2014 Volume 62 Issue 3 Pages 304-307
Dehydroxymethylepoxyquinomycin (DHMEQ, 1) is well known to inhibit nuclear factor-kappa B (NF-κB), which is closely associated with immune, inflammatory, and apoptotic processes as an inducible transcription factor. The inhibitory effect seems to be the result of the ring opening of an epoxide of 1 with Cys38 of p65. We have synthesized an analog 4 containing a cyclopropanated quinol skeleton and examined its ability to inhibit NF-κB. Surprisingly, 4 showed no remarkable NF-κB inhibitory activity as determined through expression of cyclooxygenase-2 (COX-2) in an RAW264.7 macrophage cell line.
The recent growth in molecular biology has generated new drug targets causing a dramatic paradigm shift in the chemotherapeutics of cancer (e.g., molecularly targeted therapeutics). Recently, much attention has been concentrated on nuclear factor-kappa B (NF-κB), which belongs to a family of transcription factors associated with immune and inflammatory processes. NF-κB is a homo- or heterodimer of Rel family proteins including p65 (RelA), p50, RelB, c-Rel, and p52. Among them, p65 (RelA), c-Rel, and p52 have a transactivation domain. Continuous activation of NF-κB is known to occur in various types of human tumors, and is correlated with inactivation of apoptosis via transcriptional misregulation. NF-κB inhibitors are thus expected to be prominent candidates for anticancer drugs.1) Dehydroxymethyl-epoxyquinomycin (DHMEQ, 1), first synthesized by Umezawa and colleagues on the basis of a structural modification of epoxyquinomycin C (Chart 1), shows a potent NF-κB inhibitory effect with little toxicity.2) The careful matrix assisted laser desorption ionization-time of flight (MALDI-TOF)-MS analysis by Umezawa and colleagues of chymotrypsin-treated p65 peptide clarified that the bioactivity of 1 is a result of coupling with Cys38 of p65.3) To specify the covalent bond formed, they conducted a reaction of 1 with N-Boc-L-Cys methyl ester. The resulting conjugate was identified as 2 based on its mass and NMR spectra. Although the regioselective ring opening of the epoxide is essential for the additional inhibitory effect of 1 against p65, it has not yet been completely confirmed whether a similar chemical binding occurs in biological processes. In this paper, we describe the synthesis of a new DHMEQ analog 4 with a cyclopropane ring instead of an epoxide and the evaluation of its NF-κB inhibitory activity to clarify the significance of the epoxide in 1 using a structure–activity relationship study.
Because 3b also inhibits NF-κB activation,4) synthesis of its analog 8 was initially conducted as shown in Chart 2. Dienone 5 was prepared from 2,5-dimethoxyaniline following a known procedure.5) Treatment of dienone 5 with trimethylsulfoxonium iodide in the presence of NaH resulted in selective cyclopropanation at the C4,5 double bond to generate 6 in 77% yield.6,7) Deacetalization of 6 with pyridinium p-toluenesulfonate (PPTS) afforded the diketone 7 in excellent yield. Finally, reduction of 7 with L-selectride® proceeded stereoselectively to give 8 with cis-configuration. Next, we attempted the conversion of 8 into the second target 4. After protection of the alcohol 8 with an acetyl group, the resulting acetate 9 was subjected to removal of an alloc group upon treatment with Pd(PPh3)4 and dimedone. Unfortunately, although deprotection of 9 proceeded smoothly to give amine 10, a partial O→N acetyl migration occurred to also generate alcohol 11. We thus decided to synthesize 4 from the dienone 12, which can be prepared in two steps from 2,5-dimethoxyaniline.8) Cyclopropanation of 12 with trimethylsulfoxonium iodide proceeded regioselectively to give 13 in good yield. Finally, 13 was successfully converted into the target 4 by removal of dimethyl acetal and subsequent stereoselective reduction. The stereochemistry of the alcohol was verified by nuclear Overhauser effect (NOE) measurement.
It is reported that DHMEQ reacts rapidly with nucleophiles like benzylthiol and cysteine derivatives to give DHMEQ–cysteine adducts in dimethyl sulfoxide (DMSO)/phosphine buffer.3) Although compound 4 was submitted to the same reaction, no adduct formation was observed, even after a long reaction time (Chart 3).
Next, its inhibitory activity on NF-κB was examined in an RAW264.7 macrophage cell line. It is known that cyclooxygenase-2 (COX-2) is expressed in the cell when NF-κB is activated.9) Compounds 1 and 4 were dissolved in DMSO at 1 mg/mL, respectively, and 10 µL was added to the cell culture. After 1 h, the medium was washed off. The cells were treated with lipopolysaccharide (LPS) and cultured for 8 h. Subsequently, the cells were lysed and the inhibitory activity on NF-κB was assesed by estimating the expression of COX-2. The production level of COX-2 was inhibited in cells treated with DHMEQ (1), but cells treated with compound 4 produced COX-2 at almost the same level as the control group (Chart 4). Therefore, we concluded that compound 4 has little or no inhibitory activity on NF-κB in RAW264.7 cells.
Cells were treated with or without chemicals at indicated concentrations for 1 h and stimulated with or without LPS at indicated concentrations for 8 h. COX-2 expression was evaluated by Western blotting using anti-COX-2 antibody (BD, cat. No. C22420) according to the manufacturer’s instructions.
We have achieved the synthesis of 4, a cyclopropane derivative of DHMEQ (1), from commercially available 2,5-dimethoxyaniline in 5 steps (total yield 29%). We showed that 4 has no recognizable inhibitory activity on NF-κB in an RAW264.7 macrophage cell line. These results strongly support the suggestion by Umezawa and colleagues, that is, the covalent binding of the thiol group in Cys38 of p65 with the epoxide of DHMEQ is closely correlated with its inhibitory activity.
1H- and 13C-NMR spectra were recorded on a JEOL JNM-ECX-400 spectrometer at 400 and 100 MHz, respectively. Chemical shifts were expressed in δ parts per million with tetramethylsilane as internal standard (δ=0 ppm) for 1H-NMR. Chemical shifts of carbon signals were referenced to CDCl3 (δC=77.0 ppm). The following abbreviations are used: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, and br=broad. IR spectra were recorded on a Shimadzu IR Prestige-21. Mass spectra were recorded on a JEOL JMS-GCmate II. Melting points were determined on a Yanaco MP-S3 micro melting point apparatus and were uncorrected.
Trimethylsulfoxonium iodide (12.9 g, 57.4 mmol) was dissolved in DMSO (31.5 mL) and stirred at room temperature under an argon atmosphere. To this solution was added sodium hydride (50–72% in mineral oil, 2.25 g). After stirring for 30 min, dienone 5 (0.73 g, 2.87 mmol) in DMSO (31.5 mL) was added and stirred for 17 h. To this reaction mixture was added aqueous saturated NH4Cl and extracted with ethyl acetate for 3 times. The organic phase was dried over Na2SO4 and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (hexane–ethyl acetate=1 : 1) to give 6 (0.59 g, 77%).
(2,2-Dimethoxy-5-oxobicyclo[4.1.0]hept-3-en-3-yl)carbamic Acid Allyl Ester 6: 1H-NMR (CDCl3, 400 MHz) δ: 7.28 (br s, 1H), 6.65 (d, J=1.2 Hz, 1H), 5.94 (m, 1H), 5.36 (dq, J=17.2, 1.6 Hz, 1H), 5.28 (dd, J=10.4, 1.2 Hz, 1H), 4.64 (q, J=1.6 Hz, 1H), 4.62 (q, J=1.6 Hz, 1H), 3.56 (s, 3H), 3.27 (s, 3H), 2.08 (m, 1H), 2.00 (m, 1H), 1.29 (m, 1H), 0.96 (q, J=4.8 Hz, 1H); 13C-NMR (CDCl3, 100 MHz) δ: 196.05, 152.01, 144.32, 131.54, 118.81, 107.01, 96.63, 66.26, 50.96, 49.42, 23.16, 18.15, 13.01; IR (film) 1744, 1659, 1628, 1512, 1211 cm−1; high resolution (HR)-MS (electron ionization (EI)) Calcd for C13H17NO5 (M+) 267.1107, Found 267.1122.
Dimethyl acetal 6 (0.28 g, 1.06 mmol) was dissolved in acetone (10 mL) and stirred at room temperature. To this solution was added H2O (1 mL) and (PPTS, 0.27 g, 1.06 mmol). After stirring for 24 h, aqueous saturated NaHCO3 was added and extracted with ethyl acetate for 3 times. The organic phase was dried over Na2SO4 and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (hexane–ethyl acetate=1 : 1) to give diketone 7 (0.23 g, 97%).
(2,5-Dioxobicyclo[4.1.0]hept-3-en-3-yl)carbamic Acid Allyl Ester 7: 1H-NMR (CDCl3, 400 MHz) δ: 7.57 (br s, 1H), 7.02 (d, J=2.0 Hz, 1H), 5.92 (m, 1H), 5.93 (ddt, J=17.6, 10.8, 6.0 Hz, 1H), 5.37 (dq, J=17.6, 1.2 Hz, 1H), 5.29 (dq, J=10.0, 1.2 Hz, 1H), 4.66 (dt, J=6.0, 1.2 Hz, 1H), 2.62–2.52 (m, 2H), 1.75 (dt, J=8.8, 5.2 Hz, 1H), 1.66 (q, J=5.2 Hz, 1H); 13C-NMR (CDCl3, 100 MHz) δ: 194.18, 190.52, 151.92, 138.67, 131.33, 119.14, 113.69, 66.71, 26.98, 25.40, 21.16; IR (film) 1744, 1658, 1512, 1203 cm−1; HR-MS (EI) Calcd for C11H11NO4 (M+) 221.0688, Found 221.0688.
Diketone 7 (0.10 g, 0.46 mmol) was dissolved in tetrahydrofuran (THF) (5 mL) and stirred at −38°C under an argon atmosphere. To this solution was added L-selectride® (1.0 M in THF solution, 0.47 mL). After stirring for 3 min, aqueous 10% HCl was added and extracted with ethyl acetate for 3 times. The organic phase was dried over Na2SO4 and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (hexane–acetone=2.5 : 1) to give alcohol 8 (92.0 mg, 90%). The resulting solid was recrystallized from hexane–ethyl acetate to give colourless crystal.
(2-Hydroxy-5-oxobicyclo[4.1.0]hept-3-en-3-yl)carbamic Acid Allyl Ester 8: mp 94–95°C; 1H-NMR (CDCl3, 400 MHz) δ: 7.56 (s, 1H), 6.44 (t, J=1.2 Hz, 1H), 5.92 (ddt, J=17.2, 10.8, 4.8 Hz, 1H), 5.35 (dq, J=17.6, 1.2 Hz, 1H), 5.27 (dq, J=10.4, 1.2 Hz, 1H), 4.81 (dt, J=7.6, 1.2 Hz, 1H), 4.63 (dt, J=5.6, 1.2 Hz, 2H), 3.94 (d, J=7.6 Hz, 1H), 1.95–2.09 (m, 2H), 1.25 (m, 1H), 1.06 (q, J=5.2 Hz, 1H); 13C-NMR (CDCl3, 100 MHz) δ: 198.02, 152.26, 149.22, 131.60, 118.94, 104.24, 66.46, 64.41, 23.62, 17.56, 12.55; IR (KBr) 3225, 1744, 1620, 1551, 1227 cm−1; HR-MS (EI) Calcd for C11H13NO4 (M+) 223.0845, Found 223.0844.
Trimethylsulfoxonium iodide (0.40 g, 1.80 mmol) was dissolved in DMSO (1.6 mL) and stirred at 0°C under an argon atmosphere. To this solution was added sodium hydride (50–72% in mineral oil, 86.0 mg). After stirring for 20 min at room temperature, dienone 12 (0.12 g, 0.36 mmol) in DMSO (2 mL) was added and stirred for 1.5 h. To this reaction mixture was added aqueous saturated NH4Cl and extracted with ethyl acetate for 3 times. The organic phase was dried over Na2SO4 and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (hexane–ethyl acetate=1 : 1) to give 13 (0.10 g, 92%).
N-(2,2-Dimethoxy-5-oxobicyclo[4.1.0]hept-3-en-3-yl)-2-hydroxy-benzamide 13: Amorphous 1H-NMR (CDCl3, 400 MHz) δ: 11.47(br s, 1H), 8.79 (s, 1H), 7.45 (m, 2H), 7.11 (d, J=1.2, 1H), 7.02 (dd, J=8.0, 0.8 Hz, 1H), 6.94 (dt, J=8.0, 0.8 Hz, 1H), 3.66 (s, 3H), 3.31 (s, 3H), 2.16 (m, 1H), 2.06 (dt, J=7.6, 6.4 Hz, 1H), 1.35 (dt, J=8.4, 5.2 Hz, 1H), 1.05 (q, J=5.2 Hz, 1H); 13C-NMR (CDCl3, 100 MHz) δ: 196.59, 168.93, 161.58, 142.99, 135.31, 125.78, 119.28, 118.96, 114.65, 110.25, 97.10, 51.18, 49.97, 23.49, 18.32, 13.23; IR (film) 3248, 1659, 1636, 1605, 1520, 1211, 1065 cm−1; HR-MS (EI) Calcd for C15H13NO4 ([M−CH3OH]+) 271.0845, Found 271.0845.
Dimethyl acetal 13 (0.80 g, 2.62 mmol) was dissolved in acetone (27 mL) and stirred at room temperature. To this solution was added H2O (2.6 mL) and PPTS (0.66 g, 2.62 mmol). After stirring for 22 h, aqueous saturated NaHCO3 was added and extracted with ethyl acetate for 3 times. The organic phase was dried over Na2SO4 and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (hexane–ethyl acetate=3 : 2) to give diketone (0.62 g, 92%). Diketone (0.62 g, 2.42 mmol) was dissolved in THF (25 mL) and stirred at −20°C under an argon atmosphere. To this solution was added L-selectride® (1.0 M in THF solution, 4.9 mL). After stirring for 1.5 h, aqueous 10% HCl was added and extracted with ethyl acetate for 3 times. The organic phase was dried over Na2SO4 and concentrated in vacuo to give alcohol 4 (0.41 g, 65%). The resulting solid was recrystallized from EtOH to give colourless crystal.
2-Hydroxy-N-(2-hydroxy-5-oxobicyclo[4.1.0]hept-3-en-3-yl)benzamide 4: mp 212°C (dec.); 1H-NMR (DMSO-d6, 400 MHz) δ: 11.70 (s, 1H), 10.95 (s, 1H), 7.91 (dd, J=7.6, 2.0 Hz, 1H), 7.41 (dt, J=6.4, 2.0 Hz, 1H), 6.97 (t, J=8.0 Hz, 1H), 6.94 (dd, J=8.0, 0.8 Hz, 1H), 6.74 (t, J=1.6 Hz, 1H), 6.05 (d, J=8.0 Hz, 1H), 4.79 (dt, J=7.2, 1.6 Hz, 1H), 1.93 (m, 1H), 1.79 (m, 1H), 1.16 (dt, J=8.0, 4.0 Hz, 1H), 0.93 (q, J=4.8 Hz, 1H); 13C-NMR (DMSO-d6, 100 MHz) δ: 197.04, 164.69, 156.03, 149.95, 134.08, 131.10, 119.85, 118.47, 116.90, 105.80, 63.47, 23.24, 17.42, 12.07; IR (KBr) 3364, 1659, 1605, 1528 cm−1; HR-MS (EI) Calcd for C14H13NO4 (M+) 259.0845, Found 259.0845.
