these organic compounds to exhibit STAT3 phosphorylation inhibitory activity (Non-Patent Document 12). As a result, the representative naphthoquinone compounds of formulas 1 to 4 were able to be evaluated to a certain extent for their ability to inhibit cancer cell proliferation, but this was not yet sufficient.

[0012] Our previous results also indicated that the presence of a phenolic hydroxy group at C-5 is crucial for enhancing the antiproliferative effect. However, the relationship between the presence of the phenolic hydroxy group and STAT3 inhibitory activity remained unclear (Non-Patent Document 13). Furthermore, sulfonamide derivatives have been reported to be promising STAT3 phosphorylation inhibitors (Non-Patent Document 1(b)). In fact, Kong et al. reported that naphthoquinones containing a sulfonamide group adjacent to the quinone carbonyl group are orally available, potent, and selective STAT3 inhibitors (Non-Patent Document 14).

[0013] Therefore, the inventors conceived the idea that ortho- and para-furanonaphthoquinone compounds, in which a phenolic hydroxy group or a sulfonamide group is introduced into the furanonaphthoquinone skeleton, could be novel, potent STAT3 inhibitors with higher efficacy than conventional STAT3 inhibitors such as those of formulas (1) to (4) (see Figure 7).

[0014] Based on the above results, the inventors decided to investigate the relationship between the synthesis of ortho- and para-furanonaphthoquinones having a phenolic hydroxy group or a sulfonamide group, such as those represented by formulas (5) to (8) (see Figure 7), and their STAT3 inhibitory activity, and sought novel compounds having a furanonaphthoquinone skeleton. [Means for solving the problem]

[0015] As described above, the present invention was completed as a result of testing naphthoquinone compounds having superior STAT3 inhibitory activity to conventional compounds.

[0016] More specifically, the STAT3 inhibitor contains as an active ingredient at least one naphthoquinone compound represented by formula (21), (21S), (21R), (22), (23), (18), or (14).

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[0023] [ka] [Effects of the Invention]

[0024] The STAT3 inhibitor according to the present invention, which contains as an active ingredient a naphthoquinone compound represented by formula (21), (21S), (21R), (22), (23), (18), or (14), can effectively inhibit the phosphorylation of STAT3 and suppress the proliferation of cancer cells. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 shows a synthesis scheme for compounds of formulas (14) and (18). [Figure 2] FIG. 1 shows a synthesis scheme for compounds of formulae (21) and (22). [Figure 3] FIG. 1 is a diagram showing a synthesis scheme for a compound of formula (23). [Figure 4] FIG. 1 shows the results of Western blotting in which the phosphorylation inhibitory properties of the novel substance according to the present invention and conventional compounds were examined. [Figure 5] FIG. 1 shows the results of a binding simulation between STAT3 and the compound of formula (21). [Figure 6] FIG. 1 shows the structures of conventional STAT3 inhibitors. [Figure 7] FIG. 1 shows the structures of ortho- and para-furanonaphthoquinones with phenolic hydroxy groups or sulfonamide groups compared with conventional STAT3 inhibitors. DETAILED DESCRIPTION OF THE INVENTION

[0026] The naphthoquinone compounds represented by formula (21), (22), (23), (18), or (14) according to the present invention and STAT3 inhibitors using the same will be described below. Note that the following description is an example of one embodiment and one example of the present invention, and the present invention is not limited to the following description. The following embodiment can be modified without departing from the spirit of the present invention.

[0027] The naphthoquinone compound according to the present invention is a naphthoquinone compound represented by formula (21), (21S), (21R), (22), (23), (18), or (14). The numbers in the structural formula are numbers for identifying the compound and are not constituent elements of the compound.

[0028] The compounds according to the present invention can be converted into salts by mixing with a pharmaceutically acceptable acid in a solvent such as water, methanol, ethanol, acetone, etc. Examples of pharmaceutically acceptable acids include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfate, phosphoric acid, and nitric acid, and organic acids such as acetic acid, propionic acid, oxalic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, maleic acid, fumaric acid, methanesulfonic acid, p-toluenesulfonic acid, and ascorbic acid.

[0029] When used for treatment, the compounds of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, or intramuscularly, topically, rectally, transdermally, or intranasally) as pharmaceutical compositions. Compositions for oral administration include, for example, tablets, capsules, pills, granules, powders, liquids, and suspensions. Compositions for parenteral administration include, for example, aqueous or oily solutions for injection, ointments, creams, lotions, aerosols, suppositories, and patches. These formulations are prepared using conventionally known techniques and may contain non-toxic and inert carriers or excipients commonly used in the pharmaceutical field.

[0030] The therapeutically effective amount will vary depending on factors such as the severity of the infection, age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimal therapeutic response. For example, several divided doses can be administered daily, or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. [Example]

[0031] In the following examples, the analyzers such as NMR were as follows: 1 H- and 13C-NMR spectra were obtained using a Bruker-Biospin Avance III 400 MHz NMR spectrometer and acquired in CDCl3 unless otherwise noted. Chemical shift values ​​are expressed in ppm relative to internal tetramethylsilane. Coupling constant J values ​​are expressed in Hz. Abbreviations are as follows: s, singlet; d, doublet; t, triplet; m, multiplet. IR spectra were recorded on a Shimadzu IR Affinty-1S spectrometer.

[0032] IR spectroscopy of the oil samples was measured as neat liquid films. The wavenumber of the maximum absorption peak in IR spectroscopy is in cm. -1 The extract was washed with brine and then dried over sodium sulfate. Silica gel column chromatography was used for purification. NQ801 (compound of formula (1): see Figure 7) was synthesized according to a known literature procedure.

[0033] Scheme 1 Compounds (14) and (18) were synthesized according to Scheme 1 in FIG.

[0034] <2,6-Dihydroxy-3-iodonaphthalene-1,4-dione Synthesis of compound of formula (10) The compound of formula (9) (Non-Patent Document 23) (1.8 g, 9.34 mmol) and K2CO3 (3.9 g, 28.0 mmol) were added to distilled water (40 mL), followed by the addition of morpholine-iodine complex (3.5 g, 10.3 mmol). After stirring at room temperature for 1.5 hours, the reaction was quenched with 25% phosphoric acid and cooled to 0 °C in an ice bath. The resulting solid was collected by suction filtration. The filtrate was extracted with EtOAc. The combined organic extracts were washed with brine, dried over Na2SO4, and then concentrated. The resulting solid was dried under vacuum. A brown solid was obtained in 56% yield (1.7 g). This solid was dissolved in EtOAc and subjected to thin-layer chromatography using a 50:1 mixture of EtOAc and MeOH as the mobile phase. The Rf value was 0.2. This compound was used in the next step without further purification due to its instability.

[0035] <Synthesis of the compound 46-Hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-dione (11)> A mixture of CuO (636 mg, 4.45 mmol), 3-butyn-2-ol (1.74 mL, 1.50 mmol), and pyridine (42 mL) was stirred at room temperature for 1 hour under an Ar atmosphere. A solution of the compound of formula (10) (1.4 g, 4.43 mmol) and Pd(OAc) (30 mg, 0.13 mmol) in DMF (40 mL) was added to the reaction mixture, and the reaction mixture was stirred at 80 °C for 1 hour.

[0036] After cooling to room temperature, the mixture was filtered through a pad of Celite. The Celite was washed with EtOAc, and the combined organics were concentrated. The crude product was diluted with 10% HCl and extracted with EtOAc. The organic phase was washed with H2O and brine, dried over Na2SO4, and then concentrated. The crude product was chromatographed on silica gel. Yield 17% (62 mg). A yellow solid was obtained. This solid was dissolved in EtOAc and subjected to thin-layer chromatography using a 1:1 mixture of hexane / EtOAc as the mobile phase, giving an Rf value of 0.3.

[0037] The solid NMR spectrum was as follows. 1 1H NMR (DMSO): δ 10.95 (s, 1H), 7.88 (d, J = 8.4, 1H), 7.31 (s, 1H), 7.08 (d, J = 8.4, 1H), 6.78 (s, 1H), 5.72 (d, J = 4.5, 1H), 4.79 (dd, J = 4.5, 6.6, 1H), 1.39 (d, J = 6.6, 3H). 13 13C NMR (DMSO): δ 180.9 (C), 172.7 (C), 166. (C), 163.3 (C), 152.1 (C), 135.6 (C), 130.7 (C), 129.7 (CH), 124.4 (C), 120.7 (CH), 113.4 (CH), 103.6 (CH), 62.4 (CH), 22.0 (CH3). IR (KBr): 2924, 1720, 1373, 1026. HRMS (ESI) m / z: [M+Na] + calcd for [C 14 H 10 O5Na] + , 281.0426; Found, 281.0435. From the above, it was confirmed that the compound was the one of formula (11).

[0038] <O-(2-(1-hydroxyethyl)-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-6-yl)dimethylcarbamothioate (Synthesis of the compound of formula (12))> To a stirred solution of the compound of formula (11) (100 mg, 0.41 mmol) and dimethylthiocarbamoyl chloride (77 mg, 0.62 mmol) in dry DMF (10 mL) was added DABCO (93 mg, 0.82 mmol). After stirring at room temperature for 2 hours, the reaction was quenched by the addition of H2O and extracted with EtOAc. The organic extract was washed with brine, dried over Na2SO4, and then concentrated.

[0039] The crude product was chromatographed on silica gel. A yellow solid with a yield of 55% (80 mg) was obtained. This solid was dissolved in EtOAc, and thin-layer chromatography was performed using a solution of hexane / EtOAc mixed 1:1 as the mobile phase, and the Rf value was 0.15.

[0040] The NMR spectrum of this solid was as follows. 1 1H NMR (CDCl3): δ 8.24 (d, J = 8.3, 1H), 7.83 (d, J = 2.4, 1H), 7.44 (dd, J = 2.4, 8.3, 1H), 6.84 (s, 1H), 5.04 (q, J = 6.5, 1H), 3.47 (s, 3H), 3.39 (s, 3H), 1.65 (d, J = 6.5, 3H). 13 13C NMR (CDCl3): δ 186.3 (C), 179.9 (C), 172.6 (C), 165.3 (C), 158.2 (C), 152.0 (C), 134.7 (C), 131.4 (C), 129.9 (C), 128.5 (CH), 128.3 (CH), 121.6 (C), 103.79 (CH), 63.9 (CH), 43.4 (CH3), 39.0 (CH3), 21.5 (CH3). IR (KBr): 1666, 1527, 1381, 1226, 1118, 948. HRMS (ESI) m / z: [M+Na] + calcd for [C 17 H 15 NO5SNa] + , 368.0569, Found, 368.0579. From the above, it was confirmed that it is the compound of formula (12).

[0041] <S-(2-(1-hydroxyethyl)-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-6-yl)dimethylcarbamothioate Synthesis of the compound of formula (13)> The compound of formula (12) (75 mg, 0.21 mmol) was dissolved in DMA (6.0 mL) and heated to 200 °C in a microwave (200 W) for 30 min. After cooling to room temperature, the solvent was removed in vacuo. The crude product was chromatographed on silica gel to give a yellow solid in 84% yield (67 mg). This solid was dissolved in EtOAc and analyzed by thin-layer chromatography using a 1:1 mixture of hexane / EtOAc as the mobile phase, giving an Rf value of 0.3.

[0042] The NMR spectrum of this solid was as follows: 1 H NMR(CDCl3):δ 8.22(d,J=1.6,1H),8.15(d,J=8.0,1H),7.85(dd,J=1.6,8.0,1H),6.80( s,1H),4.99(q,J=6.7,1H),3.11(s,3H),3.04(s,3H),1.61(d,J=6.7,3H). 13 C NMR(CDCl3):δ 179.8(C),172.7(C),165.6(C),164.9(C),151.8(C),140.1(CH),136.7(C),133.2(CH),133.0 (C),132.2(C),131.2(C),127.0(C),103.8(CH),63.7(CH),37.2(CH3),37.0(CH3),21.5(CH3). IR(KBr):1666,1581,1365,1095,972,740. HRMS(ESI) m / z:[M+Na] + calcd for [C 17 H 15 NO5SNa] + ,368.0569,Found,368.0551. From the above, it was confirmed that the compound was of formula (13).

[0043] <Synthesis of the compound of formula 2-(1-hydroxyethyl)-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-6-sulfonamide (14)> The compound of formula (13) (38 mg, 0.11 mmol) and NCS (N-chlorosuccinimide) (88 mg, 0.66 mmol) were stirred in a dry CH3CN (10 mL) solution and the temperature was adjusted to 0 °C, and 10% HCl (100 μL) was added to obtain a reaction mixture.

[0044] After stirring at room temperature for 0.5 h, 28% NHOH (200 μL) was added to the reaction mixture. After stirring at room temperature for an additional 0.5 h, the reaction mixture was concentrated in vacuo and extracted with EtOAc to obtain the organic extract. The organic extract was washed with NaHCO and saturated brine, dried over NaSO, and then concentrated. The crude product was chromatographed on silica gel to obtain a yellow solid in 62% yield (22 mg). This solid was dissolved in EtOAc and subjected to thin-layer chromatography using a 1:1 mixture of hexane / EtOAc as the mobile phase, giving an Rf value of 0.2.

[0045] The NMR spectrum of this solid was as follows: 1 H NMR(DMSO):δ 8.52(d,J=1.8,1H),8.34(d,J=8.0,1H),8.30(dd,J=1.8,8.0,1H),7.80(s,2H), 7.03(s,1H),5.90(d,J=5.6,1H),4.95(qd,J=5.6,6.6,1H),1.54(d,J=6.6,3H). 13 C NMR(DMSO):δ 179.8(C),172.2(C),167.5(C),151.9(C),148.9(C),134.8(C),133.9(C),13 1.6(CH),131.1(C),127.9(CH),124.0(CH),104.1(CH),62.7(CH),22.7(CH3). IR(KBr):1658,1527,1149,972,478. HRMS(ESI) m / z:[M+Na] + calcd for [C 14 H 11 NO6SNa] + ,344.0205,Found,344.0211. From the above, it was confirmed that the compound was of formula (14).

[0046] <Synthesis of the compound 2-Acetyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-6-sulfonamide (18)> To a solution of the compound (14) (20 mg, 0.062 mmol) in dry CHCl (3.0 mL) was added pyridinium dichromate (PDC) (234 mg, 0.31 mmol) at 0°C. After stirring at room temperature for 2 h, the mixture was filtered through a pad of Celite. The Celite was washed with CHCl, and the filtrate and the CHCl from the Celite wash were combined and concentrated. The crude product was chromatographed on silica gel to give a yellow solid in 68% yield (13 mg). This solid was dissolved in EtOAc and subjected to thin-layer chromatography using a 1:1 mixture of hexane / EtOAc as the mobile phase; the Rf value was 0.4.

[0047] The NMR spectrum of this solid was as follows: 1 H-NMR (DMSO): δ 8.44(s,1H),8.26(d,J=8.1,1H),8.21(dd,J=1.7,8.1,1H),8.03(d,J=1.7,1H),7.74(s,2H),2.55(s,3H). 13 C-NMR (DMSO):δ 187.8(C),179.2(C),173.3(C),154.7(C),153.8(C),149.2(C),135.0(C),13 4.1(C),131.3(CH),130.7(C),128.2(CH),124.1(CH),115.0(CH),27.2(CH3). IR(KBr):2916,2846,1674,1257,1087,802,617. HRMS(ESI) m / z:[M+Na] + calcd for [C 14 H9NO6SNa] + ,342.0048,Found,342.0062. It was confirmed that the compound was the one of formula (18).

[0048] Scheme 2 Next, the compounds of formula (21) and formula (22) were synthesized according to the procedure of Scheme 2 in Figure 2.

[0049] <Synthesis of O-(2-(1-hydroxyethyl)-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-5-yl)dimethylcarbamothioate (compound of formula (19))> This compound was synthesized starting from NQ801 (300 mg,​​​​​​​​​​​​​​​​17 H 15 NO5SNa] + ,368.0569,Found,368.0572. From the above, it was confirmed that the compound is the one of formula (19).

[0051] <S-(2-(1-hydroxyethyl)-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-5-yl) dimethylcarbamothioate (Synthesis of the compound of formula (20)) This compound was synthesized starting from the compound of formula (19) (381 mg, 1.10 mmol) according to the existing procedure for synthesizing the compound of formula (13). As a result, a yellow solid was obtained in a yield of 76% (292 mg). This solid was dissolved in EtOAc, and thin-layer chromatography was performed using a solution of hexane / EtOAc mixed 1:1 as the mobile phase, and the Rf value was 0.1.

[0052] The NMR spectrum of this solid was as follows. 1 H NMR(CDCl3):δ 8.27(dd,J=1.3,7.8,1H),7.94(dd,J=1.3,7.8,1H),7.67(t,J=7.8,1H),6.76(d,J=0.8,1H),4.97(q,J=6.8,1H),3.25(s,3H),3.1(s,3H),2.9(s,1H),1.6(d,J=6.8,3H). 13 C NMR(CDCl3):δ 180.0(C),171.8(C),165.4(C),165.2(C),149.8(C),141.6(CH),133.8(C),132.2(C),132.1(C),131.8(CH),131.6(C),127.3(CH),103.4(CH),63.1(CH),36.8(CH3),36.5(CH3),20.9(CH3) . IR(KBr):3448,1658,1381,1095. HRMS(ESI) m / z:[M+H] + calcd for [C17 H 16 NO5S] + ,346.0749,Found,346.0753. From the above, it was confirmed that the compound was of formula (20).

[0053] <Synthesis of the compound of formula 8-(1-Hydroxyethyl)-6H-furo[3',2':2,3]naphtho[1,8-cd]isothiazol-6-one 2,2-dioxide (21)> This compound was synthesized starting from compound (20) (30 mg, 0.086 mmol) according to a known procedure for the synthesis of compound (14). A yellow solid was obtained in 53% yield (14 mg). This solid was dissolved in EtOAc and subjected to thin-layer chromatography using a 1:1 mixture of hexane / EtOAc as the mobile phase, resulting in an Rf value of 0.3. Formulas (21S) and (21R) were also synthesized independently by using (S)-3-butyn-2-ol and (R)-3-butyn-2-ol, respectively, in the synthesis of compound (11) (see Figure 1).

[0054] The NMR spectrum of this solid was as follows: 1 H NMRCDCl3):δ 8.31(d,J=7.7,1H),8.10(d,J=7.7,1H),7.97(t,J=7.7,1H),7.04(s,1H),5.10(q,J=6.9,1H),1.69(d,J=6.9,3H). 13 C NMR(CDCl3):δ 168.5(C),164.7(C),155.4(C),150.7(C),136.0(C),132.6(CH),128.0(CH), 127.6(C),125.8(C),124.1(C),123.4(CH),101.7(CH),61.7(CH),19.5(CH3). IR(KBr):3448,1674,1512,1342,1165,987,732. HRMS(ESI) m / z:[M+Na] + calcd for [C14 H8NO5SNa] + ,326.0099,Found,326.0093. From the above, it was confirmed that the compound was of formula (21).

[0055] <Synthesis of the compound 8-Acetyl-6H-furo[3',2':2,3]naphtho[1,8-cd]isothiazol-6-one 2,2-dioxide (22)> This compound was synthesized starting from compound (21) (14 mg, 0.046 mmol) according to a published procedure for the synthesis of compound (18). A yellow solid was obtained in 61% yield (8.5 mg). The solid was dissolved in EtOAc and subjected to thin-layer chromatography (TLC) using a 1:1 mixture of hexane / EtOAc as the mobile phase, resulting in an Rf value of 0.3.

[0056] The NMR spectrum of this solid was as follows: 1H NMR (CDCl3): δ 8.40(d,J=7.5,1H),8.23(d,J=7.5,1H),8.06(t,J=7.5,1H),7.81(s,1H),2.72(s,3H). 13 C NMR(CDCl3):δ 191.6(C),175.8(C),162.0(C),160.6(C),158.6(C),143.0(C),140.1(CH),1 35.3(CH),134.5(C),132.9(C),131.1(CH),130.2(C),117.2(CH),31.6(CH3). IR(KBr):1681,1581,1356,1257,1141. HRMS(ESI) m / z:[M+Na] + calcd for [C 14 H7NO5SNa] + ,323.9943,Found,323.9951. From the above, it was confirmed that the compound was of formula (22).

[0057] Scheme 3 Next, the compound of formula (23) was synthesized according to the method of Scheme 3.

[0058] <Synthesis of the compound of formula 2-(1-hydroxyethyl)-N,N-dimethyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-5-sulfonamide (23)> This compound was synthesized according to the existing procedure for the synthesis of compound (14), starting from compound (20) (30 mg, 0.086 mmol). Dimethylamine (2.0 M in THF, 1.0 mL) was used instead of NH4OH. This resulted in a 53% yield (16 mg) of a yellow solid. This solid was dissolved in EtOAc and subjected to thin-layer chromatography using a 1:1 mixture of hexane / EtOAc as the mobile phase, giving an Rf value of 0.15.

[0059] The NMR spectrum of this solid was as follows: 1H NMR(CDCl3):δ 8.49(d,J=7.8,1H),8.24(d,J=7.8,1H),7.86(t,J=7.8,1H),6.87(s,1 H),5.05(d,J=6.5,1H),3.07(s,6H),2.80(s,1H),1.65(d,J=6.5,3H). 13 C NMR(CDCl3):δ 178.0(C),171.3(C),166.3(C),149.7(C),140.9(C),135.1(C),134.9(CH),133.1(CH),1 32.4(C),131.8(CH),131.0(C),104.4(CH),63.7(CH),37.8(CH3),37.8(CH3),21.4(CH3). IR(KBr):1681,1543,1373,1211,1157,972,794. HRMS(ESI) m / z:[M+Na] + calcd for [C 14 H9NO5S] + ,372.0518,Found,372.0522. From the above, it was confirmed that the compound was of formula (23).

[0060] <Inhibitory effect on cancer cell proliferation> MDA-MB-231 (breast cancer cells), A549 (lung cancer cells), and Raw264.7 (mouse-derived macrophage cells) were purchased from KAC Co. Ltd. (Japan). All cells were cultured in DMEM supplemented with 10% (vol / vol) FBS and 1% penicillin / streptomycin / amphotericin B (Nacalai tesque) at 37°C in a humidified incubator with 5% CO2. The antiproliferative activity of each compound was examined using these cells.

[0061] Cell proliferation was measured using a WST8 assay kit (Dojindo) according to the manufacturer's instructions. Briefly, 5 × 10 cells (MDA-MB-231, A549, or Raw264.7) were cultured at 10°C for 1 hour. 3 Cells were seeded into 96-well microplates at a density of 1000 cells / well. After overnight incubation, cells were treated with test compounds (0-30 μM) for 72 hours. Absorbance was measured at 450 nm, and viability was calculated as a percentage of cell proliferation relative to the compound-untreated control (0.1% DMSO).

[0062] WST8 assays were performed in triplicate. IC 50 Values ​​were calculated by nonlinear regression against compound-untreated controls using GraphPad Prism software v7.0, and the results for each concentration were expressed as the mean. The results are shown in Tables 1 and 2.

[0063] [Table 1]

[0064] [Table 2]

[0065] As a result of the antiproliferative activity of ortho-naphthoquinones against A549 cells and MDA-MB-231 cells, compounds of formulas (6)-(8) containing a phenolic hydroxyl group showed higher antiproliferative activity than compound of formula (4) and β-lapachon (compound of formula (3)) which do not contain a phenolic hydroxyl group (see Table 2, entries 5-8 and 14).

[0066] Among the paranaphthoquinone and tetracyclic compounds tested, compounds with fused sulfonamide structures (21), (21S), (21R), and (22) showed significantly higher activity than the parent compound NQ801 (see Table 1, entries 1-4, and Table 2, entry 12). Furthermore, compounds with (21), (21S), and (21R) were 10 times less sensitive to cancer cells than to normal cells, Raw264.7, demonstrating high selectivity for cancer cells.

[0067] On the other hand, the antiproliferative activity of compound (23), in which a dimethylamino group was introduced to prevent the condensation reaction between the sulfonamide group and the adjacent carbonyl group, was reduced (see Table 2, entry 9). Furthermore, the antiproliferative activity of compounds (14) and (18), in which a sulfonamide group was introduced at C6, was comparable to that of the parent NQ801, but was lower than that of compounds in which a sulfonamide group was introduced at C5 (see Table 2, entries 11, 10, and 12).

[0068] The difference in the substituents (e.g., hydroxyl group and carbonyl group) on the furan ring side chain did not significantly affect the activity, and the activity was maintained at a high level (Table 1, entries 1, 2, and 3 vs. 4; Table 2, entry 11 vs. 10).

[0069] To examine whether these synthetic compounds, which exhibit potent antiproliferative activity against cancer cells, can inhibit the phosphorylation of STAT3 at Y705, A549 cells were treated with these compounds and Western blot (WB) analysis was performed to evaluate their inhibitory effect on STAT3 phosphorylation.

[0070] Western blot analysis Western blot (WB) analysis was performed as follows. The following antibodies were used: pTyr705-STAT3 (#9145) and STAT3 (#9139) were purchased from Cell Signaling Technology. Actin (#GTX629630) was purchased from GeneTex. Anti-mouse IgG (H+L), HRP conjugate (#W4021), anti-rabbit IgG (H+L), and HRP conjugate (#4011) were purchased from Promega.

[0071] The procedure was as follows: 2 × 10 A549 cells were plated in a 6-well microplate. 6 Cells were seeded at a density of 1000 cells / well. After overnight incubation, the growth medium was replaced and the cells were incubated for 24 hours at 37°C under serum-free conditions. Test compounds (or DMSO) were added to the wells and incubated for 1 hour before stimulation with IL-6 (final concentration 10 ng / mL).

[0072] After 1 h of incubation, cells were lysed using lysis buffer, and proteins were extracted for 30 min at 4°C and centrifuged at 10,000 rpm for 30 min at 4°C. Denatured proteins (14 μg / lane) were electrophoresed on 6–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to PVDF (polyvinylidene difluoride) membranes (Bio-Rad).

[0073] After blocking with Bullet Blocking One (Nacalai Tesque) for 10 minutes, the membrane was incubated with the primary antibody of interest for 1 hour at room temperature, followed by incubation with an HRP (horseradish peroxidase)-conjugated secondary antibody (1:20,000) for 1 hour at room temperature. Signals were visualized using ECL Western blotting substrate (Chemi-Lumi One, Nacalai Tesque). The results are shown in Figure 4.

[0074] Referring to Figure 4, Figure 4(A) shows the results when compounds of formulas (6), (7), (8), and (4) were used. This shows the STAT3 phosphorylation inhibitory activity of four ortho-naphthoquinones. Figure 4(B) shows the results when compounds of formulas (21), (22), (23), and (18) were used. This shows the STAT3 phosphorylation inhibitory activity of sulfonamides and tetracyclic compounds. In the figure, "D" represents DMSO, and "b" represents baricitinib (JAK inhibitor). The "Intensity" value represents the ratio of the intensity of the p-STAT3 band to the actin band.

[0075] Referring to Figure 4(A), compounds of formula (7) and formula (8) completely inhibited STAT3 phosphorylation at concentrations of <1.0 μM and significantly reduced IL-6-stimulated STAT3 phosphorylation without affecting the total amount of STAT3 protein.

[0076] Furthermore, isonapabucasin (4), previously reported by Muller et al. as a potent STAT3 inhibitor (see Figure 7), exhibited significantly lower STAT3 phosphorylation inhibitory activity than compounds (7) and (8) (see Figure 7).

[0077] Furthermore, compound (6) showed superior inhibitory potency to β-lapachone (compound (3)). In a previous study, β-lapachone (compound (3)) did not inhibit STAT3 phosphorylation even at a concentration of 10 μM (Non-Patent Document 12). These results clearly demonstrate that the presence of a hydroxy group on the aromatic ring is an important factor for the potent inhibitory activity of STAT3 phosphorylation.

[0078] As shown in Figure 4(B), compounds (21) and (22), which were found to have more potent antiproliferative effects, were evaluated at lower concentrations (0.01–0.3 μM). These compounds completely inhibited STAT3 phosphorylation at concentrations <0.1 μM and significantly reduced IL-6-stimulated STAT3 phosphorylation without affecting the total amount of STAT3 protein.

[0079] On the other hand, the STAT3 phosphorylation inhibitory activity of compound (23), which does not have a tetracyclic skeleton, was significantly reduced, revealing the important role of the tetracyclic skeleton in STAT3 phosphorylation inhibitory activity. The above Western blot (WB) results revealed that the compounds that showed potent antiproliferative activity also potently inhibited STAT3 phosphorylation, suggesting that the enhanced inhibitory activity of STAT3 phosphorylation also contributes to the enhanced antiproliferative activity.

[0080] <Bonding simulation> To gain further insight into the binding mode and understand the differences in activity between the synthesized compounds, molecular modeling experiments were performed using AutoDock Vina software. The protein was constructed based on the X-ray structure of STAT3 available through the RCSB Protein Data Bank (PDB code: 1BG1). Unless otherwise specified, default parameters were used according to the instructions in the AutoDock manual.

[0081] The results of molecular modeling are shown in Figure 5. Figure 5 shows the binding mode of compound (21) to the hinge region of STAT3. The hinge region is indicated by a box, and compound (21) is indicated by "21." As shown in Figure 5A, it was revealed that the synthesized compound (21) can bind to the pocket of STAT3.

[0082] The predicted docking pose of compound (21) is shown in Figure 5B. Compound (21) could potentially form hydrogen bonds with His332, Arg335, Asp566, and Lys573 amino acid residues, with a calculated average binding energy of -8.0 kcal / mol. It was also suggested that hydrogen bonds between the oxygen atom of the sulfonyl group and the adjacent polar amino acid residues could improve the binding affinity of compound (21).

[0083] Previously, Li et al. reported that napabucasin binds to a small pocket between the linker and DNA-binding domain in the STAT3 crystal structure, but many studies have suggested that napabucasin inhibits STAT3 phosphorylation by binding to the SH2 domain. The docking simulation results shown in Figure 5 are in good agreement with these X-ray crystallography data, indicating that the inhibitory mechanism of compound (21) is similar to that of napabucasin.

[0084] In conclusion, novel ortho- and para-naphthoquinone derivatives bearing phenolic hydroxy or sulfonamide moieties were synthesized and evaluated for their antiproliferative and STAT3 phosphorylation inhibitory activities. Among the synthesized compounds, compounds (21) and (22) showed the most potent antiproliferative activity in the nanomolar range against two cancer cell lines.

[0085] Furthermore, compounds of formulas (21) and (22) significantly reduced IL-6-induced STAT3 phosphorylation without affecting the total amount of STAT3 protein. [Industrial Applicability]

[0086] The present invention can be suitably used as a STAT3 phosphorylation inhibitor.

Claims

1. A STAT3 inhibitor comprising, as an active ingredient, at least one naphthoquinone compound represented by formula (21), formula (21S), formula (21R), formula (22), formula (23), formula (18), or formula (14). 【Chemistry 100】 【Chemistry 101】 【Chemistry 102】 【Chemistry 103】 【Chemistry 104】 【Chemistry 105】 【Chemistry 106】

2. 10. A STAT3 phosphorylation inhibitor comprising at least one compound selected from the compounds of claim 1 as an active ingredient.

3. 10. A cancer cell proliferation inhibitor comprising at least one compound selected from the compounds of claim 1 as an active ingredient.

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