Semisynthetic Derivatives of Fradcarbazole A and ... - ACS Publications

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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Semisynthetic Derivatives of Fradcarbazole A and Their Cytotoxicity against Acute Myeloid Leukemia Cell Lines Mingpeng Li,†,§,⊥,∥ Yanchao Xu,†,§,⊥,∥ Mingxing Zuo,†,§,⊥ Wen Liu,†,⊥ Liping Wang,*,†,§,⊥ and Weiming Zhu*,†,‡ †

State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550014, China Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China § School of Pharmaceutical Sciences, Guizhou Medical University, Guiyang 550025, China ⊥ Key Laboratory of Chemistry for Natural Products of Guizhou Province, Chinese Academy of Sciences, Guiyang 550014, China

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S Supporting Information *

ABSTRACT: Fourteen derivatives of the marine-derived fradcarbazole A were synthesized from staurosporine. Their structures were identified by NMR and high-resolution electrospray ionization mass spectrometry (HRESIMS). The derivatives were screened in vitro for antiproliferative activity against three human leukemic cell lines (MV4−11, HL-60, K562). All of the derivatives displayed cytotoxicity against the human FLT-3 internal tandem duplication (ITD) mutant acute myeloid leukemia (AML) cell line MV4−11 with IC50 values of 0.32−0.96 μM. The mechanism of action studies indicated that the most effective 3-chloro-5‴-fluorofradcarbazole A (6) induced apoptosis of the MV4−11 cells and arrested the cell cycle at the G0/G1 phase. Furthermore, compound 6 can reduce the expression of FLT-3, CDK2, and c-kit. The results suggest that 3-chloro-5‴-fluorofradcarbazole A (6) is a potential candidate for developing novel anti-AML agents in the future.

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ince staurosporine was isolated from Streptomyces staurosporeus in 1977,1 it and related indolocarbazole alkaloids have received great attention due to their unusual structures and antitumor activities.2−4 Among them, PKC-412, which is a staurosporine derivative, has been approved by the Food and Drug Administration (FDA) for the treatment of adult patients with newly diagnosed, Feline McDonough Sarcoma (FMS)like tyrosine kinase 3 (FLT-3) mutation-positive AML.5 Fradcarbazole A with a novel staurosporine-thiazole-indole skeleton was isolated from a mutant strain of the marinederived Streptomyces f radiae 007M135 and semisynthesized from staurosporine by our group.6,7 To find new anti-AML drugs, 14 fradcarbazole A derivatives were synthesized. All of the compounds (1−14) and fradcarbazole A displayed selective cytotoxicity against the human FLT-3 ITD mutant AML cell line MV4−11. The preliminary mechanism of action of compound 6 on the MV4−11 cell line was also studied.

The 2-oxotryptamine derivatives (15−19) were separately synthesized from tryptamine, 5-fluorotryptamine, 5-chlorotryptamine, 5-bromotryptamine, and 5-methoxytryptamine by selective tert-butyloxycarbonyl (Boc) protection (83%−99% yield), 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) oxidation (58%−81% yield), and trifluoroacetic acid (TFA) deprotection (72%−94% yield) (Scheme 2). 3-Chlorostaurosporine (23) and 3-bromostaurosporine (24) were synthesized from staurosporine by Boc protection (76% yield), halogenation with N-chlorosuccinimide (NCS)8 (48% yield) or N-bromosuccinimide (NBS)8 (94% yield) and TFA deprotection (93% yield for 23, 50% yield for 24). Reaction of staurosporine or a halogenated staurosporine with thiocarbonyldiimidazole gave 3′-N-(1H-imidazole-1-carbamothioyl) staurosporine (25, 82% yield) and its halogenated derivatives (26, 71% yield and 27, 88% yield). Alkylation of 25−27 with MeI in MeCN gave the thiocarbamoyl imidazolium iodides (28−30, 68%−74% yield). The nucleophilic reactions of 15−19 with the thiocarbamoyl imidazolium iodides (28−30) afforded the thiourea intermediates 31−44 in 38%−56% yield. The intramolecular cyclization/dehydration

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RESULTS AND DISCUSSION The strategy that we proposed in our previous work7 was used to synthesize the fradcarbazole A derivatives (Scheme 1). The thiourea intermediates were synthesized from staurosporine or halogenated staurosporine and 2-oxotryptamine derivatives. The thiourea intermediates further underwent intramolecular cyclization/dehydration to yield fradcarbazole A derivatives. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 16, 2019

A

DOI: 10.1021/acs.jnatprod.9b00468 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Scheme 1. Synthetic Route to Fradcarbazole A Derivatives

Scheme 2. Synthesis of 2-Oxotryptamine Derivatives (15−19)

Analytical). IR spectra were taken on a Nicolet Nexus 470 spectrophotometer (Thermo Nicolet Corporation) as KBr disks. NMR spectra were recorded on a Bruker 600 MHz spectrometer with tetramethylsilane (TMS) as the internal standard. Electrospray ionization mass spectrometry (ESIMS) and HRESIMS measurements were performed on a Waters 2695 LCQ-MS liquid chromatography mass spectrometer. Semipreparative high-performance liquid chromatography (HPLC) separations were performed using a Hitachi Primaide Organizer HPLC system with a YMC-Pack ODS-A column (S-5 μm, 12 nm, 250 × 10 mm). Thin layer chromatography (TLC) and column chromatography (CC) were performed on plates precoated with silica gel GF 254 (10−40 μm) and over silica gel (200−300 mesh, Qingdao Marine Chemical Factory). Chromatographic grade MeOH for HPLC was from Tianjin Siyou Fine Chemicals Co., Ltd. Chemistry. Synthesis of 5‴-Fluorofradcarbazole A (1). Et3N (0.5 mL) and Boc2O (488 mg, 2.25 mmol) were added to a solution of 5fluorotryptamine (400 mg, 2.25 mmol) in tetrahydrofuran (THF, 15 mL) at 10 °C. After it was stirred for 1 h at 10 °C, the reaction mixture was poured into ice water (50 mL) and extracted with ethyl acetate (EtOAc, 3 × 50 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by flash column chromatography (FCC) eluting with petroleum ether (PE)−EtOAc (3:1) to provide 16a (486 mg, 83% yield) as an amorphous white solid; 1H NMR (600 MHz, deuterated dimethyl sulfoxide (DMSO-d6)) δ 10.91 (s, 1H, NH), 7.21−7.33 (m, 3H, ArH), 6.87−6.92 (m, 2H, ArH, NH), 3.14− 3.19 (m, 2H, CH2), 2.76 (t, J = 7.4 Hz, 2H, CH2), 1.37 (s, 9H, 3 × CH3); 13C NMR (150 MHz, DMSO-d6) δ 156.6 (d, 1JC−F = 230.6 Hz), 155.6, 132.9, 127.6 (d, 3JC−F = 9.7 Hz), 124.8, 112.3 (d, 3JC−F = 9.7 Hz), 112.2(d, 4JC−F = 4.5 Hz), 109.0 (d, 2JC−F = 25.2 Hz), 103.0 (d, 2JC−F = 25.2 Hz), 77.5, 40.8, 28.3 (3 × C), 25.4; ESIMS m/z 301.0 [M + Na]+. Compound 16a (100 mg, 0.31 mmol) was dissolved in THF/H2O (10:1, 11 mL), and then DDQ (144 mg, 0.62 mmol) was added at 0 °C. After it was stirred for 2 h at 0 °C, the reaction mixture was poured into EtOAc (100 mL) and washed extensively with saturated aqueous NaHCO3 (4 × 100 mL) to remove the excess DDQ. The EtOAc layer was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by FCC eluting with PE−EtOAc (2:1) to provide 16b (70 mg, 67% yield) as a colorless solid; IR (KBr) 3325, 3259, 2984, 1693, 1648, 1535, 1470, 1431, 1370, 1295, 1258, 1227, 1178, 874, 809, 638 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 8.47 (s, 1H, ArH), 7.81−7.84 (m, 1H, ArH), 7.48−7.51 (m, 1H, ArH), 7.06−7.10 (m, 1H, ArH), 7.03 (t, J = 5.9

of 31−44 with the (CF3CO)2O/EtOH system formed 1−14 in 47%−81% yields (Scheme 3). Derivatives 1−14 and fradcarbazole A were evaluated for their cytotoxicities against the human cancer cell lines, FLT-3 ITD mutant AML cell (MV4−11), chronic myelogenous leukemia cell (K562), acute promyelocytic leukemia cell (HL60), as well as the normal human peripheral blood mononuclear cell line (PBMC), with the positive control 3′N-benzoylstaurosporine (PKC-412) in each panel. All of the tested compounds selectively inhibit the MV4−11 cell line with IC50 values of 0.32−0.96 μM but are inact toward the K562, HL-60, and PBMC cell lines with IC50 values greater than 10 μM (Table 1). Apoptosis or programmed cell death is essential for the normal functioning and survival of most multicellular organisms.9 Inducing tumor cells to recover their apoptotic ability is an effective antitumor strategy.10 Compound 6 can induce apoptosis in MV4−11 cells at 0.3 μM (Figure 1). The effects of compound 6 on the cell cycle phase in MV4−11 cells were also evaluated. After treatment with 0.15, 0.30, and 0.60 μM of compound 6 for 24 h, cells were harvested and analyzed by flow cytometry. The results showed that compound 6 had a significant effect on the cell cycle redistribution and arrested the MV4−11 cell cycle in the G0/G1 phase (Figure 2). FMS-like tyrosine kinase-3 (FLT-3) and c-kit are class III receptor tyrosine kinases, which result in pro-growth and prosurvival signaling through STAT-5, Ras/Raf/ERK1/2, and PI3K/AKT pathways.11,12 FLT-3 and c-kit are overexpressed in AML cells. Inhibition of FLT-3 and c-kit expression can induce apoptosis of the AML cells.12,13 Compound 6 downregulated p-FLT3, FLT3, and c-kit in a dose-dependent manner and led to apoptosis in MV4−11 cells (Figure 3). In addition, cyclin-dependent kinase 2 (CDK2, a cell cycle protein) was also decreased, which may lead to arrest of the cell cycle at the G0/G1 phase. The results suggest that the derivatives of fradcarbazole A are potential candidates for developing novel anti-AML agents in the future.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using an AUTOPOL1 polarimeter (Rudolph Research B



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Scheme 3. Synthesis of Fradcarbazole A Derivatives (1−14)

1343, 1312, 1226, 1176, 1149, 1119, 1075, 745 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 12.15 (s, 1H, NH), 9.29 (d, J = 7.9 Hz, 1H, ArH), 8.61 (s, 1H, NH), 8.58 (s, 1H, ArH), 8.06 (d, J = 7.7 Hz, 1H, ArH), 8.01 (d, J = 8.5 Hz, 1H, ArH), 7.94 (brs, 1H, NH), 7.85 (dd, J = 9.8, 2.4 Hz, 1H, ArH), 7.75 (d, J = 8.2 Hz, 1H, ArH), 7.48−7.54 (m, 3H, ArH), 7.37 (t, J = 7.4 Hz, 1H, ArH), 7.31 (t, J = 7.4 Hz, 1H, ArH), 7.08−7.12 (m, 2H, ArH, H-1′), 5.92 (brs, 1H, H-3′), 4.95−5.05 (m, 4H, H-7, H-3″), 4.53 (brs, 1H, H-4′), 2.95 (s, 3H, 3′-NCH3), 2.87 (s, 3H, 4′-OCH3), 2.71−2.76 (m, 1H, H-2′a), 2.36 (s, 3H, 6′-CH3), 2.27−2.33 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 190.2, 182.5, 172.0, 158.6 (d, 1JC−F = 233.5 Hz), 139.1, 136.4, 134.8, 133.0, 132.8, 129.1, 126.0 (d, 3JC−F = 10.0 Hz), 125.7, 125.4, 125.1, 125.0, 123.8, 122.7, 121.5, 120.4, 119.5, 119.4, 115.3, 114.3 (d, 4JC−F = 4.2 Hz), 114.2, 113.9, 113.5 (d, 3JC−F = 10.0 Hz), 111.1 (d, 2JC−F = 24.0 Hz), 109.2, 106.0 (d, 2JC−F = 24.0 Hz), 95.0, 82.9, 82.4, 60.4, 54.3, 51.9, 45.5, 32.8, 29.6, 27.7; HRESIMS m/z 699.2193 [M − H]− (calcd for C39H32FN6O4S, 699.2184). EtOH (200 μL) and (CF3CO)2O (400 μL) were added sequentially to a solution of 31 (55 mg, 0.079 mmol) in CH2Cl2 (6 mL) at 0 °C. After it was stirred for 1 h at 0 °C, the reaction was quenched by adding 6 mL of saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers

Hz, 1H, NH), 4.28 (d, J = 5.9 Hz, 2H, CH2), 1.40 (s, 9H, 3 × CH3); 13 C NMR (150 MHz, DMSO-d6) δ 190.9, 158.6 (d, 1JC−F = 235.5 Hz), 156.0, 134.9, 133.0, 126.0 (d, 3JC−F = 10.8 Hz), 114.1(d, 4JC−F = 4.0 Hz), 113.5 (d, 3JC−F = 10.8 Hz), 111.1 (d, 2JC−F = 26.0 Hz), 106.0 (d, 2JC−F = 26.0 Hz), 77.9, 46.8, 28.3 (3 × C); HRESIMS m/z 315.1113 [M + Na]+ (calcd for C15H17FN2O3Na, 315.1115). The ketone intermediate 16b (300 mg, 0.73 mmol) was treated with TFA (5 mL) at 10 °C. After the resultant solution was stirred for 1 h at 10 °C, the solvent was removed in vacuo (using several benzene coevaporations to completely remove TFA) to give the keto tryptamine TFA salt 16 (185 mg, 72% yield) as a white solid, which was used immediately. The thiocarbonylimidazolium salt 28 was synthesized from staurosporine using our reported method.7 To a solution of thiocarbonylimidazolium salt 28 (121 mg, 0.17 mmol) in dimethylformamide (DMF, 5 mL) was added trifluoroacetate salt 16 (147 mg, 0.51 mmol) and Et3N (0.5 mL). The reaction was stirred at room temperature (rt) for 24 h and diluted with EtOAc (10 mL). The organic layer was washed with 1 N HCl (2 × 5 mL) and brine (10 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 31 (61 mg, 51% yield) as an amorphous white powder; [α]D18 +196 (c 0.50, CHCl3); IR (KBr) 3363, 2916, 1660, 1586, 1515, 1460, C



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ArH), 7.66−7.68 (m, 2H, ArH), 7.54−7.58 (m, 2H, ArH), 7.44−7.51 (m, 3H, ArH), 7.35 (t, J = 7.2 Hz, 1H, ArH), 7.31 (t, J = 7.3 Hz, 1H, ArH), 7.08 (t, J = 7.2 Hz, 1H, ArH), 7.04 (t, J = 8.4 Hz, 1H, H-1′), 4.96−5.01 (m, 3H, H-7, H-3′), 4.49 (s, 1H, H-4′), 2.89 (s, 3H, 3′NCH3), 2.85−2.88 (m, 1H, H-2′a), 2.71 (s, 3H, 4′-OCH3), 2.43 (s, 3H, 6′-CH3), 2.38−2.42 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 171.9, 167.5, 157.4 (d, 1JC−F = 232.7 Hz), 138.8, 136.3, 134.1, 133.2, 132.7, 129.3, 125.7, 125.4, 125.1, 125.0, 124.9 (d, 3JC−F = 9.5 Hz), 124.9, 123.8, 122.7, 121.5, 120.4, 119.9, 119.5, 119.4, 115.2, 114.2, 113.6, 113.1 (d, 3JC−F = 9.8 Hz), 110.1 (d, 2JC−F = 24.1 Hz), 109.0, 107.2 (d, 4JC−F = 4.9 Hz), 104.0 (d, 2JC−F = 24.1 Hz), 94.8, 82.5, 82.4, 60.2, 53.2, 45.5, 34.5, 29.3, 27.1; HRESIMS m/z 683.2236 [M + H]+ (calcd for C39H32FN6O3S, 683.2235). Synthesis of 5‴-Chlorofradcarbazole A (2). Et3N (0.5 mL) and Boc2O (349 mg, 1.60 mmol) were added to a solution of 5chlorotryptamine (300 mg, 1.55 mmol) in THF (15 mL) at 10 °C. After it was stirred for 1 h at 10 °C, the reaction mixture was poured into ice water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by FCC eluting with PE−EtOAc (3:1) to provide 17a (411 mg, 90% yield) as an amorphous white solid; 1H NMR (600 MHz, CDCl3) δ 8.40 (s, 1H, NH), 7.53 (d, J = 1.5 Hz, 1H, ArH), 7.24 (d, J = 8.6 Hz, 1H, ArH), 7.12 (dd, J = 8.6, 1.5 Hz, 1H, ArH), 7.00 (s, 1H, ArH), 4.65 (brs, 1H, NH), 3.35−3.44 (m, 2H, CH2), 2.88 (t, J = 6.4 Hz, 2H, CH2), 1.44 (s, 9H, 3 × CH3); 13C NMR (150 MHz, CDCl3) δ 156.0, 134.7, 128.5, 125.1, 123.4, 122.3, 118.3, 112.9, 112.2, 79.3, 41.0, 28.4(3 × C), 25.6; ESIMS m/z 317.0 [M + Na]+. Compound 17a (380 mg, 1.29 mmol) was dissolved in THF/H2O (10:1, 11 mL), and then DDQ (586 mg, 2.58 mmol) was added at 0 °C. After it was stirred for 2 h at 0 °C, the reaction mixture was poured into EtOAc (100 mL) and washed extensively with saturated aqueous NaHCO3 (4 × 100 mL). The EtOAc layer was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by FCC eluting with PE−EtOAc (1:1) to provide 17b (268 mg, 67% yield) as a colorless solid; IR (KBr) 3280, 2980, 1690, 1649, 1528, 1514, 1451, 1428, 1370, 1294, 1235, 1167, 926, 806, 628 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 12.16 (s, 1H, NH), 8.47 (s, 1H, ArH), 8.14 (s, 1H, ArH), 7.51 (d, J = 8.6 Hz, 1H), 7.24 (d, J = 8.6 Hz, 1H), 7.03 (t, J = 5.9 Hz, 1H, NH), 4.28 (d, J = 5.9 Hz, 2H, CH2), 1.40 (s, 9H, 3 × CH3); 13C NMR (150 MHz, DMSOd6) δ 190.9, 156.0, 134.9, 134.7, 126.5, 126.5, 122.9, 120.2, 113.8, 113.5, 77.9, 46.8, 28.2(3 × C); HRESIMS m/z 331.0817 [M + Na]+ (calcd for C15H17ClN2O3Na, 331.0820). The ketone intermediate 17b (230 mg, 0.75 mmol) was treated with TFA (5 mL) at 10 °C. After the resultant solution was stirred for 1 h at 10 °C, the solvent was removed in vacuo (using several benzene coevaporations to completely remove TFA) to give the keto tryptamine TFA salt 17 (202 mg, 88% yield) as a white solid, which was used immediately. To a solution of thiocarbonylimidazolium salt 28 (200 mg, 0.28 mmol) in DMF (5 mL) was added a trifluoroacetate salt 17 (85 mg, 0.28 mmol) and Et3N (0.5 mL). The

Table 1. In Vitro Antiproliferative Activity of Fradcarbazole A and Its Derivatives (IC50 μM) cell lines compounds 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Fradcarbazole A PKC-412

MV4−11

K562

HL-60

PBMC

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

>10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10

>10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10

>10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10

0.51 0.42 0.41 0.36 0.56 0.32 0.59 0.43 0.44 0.36 0.96 0.70 0.39 0.51 0.40 0.04

0.04 0.04 0.04 0.01 0.06 0.03 0.04 0.04 0.04 0.04 0.05 0.08 0.08 0.04 0.02 0.004

Figure 1. Compound 6 induces apoptotic cell death in MV4−11 cells. Cells treated with compound 6 at 0.3 μM for 24 h and stained with TUNEL to detect apoptotic cells (green). DNA (blue) was stained with 4′,6-diamidino-2-phenylindole (DAPI). were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 1 (34 mg, 63% yield) as a white powder; [α]D18 +201 (c 0.48, CHCl3); IR (KBr) 3415, 2922, 1677, 1524, 1456, 1344, 1317, 1119, 742 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.46 (s, 1H, NH), 9.32 (d, J = 7.8 Hz, 1H, ArH), 8.62 (s, 1H, NH), 8.06 (d, J = 8.0 Hz, 1H, ArH), 8.02 (d, J = 8.0 Hz, 1H,

Figure 2. Effects of compound 6 on cell cycle phase in MV4−11 cells. Cells were treated with compound 6 (0.15, 0.30, and 0.60 μM) for 24 h and then fixed and stained with PI to analyze the DNA content by flow cytometry. D



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Figure 3. Compound 6 affected FLT-3, CDK2, and c-kit in MV4−11 cells. MV4−11 cells were treated with compound 6 in different concentrations (0.15, 0.3, and 0.6 μM) for 24 h. Proteins were collected for immunoblotting analysis. The results showed that compound 6 could reduce the expression of p-FLT3, FLT3, CDK2, and c-kit. (#) P < 0.01. (△) P < 0.001. Synthesis of 5‴-Bromofradcarbazole A (3). Et3N (0.5 mL) and Boc2O (285 mg, 1.31 mmol) were added to a solution of 5bromotryptamine (300 mg, 1.26 mmol) in THF (15 mL) at 10 °C. After it was stirred for 1 h at 10 °C, the reaction mixture was poured into ice water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by FCC eluting with PE−EtOAc (3:1) to provide 18a (359 mg, 84% yield) as an amorphous white solid; 1H NMR (600 MHz, CDCl3) δ 8.44 (s, 1H, NH), 7.73 (d, J = 1.5 Hz, 1H, ArH), 7.27−7.30 (m, 1H, ArH), 7.25 (d, J = 8.6 Hz, 1H, ArH), 7.02 (s, 1H, ArH), 4.69 (brs, 1H, NH), 3.37−3.47 (m, 2H, CH2), 2.92 (t, J = 6.6 Hz, 2H, CH2), 1.48 (s, 9H, 3 × CH3); 13C NMR (150 MHz, CDCl3) δ 156.0, 135.0, 129.2, 124.8, 123.3, 121.4, 112.9, 112.6(2 × C), 79.3, 41.0, 28.4(3 × C), 25.6; ESIMS m/z 361.0 [M + Na]+. Compound 18a (100 mg, 0.30 mmol) was dissolved in THF/H2O (10:1, 11 mL), and then DDQ (134 mg, 0.60 mmol) was added at 0 °C. After it was stirred for 2 h at 0 °C, the reaction mixture was poured into EtOAc (100 mL) and washed extensively with saturated aqueous NaHCO3 (4 × 100 mL). The EtOAc layer was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by FCC eluting with PE−EtOAc (1:1) to provide 18b (61 mg, 58% yield) as a colorless solid; IR (KBr) 3288, 3168, 1689, 1655, 1528, 1441, 1372, 1313, 1143, 1110, 1026, 885, 810, 775, 636 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 8.45 (s, 1H, ArH), 8.30 (d, J = 1.5 Hz, 1H, ArH), 7.46 (d, J = 8.6 Hz, 1H), 7.35 (dd, J = 8.6, 1.5 Hz, 1H), 7.03 (t, J = 5.9 Hz, 1H, NH), 4.29 (d, J = 5.9 Hz, 2H, CH2), 1.40 (s, 9H, 3 × CH3); 13C NMR (150 MHz, DMSO-d6) δ 190.9, 156.0, 135.1, 134.5, 127.2, 125.4, 123.3, 114.6, 114.3, 113.4, 77.9, 46.8, 28.2(3 × C); HRESIMS m/z 375.0313 [M + Na]+ (calcd for C15H17BrN2O3Na, 375.0315). The ketone intermediate 18b (61 mg, 0.17 mmol) was treated with TFA (5 mL) at 10 °C. After the resultant solution was stirred for 1 h at 10 °C, the solvent was removed in vacuo (using several benzene coevaporations to completely remove TFA) to give the keto tryptamine TFA salt 18 (56 mg, 94% yield) as a white solid, which was used immediately. To a solution of thiocarbonylimidazolium salt 28 (200 mg, 0.28 mmol) in DMF (5 mL) was added a trifluoroacetate salt 18 (180 mg, 0.52 mmol) and Et3N (0.5 mL). The reaction was stirred at rt for 24 h and diluted with EtOAc (10 mL). The organic layer was washed with 1 N HCl (2 × 5 mL) and brine (10 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 33 (91 mg, 46% yield) as an amorphous white powder; [α]D18 +208 (c 0.52, CHCl3); IR (KBr) 3373, 2925, 1665, 1517, 1449, 1342, 1310, 1226, 1148, 1118, 1023, 1002, 745 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 12.22 (s, 1H, NH), 9.29 (d, J = 7.9 Hz, 1H, ArH), 8.61

reaction was stirred at rt for 24 h and diluted with EtOAc (10 mL). The organic layer was washed with 1 N HCl (2 × 5 mL) and brine (10 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/ H2O) to yield 32 (81 mg, 40% yield) as an amorphous white powder; [α]D18 +165 (c 0.51, CHCl3); IR (KBr) 3367, 2933, 1657, 1514, 1452, 1343, 1311, 1226, 1023, 746 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 12.22 (s, 1H, NH), 9.30 (d, J = 7.9 Hz, 1H, ArH), 8.61 (s, 1H, NH), 8.59 (s, 1H, ArH), 8.17 (d, J = 2.0 Hz, 1H, ArH), 8.05 (t, J = 10.3 Hz, 1H, ArH), 7.99 (t, J = 9.6 Hz, 1H, ArH), 7.95 (brs, 1H, NH), 7.74 (d, J = 8.3 Hz, 1H, ArH), 7.54 (d, J = 8.6 Hz, 1H, ArH), 7.47−7.50 (m, 2H, ArH), 7.36 (t, J = 7.5 Hz, 1H, ArH), 7.31 (t, J = 7.5 Hz, 1H, ArH), 7.26 (dd, J = 8.6, 2.0 Hz, 1H, ArH), 7.09 (t, J = 7.7 Hz, 1H, H-1′), 5.83 (brs, 1H, H-3′), 4.94−5.07 (m, 4H, H-7, H-3″), 4.53 (brs, 1H, H-4′), 2.95 (s, 3H, 3′-NCH3), 2.87 (s, 3H, 4′OCH3), 2.71−2.75 (m, 1H, H-2′a), 2.36 (s, 3H, 6′-CH3), 2.27−2.33 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 190.2, 182.5, 171.9, 139.1, 136.3, 134.9, 134.6, 132.7, 129.1, 126.6, 126.5, 125.7, 125.4, 125.0, 124.9, 123.8, 122.9, 122.7, 121.4, 120.3, 120.3, 119.5, 119.4, 115.3, 115.2, 114.2, 113.9, 113.8, 109.1, 95.0, 82.9, 82.3, 60.4, 54.3, 51.9, 45.5, 32.9, 29.5, 27.7; HRESIMS m/z 715.1885 [M − H]− (calcd for C39H32ClN6O4S, 715.1889). EtOH (200 μL) and (CF3CO)2O (400 μL) were added sequentially to a solution of 32 (60 mg, 0.084 mmol) in CH2Cl2 (6 mL) at 0 °C. After it was stirred for 1 h at 0 °C, the reaction was quenched by adding 6 mL of saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 2 (29 mg, 49% yield) as a white powder; [α]D18 +198 (c 0.52, CHCl3); IR (KBr) 3408, 2924, 1672, 1524, 1457, 1343, 1317, 1284, 1119, 744 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.57 (s, 1H, NH), 9.32 (d, J = 7.8 Hz, 1H, ArH), 8.63 (s, 1H, NH), 8.07 (d, J = 7.8 Hz, 1H, ArH), 8.03 (d, J = 8.5 Hz, 1H, ArH), 7.80 (s, 1H, ArH), 7.66−7.69 (m, 2H, ArH), 7.58 (brs, 1H, ArH), 7.47−7.51 (m, 3H, ArH), 7.36 (t, J = 7.4 Hz, 1H, ArH), 7.32 (t, J = 7.5 Hz, 1H, ArH), 7.18−7.20 (m, 1H, ArH), 7.09 (t, J = 7.4 Hz, 1H, H-1′), 4.97−5.01 (m, 3H, H-7, H-3′), 4.50 (s, 1H, H-4′), 2.91 (s, 3H, 3′-NCH3), 2.85−2.90 (m, 1H, H-2′a), 2.71 (s, 3H, 4′-OCH3), 2.43 (s, 3H, 6′-CH3), 2.39−2.43 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 171.9, 167.7, 138.7, 136.3, 135.0, 134.5, 132.7, 129.2, 125.9, 125.7, 125.4, 125.1, 125.0, 124.6, 124.3, 123.8, 122.7, 121.9, 121.5, 120.3, 119.5(2 × C), 119.4, 118.2, 115.2, 114.1, 113.6(2 × C), 109.0, 106.8, 94.8, 82.5, 82.4, 60.2, 53.2, 45.4, 34.5, 29.2, 27.1; HRESIMS m/z 699.1940 [M + H]+ (calcd for C39H32ClN6O3S, 699.1940). E



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(s, 1H, NH), 8.56 (s, 1H, ArH), 8.31 (s, 1H, ArH), 8.05 (d, J = 7.7 Hz, 1H, ArH), 8.00 (d, J = 8.5 Hz, 1H, ArH), 7.95 (brs, 1H, NH), 7.74 (d, J = 8.3 Hz, 1H, ArH), 7.48−7.50 (m, 3H, ArH), 7.35−7.38 (m, 2H, ArH), 7.30 (t, J = 7.5 Hz, 1H, ArH), 7.08 (t, J = 7.7 Hz, 1H, H-1′), 5.90 (brs, 1H, H-3′), 4.92−5.06 (m, 4H, H-7, H-3″), 4.52 (brs, 1H, H-4′), 2.94 (s, 3H, 3′-NCH3), 2.87 (s, 3H, 4′-OCH3), 2.70−2.74 (m, 1H, H-2′a), 2.35 (s, 3H, 6′-CH3), 2.26−2.32 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 190.3, 182.5, 172.0, 139.1, 136.4, 135.2, 134.5, 132.8, 129.1, 127.3, 125.7, 125.5, 125.4, 125.1, 124.9, 123.8, 123.3, 122.7, 121.5, 120.4, 119.5, 119.4, 115.3, 114.6, 114.4, 114.2, 113.9, 113.7, 109.2, 95.0, 82.9, 82.4, 60.5, 54.3, 51.9, 45.5, 32.9, 29.7, 27.7; HRESIMS m/z 759.1387 [M − H]− (calcd for C39H32BrN6O4S, 759.1384). EtOH (200 μL) and (CF3CO)2O (400 μL) were added sequentially to a solution of 33 (42 mg, 0.055 mmol) in CH2Cl2 (6 mL) at 0 °C. After it was stirred for 1 h at 0 °C, the reaction was quenched by adding 6 mL of saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 3 (28 mg, 69% yield) as a white powder; [α]D18 +192 (c 0.52, CHCl3); IR (KBr) 3417, 2925, 1672, 1524, 1456, 1343, 1317, 1119, 744 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.58 (s, 1H, NH), 9.32 (d, J = 3.0 Hz, 1H, ArH), 8.62 (s, 1H, NH), 8.06 (d, J = 7.4 Hz, 1H, ArH), 8.02 (d, J = 7.9 Hz, 1H, ArH), 7.93 (s, 1H, ArH), 7.65−7.68 (m, 2H, ArH), 7.56 (s, 1H, ArH), 7.46−7.51 (m, 2H, ArH), 7.43 (d, J = 8.5 Hz, 1H, ArH), 7.35 (t, J = 6.3 Hz, 1H, ArH), 7.29−7.32 (m, 2H, ArH), 7.09 (brs, 1H, H1′), 4.98−5.01 (m, 3H, H-7, H-3′), 4.49 (s, 1H, H-4′), 2.91 (s, 3H, 3′-NCH3), 2.85−2.90 (m, 1H, H-2′a), 2.71 (s, 3H, 4′-OCH3), 2.43 (s, 3H, 6′-CH3), 2.38−2.43 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 171.9, 167.8, 138.7, 136.3, 135.2, 134.5, 132.7, 129.2, 126.6, 125.7, 125.4, 125.1, 125.0, 124.5, 124.4, 123.8, 122.7, 121.5, 121.2, 120.3, 119.5, 119.4 (2 × C), 115.2, 114.2, 114.0, 113.5, 112.3, 109.0, 106.6, 94.8, 82.5, 82.4, 60.2, 53.2, 45.4, 34.5, 29.2, 27.1; HRESIMS m/z 743.1432 [M + H]+ (calcd for C39H32BrN6O3S, 743.1434). Synthesis of 5‴-Methoxyfradcarbazole A (4). Et3N (1.5 mL) and Boc2O (1.37 g, 6.32 mmol) were added to a solution of 5methoxytryptamine (1.0 g, 5.26 mmol) in THF (15 mL) at 10 °C. After it was stirred for 1 h at 10 °C, the reaction mixture was poured into ice water (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by FCC eluting with PE−EtOAc (5:1) to provide 19a (1.51 g, 99% yield) as an amorphous white solid; 1H NMR (600 MHz, CDCl3) δ 8.18 (s, 1H, NH), 7.27 (d, J = 8.8 Hz, 1H, ArH), 7.06 (s, 1H, ArH), 7.01 (s, 1H, ArH), 6.89 (dd, J = 8.8, 1.5 Hz, 1H, ArH), 4.61 (brs, 1H, NH), 3.89 (s, 3H, OMe), 3.46−3.50 (m, 2H, CH2), 2.94 (t, J = 6.4 Hz, 2H, CH2), 1.46 (s, 9H, 3 × CH3); 13C NMR (150 MHz, CDCl3) δ 156.0, 154.0, 131.5, 127.7, 122.8, 112.8, 112.3, 111.9, 100.6, 79.1, 55.9, 40.7, 28.4(3 × C), 25.8; ESIMS m/z 313.1 [M + Na]+. Compound 19a (1.3 g, 4.48 mmol) was dissolved in THF/H2O (10:1, 55 mL), and then DDQ (2.0 g, 8.96 mmol) was added at 0 °C. After it was stirred for 2 h at 0 °C, the reaction mixture was poured into EtOAc (100 mL) and washed extensively with saturated aqueous NaHCO3 (4 × 100 mL). The EtOAc layer was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by FCC eluting with PE−EtOAc (1:1) to provide 19b (1.1 g, 55% yield) as a colorless solid; IR (KBr) 3365, 3127, 2978, 1687, 1616, 1530, 1463, 1433, 1367, 1293, 1275, 1253, 1213, 1165, 1095, 1026, 918, 798, 627, 582 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.86 (brs, 1H, NH), 8.33 (d, J = 3.1 Hz, 1H, ArH), 7.67 (d, J = 2.5 Hz, 1H, ArH), 7.37 (d, J = 8.8 Hz, 1H), 6.97 (t, J = 5.9 Hz, 1H, NH), 6.85 (dd, J = 8.8, 2.5 Hz, 1H, ArH), 4.26 (d, J = 5.9 Hz, 2H, CH2), 3.78 (s, 3H, OMe), 1.41 (s, 9H, 3 × CH3); 13C NMR (150 MHz, DMSO-d6) δ 190.7, 156.0, 155.4, 133.4, 131.2, 126.2, 113.8, 112.9, 112.7, 102.9, 77.8, 55.2, 46.7, 28.3(3 × C); HRESIMS m/z 327.1314 [M + Na]+ (calcd for C16H20N2O4Na, 327.1315).

The ketone intermediate 19b (1.1 g, 3.62 mmol) was treated with TFA (10 mL) at 10 °C. After the resultant solution was stirred for 1 h at 10 °C, the solvent was removed in vacuo by coevaporation with benzene to give the keto tryptamine TFA salt 19 (0.9 g, 83% yield) as a white solid, which was used immediately. To a solution of thiocarbonylimidazolium salt 28 (200 mg, 0.28 mmol) in DMF (5 mL) was added a trifluoroacetate salt 19 (169 mg, 0.56 mmol) and Et3N (0.5 mL). The reaction was stirred at rt for 24 h and diluted with EtOAc (10 mL). The organic layer was washed with 1 N HCl (2 × 5 mL) and brine (10 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 34 (96 mg, 48% yield) as an amorphous white powder; [α]D18 +179 (c 0.50, CHCl3); IR (KBr) 3250, 3939, 1665, 1586, 1514, 1461, 1343, 1311, 1211, 1119, 1024, 746 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.93 (d, J = 3.0 Hz, 1H, NH), 9.30 (d, J = 8.0 Hz, 1H, ArH), 8.63 (s, 1H, NH), 8.45 (d, J = 3.0 Hz, 1H, ArH), 8.06 (d, J = 7.8 Hz, 1H, ArH), 8.01 (d, J = 8.5 Hz, 1H, ArH), 7.90 (brs, 1H, NH), 7.74 (d, J = 8.3 Hz, 1H, ArH), 7.70 (d, J = 2.5 Hz, 1H, ArH), 7.46−7.51 (m, 2H, ArH), 7.41 (d, J = 8.8 Hz, 1H, ArH), 7.36 (t, J = 7.4 Hz, 1H, ArH), 7.31 (t, J = 7.5 Hz, 1H, ArH), 7.10 (t, J = 7.6 Hz, 1H, H-1′), 6.87 (dd, J = 8.8, 2.5 Hz, 1H, ArH), 5.94 (brs, 1H, H-3′), 4.94−5.05 (m, 4H, H-7, H-3″), 4.52 (brs, 1H, H-4′), 3.79 (s, 3H, 5‴-OMe), 2.96 (s, 3H, 3′-NCH3), 2.85 (s, 3H, 4′-OCH3), 2.71−2.76 (m, 1H, H-2′a), 2.37 (s, 3H, 6′-CH3), 2.27−2.30 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 190.0, 182.4, 172.0, 155.5, 139.0, 136.4, 133.5, 132.8, 131.3, 129.2, 126.3, 125.7, 125.4, 125.0, 125.0, 123.8, 122.7, 121.5, 120.4, 119.5, 119.4, 115.3, 114.2, 114.0, 113.9, 113.0, 112.7, 109.2, 102.9, 95.0, 83.0, 82.4, 60.4, 55.3, 54.3, 51.9, 45.5, 32.8, 29.5, 27.7; HRESIMS m/z 711.2391 [M − H]− (calcd for C40H35N6O5S, 711.2384). EtOH (200 μL) and (CF3CO)2O (400 μL) were added sequentially to a solution of 34 (38 mg, 0.053 mmol) in CH2Cl2 (6 mL) at 0 °C. After it was stirred for 1 h at 0 °C, the reaction was quenched by adding 6 mL of saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 4 (30 mg, 79% yield) as a white powder; [α]D18 +180 (c 0.51, CHCl3); IR (KBr) 3408, 2925, 1676, 1524, 1456, 1344, 1318, 1119, 744 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.23 (s, 1H, NH), 9.31 (d, J = 8.0 Hz, 1H, ArH), 8.65 (s, 1H, NH), 8.06 (d, J = 7.7 Hz, 1H, ArH), 8.02 (d, J = 8.5 Hz, 1H, ArH), 7.67 (d, J = 8.2 Hz, 1H, ArH), 7.57 (s, 1H, ArH), 7.53 (d, J = 2.0 Hz, 1H, ArH), 7.45−7.51 (m, 2H, ArH), 7.30−7.36 (m, 3H, ArH), 7.26 (brs, 1H, ArH), 7.07−7.10 (m, 1H, H-1′), 6.84 (dd, J = 8.8, 2.0 Hz, 1H, ArH), 5.02 (s, 2H, H-7), 4.97 (d, J = 12.2 Hz, 1H, H3′), 4.50 (brs, 1H, H-4′), 3.84 (s, 3H, 5‴-OMe), 2.91 (s, 3H, 3′NCH3,), 2.84−2.89 (m, 1H, H-2′a), 2.70 (s, 3H, 4′-OMe), 2.44 (s, 3H, 6′-CH3), 2.38−2.42 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 172.0, 167.3, 154.0, 138.8, 136.3, 133.7, 132.7, 131.6, 129.3, 125.9,125.8, 125.4, 125.2, 125.0, 123.8, 123.6, 122.7, 121.5, 120.7, 120.4, 119.6, 119.5, 115.2, 114.2, 113.6, 112.8, 112.1, 109.1, 106.7, 100.8, 94.8, 82.5, 82.4, 60.2, 55.4, 53.3, 45.5, 34.5, 29.3, 27.1; HRESIMS m/z 695.2433 [M + H]+ (calcd for C40H35N6O4S, 695.2435). Synthesis of 3-Chlorofradcarbazole A (5). Et3N (2.0 mL) and Boc2O (1.3 g, 6.0 mmol) were added to a solution of staurosporine (2.33 g, 5.0 mmol) in THF (50 mL) at 10 °C. After it was stirred for 30 min at 0 °C, the reaction mixture was poured into ice water (100 mL) and extracted with CH2Cl2 (3 × 100 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by FCC eluting with CH2Cl2−MeOH (30:1) to provide 20 (2.18 g, 76% yield) as a white solid; [α]D18 +92 (c 0.50, CHCl3); IR (KBr) 3416, 2931, 1687, 1587, 1455, 1345, 1312, 1285, 1225, 1143, 1027, 747 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 9.32 (d, J = 7.9 Hz, 1H, ArH), 8.62 (s, 1H, NH), 7.99−8.08 (m, 2H, ArH), 7.64 (d, J = 8.2 Hz, 1H, ArH), 7.46−7.51 (m, 2H, ArH), 7.29−7.37 (m, 2H, ArH), 7.00 (t, J = 7.6 Hz, 1H, H-1′), 5.01 (s, 2H, H-7), 4.48−4.65 (m, 1H, H-3′), 4.28 (s, 1H, H-4′), 2.72−2.83 (m, 4H, 3′-NCH3, H-2′a), 2.64 F



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was stirred at rt for 24 h. The solvent was removed in vacuo, and the residue was washed with 50 mL of PE-CH2Cl2 (4:1) to provide the salt 29 (300 mg, 68% yield) as a light yellow powder; [α]D18 +258 (c 0.52, CHCl3); IR (KBr) 2919, 1672, 1584, 1499, 1459, 1397, 1343, 1287, 1223, 1169, 1045, 743 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 9.69 (s, 1H, ArH), 9.35 (d, J = 1.8 Hz, 1H, ArH), 8.73 (s, 1H, NH), 8.14 (brs, 1H, ArH), 8.10 (d, J = 7.7 Hz, 1H, ArH), 8.07 (d, J = 8.3 Hz, 1H, ArH), 7.87 (brs, 1H, ArH), 7.69 (d, J = 8.0 Hz, 1H, ArH), 7.52−7.55 (m, 2H, ArH), 7.39 (t, J = 7.5 Hz, 1H, ArH), 7.19 (brs, 1H, H-1′), 5.45 (d, J = 11.3 Hz, 1H, H-3′), 5.02 (s, 2H, H-7), 4.75 (brs, 1H, H-4′), 3.90 (s, 3H, 5″-NCH3), 3.05−3.11 (m, 4H, 3′NCH3, H-2′a), 2.69 (s, 3H, 4′-OMe), 2.43−2.48 (m, 4H, 6′-CH3, H2′b); 13C NMR (150 MHz, DMSO-d6) δ 174.3, 171.7, 138.7, 138.0, 134.6, 133.1, 129.1, 126.0, 125.5, 125.2, 124.7, 123.9, 123.8, 123.7(2 × C), 121.8, 121.2, 120.7, 119.6, 114.8, 114.3, 113.5, 110.6, 94.7, 81.9, 81.2, 60.5, 59.1, 45.6, 38.5, 36.5, 29.2, 26.7; HRESIMS m/z 625.1791 [M-I]+ (calcd for C33H30ClN6O3S, 625.1783). The trifluoroacetate salt 15 was synthesized from tryptamine using our reported method.7 To a solution of thiocarbonylimidazolium salt 29 (280 mg, 0.37 mmol) in DMF (10 mL) was added a trifluoroacetate salt 15 (200 mg, 0.74 mmol) and Et3N (1.0 mL). The reaction was stirred at rt for 24 h and diluted with EtOAc (20 mL). The organic layer was washed with 1 N HCl (2 × 10 mL) and brine (20 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 35 (130 mg, 49% yield) as an amorphous white powder; [α]D18 +169 (c 0.52, CHCl3); IR (KBr) 3244, 2922, 1671, 1519, 1458, 1341, 1287, 1223, 1024, 1003, 746 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 12.03 (s, 1H, NH), 9.33 (d, J = 2.0 Hz, 1H, ArH), 8.70 (s, 1H, NH), 8.50 (d, J = 1.7 Hz, 1H, ArH), 8.18 (d, J = 7.3 Hz, 1H, ArH), 8.07 (d, J = 7.8 Hz, 1H, ArH), 8.02 (d, J = 8.5 Hz, 1H, ArH), 7.92 (brs, 1H, NH), 7.79 (d, J = 8.7 Hz, 1H, ArH), 7.49− 7.53 (m, 3H, ArH), 7.37 (t, J = 7.4 Hz, 1H, ArH), 7.20−7.25 (m, 2H, ArH), 7.10 (t, J = 7.7 Hz, 1H, H-1′), 5.92 (brs, 1H, H-3′), 4.97−5.08 (m, 4H, H-7, H-3″), 4.52 (brs, 1H, H-4′), 2.96 (s, 3H, 3′-NCH3), 2.85 (s, 3H, 4′-OCH3), 2.71−2.76 (m, 1H, H-2′a), 2.36 (s, 3H, 6′CH3), 2.26−2.32 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 190.0, 182.4, 171.8, 139.1, 136.4, 134.7, 133.3, 133.2, 128.9, 125.8, 125.4, 125.3, 125.1, 124.5, 123.8, 123.7(2 × C), 122.9, 121.8, 121.5, 121.2, 120.5, 119.4, 114.7, 114.2, 114.1, 113.9, 112.2, 110.9, 95.1, 82.8, 82.5, 60.4, 54.2, 51.9, 45.6, 32.8, 29.5, 27.6; HRESIMS m/z 715.1898 [M − H]− (calcd for C39H32ClN6O4S, 715.1889). EtOH (200 μL) and (CF3CO)2O (400 μL) were added sequentially to a solution of 35 (61 mg, 0.085 mmol) in CH2Cl2 (6 mL) at 0 °C. After it was stirred for 1 h at 0 °C, the reaction was quenched by adding 6 mL of saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 5 (43 mg, 73% yield) as a white powder; [α]D18 +208 (c 0.52, CHCl3); IR (KBr) 3219, 2922, 1676, 1521, 1457, 1343, 1288, 742 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.36 (s, 1H, NH), 9.35 (s, 1H, ArH), 8.71 (s, 1H, NH), 8.07 (d, J = 7.8 Hz, 1H, ArH), 8.04 (d, J = 8.6 Hz, 1H, ArH), 7.82 (d, J = 7.8 Hz, 1H, ArH), 7.2 (d, J = 7.8 Hz, 1H, ArH), 7.58 (brs, 1H, ArH), 7.55 (brs, 1H, ArH), 7.52 (dd, J = 8.7, 2.1 Hz, 1H, ArH), 7.49 (t, J = 7.8 Hz, 1H, ArH), 7.45 (d, J = 8.1 Hz, 1H, ArH), 7.37 (t, J = 7.4 Hz, 1H, ArH), 7.18 (t, J = 7.5 Hz, 1H, ArH), 7.08−7.13 (m, 2H, ArH, H-1′), 5.02 (s, 2H, H-7), 4.97 (d, J = 12.0 Hz, 1H, H-3′), 4.49 (brs, 1H, H4′), 2.91 (s, 3H, 3′-NCH3), 2.84−2.90 (m, 1H, H-2′a), 2.70 (s, 3H, 4′-OCH3), 2.43 (s, 3H, 6′-CH3), 2.37−2.42 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 171.8, 167.4, 138.8, 136.5, 134.7, 133.9, 133.1, 129.1, 125.9, 125.3, 125.1, 124.8, 124.6, 123.8, 123.7(2 × C), 122.8, 121.8, 121.6, 120.6, 120.5, 119.7, 119.4, 119.2, 114.7, 114.2, 113.6, 112.0, 110.8, 106.9, 94.9, 82.5(2 × C), 60.2, 53.1, 45.6, 34.5, 29.2, 27.0; HRESIMS m/z 699.1938 [M + H]+ (calcd for C39H32ClN6O3S, 699.1940). Synthesis of 3-Chloro-5‴-fluorofradcarbazole A (6). To a solution of thiocarbonylimidazolium salt 29 (300 mg, 0.40 mmol) in DMF (10 mL) was added trifluoroacetate salt 16 (116 mg, 0.40

(s, 3H, 4′-OCH3), 2.34 (s, 3H, 6′-CH3), 2.14−2.21 (m, 1H, H-2′b), 1.57/1.46 (s, 9H, 3 × 4″-CH3); 13C NMR (150 MHz, DMSO-d6) δ 171.9, 154.9/154.2, 139.0/138.8, 136.2, 132.7, 129.1, 125.7, 125.3, 124.9(2 × C), 123.7, 122.6, 121.4, 120.3, 119.5, 119.4, 115.2, 114.1, 113.7, 108.9, 94.6, 84.0/83.3, 82.2, 79.6/79.3, 60.5, 50.5/49.7, 45.4, 30.1, 29.5, 28.1(3 × C), 27.0; ESIMS m/z 589.2[M + Na]+. NCS (745 mg, 5.6 mmol) was added to a solution of 20 (2.0 g, 3.54 mmol) in CH2Cl2−MeOH (1:1, 50 mL) at rt. After it was stirred for 6 h at rt, the reaction mixture was poured into ice water (100 mL) and extracted with CH2Cl2 (3 × 100 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by FCC eluting with CH2Cl2−MeOH (30:1) to provide 21 (1.03 g, 48% yield) as a white solid; [α]D18 +102 (c 0.51, CHCl3); IR (KBr) 2925, 1687, 1458, 1344, 1288, 1142, 778, 743, 726 cm−1; 1H NMR (600 MHz, DMSOd6) δ 9.32 (d, J = 2.0 Hz, 1H, ArH), 8.70 (s, 1H, NH), 8.01−8.10 (m, 2H, ArH), 7.70 (d, J = 8.0 Hz, 1H, ArH), 7.47−7.53 (m, 2H, ArH), 7.35 (t, J = 8.0 Hz, 1H, ArH), 7.01 (t, J = 7.6 Hz, 1H, H-1′), 5.01 (s, 2H, H-7), 4.44−4.62 (m, 1H, H-3′), 4.27 (brs, 1H, H-4′), 2.76 (s, 3H, 3′-NCH3), 2.60−2.68 (m, 4H, 4′-OCH3, H-2′a), 2.33 (s, 3H, 6′CH3), 2.11−2.20 (m, 1H, H-2′b), 1.56/1.45 (m, 9H, 3 × 4″-CH3); 13 C NMR (150 MHz, DMSO-d6) δ 171.8, 154.9/154.2, 138.8, 134.6, 133.2, 128.9, 125.7, 125.2, 125.1, 124.6, 123.8, 123.7, 123.6, 121.6, 120.5, 119.4, 114.6, 114.2, 113.9/113.8, 110.7, 94.7, 83.9/83.2, 82.3, 79.7/79.4, 60.5, 50.4/49.6, 45.6, 30.2, 29.5, 28.1(3 × C), 27.0; HRESIMS m/z 623.2028 [M + Na]+ (calcd for C33H33ClN4O5Na, 623.2032). TFA (10 mL) was added to a solution of 21 (1.03 g, 1.72 mmol) in CH2Cl2 (10 mL) at 0 °C. After the resultant solution was stirred for 1 h at 0 °C, the solvent was removed in vacuo. The residue was treated with saturated aqueous NaHCO3 (50 mL) for 30 min and then extracted with CH2Cl2 (5 × 100 mL). The CH2Cl2 extracts were purified by FCC eluting with CH2Cl2−EtOAc (5:1) to provide 23 (0.8 g, 93% yield) as a white solid; [α]D18 +39 (c 0.48, CHCl3); IR (KBr) 3414, 1667, 1458, 1401, 1352, 1266, 1225, 1103, 1029, 788, 741 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 9.32 (d, J = 2.1 Hz, 1H, ArH), 8.61 (s, 1H, NH), 7.96−7.99 (m, 2H, ArH), 7.64 (d, J = 8.0 Hz, 1H, ArH), 7.46 (dd, J = 8.0, 2.0 Hz, 1H, ArH), 7.42 (t, J = 8.0 Hz, 1H, ArH), 7.28 (t, J = 8.0 Hz, 1H, ArH), 6.69−6.71 (m, 1H, H-1′), 4.96 (s, 2H, H-7), 4.05 (d, J = 3.5 Hz, 1H, H-3′), 3.85−3.87 (m, 1H, H-4′), 3.34 (s, 3H, 4′-OCH3), 3.23−3.26 (m, 1H, H-3′), 2.47−2.52 (m, 1H, H-2′a), 2.28−2.32 (m, 4H, H-2′b, 3′-NCH3), 1.42 (s, 3H, 6′CH3); 13C NMR (150 MHz, DMSO-d6) δ 172.1, 139.5, 134.7, 132.5, 129.9, 127.6, 124.5(2 × C), 124.4, 123.7, 123.5, 123.3, 120.9, 119.8, 118.7, 115.4, 113.9, 113.0, 110.0, 91.1, 82.7, 80.0, 57.2, 49.8, 45.5, 33.2, 29.7, 29.3; HRESIMS m/z 501.1688 [M + H]+ (calcd for C28H26ClN4O3, 501.1688). Et3N (2.0 mL) and N,N′-thiocarbonyldiimidazole (502 mg, 2.82 mmol) were added sequentially to a solution of 23 (470 mg, 0.94 mmol) in CH2Cl2 (20 mL) at rt. After it was stirred overnight, the reaction flask contents were poured into cold water (20 mL) and extracted with CH2Cl2 (3 × 50 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by FCC eluting with CH2Cl2−MeOH (30:1) to provide 26 (410 mg, 71% yield) as a light yellow solid; [α]D18 +278 (c 0.49, CHCl3); IR (KBr) 3413, 1677, 1458, 1398, 1344, 1288, 1224, 1026, 1004, 744 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 9.34 (s, 1H, ArH), 8.71 (s, 1H, NH), 8.11 (s, 1H, ArH), 8.07 (d, J = 7.8 Hz, 1H, ArH), 8.03 (d, J = 8.3 Hz, 1H, ArH), 7.51−7.68 (m, 4H, ArH), 7.38 (t, J = 7.4 Hz, 1H, ArH), 7.14 (brs, 1H, ArH), 7.05 (brs, 1H, H-1′), 5.46 (brs, 1H, H-3′), 5.01 (s, 2H, H7), 4.78 (brs, 1H, H-4′), 3.03−3.07 (m, 4H, 3′-NCH3, H-2′a), 2.70 (s, 3H, 4′-OMe), 2.39−2.46 (m, 4H, 6′-CH3, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 179.4, 171.8, 138.8, 137.7, 134.6, 133.1, 129.1, 129.0, 126.0, 125.5, 125.2, 124.7, 123.9, 123.8, 123.7, 121.7, 120.7, 119.9, 119.6, 114.8, 114.3, 113.5, 110.6, 94.9, 82.0, 81.7, 60.4, 58.2, 45.6, 38.2, 29.4, 27.0; HRESIMS m/z 633.1453 [M + Na]+ (calcd for C32H27ClN6O3SNa, 633.1446). Compound 26 (360 mg, 0.59 mmol) was dissolved in 10 mL of CH3CN and treated with MeI (0.5 mL, 1.39 mmol) at rt. The mixture G



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82.8, 82.5, 60.4, 54.2, 51.9, 45.6, 32.8, 29.5, 27.6; HRESIMS m/z 749.1506 [M − H]− (calcd for C39H31Cl2N6O4S, 749.1499). EtOH (200 μL) and (CF3CO)2O (400 μL) were added sequentially to a solution of 37 (27 mg, 0.036 mmol) in CH2Cl2 (6 mL) at 0 °C. After it was stirred for 1 h at 0 °C, the reaction was quenched by adding 6 mL of saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 7 (14 mg, 53% yield) as a white powder; [α]D18 +139 (c 0.52, CHCl3); IR (KBr) 3417, 2924, 1677, 1524, 1460, 1344, 1288, 1125, 1099, 793, 741 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.70 (s, 1H, NH), 9.35 (s, 1H, ArH), 8.71 (s, 1H, NH), 8.08 (d, J = 7.7 Hz, 1H, ArH), 8.05 (d, J = 8.4 Hz, 1H, ArH), 7.78 (brs, 1H, ArH), 7.73 (d, J = 8.6 Hz, 1H, ArH), 7.66 (brs, 1H, ArH), 7.45−7.56 (m, 4H, ArH), 7.37 (t, J = 7.0 Hz, 1H, ArH), 7.18 (d, J = 8.6 Hz, 1H, ArH), 7.10 (t, J = 7.0 Hz, 1H, H-1′), 5.03 (s, 2H, H-7), 4.98 (d, J = 13.0 Hz, 1H, H-3′), 4.49 (brs, 1H, H-4′), 2.91 (s, 3H, 3′-NCH3), 2.86−2.90 (m, 1H, H-2′a), 2.70 (s, 3H, 4′-OCH3), 2.42 (s, 3H, 6′-CH3), 2.38−2.42 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 171.8, 167.7, 138.8, 135.0, 134.7, 134.4, 133.1, 129.1, 128.5, 125.9(2 × C), 125.3, 125.1, 124.7, 124.6, 124.3, 123.8, 123.7, 123.6, 121.8, 121.6, 120.5, 119.6, 119.4, 118.2, 114.7, 114.2, 113.6(2 × C), 106.7, 94.9, 82.5, 82.4, 60.2, 53.1, 45.6, 34.5, 29.2, 27.0; HRESIMS m/z 755.1368 [M + H]+ (calcd for C39H30 Cl2N6O3SNa, 755.1369). Synthesis of 5‴-Bromo-3-chlorofradcarbazole A (8). To a solution of thiocarbonylimidazolium salt 29 (110 mg, 0.15 mmol) in DMF (10 mL) was added trifluoroacetate salt 18 (52 mg, 0.15 mmol) and Et3N (1.0 mL). The reaction was stirred at rt for 24 h and diluted with EtOAc (20 mL). The organic layer was washed with 1 N HCl (2 × 10 mL) and brine (20 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 38 (45 mg, 38% yield) as an amorphous white powder; [α]D18 +167 (c 0.48, CHCl3); IR (KBr) 3373, 2925, 1677, 1514, 1458, 1343, 1289, 1224, 1149, 1047, 794, 744, 727 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 12.22 (s, 1H, NH), 9.33 (s, 1H, ArH), 8.70 (s, 1H, NH), 8.57 (s, 1H, ArH), 8.31 (s, 1H, ArH), 8.07 (d, J = 8.0 Hz, 1H, ArH), 8.02 (d, J = 8.6 Hz, 1H, ArH), 7.95 (brs, 1H, NH), 7.79 (d, J = 8.6 Hz, 1H, ArH), 7.36− 7.52 (m, 5H, ArH), 7.10 (t, J = 7.5 Hz, 1H, H-1′), 5.90 (brs, 1H, H3′), 4.92−5.05 (m, 4H, H-7, H-3″), 4.52 (brs, 1H, H-4′), 2.95 (s, 3H, 3′-NCH3), 2.86 (s, 3H, 4′-OCH3), 2.71−2.74 (m, 1H, H-2′a), 2.35 (s, 3H, 6′-CH3), 2.26−2.31 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 190.2, 182.5, 171.8, 139.1, 135.1, 134.7, 134.5, 133.2, 128.9, 127.2, 125.7, 125.5, 125.3, 125.1, 124.5, 123.8, 123.7(2 × C), 123.3, 121.5, 120.5, 119.4, 114.7, 114.6, 114.3, 114.2, 114.0, 113.6, 110.9, 95.1, 82.8, 82.5, 60.4, 54.2, 51.9, 45.6, 32.8, 29.5, 27.6; HRESIMS m/z 793.1000 [M − H]− (calcd for C39H31BrClN6O4S, 793.0994). EtOH (200 μL) and (CF3CO)2O (400 μL) were added sequentially to a solution of 38 (30 mg, 0.038 mmol) in CH2Cl2 (6 mL) at 0 °C. After it was stirred for 1 h at 0 °C, the reaction was quenched by adding 6 mL of saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 8 (14 mg, 47% yield) as a white powder; [α]D18 +165 (c 0.51, CHCl3); IR (KBr) 3421, 2924, 1670, 1521, 1458, 1418, 1342, 1288, 669 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.58 (s, 1H, NH), 9.35 (d, J = 1.8 Hz, 1H, ArH), 8.71 (s, 1H, NH), 8.08 (d, J = 7.8 Hz, 1H, ArH), 8.04 (d, J = 8.5 Hz, 1H, ArH), 7.92 (brs, 1H, ArH), 7.73 (d, J = 8.7 Hz, 1H, ArH), 7.65 (d, J = 2.4 Hz, 1H, ArH), 7.56 (brs, 1H, ArH), 7.48−7.54 (m, 2H, ArH), 7.42 (d, J = 8.6 Hz, 1H, ArH), 7.37 (t, J = 7.4 Hz, 1H, ArH), 7.29 (dd, J = 8.6, 1.8 Hz, 1H, ArH), 7.09−7.11 (m, 1H, H-1′), 5.03 (s, 2H, H7), 4.96−4.99 (m, 1H, H-3′), 4.49 (s, 1H, H-4′), 2.91 (s, 3H, 3′NCH3), 2.86−2.90 (m, 1H, H-2′a), 2.70 (s, 3H, 4′-OCH3), 2.43 (s, 3H, 6′-CH3), 2.38−2.42 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 171.8, 167.8, 138.8, 135.2, 134.7, 134.5, 133.1, 129.1,

mmol) and Et3N (1.0 mL). The reaction was stirred at rt for 24 h and diluted with EtOAc (20 mL). The organic layer was washed with 1 N HCl (2 × 10 mL) and brine (20 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 36 (150 mg, 50% yield) as an amorphous white powder; [α]D18 +173 (c 0.51, CHCl3); IR (KBr) 3254, 2916, 1671, 1519, 1459, 1342, 1288, 1224, 1176, 1025, 802, 779, 742 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 12.16 (s, 1H, NH), 9.33 (d, J = 2.1 Hz, 1H, ArH), 8.71 (s, 1H, NH), 8.58 (s, 1H, ArH), 8.07 (d, J = 7.7 Hz, 1H, ArH), 8.02 (d, J = 8.5 Hz, 1H, ArH), 7.95 (brs, 1H, NH), 7.85 (dd, J = 9.8, 2.1 Hz, 1H, ArH), 7.80 (d, J = 8.7 Hz, 1H, ArH), 7.50−7.54 (m, 3H, ArH), 7.38 (t, J = 7.4 Hz, 1H, ArH), 7.09−7.12 (m, 2H, ArH, H-1′), 5.92 (brs, 1H, H-3′), 4.95−5.05 (m, 4H, H-7, H-3″), 4.52 (brs, 1H, H-4′), 2.95 (s, 3H, 3′NCH3), 2.86 (s, 3H, 4′-OCH3), 2.72−2.76 (m, 1H, H-2′a), 2.36 (s, 3H, 6′-CH3), 2.26−2.32 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 190.1, 182.5, 171.8, 158.6 (d, 1JC−F = 234.1 Hz), 139.1, 134.8 (2 × C), 133.2, 133.0, 128.9, 126.0 (d, 3JC−F = 10.0 Hz), 125.8, 125.3, 125.1, 124.5, 123.8, 123.7(2 × C), 121.6, 120.5, 119.4, 114.7, 114.3 (d, 4JC−F = 4.0 Hz), 114.3, 114.0, 113.5 (d, 3JC−F = 10.0 Hz), 111.1 (d, 2JC−F = 23.8 Hz), 110.9, 106.0 (d, 2JC−F = 23.8 Hz), 95.1, 82.8, 82.5, 60.4, 54.3, 51.9, 45.6, 32.8, 29.5, 27.6; HRESIMS m/z 733.1803 [M − H]− (calcd for C39H31ClFN6O4S, 733.1795). EtOH (200 μL) and (CF3CO)2O (400 μL) were added sequentially to a solution of 36 (50 mg, 0.068 mmol) in CH2Cl2 (6 mL) at 0 °C. After it was stirred for 1 h at 0 °C, the reaction was quenched by adding 6 mL of saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 6 (30 mg, 62% yield) as a white powder; [α]D18 +141 (c 0.51, CHCl3); IR (KBr) 3413, 3174, 2908, 1661, 1525, 1463, 1347, 1288, 1025, 787 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.47 (s, 1H, NH), 9.34 (s, 1H, ArH), 8.71 (s, 1H, NH), 8.02−8.08 (m, 2H, ArH), 7.72 (t, J = 7.9 Hz, 1H, ArH), 7.65 (brs, 1H, ArH), 7.43−7.56 (m, 5H, ArH), 7.35−7.38 (m, 1H, ArH), 7.07−7.10 (m, 1H, ArH), 7.03 (t, J = 9.0 Hz, 1H, H-1′), 5.02 (s, 2H, H-7), 4.96 (d, J = 12.7 Hz, 1H, H-3′), 4.49 (brs, 1H, H-4′), 2.90 (s, 3H, 3′-NCH3), 2.85−2.89 (m, 1H, H-2a), 2.70 (s, 3H, 4′-OCH3), 2.43 (s, 3H, 6′-CH3), 2.37−2.41 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 171.8, 167.6, 157.4 (d, 1JC−F = 232.8 Hz), 138.8, 134.7, 134.1, 133.2, 133.1, 129.1, 126.0, 125.3, 125.2, 124.9, 124.9 (d, 3 JC−F = 9.7 Hz), 124.6, 123.8 (2 × C), 123.7, 121.6, 120.6, 120.0, 119.4, 114.7, 114.2, 113.7, 113.1 (d, 3JC−F = 9.7 Hz), 110.8, 110.1(d, 2 JC−F = 26.2 Hz), 107.2 (d, 4JC−F = 4.4 Hz), 104.0 (d, 2JC−F = 26.2 Hz), 94.9, 82.5, 82.5, 60.2, 53.2, 45.6, 34.5, 29.2, 27.0; HRESIMS m/z 717.1843 [M + H]+ (calcd for C39H31ClFN6O3S, 717.1845). Synthesis of 3,5‴-Dichlorofradcarbazole A (7). To a solution of thiocarbonylimidazolium salt 29 (110 mg, 0.15 mmol) in DMF (10 mL) was added trifluoroacetate salt 17 (46 mg, 0.15 mmol) and Et3N (1.0 mL). The reaction was stirred at rt for 24 h and diluted with EtOAc (20 mL). The organic layer was washed with 1 N HCl (2 × 10 mL) and brine (20 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 37 (50 mg, 45% yield) as an amorphous white powder; [α]D18 +158 (c 0.32, CHCl3); IR (KBr) 3374, 2925, 1677, 1514, 1458, 1343, 1289, 1224, 1149, 802, 781, 744 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 9.32 (d, J = 2.0 Hz, 1H, ArH), 8.71 (s, 1H, NH), 8.58 (s, 1H, ArH), 8.16 (d, J = 2.0 Hz, 1H, ArH), 8.07 (d, J = 7.8 Hz, 1H, ArH), 8.02 (d, J = 8.5 Hz, 1H, ArH), 7.96 (brs, 1H, NH), 7.79 (d, J = 8.7 Hz, 1H, ArH), 7.49−7.55 (m, 3H, ArH),7.37 (t, J = 7.4 Hz, 1H, ArH), 7.26 (dd, J = 8.6, 2.1 Hz, 1H, ArH), 7.10 (t, J = 7.7 Hz, 1H, H-1′), 5.91 (brs, 1H, H-3′), 4.94−5.07 (m, 4H, H-7, H-3″), 4.52 (brs, 1H, H-4′), 2.95 (s, 3H, 3′-NCH3), 2.85 (s, 3H, 4′-OCH3), 2.72−2.76 (m, 1H, H-2′a), 2.36 (s, 3H, 6′CH3), 2.26−2.32 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 190.2, 182.5, 171.8, 139.1, 135.0, 134.7, 133.2, 128.9, 126.6, 126.5, 125.7, 125.3, 125.1, 124.5, 123.8, 123.7(2 × C), 122.9, 121.7, 121.5, 120.5, 120.3, 119.4, 114.7, 114.2, 114.0, 113.9, 113.8, 110.9, 95.1, H



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126.6, 125.9, 125.3, 125.1, 124.5(2 × C), 124.4, 123.8, 123.7, 123.6, 121.6, 121.2, 120.5, 119.5, 119.4, 114.7, 114.2, 114.0, 113.6, 112.2, 110.8, 106.6, 94.9, 82.5, 82.4, 60.2, 53.1, 45.6, 34.5, 29.2, 27.0; HRESIMS m/z 777.1042 [M + H]+ (calcd for C39H31BrClN6O3S, 777.1045). Synthesis of 3-Chloro-5‴-methoxyfradcarbazole A (9). To a solution of thiocarbonylimidazolium salt 29 (280 mg, 0.37 mmol) in DMF (10 mL) was added trifluoroacetate salt 19 (111 mg, 0.37 mmol) and Et3N (1.0 mL). The reaction was stirred at rt for 24 h and diluted with EtOAc (20 mL). The organic layer was washed with 1 N HCl (2 × 10 mL) and brine (20 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 39 (119 mg, 43% yield) as an amorphous white powder; [α]D18 +188 (c 0.51, CHCl3); IR (KBr) 3250, 2925, 1671, 1516, 1450, 1342, 1288, 1212, 1025, 1004, 777, 746, 667 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.92 (s, 1H, NH), 9.33 (s, 1H, ArH), 8.70 (s, 1H, NH), 8.43 (s, 1H, ArH), 8.07 (d, J = 7.8 Hz, 1H, ArH), 8.02 (d, J = 8.5 Hz, 1H, ArH), 7.90 (brs, 1H, NH), 7.79 (d, J = 8.7 Hz, 1H, ArH), 7.69 (s, 1H, ArH), 7.48−7.52 (m, 2H, ArH), 7.36−7.40 (m, 2H, ArH), 7.10 (t, J = 7.6 Hz, 1H, H-1′), 6.87 (dd, J = 8.7, 2.4 Hz, 1H, ArH), 5.93 (brs, 1H, H3′), 4.95−5.03 (m, 4H, H-7, H-3″), 4.50 (brs, 1H, H-4′), 3.78 (s, 3H, 5‴-OCH3), 2.95 (s, 3H, 3′-NCH3), 2.83 (s, 3H, 4′-OCH3), 2.71− 2.75 (m, 1H, H-2′a), 2.36 (s, 3H, 6′-CH3), 2.26−2.32 (m, 1H, H2′b); 13C NMR (150 MHz, DMSO-d6) δ 189.9, 182.4, 171.8, 155.5, 139.1, 134.8, 133.5, 133.2, 131.3, 129.0, 126.3, 125.8, 125.3, 125.1, 124.5, 123.8, 123.7(2 × C), 121.6, 120.5, 119.4, 114.7, 114.3, 114.0(2 × C), 113.0, 112.7, 110.9, 102.9, 95.1, 82.9, 82.5, 60.4, 55.3, 54.2, 51.8, 45.6, 32.7, 29.4, 27.6; HRESIMS m/z 745.2003 [M − H]− (calcd for C40H34ClN6O5S, 745.1994). EtOH (200 μL) and (CF3CO)2O (400 μL) were added sequentially to a solution of 39 (35 mg, 0.047 mmol) in CH2Cl2 (6 mL) at 0 °C. After it was stirred for 1 h at 0 °C, the reaction was quenched by adding 6 mL of saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 9 (16 mg, 47% yield) as a white powder; [α]D18 +180 (c 0.49, CHCl3); IR (KBr) 3410, 2933, 1676, 1522, 1450, 1344, 1288, 1224, 787 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.20 (s, 1H, NH), 9.34 (s, 1H, ArH), 8.71 (s, 1H, NH), 8.08 (d, J = 7.8 Hz, 1H, ArH), 8.05 (d, J = 8.6 Hz, 1H, ArH), 7.72 (dd, J = 8.7, 2.0 Hz, 1H, ArH), 7.55 (s, 1H, ArH), 7.48−7.53(m, 3H, ArH), 7.3−7.38 (m, 2H, ArH), 7.24 (brs, 1H, ArH), 7.08−7.11 (m, 1H, H-1′), 6.83 (dd, J = 8.8, 2.0 Hz, 1H, ArH), 5.02 (s, 2H, H-7), 4.97 (d, J = 12.0 Hz, 1H, H-3′), 4.50 (s, 1H, H-4′), 3.83 (s, 3H,5‴OCH3), 2.90 (s, 3H, 3′-NCH3), 2.86−2.89 (m, 1H, H-2′a), 2.67 (s, 3H, 4′-OCH3), 2.43 (s, 3H, 6′-CH3), 2.38−2.42 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 171.8, 167.3, 153.9, 138.8, 134.7, 133.7, 133.1, 131.6, 129.1, 125.9, 125.3, 125.1(2 × C), 124.6, 123.8, 123.7, 123.6, 123.5, 121.6, 120.7, 120.5, 119.4, 114.6, 114.2, 113.6, 112.7, 112.0, 110.8, 106.7, 100.8, 94.9, 82.5(2 × C), 60.2, 55.4, 53.1, 45.6, 34.5, 29.2, 27.0; HRESIMS m/z 729.2040 [M + H]+ (calcd for C40H34ClN6O4S, 729.2045). Synthesis of 3-Bromofradcarbazole A (10). NBS (347 mg, 1.95 mmol) was added to a solution of 20 (1.0 g, 1.77 mmol) in CH2Cl2− MeOH (1:1, 30 mL) at 0 °C. After it was stirred for 30 min at 0 °C, the reaction mixture was poured into ice water (100 mL) and extracted with CH2Cl2 (3 × 100 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by FCC eluting with PE−EtOAc (1:1) to provide 22 (1.07 g, 94% yield) as a white solid; [α]D18 +93 (c 0.52, CHCl3); IR (KBr) 2928, 1687, 1457, 1344, 1288, 1222, 1144, 743 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 9.48 (s, 1H, ArH), 8.71 (s, 1H, NH), 8.00−8.10 (m, 2H, ArH), 7.60−7.66 (m, 2H, ArH), 7.50 (brs, 1H, ArH), 7.36 (t, J = 7.4 Hz, 1H, ArH), 7.01 (t, J = 7.5 Hz, 1H, H-1′), 5.01 (s, 2H, H-7), 4.44−4.63 (m, 1H, H-3′), 4.28 (brs, 1H, H-4′), 2.76 (s, 3H, 3′-NCH3), 2.60−2.67 (m, 4H, 4′-OCH3, H-2′a), 2.33 (s, 3H, 6′-CH3), 2.11−2.20 (m, 1H, H2′b), 1.56/1.46 (m, 9H, 3 × 4″-CH3); 13C NMR (150 MHz, DMSO-

d6) δ 171.8, 154.8/154.2, 138.8, 134.9, 133.2, 128.8, 127.6, 127.5, 125.5, 125.2, 124.3, 123.6, 121.6, 120.4, 119.4, 114.6, 114.0, 113.8/ 113.7, 111.6, 111.1, 94.7, 83.9/83.2, 82.3, 79.7/79.3, 60.5, 50.4/49.6, 45.6, 30.1, 29.5, 28.1(3 × C), 26.9; HRESIMS m/z 667.1520 [M + Na]+ (calcd for C33H33BrN4O5Na, 667.1527). TFA (10 mL) was added to a solution of 22 (1.07 g, 1.66 mmol) in CH2Cl2 (10 mL) at 0 °C. After the resultant solution was stirred for 1 h at 0 °C, the solvent was removed in vacuo. The residue was treated with saturated aqueous NaHCO3 (50 mL) for 30 min and then extracted with CH2Cl2 (5 × 100 mL). The CH2Cl2 extracts were purified by FCC eluting with CH2Cl2−EtOAc (5:1) to provide 24 (0.45 g, 50% yield) as a white solid; [α]D18 +49 (c 0.49, CHCl3); IR (KBr) 3418, 1677, 1456, 1400, 1350, 1285, 1224, 1134, 1105, 740 cm−1; 1H NMR (600 MHz, CDCl3) δ 9.56 (d, J = 2.0 Hz, 1H, ArH), 7.92 (d, J = 8.0 Hz, 1H, ArH), 7.89 (d, J = 8.0 Hz, 1H, ArH), 7.54 (d, J = 8.0, 2.0 Hz, 1H, ArH), 7.41−7.44 (m, 1H, ArH), 7.31−7.34 (m, 1H, ArH), 7.16 (d, J = 8.0 Hz, 1H, ArH), 6.49−6.51 (m, 1H, H-1′), 6.45 (s, 1H, NH), 5.01 (s, 2H, H-7), 3.85−3.87 (m, 1H, H-4′), 3.42 (s, 3H, 4′-OCH3), 3.33−3.35 (m, 1H, H-3′), 2.69−2.72 (m, 1H, H2′a), 2.34−2.39 (m, 4H, H-2′b, 3′-NCH3), 1.50 (s, 3H, 6′-CH3); 13C NMR (150 MHz, CDCl3) δ 173.3, 139.8, 135.1, 132.5, 130.6, 128.9, 127.7, 127.7, 124.0, 124.4, 124.3, 120.6, 120.1, 118.4, 115.3, 114.4, 114.3, 112.6, 108.3, 91.0, 84.1, 80.1, 57.2, 50.1, 45.9, 33.3, 30.1, 29.7; ESIMS m/z 567.1[M + Na]+. Et3N (3.0 mL) and N,N′-thiocarbonyldiimidazole (587 mg, 3.3 mmol) were added sequentially to a solution of 24 (600 mg, 1.1 mmol) in CH2Cl2 (30 mL) at rt. After it was stirred overnight, the reaction flask contents were poured into cold water (30 mL) and extracted with CH2Cl2 (3 × 60 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by FCC eluting with CH2Cl2−MeOH (30:1) to provide 27 (631 mg, 88% yield) as a light yellow solid; [α]D18 +235 (c 0.51, CHCl3); IR (KBr) 3385, 1682, 1457, 1397, 1343, 1288, 1222, 1076, 1045, 744 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 9.45 (s, 1H, ArH), 8.71 (s, 1H, NH), 8.12 (brs, 1H, ArH), 8.08 (d, J = 7.8 Hz, 1H, ArH), 8.04 (d, J = 7.8 Hz, 1H, ArH), 7.64 (brs, 1H, ArH), 7.62−7.66 (m, 2H, ArH), 7.53 (t, J = 7.8 Hz, 1H, ArH), 7.38 (t, J = 7.8 Hz, 1H, ArH), 7.15 (brs, 1H, ArH), 7.05 (brs, 1H, H-1′), 5.46 (brs, 1H, H-3′), 5.03 (s, 2H, H-7), 4.78 (brs, 1H, H-4′), 3.00−3.08 (m, 4H, 3′-NCH3, H-2′a), 2.76 (s, 3H, 4′OMe), 2.38−2.46 (m, 4H, 6′-CH3, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 179.4, 171.7, 138.9, 137.7, 134.9, 133.2, 129.1, 129.0, 127.7, 127.6, 125.8, 125.5, 124.4, 123.7, 121.7, 120.7, 119.9, 119.6, 114.8, 114.2, 113.6, 111.8, 111.1, 94.9, 82.0, 81.7, 60.4, 58.1, 45.6, 38.2, 29.4, 26.9; HRESIMS m/z 677.0934 [M + Na]+ (calcd for C32H27BrN6O3SNa, 677.0941). Compound 27 (583 mg, 0.89 mmol) was dissolved in 45 mL of CH3CN and treated with MeI (1.0 mL) at rt. The mixture was stirred at rt for 24 h. The solvent was removed in vacuo, and the residue was washed with 50 mL of PE−CH2Cl2 (4:1) to provide the salt 30 (521 mg, 74% yield) as a light yellow powder; [α]D18 +269 (c 0.49, CHCl3); IR (KBr) 2920, 1672, 1584, 1493, 1454, 1397, 1342, 1287, 1222, 1167, 1071, 1044, 743 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 9.69 (s, 1H, ArH), 9.50 (s, 1H, ArH), 8.71 (s, 1H, NH), 8.14 (brs, 1H, ArH), 8.09 (d, J = 7.7 Hz, 1H, ArH), 8.06 (d, J = 8.4 Hz, 1H, ArH), 7.86 (brs, 1H, ArH), 7.63−7.67 (m, 2H, ArH), 7.54 (t, J = 7.6 Hz, 1H, ArH), 7.39 (t, J = 7.6 Hz, 1H, ArH), 7.17−7.20 (m, 1H, H1′), 5.39 (d, J = 12.4 Hz, 1H, H-3′), 5.02 (s, 2H, H-7), 4.75 (brs, 1H, H-4′), 3.92 (s, 3H, 5″-NCH3), 3.06−3.11 (m, 4H, 3′-NCH3, H-2′a), 2.71 (s, 3H, 4′-OMe), 2.43−2.47 (m, 4H, 6′-CH3, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 174.3, 171.7, 138.7, 138.0, 134.9, 133.1, 129.1, 127.7, 127.6, 125.8, 125.5, 124.4, 123.7, 123.1, 121.8, 121.2, 120.8, 119.6, 114.9, 114.2, 113.5, 111.8, 111.1, 94.7, 81.9, 81.2, 60.5, 59.1, 45.6, 38.5, 36.5, 29.2, 26.7; HRESIMS m/z 669.1288 [M − I]+ (calcd for C33H30BrN6O3S, 669.1278). To a solution of thiocarbonylimidazolium salt 30 (110 mg, 0.14 mmol) in DMF (10 mL) was added trifluoroacetate salt 15 (38 mg, 0.14 mmol) and Et3N (1.0 mL). The reaction was stirred at rt for 24 h and diluted with EtOAc (20 mL). The organic layer was washed with 1 N HCl (2 × 10 mL) and brine (20 mL), dried over anhydrous I



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EtOH (200 μL) and (CF3CO)2O (400 μL) were added sequentially to a solution of 41 (50 mg, 0.064 mmol) in CH2Cl2 (6 mL) at 0 °C. After it was stirred for 1 h at 0 °C, the reaction was quenched by adding 6 mL of saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 11 (24 mg, 50% yield) as a white powder; [α]D18 +139 (c 0.49, CHCl3); IR (KBr) 3415, 2922, 1666, 1525, 1460, 1344, 1288, 1223, 1104, 1024, 785, 744 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.49 (s, 1H, NH), 9.49 (s, 1H, ArH), 8.71 (s, 1H, NH), 8.07 (d, J = 7.8 Hz, 1H, ArH), 8.03 (d, J = 8.5 Hz, 1H, ArH), 7.62−7.68 (m, 3H, ArH), 7.53−7.56 (m, 2H, ArH), 7.48 (t, J = 7.8 Hz, 1H, ArH), 7.44−7.46 (m, 1H, ArH), 7.36 (t, J = 7.4 Hz, 1H, ArH), 7.01−7.10 (m, 2H, ArH, H-1′), 5.02 (s, 2H, H-7), 4.96 (d, J = 12.0 Hz, 1H, H-3′), 4.48 (brs, 1H, H-4′), 2.88 (s, 3H, 3′-NCH3), 2.84−2.88 (m, 1H, H-2′a), 2.69 (s, 3H, 4′-OCH3), 2.43 (s, 3H, 6′CH3), 2.37−2.41 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 171.8, 167.5, 157.4 (d, 1JC−F = 231.8 Hz), 138.8, 135.0, 134.1, 133.2, 133.1, 129.0, 127.7, 127.6, 125.8, 125.3, 124.9 (d, 3JC−F = 9.8 Hz), 124.9, 124.3, 123.6, 121.6, 120.5, 120.0, 119.4, 114.7, 114.1, 113.6, 113.1 (d, 3JC−F = 9.8 Hz), 111.7, 111.2, 110.1(d, 2JC−F = 25.4 Hz), 107.2 (d, 4JC−F = 4.5 Hz), 103.9 (d, 2JC−F = 25.4 Hz), 94.9, 82.5, 82.4, 60.2, 53.1, 45.6, 34.5, 29.2, 27.0; HRESIMS m/z 761.1337 [M + H]+ (calcd for C39H31BrFN6O3S, 761.1340). Synthesis of 3-Bromo-5‴-chlorofradcarbazole A (12). To a solution of thiocarbonylimidazolium salt 30 (200 mg, 0.25 mmol) in DMF (10 mL) was added trifluoroacetate salt 17 (76 mg, 0.25 mmol) and Et3N (1.0 mL). The reaction was stirred at rt for 24 h and diluted with EtOAc (20 mL). The organic layer was washed with 1 N HCl (2 × 10 mL) and brine (20 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 42 (90 mg, 44% yield) as an amorphous white powder; [α]D18 +165 (c 0.51, CHCl3); IR (KBr) 3374, 2922, 1677, 1514, 1455, 1343, 1288, 1223, 1150, 794, 775, 744 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 12.21 (s, 1H, NH), 9.47 (d, J = 2.0 Hz, 1H, ArH), 8.70 (s, 1H, NH), 8.58 (s, 1H, ArH), 8.16 (d, J = 2.0 Hz, 1H, ArH), 8.07 (d, J = 7.8 Hz, 1H, ArH), 8.02 (d, J = 8.5 Hz, 1H, ArH), 7.95 (brs, 1H, NH), 7.75 (d, J = 8.7 Hz, 1H, ArH), 7.63 (dd, J = 8.6, 2.0 Hz, 1H, ArH), 7.54 (d, J = 8.6 Hz, 1H, ArH), 7.51 (t, J = 7.7 Hz, 1H, ArH), 7.37 (t, J = 7.4 Hz, 1H, ArH), 7.26 (dd, J = 8.6, 2.1 Hz, 1H, ArH), 7.09 (t, J = 7.7 Hz, 1H, H-1′), 5.90 (brs, 1H, H-3′), 4.93−5.05 (m, 4H, H-7, H-3″), 4.52 (s, 1H, H-4′), 2.95 (s, 3H, 3′-NCH3), 2.86 (s, 3H, 4′-OCH3), 2.71−2.75 (m, 1H, H-2′a), 2.35 (s, 3H, 6′-CH3), 2.26−2.32 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 190.2, 182.5, 171.8, 139.1, 135.0, 134.9, 134.7, 133.2, 128.9, 127.7, 127.5, 126.6, 126.5, 125.6, 125.3, 124.3, 123.7, 122.9, 121.6, 120.5, 120.3, 119.4, 114.7, 114.1, 114.0, 113.9, 113.8, 111.6, 111.3, 95.1, 82.8, 82.5, 60.4, 54.2, 51.9, 45.6, 32.8, 29.5, 27.5; HRESIMS m/z 793.0995 [M − H]− (calcd for C39H31BrClN6O4S, 793.0994). EtOH (200 μL) and (CF3CO)2O (400 μL) were added sequentially to a solution of 42 (38 mg, 0.048 mmol) in CH2Cl2 (6 mL) at 0 °C. After it was stirred for 1 h at 0 °C, the reaction was quenched by adding 6 mL of saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 12 (18 mg, 48% yield) as a white powder; [α]D18 +163 (c 0.49, CHCl3); IR (KBr) 3422, 2925, 1677, 1524, 1460, 1344, 1288, 1223, 1103, 787, 741 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.58 (s, 1H, NH), 9.50 (s, 1H, ArH), 8.71 (s, 1H, NH), 8.06 (t, J = 7.5 Hz, 1H, ArH), 8.03 (d, J = 8.4 Hz, 1H, ArH), 7.79 (brs, 1H, ArH), 7.62−7.68 (m, 3H, ArH), 7.56 (s, 1H, ArH), 7.46−7.50 (m, 2H, ArH), 7.36 (t, J = 7.5 Hz, 1H, ArH), 7.18 (dd, J = 8.4, 1.9 Hz, 1H, ArH), 7.09 (brs, 1H, H-1′), 5.02 (s, 2H, H7), 4.95−4.99 (m, 1H, H-3′), 4.48 (s, 1H, H-4′), 2.90 (s, 3H, 3′NCH3), 2.85−2.89 (m, 1H, H-2′a), 2.69 (s, 3H, 4′-OCH3), 2.43 (s, 3H, 6′-CH3), 2.37−2.42 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 171.8, 167.7, 138.8, 134.9(2 × C), 134.5, 133.1, 129.0,

Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 40 (52 mg, 49% yield) as an amorphous white powder; [α]D18 +200 (c 0.52, CHCl3); IR (KBr) 3373, 2937, 1676, 1514, 1458, 1342, 1288, 1223, 1126, 745 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 12.03 (d, J = 2.4 Hz, 1H, NH), 9.48 (d, J = 2.0 Hz, 1H, ArH), 8.70 (s, 1H, NH), 8.50 (d, J = 3.0 Hz, 1H, ArH), 8.18 (d, J = 7.5 Hz, 1H, ArH), 8.07 (d, J = 7.8 Hz, 1H, ArH), 8.02 (d, J = 8.5 Hz, 1H, ArH), 7.92 (brs, 1H, NH), 7.75 (d, J = 8.7 Hz, 1H, ArH), 7.63 (dd, J = 8.6, 2.0 Hz, 1H, ArH), 7.49− 7.52 (m, 2H, ArH), 7.37 (t, J = 7.4 Hz, 1H, ArH), 7.19−7.25 (m, 2H, ArH), 7.09 (t, J = 8.0 Hz, 1H, H-1′), 5.92 (brs, 1H, H-3′), 4.97−5.07 (m, 4H, H-7, H-3′), 4.52 (brs, 1H, H-4′), 2.95 (s, 3H, 3′-NCH3), 2.85 (s, 3H, 4′-OCH3), 2.71−2.75 (m, 1H, H-2′a), 2.36 (s, 3H, 6′CH3), 2.26−2.31 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 190.0, 182.4, 171.8, 139.1, 136.4, 135.0, 133.3, 133.2, 128.9, 127.7, 127.5, 125.6, 125.4, 125.2, 124.3, 123.6, 122.9, 121.8, 121.6, 121.2, 120.5, 119.4, 114.7, 114.1(2 × C), 114.0, 112.2, 111.6, 111.3, 95.1, 82.8, 82.5, 60.4, 54.2, 51.9, 45.6, 32.6, 29.5, 27.5; HRESIMS m/z 759.1392 [M − H]− (calcd for C39H32BrN6O4S, 759.1384). EtOH (200 μL) and (CF3CO)2O (400 μL) were added sequentially to a solution of 40 (36 mg, 0.047 mmol) in CH2Cl2 (6 mL) at 0 °C. After it was stirred for 1 h at 0 °C, the reaction was quenched by adding 6 mL of saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 10 (19 mg, 55% yield) as a white powder; [α]D18 +178 (c 0.49, CHCl3); IR (KBr) 3412, 2922, 1676, 1525, 1458, 1344, 1288, 1125, 744 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.38 (s, 1H, NH), 9.50 (s, 1H, ArH), 8.71 (s, 1H, NH), 8.07 (d, J = 7.6 Hz, 1H, ArH), 8.04 (d, J = 8.3 Hz, 1H, ArH), 7.82 (d, J = 7.7 Hz, 1H, ArH), 7.62−7.69 (m, 2H, ArH), 7.58 (s, 1H, ArH), 7.55 (s, 1H, ArH), 7.49 (t, J = 7.7 Hz, 1H, ArH), 7.45 (d, J = 8.0 Hz, 1H, ArH), 7.37 (t, J = 7.2 Hz, 1H, ArH), 7.18 (t, J = 7.4 Hz, 1H, ArH), 7.08−7.13 (m, 2H, ArH, H-1′), 5.03 (s, 2H, H-7), 4.97 (d, J = 12.8 Hz, 1H, H-3′), 4.49 (s, 1H, H-4′), 2.91 (s, 3H, 3′-NCH3), 2.85−2.89 (m, 1H, H-2′a), 2.70 (s, 3H, 4′-OCH3), 2.43 (s, 3H, 6′CH3), 2.37−2.42 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 171.8, 167.4, 138.8, 136.5, 135.0, 133.9, 133.1, 129.0, 127.7, 127.6, 125.8, 125.3, 124.8, 124.3, 123.6, 122.8, 121.8, 121.6, 120.6, 120.5, 119.6, 119.4, 119.1, 114.7, 114.0, 113.6, 112.0, 111.6, 111.2, 106.9, 94.9, 82.5, 82.5, 60.2, 53.1, 45.6, 34.5, 29.2, 27.0; HRESIMS m/z 743.1431 [M + H]+ (calcd for C39H32BrN6O3S, 743.1434). Synthesis of 3-Bromo-5‴-fluorofradcarbazole A (11). To a solution of thiocarbonylimidazolium salt 30 (228 mg, 0.29 mmol) in DMF (10 mL) was added trifluoroacetate salt 16 (84 mg, 0.29 mmol) and Et3N (1.0 mL). The reaction was stirred at rt for 24 h and diluted with EtOAc (20 mL). The organic layer was washed with 1 N HCl (2 × 10 mL) and brine (20 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 41 (101 mg, 45% yield) as an amorphous white powder; [α]D18 +180 (c 0.49, CHCl3); IR (KBr) 3250, 2912, 1671, 1519, 1458, 1342, 1288, 1223, 1176, 1025, 1004, 775, 742 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 12.16 (s, 1H, NH), 9.48 (s, 1H, ArH), 8.70 (s, 1H, NH), 8.57 (s, 1H, ArH), 8.07 (d, J = 7.8 Hz, 1H, ArH), 8.01 (d, J = 8.5 Hz, 1H, ArH), 7.94 (s, 1H, NH), 7.85 (d, J = 9.8 Hz, 1H, ArH), 7.74 (d, J = 8.7 Hz, 1H, ArH), 7.62 (dd, J = 8.6, 2.0 Hz, 1H, ArH), 7.48−7.54 (m, 2H, ArH), 7.37 (t, J = 7.4 Hz, 1H, ArH), 7.07−7.11 (m, 2H, ArH, H-1′), 5.91 (brs, 1H, H-3′), 4.95−5.05 (m, 4H, H-7, H-3″), 4.51 (brs, 1H, H-4′), 2.95 (s, 3H, 3′-NCH3), 2.84 (s, 3H, 4′-OCH3), 2.71−2.75 (m, 1H, H2′a), 2.36 (s, 3H, 6′-CH3), 2.26−2.33 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 190.2, 182.5, 171.8, 158.6 (d, 1JC−F = 234.8 Hz), 139.1, 135.0, 134.9, 133.3, 133.1, 128.9, 127.7, 127.6, 126.1 (d, 3 JC−F = 10.8 Hz), 125.6, 125.3, 124.4, 123.7, 121.6, 120.5, 119.4, 114.8, 114.3 (d, 4JC−F = 4.2 Hz), 114.2, 114.0, 113.5 (d, 3JC−F = 10.8 Hz), 111.7, 111.4, 111.1 (d, 2JC−F = 24.0 Hz), 106.0 (d, 2JC−F = 24.0 Hz), 95.1, 82.9, 82.5, 60.4, 54.3, 51.9, 45.7, 32.8, 29.5, 27.6; HRESIMS m/z 777.1296 [M − H]− (calcd for C39H31BrFN6O4S, 777.1289). J



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127.7, 127.6, 125.9, 125.8, 125.3, 124.7, 124.3(2 × C), 123.6, 121.9, 121.6, 120.5, 119.6, 119.4, 118.2, 114.7, 114.0, 113.6(2 × C), 111.7, 111.2, 106.8, 94.9, 82.5, 82.4, 60.2, 53.1, 45.6, 34.5, 29.2, 27.0; HRESIMS m/z 777.1044[M + H]+ (calcd for C39H31BrClN6O3S, 777.1045). Synthesis of 3,5‴-Dibromofradcarbazole A (13). To a solution of thiocarbonylimidazolium salt 30 (100 mg, 0.13 mmol) in DMF (10 mL) was added trifluoroacetate salt 18 (45 mg, 0.13 mmol) and Et3N (1.0 mL). The reaction was stirred at rt for 24 h and diluted with EtOAc (20 mL). The organic layer was washed with 1 N HCl (2 × 10 mL) and brine (20 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 43 (45 mg, 42% yield) as an amorphous white powder; [α]D18 +185 (c 0.50, CHCl3); IR (KBr) 3385, 2924, 1677, 1517, 1457, 1343, 1288, 1223, 1151, 788, 779, 746 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 9.48 (d, J = 1.8 Hz, 1H, ArH), 8.71 (s, 1H, NH), 8.56 (s, 1H, ArH), 8.31 (d, J = 1.6 Hz, 1H, ArH), 8.07 (d, J = 7.8 Hz, 1H, ArH), 8.02 (d, J = 8.5 Hz, 1H, ArH), 7.97 (brs, 1H, NH), 7.75 (d, J = 8.7 Hz, 1H, ArH), 7.63 (dd, J = 8.6, 1.8 Hz, 1H, ArH), 7.49−7.52 (m, 2H, ArH), 7.36−7.38 (m, 2H, ArH), 7.09 (t, J = 7.7 Hz, 1H, H-1′), 5.90 (s, 1H, H-3′), 4.92−5.07 (m, 4H, H-7, H-3″), 4.52 (brs, 1H, H-4′), 2.95 (s, 3H, 3′-NCH3), 2.86 (s, 3H, 4′-OCH3), 2.71−2.74 (m, 1H, H-2′a), 2.35 (s, 3H, 6′CH3), 2.26−2.31 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 190.2, 182.5, 171.8, 139.1, 135.3, 135.0, 134.6, 133.2, 128.9, 127.7, 127.5, 127.3, 125.6, 125.4, 125.3, 124.3, 123.7, 123.3, 121.6, 120.5, 119.4, 114.7, 114.5, 114.4, 114.1, 114.0, 113.6, 111.6, 111.3, 95.1, 82.8, 82.5, 60.4, 54.2, 51.9, 45.6, 32.8, 29.5, 27.5; HRESIMS m/z 837.0497 [M − H]− (calcd for C39H31Br2N6O4S, 837.0489). EtOH (200 μL) and (CF3CO)2O (400 μL) were added sequentially to a solution of 43 (50 mg, 0.060 mmol) in CH2Cl2 (6 mL) at 0 °C. After it was stirred for 1 h at 0 °C, the reaction was quenched by adding 6 mL of saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 13 (26 mg, 53% yield) as a white powder; [α]D18 +169 (c 0.50, CHCl3); IR (KBr) 3417, 2922, 1676, 1524, 1456, 1343, 1288, 1223, 1126, 1103, 789, 741 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.66 (s, 1H, NH), 9.49 (s, 1H, ArH), 8.71 (s, 1H, NH), 8.07 (d, J = 7.7 Hz, 1H, ArH), 8.04 (d, J = 8.5 Hz, 1H, ArH), 7.92 (s, 1H, ArH), 7.62−7.69 (m, 3H, ArH), 7.55 (s, 1H, ArH), 7.49 (d, J = 8.0 Hz, 1H, ArH), 7.43 (d, J = 8.5 Hz, 1H, ArH), 7.37 (t, J = 7.4 Hz, 1H, ArH), 7.28−7.30 (m, 1H, ArH), 7.08−7.11 (m, 1H, H-1′), 5.04 (s, 2H, H-7), 4.97 (d, J = 12.7 Hz, 1H, H-3′), 4.49 (brs, 1H, H-4′), 2.91 (s, 3H, 3′-NCH3), 2.85−2.89 (m, 1H, H2′a), 2.69 (s, 3H, 4′-OCH3), 2.43 (s, 3H, 6′-CH3), 2.36−2.41 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 171.8, 167.8, 138.8, 135.2, 135.0, 134.5, 133.1, 129.1, 127.7, 127.6, 126.6, 125.8, 125.3, 124.5, 124.4, 124.3, 123.6, 121.6, 121.2, 120.5, 119.5, 119.4, 114.7, 114.1(2 × C), 113.6, 112.2, 111.7, 111.2, 106.6, 94.9, 82.5, 82.4, 60.2, 53.1, 45.6, 34.5, 29.2, 27.0; HRESIMS m/z 821.0539 [M + H]+ (calcd for C39H31Br2N6O3S, 821.0539). Synthesis of 3-Bromo-5‴-methoxyfradcarbazole A (14). To a solution of thiocarbonylimidazolium salt 30 (200 mg, 0.25 mmol) in DMF (10 mL) was added a trifluoroacetate salt 19 (75 mg, 0.25 mmol) and Et3N (1.0 mL). The reaction was stirred at rt for 24 h and diluted with EtOAc (20 mL). The organic layer was washed with 1 N HCl (2 × 10 mL) and brine (20 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 44 (109 mg, 56% yield) as an amorphous white powder; [α]D18 +185 (c 0.50, CHCl3); IR (KBr) 3385, 2924, 1677, 1517, 1457, 1343, 1288, 1223, 1151, 788, 779, 746 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.92 (s, 1H, NH), 9.47 (s, 1H, ArH), 8.70 (s, 1H, NH), 8.43 (d, J = 2.0 Hz, 1H, ArH), 8.07 (d, J = 7.6 Hz, 1H, ArH), 8.02 (d, J = 8.4 Hz, 1H, ArH), 7.89 (brs, 1H, NH), 7.74 (d, J = 8.6 Hz, 1H, ArH), 7.69 (s, 1H, ArH), 7.63 (d, J = 8.6 Hz, 1H, ArH), 7.50 (t, J = 7.6 Hz, 1H, ArH), 7.36− 7.41 (m, 2H, ArH), 7.09 (t, J = 7.6 Hz, 1H, H-1′), 6.87 (dd, J = 8.6, 2.1 Hz, 1H, ArH), 5.93 (brs, 1H, H-3′), 4.93−5.03 (m, 4H, H-7, H-

3″), 4.50 (brs, 1H, H-4′), 3.78 (s, 3H, 5‴-OCH3), 2.95 (s, 3H, 3′NCH3), 2.82 (s, 3H, 4′-OCH3), 2.71−2.75 (m, 1H, H-2′a), 2.36 (s, 3H, 6′-CH3), 2.26−2.32 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 189.9, 182.4, 171.8, 155.5, 139.1, 135.0, 133.4, 133.2, 131.3, 129.0, 127.7, 127.5, 126.3, 125.6, 125.3, 124.3, 123.7, 121.6, 120.5, 119.4, 114.7, 114.1, 113.9(2 × C), 113.0, 112.7, 111.6, 111.4, 102.9, 95.1, 82.9, 82.5, 60.4, 55.3, 54.2, 51.8, 45.6, 32.7, 29.4, 27.6; HRESIMS m/z 789.1499 [M − H]− (calcd for C40H34BrN6O5S, 789.1489). EtOH (200 μL) and (CF3CO)2O (400 μL) were added sequentially to a solution of 44 (56 mg, 0.071 mmol) in CH2Cl2 (6 mL) at 0 °C. After it was stirred for 1 h at 0 °C, the reaction was quenched by adding 6 mL of saturated aqueous NaHCO3 and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by semipreparative HPLC (90% MeOH/H2O) to yield 14 (32 mg, 59% yield) as a white powder; [α]D18 +177 (c 0.50, CHCl3); IR (KBr) 3411, 2925, 1677, 1524, 1459, 1343, 1288, 1223, 1103, 1024, 788, 740 cm−1; 1H NMR (600 MHz, DMSO-d6) δ 11.21 (s, 1H, NH), 9.50 (s, 1H, ArH), 8.71 (s, 1H, NH), 8.07 (d, J = 7.8 Hz, 1H, ArH), 8.04 (d, J = 8.5 Hz, 1H, ArH), 7.62−7.68 (m, 2H, ArH), 7.55 (s, 1H, ArH), 7.51 (s, 1H, ArH), 7.49 (t, J = 7.8 Hz, 1H, ArH), 7.34−7.37 (m, 2H, ArH), 7.24 (s, 1H, ArH), 7.09 (t, J = 7.6 Hz, 1H, H-1′), 6.83 (dd, J = 8.5, 1.6 Hz, 1H, ArH), 5.04 (s, 2H, H-7), 4.96 (d, J = 12.0 Hz, 1H, H-3′), 4.49 (s, 1H, H-4′), 3.83 (s, 3H, 5‴-OCH3), 2.90 (s, 3H, 3′-NCH3), 2.85− 2.89 (m, 1H, H-2′a), 2.68 (s, 3H, 4′-OCH3), 2.43 (s, 3H, 6′-CH3), 2.37−2.43 (m, 1H, H-2′b); 13C NMR (150 MHz, DMSO-d6) δ 171.8, 167.3, 153.9, 138.8, 134.9, 133.7, 133.1, 131.6, 129.1, 127.7, 127.6, 125.8, 125.3, 125.1, 124.4, 123.6, 123.5, 121.6, 120.7, 120.5, 119.4, 114.7, 114.1, 113.6, 112.7, 112.0, 111.7, 111.2, 106.7, 100.8, 94.9, 82.5, 82.5, 60.2, 55.4, 53.1, 45.6, 34.5, 29.1, 27.0; HRESIMS m/z 773.1536[M + H]+ (calcd for C40H34BrN6O4S, 773.1540). Biological Tests. Cell Culture. The cell lines (MV4−11, K562, HL-60, PBMC) were maintained in RPMI-1640 (supplemented with 10% fetal bovine serum (FBS)) and cultured at 37 °C in a CO2 incubator (5% CO2 and 95% air). Cell Titer-Glo (CTG) Assay. The inhibitory activities against the cancer and PBMC cell lines were evaluated by the CTG assay. Briefly, the cell lines were seeded in 96-well plates at a density of 2 × 103 cells/well and treated with 0.1, 0.25, 0.5, 1.25, 2.5, 5.0, and 10.0 μM doses of the compounds. After 72 h of incubation, 100 μL of CTG solution (Promega) was added into each well. The luminescence value was tested by using a microplate reader (BioTek, HM-1) after staying at rt for 10 min. Apoptosis Assay. The labeling (One Step TUNEL Apoptosis Assay Kit, Beyotime, C1086) assay was used to evaluate apoptosis induction. MV4−11 cells were treated with 0.3 μM of compound 6 for 24 h and then subjected to TUNEL assay according to the manufacturer’s instructions. The apoptotic cells with green fluorescence were detected with a fluorescence microscope. The excitation wavelength range is 450−500 nm, and the emission wavelength range is 515−565 nm. Cell Cycle Analysis. MV4−11 cells were treated with 0.15, 0.3, and 0.6 μM of compound 6 for 24 h; then, the cells were fixed by cold EtOH at −20 °C. After they were washed with phosphate-buffered solution (PBS), the cells were stained with 50 μg/mL propidium iodide (PI, Beyotime, ST511), 0.2 mg/mL RNase A (Beyotime, ST578), and 0.1% Triton X-100 (China National Medicines Corporation, 30188928) for 40 min at 37 °C. The obtained solution was centrifuged at 1500 rpm for 5 min and suspended in PBS. The cell cycle distribution was determined using a BD FACS Calibur Flow Cytometer, and the data were analyzed using Cell Quest and ModFit software. Western Blotting. The MV4−11 cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer. A total of 40 μg of protein was quantified by a bicinchoninic acid (BCA) Protein Concentration Assay Kit (Solarbio Life Sciences, PC0020) and was loaded onto a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. After electrophoresis, the proteins in the K



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gel were transferred to nitrocellulose membrane (Thermo Scientific Pierce, 88013) and incubated with primary antibodies at 4 °C overnight. The membrane was washed three times with phosphatebuffered saline with tween 20 (PBST) and incubated with horseradish peroxidase (HRP)-conjugated rabbit lgG (Beyotime, A0208) secondary antibodies for 1 h at rt. The immunoblot bands were detected by an enhanced chemiluminescence (ECL) system, and then imaged by an infrared fluorescence imaging instrument (CLiNX, 6000pro) for signal detection. Target proteins were detected using individual specific primary antibodies. Polyclonal rabbit antibodies were obtained for GAPDH, p-FLT3, FLT3, c-kit, and CDK2 from Abcam.

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(10) Zhou, L. M.; Kong, F. D.; Xie, Q. Y.; Ma, Q. Y.; Hu, Z.; Zhao, Y. X.; Luo, D. Q. Mar. Drugs 2019, 17, 219. (11) George, P.; Bali, P.; Cohen, P.; Tao, J. G.; Guo, F.; Sigua, C.; Vishvanath, A.; Fiskus, W.; Scuto, A.; Annavarapu, S.; Moscinski, L.; Bhalla, K. Cancer Res. 2004, 64, 3645−3652. (12) Ashman, L. K.; Griffith, R. Expert Opin. Invest. Drugs 2013, 22, 103−115. (13) Leung, A. Y. H.; Man, C. H.; Kwong, Y. L. Leukemia 2013, 27, 260−268.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00468.

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HRESIMS, 1H NMR, and 13C NMR spectra of compounds 1−14, 16a, 16b, 17a, 17b, 18a, 18b, 19a, 19b, 20−24, 26, 27, and 29−44 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Phone: +86-851-83809439. Fax: +86-851-83809439. E-mail: [email protected]. (L.W.) *Phone: +86-532-82031268. Fax: +86-532-82031268. E-mail: [email protected]. (W.Z.) ORCID

Weiming Zhu: 0000-0002-7591-3264 Author Contributions ∥

These authors contributed equally to the work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the NSFC (Nos. 21502034 & 30973680), the Natural Science Foundation of Guizhou (QKH J-2015-2109), the 100 Leading Talents of Guizhou Province (fund for W.Z.), the science and technology project of Guizhou (No. QKHT Z-2014-4007), the academician workstation of Guizhou (No. QKH YSZ-2015-4009), and Guizhou provincial engineering research center for natural drugs.

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