synthesis and anticancer evaluation of 2,3‐disubstituted indoles...

DOI: 10.1002/ejoc.201701001 Full Paper

C–H Functionalization | Very Important Paper |

Synthesis and Anticancer Evaluation of 2,3-DisubstitutedIndoles Derived from Azobenzenes and Internal OlefinsYongguk Oh,[a] Sang Hoon Han,[a] Neeraj Kumar Mishra,[a] Umasankar De,[a] Junho Lee,[a]Hyung Sik Kim,[a] Young Hoon Jung,[a] and In Su Kim*[a]

Abstract: Azo-directed rhodium(III)-catalysed C–H functionali-zation and intramolecular annulation reactions between azo-benzenes and internal olefins are described. This transformationleads to 2,3-disubstituted free (NH)-indoles with excellent site-selectivity and functional-group compatibility. The resultingindoles were evaluated for in-vitro anticancer activity against

IntroductionThe indole scaffold is well known as an ubiquitous structuralmotif that is found in a large number of natural products andpharmaceuticals.[1] 2,3-Disubstituted indoles have been givenconsiderable attention as a result of their interesting biologicalprofiles.[2] These compounds are known to show various biolog-ical functions; for example, they have been shown to act asglycine receptor antagonists,[2a] viral fusion inhibitors,[2b,2c] non-nucleoside transcriptase inhibitors,[2d,2e] and selective estrogen-receptor modulators (Figure 1).[2f ] The biological activities ofthese compounds are closely associated with carboxylate func-

Figure 1. Biologically relevant 2,3-disubstituted indoles.

[a] School of Pharmacy, Sungkyunkwan University,Suwon 16419, Republic of KoreaE-mail: [email protected]://pharmasyn.skku.edu/Supporting information and ORCID(s) from the author(s) for this article areavailable on the WWW under https://doi.org/10.1002/ejoc.201701001.

Eur. J. Org. Chem. 2017, 6265–6273 © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim6265

human endometrial adenocarcinoma cells (Ishikawa), triplenegative human breast cancer cells (MDA-MB-231), and humanrenal cancer cells (Caki-1). 2,3-Disubstituted indoles 3b, 3k, and5b were found to show potent cytotoxic effects that were com-petitive with the anticancer agent doxorubicin.

tionalities at the C-2 and/or C-3 positions, but they vary de-pending on the position and/or nature of the substituents onthe indole framework.

Transition-metal-catalysed C–H functionalization has becomea powerful approach for the efficient construction of structur-ally diverse molecules in organic and medicinal chemistry.[3]

The intermolecular synthesis of indoles based on catalyticC–H functionalization[4] has attracted much attention due to itsremarkable potential in terms of atom economy and environ-mental sustainability compared with traditional methods suchas the Fischer and Larock indole syntheses.[5] In pioneeringwork, Fagnou and Stuart reported an RhIII-catalysed oxidativeannulation reaction between acetanilides and internal alkynesto give 2,3-disubstituted indoles (Scheme 1).[6] Similar protocols

Scheme 1. Indole formation reactions involving C–H functionalization. 2-pyr =2-pyridyl; 2-pym = 2-pyrimidyl.

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for the formation of indoles have also been explored using 2-pyridyl, acetyl, and 2-pyrimidyl directing groups with alkynesunder PdII and RuII catalysis.[7] More recently, terminal olefinssuch as vinyl ketones, allylic alcohols, and allylic carbonateshave been used for the construction of C-2-substituted indolesunder transition-metal catalysis.[8]

Azobenzenes have recently been used in catalytic C–H func-tionalization reactions by using the azo functionality to synthe-size various N-heterocycles such as (2H)-indazoles, cinnolines,cinnolinones, and benzotriazoles.[9] Additionally, Glorius dem-onstrated a tandem RhIII-catalysed annulation reaction of azo-benzenes with terminal olefins leading to the 1-aminoindolineframework.[10] Moreover, the thermal annulation of arylhydraz-ines and dimethyl acetylenedicarboxylates has also been ex-plored for the synthesis of indoles.[11] In sharp contrast, in this

Table 1. Optimization of the reaction conditions.[a]

Entry Catalyst (mol-%) Additive (mol-%) Solvent Yield [%][b]

1 [RhCp*Cl2]2 (2.5) AgSbF6 (10) DCE trace2 [RhCp*Cl2]2 (2.5) AgSbF6 (10), DCE 42

Cu(OAc)2 (100)3 [RhCp*Cl2]2 (2.5) Cu(OAc)2 (100) DCE trace4 [RhCp*Cl2]2 (2.5) AgBF4 (10), DCE trace

Cu(OAc)2 (100)5 [RhCp*Cl2]2 (2.5) AgPF6 (10), DCE trace

Cu(OAc)2 (100)6 [RhCp*Cl2]2 (2.5) AgNTf2 (10), DCE 38

Cu(OAc)2 (100)7 [CoCp*(CO)I2] (5) AgSbF6 (10), DCE trace

Cu(OAc)2 (100)8 [Ru(p-cymene)Cl2]2 (2.5) AgSbF6 (10), DCE n.r.

Cu(OAc)2 (100)9 [IrCp*Cl2]2 (2.5) AgSbF6 (10), DCE n.r.

Cu(OAc)2 (100)10 [RhCp*Cl2]2 (2.5) AgSbF6 (10), CH2Cl2 40

Cu(OAc)2 (100)11 [RhCp*Cl2]2 (2.5) AgSbF6 (10), toluene 34

Cu(OAc)2 (100)12 [RhCp*Cl2]2 (2.5) AgSbF6 (10), MeCN n.r.

Cu(OAc)2 (100)13 [RhCp*Cl2]2 (2.5) AgSbF6 (10), dioxane 21

Cu(OAc)2 (100)14[c] [RhCp*Cl2]2 (2.5) AgSbF6 (10), DCE 65

Cu(OAc)2 (100)15[d] [RhCp*Cl2]2 (2.5) AgSbF6 (10), DCE 71


AgOAc (100)17[d] [RhCp*Cl2]2 (2.5) AgSbF6 (10), DCE trace

LiOAc (100)18[d] [RhCp*Cl2]2 (2.5) AgSbF6 (10), DCE trace

Ag2CO3 (100)19[d] [RhCp*Cl2]2 (2.5) AgSbF6 (10), DCE 70


Cu(OAc)2 (30)

[a] Reaction conditions: 1a (0.2 mmol), 2a (0.3 mmol), catalyst (quantity noted), additive (quantity noted), solvent (1 mL) under air at 80 °C for 12 h in pressuretubes. [b] Isolated yield after flash column chromatography. n.r. = no reaction. [c] 110 °C. [d] 130 °C.

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paper we report the formation of 2,3-disubstituted indolesthrough the RhIII-catalysed cross-coupling of azobenzenes andinternal olefins such as maleates and fumarates. Furthermore,the synthesized 2,3-disubstituted indole derivatives were evalu-ated for cytotoxic effects against human endometrial adenocar-cinoma cells (Ishikawa), triple negative human breast cancercells (MDA-MB-231), and human renal cancer cells (Caki-1), andwere found to have promising anticancer properties competi-tive with the anticancer agent doxorubicin.

Results and DiscussionFor our initial investigation, we chose (E)-1,2-bis[4-(trifluoro-methyl)phenyl]diazene (1a) and dibutyl maleate (2a) as modelsubstrates (Table 1).

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We were pleased to find that a combination of [RhCp*Cl2]2(2.5 mol-%) and AgSbF6 (10 mol-%) in the presence of Cu(OAc)2(100 mol-%) could be used to form 2,3-disubstituted indoleproduct 3a in 42 % yield (Table 1, Entries 1 and 2). However, inthe absence of AgSbF6, the reaction was unsuccessful (Table 1,Entry 3). Other additives including AgBF4, AgPF6, and AgNTf2were tested, but no improved results were obtained (Table 1,Entries 4–6). Moreover, changing to other catalysts such as CoIII,RuII, or IrIII was found to be ineffective in this coupling reaction(Table 1, Entries 7–9). A solvent screening showed that DCE (1,2-dichloroethane) is better than other solvents such as CH2Cl2,toluene, MeCN, and 1,4-dioxane (Table 1, Entries 10–13). In-creasing the temperature to 130 °C led to an increase in theyield of our desired adduct 3a to 71 % (Table 1, Entries 14 and15). After screening a range of additives, Cu(OAc)2 was foundto show the highest reactivity (Table 1, Entries 16–18). Interest-ingly, the use of a decreased amount (50 mol-%) of Cu(OAc)2gave a comparable yield (70 %) of 3a (Table 1, Entry 19). Finally,optimal reaction conditions were obtained, using 30 mol-% ofCu(OAc)2 under otherwise identical conditions; this gave 2,3-disubstituted indole 3a in 71 % yield (Table 1, Entry 20).

To explore the substrate scope and limitations, a range ofazobenzenes were coupled with internal olefins (maleates), as

Scheme 2. Scope of the reaction of azobenzenes with (Z)-olefins.[a] [a] Reaction conditions: 1a–1n (0.2 mmol), 2a–2c (0.3 mmol), [RhCp*Cl2]2 (2.5 mol-%),AgSbF6 (10 mol-%), Cu(OAc)2 (30 mol-%), DCE (1 mL) under air at 130 °C for 12 h in pressure tubes. [b] Isolated yield after flash column chromatography.


shown in Scheme 2. The para-substituted azobenzenes 1b and1c reacted with dibutyl maleate (2a) to give the correspondingproducts 3b and 3c in 67 and 70 % yields, respectively. Othermaleates 2b and 2c also took part in the coupling reaction todeliver the desired products 3d and 3e in moderate to goodyields. In addition, para-substituted azobenzenes 1d–1f cou-pled with 2a to give the corresponding indoles 3f–3h in moder-ate yields. To our delight, azobenzenes containing electron-richand electron-deficient substituents at the meta position alsoshowed good reactivity, and indole products 3i–3m wereformed in moderate to good yields. The reactions took placeregioselectively at the less hindered C–H bond, and it is alsosignificant that nitro, acetyl, and chloride groups were all toler-ated under the reaction conditions; these functionalities pro-vide versatile synthetic handles for further transformation. How-ever, meta-methoxy-substituted azobenzene 1l gave a regioiso-meric mixture of 3n and 3n′ in 35 and 19 % yields, respectively.Moreover, ortho-fluoro-substituted azobenzene 1m was alsotolerated in this reaction, and gave 3o in 60 % yield; ortho-bromo-substituted azobenzene 1n was less reactive and gavea lower yield of indole 3p under the current reaction conditions.

To further evaluate the substrate scope of this process, abroad range of (E)-olefins 4a–4f were screened in coupling re-

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actions with 1c and 1k, as shown in Scheme 3. We were pleasedto find that fumarates 4a and 4b and α,�-unsaturated esters4c–4e coupled with azobenzenes 1c and 1k to give the corre-sponding indoles 5a–5e, although compared to the maleatesthe reactivity was lower. In contrast, (E)-1,4-diphenylbut-2-ene-1,4-dione (4f ) proved to be a good substrate for this transfor-mation, providing 2,3-dibenzoylindole 5f in 64 % yield.

Evaluation of unsymmetrical azobenzenes 1o and 1p re-vealed that this reaction occurs predominantly on the electron-rich aromatic ring, as indoles 3h and 3g were formed as majorproducts (Scheme 4). This result indicates that the electronicenvironment of the azobenzene is crucial to tuning the siteselectivity for the formation of indoles.

To show the robustness of our synthesized 2,3-disubstitutedindoles, an annulation reaction between indole 3c andhydrazine was carried out to give pyridazinoindole derivative6a in 80 % yield. This compound is known to be a pharmaceuti-cally important heterocyclic scaffold in medicinal chemistry(Scheme 5).[12]

Scheme 3. Scope of the reaction of azobenzenes with (E)-olefins.[a] [a] Reaction conditions: 1c and 1k (0.2 mmol), 4a–4f (0.3 mmol), [RhCp*Cl2]2 (2.5 mol-%),AgSbF6 (10 mol-%), Cu(OAc)2 (30 mol-%), DCE (1 mL) under air at 130 °C for 12 h in pressure tubes. [b] Isolated yield after flash column chromatography.

Scheme 4. Reactions of unsymmetrical azobenzenes with (Z)-olefin 2a.


Scheme 5. Synthesis of pyridazinoindole derivative 6a.

A proposed reaction mechanism is outlined in Scheme 6. Acationic [Cp*RhIIIOAc][SbF6] species is generated in situ as anactive catalyst. This then coordinates to a nitrogen atom of theazo group, and a subsequent C–H activation step deliversrhodacyclic intermediate I.[13] Coordination of 2a and migratoryinsertion lead to the formation of a seven-membered rhoda-cyclic species II. Intermediate II then undergoes a rearrange-ment with the assistance of the Cu(OAc)2 additive to give amore stable six-membered coordinatively saturated RhIII speciesIII. At this stage, the presence of the electron-withdrawinggroup at the �-position possibly inhibits competitive �-hydride

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elimination that would give the corresponding Heck-type prod-uct. Additionally, the Cu(OAc)2 additive can act as Lewis acid toenhance the electrophilicity of the azo group and thus acceler-ate the nucleophilic addition of the C–Rh bond; this is in sharpcontrast with previously reported coupling reactions betweenN-Boc-azobenzenes and olefins.[10] Thus, the C–Rh bond of in-termediate III presumably undergoes nucleophilic addition tothe N=N bond to generate intermediate IV. Finally, N–N bondcleavage of IV followed by aromatization of V takes place togive free (NH)-indole 3h.

Scheme 6. Proposed reaction pathway.

The synthesized 2,3-disubstituted indoles were tested for cy-totoxic activity against human endometrial adenocarcinomacells (Ishikawa), triple negative human breast cancer cells (MDA-MB-231), and human renal cancer cells (Caki-1) (Table 2). Allhuman cancer cells were exposed to increasing concentrationsof compounds 3a–3p, 3n′, and 5a–5f for 48 h, and their survivalwas determined by an MTT assay.[14] In this experiment, repre-sentative anticancer agents such as doxorubicin and cisplatinwere selected as positive controls.[15] A number of the synthetic

Table 2. Cytotoxicity of synthetic compounds against human cancer cells.[a]

Compound IC50 [μM] Compound IC50 [μM]Ishikawa MDA-MB-231 Caki-1 Ishikawa MDA-MB-231 Caki-1

3a 3.5 18.6 10.6 3m 11.0 19.8 13.83b 2.6 9.0 5.5 3n 14.6 23.8 14.53c 5.8 9.4 5.9 3n′ 11.2 22.7 15.63d 20.5 36.7 20.2 5a >50 >50 >503e 31.0 45.7 36.6 5b 3.6 11.9 7.33f 16.8 30.6 17.4 5c >50 >50 >503g 19.7 41.8 26.2 5d >50 >50 >503h 20.1 35.6 22.8 5e >50 >50 >503i 8.8 15.7 10.4 5f >50 >50 >503j 10.3 9.6 6.9 doxorubicin 3.3 8.7 6.43k 3.6 8.2 5.2 cisplatin 30.3 >50 >503l 13.0 18.9 16.1

[a] IC50 value: substance concentration necessary for 50 % inhibition of cell viability.


compounds, including 3a–3c, 3i, 3k, and 5b, showed promisinginhibitory activity against Ishikawa, MDA-MB-231, and Caki-1cell lines. In general, compounds with electron-deficient substit-uents (3a–3c, 3i, and 3k) at the C-6 and C-5 positions or anisopropyl ester moiety (5b) at the C-2 and C-3 positions werefound to show potent cytotoxic activity. These results might bevery useful for structure–activity relationship (SAR) studies thatcould further optimize the anticancer activity. In particular,compounds 3b (IC50 = 2.6 μM against Ishikawa cells) and 3k(IC50 = 8.2 μM against MDA-MB-231 cells and IC50 = 5.2 μMagainst Caki-1 cells) showed the highest cytotoxic activity,higher than that of doxorubicin, which was used as a positivecontrol. These results suggest that 2,3-disubstituted indole de-rivatives represent a new class of powerful inhibitors againsthuman cancer cells.

The cytotoxicity results prompted further investigation intothe underlying mechanisms involving the interruption of thecellular balance of reactive oxygen species (ROS) leading to celldeath. It is known that ROS are an essential component forvarious biological processes in normal cells. However, an exces-sive production of ROS induces the extinction of cancer cells. Ithas been shown that various types of anticancer drugs kill can-cer cells through processes beginning with ROS generation.[16]

Thus, we carried out a ROS-generation experiment using 2′,7′-dichlorofluorescin diacetate (DCFDA) as a fluorescent ROS indi-cator. Generally, DCFDA penetrates into the cell cytoplasm,where it is hydrolysed by intracellular esterase and oxidized byROS to form 2′,7′-dichlorofluorescein (DCF). The resulting fluo-rescence can be measured by flow-cytometry analysis. Asshown in Figure 2, our synthetic compound 3b was found tocause an increase in ROS generation compared to the untreatedDMSO control, although it produced a lower level of ROS thanhydrogen peroxide. To confirm that the cell cytotoxicity wasderived from the ROS generation, we also carried out a ROSinhibition experiment using N-acetyl-L-cysteine (NAC) as a radi-cal scavenger.[17] As shown by the blue line in Figure 2, the ROSgeneration of 3b decreased dramatically when NAC was added.These results indicate that the ROS generation of 2,3-disubsti-tuted indoles may contribute their potent anticancer effect.Meanwhile, we pretreated Caki-1 cancer cell lines with NAC at1 and 5 mM concentrations to observe the cell viability as a

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result of ROS generation by compound 3b (Figure 3). The cyto-toxic effect of 3b was found to decrease in a NAC-concentra-tion-dependent manner. Control experiments showed that NACdid not show any cytotoxic effect at 1 and 5 mM concentrationsagainst Caki-1 cancer cell lines.

Figure 2. ROS-generation experiment with 3b in Caki-1 cancer cells.

Figure 3. ROS-inhibition experiment with 3b and NAC in Caki-1 cancer cells.

ConclusionsWe have described rhodium(III)-catalysed tandem annulationreactions between azobenzenes and internal olefins to give bio-logically important 2,3-disubstituted indoles. These transforma-tions have been applied to a wide range of (Z)- and (E)-olefinsas well as azobenzenes, and typically proceed with excellentlevels of chemoselectivity as well as with high functional-grouptolerance. In addition, all the compounds synthesized weretested for in-vitro anticancer activity against human endome-trial adenocarcinoma cells (Ishikawa), triple negative humanbreast cancer cells (MDA-MB-231), and human renal cancer cells(Caki-1). Synthesized indoles 3b, 3k, and 5b were found toshow cytotoxic effects competitive with the well-known anti-cancer agent doxorubicin. Further mechanistic investigationsindicated that the potent anticancer effect of the 2,3-disubsti-tuted indoles may result from ROS production.

Experimental SectionTypical Procedure for the Reaction of Azobenzenes with Inter-nal Olefins (Synthesis of 3a–3p and 5a–5f): (E)-1,2-Bis[4-(trifluoro-


methyl)phenyl]diazene (1a; 63.6 mg, 0.2 mmol, 100 mol-%),[RhCp*Cl2]2 (3.1 mg, 0.005 mmol, 2.5 mol-%), AgSbF6 (7.8 mg,0.02 mmol, 10 mol-%), and Cu(OAc)2 (10.9 mg, 0.06 mmol, 30 mol-%) were placed into an oven-dried sealed tube, and then dibutylmaleate (2a; 68.5 mg, 0.3 mmol, 150 mol-%) and DCE (1 mL) wereadded. The reaction mixture was stirred at 130 °C for 12 h. Thereaction mixture was then diluted with EtOAc (3 mL) and concen-trated in vacuo. The residue was purified by flash column chroma-tography (n-hexanes/EtOAc, 15:1) to give 3a (54.8 mg, 71 %).

Dibutyl 5-(Trifluoromethyl)-1H-indole-2,3-dicarboxylate (3a):White solid (54.8 mg, 71 %). M.p. 82.5–84.6 °C. 1H NMR (400 MHz,CDCl3): δ = 9.66 (br. s, 1 H), 8.37 (s, 1 H), 7.59–7.52 (m, 2 H), 4.42 (q,J = 6.4 Hz, 4 H), 1.84–1.73 (m, 4 H), 1.56–1.42 (m, 4 H), 1.01 (t, J =7.6 Hz, 3 H), 0.97 (t, J = 7.2 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3):δ = 163.6, 160.8, 135.8, 129.9, 128.7, 126.1, 126.1 (q, JC,F = 270.2 Hz),124.9 (q, JC,F = 32.4 Hz), 122.3 (q, JC,F = 3.1 Hz), 120.4 (q, JC,F =4.4 Hz), 66.2, 66.1, 30.8, 30.6, 19.3, 19.1, 13.7, 13.6 ppm. IR (KBr): ν̃ =3283, 2962, 2934, 2875, 2347, 1729, 1699, 1541, 1463, 1329, 1277,1258, 1160, 1109, 1078, 942, 899, 816 cm–1. HRMS (quadrupole, EI):calcd. for C19H22F3NO4 [M]+ 385.1501; found 385.1502.

2,3-Dibutyl 5-Ethyl 1H-indole-2,3,5-tricarboxylate (3b): Pale redsolid (52.2 mg, 67 %). M.p. 75.8–77.9 °C. 1H NMR (400 MHz, CDCl3):δ = 9.68 (br. s, 1 H), 8.78 (s, 1 H), 8.04 (dd, J = 8.8, 1.6 Hz, 1 H), 7.46(d, J = 8.4 Hz, 1 H), 4.44–4.37 (m, 6 H), 1.85–1.80 (m, 2 H), 1.78–1.72(m, 2 H), 1.58–1.45 (m, 4 H), 1.41 (t, J = 7.2 Hz, 3 H), 1.01 (t, J =7.6 Hz, 3 H), 0.96 (t, J = 7.2 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3):δ = 166.9, 163.8, 160.8, 137.1, 129.6, 126.7, 126.2, 125.6, 124.8, 113.4,111.8, 66.0, 64.9, 60.9, 30.8, 30.5, 19.3, 19.1, 14.4, 13.7, 13.6 ppm. IR(KBr): ν̃ = 3310, 2958, 2929, 2872, 2348, 1686, 1536, 1458, 1335,1242, 1154, 1063, 800, 738 cm–1. HRMS (quadrupole, EI): calcd. forC21H27NO6 [M]+ 389.1838; found 389.1835.

Dibutyl 5-(Trifluoromethoxy)-1H-indole-2,3-dicarboxylate (3c):Dark brown solid (56.3 mg, 70 %). M.p. 74.2–76.8 °C. 1H NMR(400 MHz, CDCl3): δ = 9.57 (br. s, 1 H), 7.93 (s, 1 H), 7.44 (d, J =8.8 Hz, 1 H), 7.22 (d, J = 8.8, 1.6 Hz, 1 H), 4.39 (td, J = 6.8, 2.0 Hz, 4H), 1.83–1.73 (m, 4 H), 1.55–1.41 (m, 4 H), 1.00 (t, J = 7.6 Hz, 3 H),0.96 (t, J = 7.2 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 163.6,160.8, 144.8 (q, JC,F = 2.2 Hz), 132.8, 130.2, 127.1, 120.0, 119.4 (q,JC,F = 254.9 Hz), 114.9, 112.9, 112.3, 60.1, 64.9, 30.8, 30.6, 19.3, 19.1,13.7, 13.6 ppm. IR (KBr): ν̃ = 3291, 2962, 2933, 2875, 2347, 1729,1689, 1530, 1457, 1250, 1157, 1069, 739 cm–1. HRMS (quadrupole,EI): calcd. for C19H22F3NO5 [M]+ 401.1450; found 401.1453.

Diethyl 5-(Trifluoromethoxy)-1H-indole-2,3-dicarboxylate (3d):Dark brown solid (41.3 mg, 60 %). M.p. 85.6–88.4 °C. 1H NMR(400 MHz, CDCl3): δ = 9.68 (br. s, 1 H), 7.94 (s, 1 H), 7.44 (d, J =9.2 Hz, 1 H), 7.22 (d, J = 8.8 Hz, 1 H), 4.44 (q, J = 6.8 Hz, 4 H), 1.44(t, J = 7.2 Hz, 3 H), 1.41 (t, J = 7.2 Hz, 3 H) ppm. 13C NMR (100 MHz,CDCl3): δ = 163.6, 160.8, 144.8 (q, JC,F = 2.1 Hz), 132.9, 130.2, 127.2,119.9, 119.4 (q, JC,F = 254.8 Hz), 114.9, 113.0, 112.2, 62.2, 60.9, 14.3,14.1 ppm. IR (KBr): ν̃ = 3301, 3054, 2984, 2361, 1755, 1689, 1535,1457, 1363, 1337, 1249, 1155, 1062, 1021, 987, 764, 743 cm–1. HRMS(quadrupole, EI): calcd. for C15H14F3NO5 [M]+ 345.0824; found345.0828.

Dimethyl 5-(Trifluoromethoxy)-1H-indole-2,3-dicarboxylate(3e): Dark brown solid (35.7 mg, 56 %). M.p. 177.8–179.9 °C. 1H NMR(400 MHz, CDCl3): δ = 9.75 (br. s, 1 H), 7.93 (s, 1 H), 7.45 (d, J =8.8 Hz, 1 H), 7.23 (dd, J = 8.8, 1.6 Hz, 1 H), 3.99 (s, 3 H), 3.98 (s, 3 H)ppm. 13C NMR (100 MHz, CDCl3): δ = 164.0, 161.1, 144.8 (q, JC,F =1.9 Hz), 132.9, 130.0, 127.0, 120.2, 119.4 (q, JC,F = 254.8 Hz), 115.2,113.1, 111.9, 52.9, 52.0 ppm. IR (KBr): ν̃ = 3308, 3056, 2853, 2363,1752, 1691, 1539, 1456, 1380, 1344, 1245, 1211, 1151, 1065, 967,

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905, 883, 764, 741 cm–1. HRMS (quadrupole, EI): calcd. forC13H10F3NO5 [M]+ 317.0511; found 317.0513.

Dibutyl 5-Chloro-1H-indole-2,3-dicarboxylate (3f): Dark brownsolid (38.6 mg, 55 %). M.p. 69.1–71.9 °C. 1H NMR (400 MHz, CDCl3):δ = 9.52 (br. s, 1 H), 8.03 (s, 1 H), 7.36 (d, J = 8.8 Hz, 1 H), 7.30 (d,J = 8.8 Hz, 1 H), 4.41–4.36 (m, 4 H), 1.83–1.72 (m, 4 H), 1.52–1.43(m, 4 H), 0.99 (t, J = 7.2 Hz, 3 H), 0.96 (t, J = 7.2 Hz, 3 H) ppm. 13CNMR (100 MHz, CDCl3): δ = 163.7, 160.9, 133.0, 129.5, 128.3, 127.8,126.3, 122.1, 113.0, 111.6, 65.9, 64.8, 30.8, 30.6, 19.3, 19.1, 13.7,13.6 ppm. IR (KBr): ν̃ = 2959, 2933, 2873, 2363, 1698, 1637, 1445,1333, 1246, 1212, 1179, 1081, 936, 874, 804, 770 cm–1. HRMS (quad-rupole, EI): calcd. for C18H22ClNO4 [M]+ 351.1237; found 351.1237.

Dibutyl 5-Methyl-1H-indole-2,3-dicarboxylate (3g): White solid(33.4 mg, 50 %). M.p. 47.7–49.5 °C. 1H NMR (400 MHz, CDCl3): δ =9.10 (br. s, 1 H), 7.81 (s, 1 H), 7.31 (d, J = 8.8 Hz, 1 H), 7.19 (d, J =6.8 Hz, 1 H), 4.38 (q, J = 6.8 Hz, 4 H), 2.46 (s, 3 H), 1.83–1.73 (m, 4H), 1.55–1.42 (m, 4 H), 1.01–0.96 (m, 6 H) ppm. 13C NMR (100 MHz,CDCl3): δ = 164.5, 161.0, 133.1, 132.0, 127.9, 127.8, 127.2, 121.8,111.8, 111.4, 65.6, 64.7, 30.9, 30.7, 21.6, 19.3, 19.1, 13.8, 13.7 ppm.IR (KBr): ν̃ = 3310, 2958, 2929, 2872, 2348, 1686, 1536, 1458, 1335,1273, 1242, 1164, 1063, 940, 800, 738 cm–1. HRMS (quadrupole, EI):calcd. for C19H25NO4 [M]+ 331.1784; found 331.1785.

Dibutyl 1H-Indole-2,3-dicarboxylate (3h): White solid (33.6 mg,53 %). M.p. 60.2–62.0 °C. 1H NMR (400 MHz, CDCl3): δ = 9.37 (br. s,1 H), 8.02 (d, J = 8.0 Hz, 1 H), 7.45 (d, J = 8.0 Hz, 1 H), 7.38–7.34 (m,1 H), 7.28–7.24 (m, 1 H), 4.41–4.37 (m, 4 H), 1.83–1.73 (m, 4 H), 1.53–1.44 (m, 4 H), 1.00–0.95 (m, 6 H) ppm. 13C NMR (100 MHz, CDCl3):δ = 164.3, 161.1, 134.7, 128.1, 126.8, 125.8, 122.6, 122.6, 112.3, 111.9,65.7, 64.7, 30.8, 30.6, 19.3, 19.1, 13.8, 13.7 ppm. IR (KBr): ν̃ = 3313,2959, 2933, 2873, 1692, 1636, 1451, 1432, 1329, 1245, 1213, 1179,1119, 1067, 938, 747 cm–1. HRMS (quadrupole, EI): calcd. forC18H23NO4 [M]+ 317.1627; found 317.1626.

Dibutyl 6-(Trifluoromethyl)-1H-indole-2,3-dicarboxylate (3i):White solid (50.3 mg, 65 %). M.p. 103.6–105.1 °C. 1H NMR (400 MHz,CDCl3): δ = 9.90 (br. s, 1 H), 8.14 (d, J = 8.6 Hz, 1 H), 7.76 (s, 1 H),7.47 (dd, J = 8.6, 1.2 Hz, 1 H), 4.42–4.39 (m, 4 H), 1.84–1.73 (m, 4 H),1.54–1.41 (m, 4 H), 1.00 (t, J = 7.3 Hz, 3 H), 0.96 (t, J = 7.3 Hz, 3 H)ppm. 13C NMR (100 MHz, CDCl3): δ = 163.7, 160.9, 133.6, 130.7,128.8, 127.7 (q, JC,F = 32.1 Hz), 124.4 (q, JC,F = 270.3 Hz), 123.4, 118.8(q, JC,F = 3.14 Hz), 112.0, 109.7 (q, JC,F = 4.4 Hz), 66.2, 65.0, 30.8,30.5, 19.3, 19.0, 13.7, 13.6 ppm. IR (KBr): ν̃ = 3317, 2960, 2871, 2356,2342, 1676, 1438, 1342, 1260, 1243, 1161, 1110, 1076, 881, 824,773 cm–1. HRMS (quadrupole, EI): calcd. for C19H22F3NO4 [M]+

385.1501; found 385.1503.

Dibutyl 6-Nitro-1H-indole-2,3-dicarboxylate (3j): Orange solid(39.4 mg, 54 %). M.p. 110.3–112.9 °C. 1H NMR (400 MHz, CDCl3): δ =10.1 (br. s, 1 H), 8.44 (s, 1 H), 8.14–8.13 (m, 2 H), 4.45–4.40 (m, 4 H),1.82–1.76 (m, 4 H), 1.52–1.44 (m, 4 H), 1.00 (t, J = 7.2 Hz, 3 H), 0.96(t, J = 7.2 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 163.3, 160.5,145.7, 133.2, 133.1, 130.9, 123.3, 117.2, 112.2, 108.9, 66.6, 65.2, 30.8,30.5, 19.3, 19.1, 13.8, 13.6 ppm. IR (KBr): ν̃ = 3298, 2962, 2346, 1671,1612, 1322, 1266, 1188, 1078, 740 cm–1. HRMS (quadrupole, EI):calcd. for C18H22N2O6 [M]+ 362.1478; found 362.1481.

Dibutyl 6-Acetyl-1H-indole-2,3-dicarboxylate (3k): Pale brownsolid (31.7 mg, 44 %). M.p. 95.7–97.0 °C. 1H NMR (400 MHz, CDCl3):δ = 9.72 (br. s, 1 H), 8.13–8.08 (m, 2 H), 7.86 (dd, J = 8.4, 1.2 Hz, 1H), 4.40 (td, J = 6.8, 1.2 Hz, 4 H), 2.67 (s, 3 H), 1.84–1.73 (m, 4 H),1.54–1.42 (m, 4 H), 1.00 (t, J = 7.2 Hz, 3 H), 0.97 (t, J = 7.2 Hz, 3 H)ppm. 13C NMR (100 MHz, CDCl3): δ = 197.8, 163.8, 160.7, 134.5,134.2, 131.4, 130.2, 122.6, 122.1, 112.9, 112.1, 66.1, 64.9, 30.9, 30.6,26.9, 19.3, 19.1, 13.8, 13.6 ppm. IR (KBr): ν̃ = 3305, 2959, 2360, 1725,


1683, 1525, 1435, 1324, 1274, 1236, 1179, 1145, 1073, 931, 900, 810,743 cm–1. HRMS (quadrupole, EI): calcd. for C20H25NO5 [M]+

359.1733; found 359.1730.

Dibutyl 6-Chloro-1H-indole-2,3-dicarboxylate (3l): Pale red solid(45.5 mg, 64 %). M.p. 91.9–93.9 °C. 1H NMR (400 MHz, CDCl3): δ =9.49 (br. s, 1 H), 7.94 (d, J = 8.4 Hz, 1 H), 7.44 (s, 1 H), 7.21 (dd, J =8.4, 1.6 Hz, 1 H), 4.39 (t, J = 6.8 Hz, 4 H), 1.82–1.72 (m, 4 H), 1.52–1.43 (m, 4 H), 1.00–0.95 (m, 6 H) ppm. 13C NMR (100 MHz, CDCl3):δ = 163.9, 160.9, 135.0, 131.8, 128.8, 125.4, 123.7, 123.4, 112.4, 111.7,65.9, 64.9, 30.8, 30.6, 19.3, 19.1, 13.8, 13.7 ppm. IR (KBr): ν̃ =3304, 2960, 2359, 1676, 1525, 1429, 1318, 1274, 1243, 1181, 1077,736 cm–1. HRMS (quadrupole, EI): calcd. for C18H22ClNO4 [M]+

351.1237; found 351.1235.

Dibutyl 6-Ethyl-1H-indole-2,3-dicarboxylate (3m): Brown stickysolid (37.4 mg, 54 %). 1H NMR (500 MHz, CDCl3): δ = 9.19 (br. s, 1H), 7.92 (d, J = 8.5 Hz, 1 H), 7.23 (s, 1 H), 7.13 (dd, J = 8.5, 1.0 Hz, 1H), 4.40–4.36 (m, 4 H), 2.76 (q, J = 7.5 Hz, 2 H), 1.81–1.73 (m, 4 H),1.52–1.44 (m, 4 H), 1.28 (t, J = 7.5 Hz, 3 H), 1.00–0.96 (m, 6 H) ppm.13C NMR (125 MHz, CDCl3): δ = 164.4, 161.1, 142.7, 135.2, 127.5,125.0, 123.6, 122.4, 112.4, 110.1, 65.6, 64.7, 30.9, 30.6, 29.2, 19.3,19.1, 15.7, 13.8, 13.7 ppm. IR (KBr): ν̃ = 3307, 2959, 2927, 2872,2347, 1698, 1535, 1428, 1261, 1214, 1181, 1135, 1067, 964, 819,771 cm–1. HRMS (quadrupole, EI): calcd. for C20H27NO4 [M]+

345.1940; found 345.1942.

Dibutyl 6-Methoxy-1H-indole-2,3-dicarboxylate (3n): Pale brownsolid (24.6 mg, 35 %). M.p. 52.6–54.2 °C. 1H NMR (400 MHz, CDCl3):δ = 9.17 (br. s, 1 H), 7.87 (d, J = 9.2 Hz, 1 H), 6.91 (dd, J = 8.8, 2.0 Hz,1 H), 6.82 (s, 1 H), 4.40–4.35 (m, 4 H), 3.84 (s, 3 H), 1.82–1.72 (m, 4H), 1.52–1.43 (m, 4 H), 1.00–0.95 (m, 6 H) ppm. 13C NMR (100 MHz,CDCl3): δ = 164.3, 160.9, 159.0, 135.8, 126.7, 123.5, 121.3, 113.8,112.8, 93.5, 65.5, 64.7, 55.5, 30.9, 30.7, 19.3, 19.1, 13.8, 13.7 ppm. IR(KBr): ν̃ = 3315, 2958, 2363, 2342, 1698, 1627, 1541, 1426, 1321,1248, 1119, 1067, 1030, 823, 739 cm–1. HRMS (quadrupole, EI): calcd.for C19H25NO5 [M]+ 347.1733; found 347.1731.

Dibutyl 4-Methoxy-1H-indole-2,3-dicarboxylate (3n′): Whitesolid (13.4 mg, 19 %). M.p. 106.6–107.6 °C. 1H NMR (400 MHz,CDCl3): δ = 8.92 (br. s, 1 H), 7.27–7.23 (m, 1 H), 6.98 (d, J = 8.4 Hz,1 H), 6.51 (dd, J = 8.0 Hz, 1 H), 4.38 (t, J = 6.4 Hz, 2 H), 4.32 (t, J =6.8 Hz, 2 H), 3.89 (s, 3 H), 1.78–1.70 (m, 4 H), 1.50–1.42 (m, 4 H),0.98 (t, J = 7.6 Hz, 3 H), 0.95 (t, J = 7.6 Hz, 3 H) ppm. 13C NMR(100 MHz, CDCl3): δ = 166.6, 160.9, 154.3, 136.7, 127.2, 122.9, 116.5,114.5, 104.6, 100.6, 65.4, 65.3, 55.5, 30.7, 30.6, 19.2, 19.1, 13.8,13.7 ppm. IR (KBr): ν̃ = 3308, 2956, 2870, 2358, 1735, 1697, 1541,1458, 1355, 1247, 1192, 1105, 1066, 936, 782, 738, 684 cm–1. HRMS(quadrupole, EI): calcd. for C19H25NO5 [M]+ 347.1733; found347.1731.

Dibutyl 7-Fluoro-1H-indole-2,3-dicarboxylate (3o): Orange solid(40.6 mg, 60 %). M.p. 86.1–88.7 °C. 1H NMR (400 MHz, CDCl3): δ =9.42 (br. s, 1 H), 7.78 (d, J = 8.4 Hz, 1 H), 7.19–7.14 (m, 1 H), 7.05(dd, J = 10.8, 7.6 Hz, 1 H), 4.42–4.38 (m, 4 H), 1.83–1.74 (m, 4 H),1.54–1.42 (m, 4 H), 1.00 (t, J = 7.2 Hz, 3 H), 0.97 (t, J = 7.2 Hz, 3 H)ppm. 13C NMR (100 MHz, CDCl3): δ = 163.8, 160.6, 149.3 (d, JC,F =245.8 Hz), 129.9 (d, JC,F = 4.4 Hz), 128.9, 123.7 (d, JC,F = 14.5 Hz),122.5 (d, JC,F = 5.9 Hz), 118.3 (d, JC,F = 4.0 Hz), 112.9 (d, JC,F = 2.2 Hz),109.9 (d, JC,F = 15.3 Hz), 65.9, 64.9, 30.8, 30.6, 19.3, 19.1, 13.8,13.7 ppm. IR (KBr): ν̃ = 3285, 2958, 2931, 2870, 2349, 1722, 1686,1584, 1539, 1439, 1401, 1328, 1257, 1177, 1095, 1040, 936, 879,781 cm–1. HRMS (quadrupole, EI): calcd. for C18H22FNO4 [M]+

335.1533; found 335.1533.

Dibutyl 7-Bromo-1H-indole-2,3-dicarboxylate (3p): Orange oil(30.4 mg, 38 %). 1H NMR (400 MHz, CDCl3): δ = 9.23 (br. s, 1 H), 7.97

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(d, J = 8.4 Hz, 1 H), 7.51 (d, J = 7.6 Hz, 1 H), 7.14 (t, J = 7.6 Hz, 1 H),4.42–4.37 (m, 4 H), 1.82–1.74 (m, 4 H), 1.53–1.42 (m, 4 H), 1.00 (t,J = 7.6 Hz, 3 H), 0.96 (t, J = 7.2 Hz, 3 H) ppm. 13C NMR (100 MHz,CDCl3): δ = 163.8, 160.6, 133.5, 128.8, 127.9, 127.7, 123.5, 121.9,113.2, 105.1, 66.0, 64.9, 30.8, 30.6, 19.3, 19.1, 13.8, 13.7 ppm. IR (KBr):ν̃ = 3286, 2958, 2930, 2872, 2361, 2340, 1697, 1561, 1540, 1447,1309, 1239, 1174, 1138, 1070, 937, 798, 778 cm–1. HRMS (quadru-pole, EI): calcd. for C18H22BrNO4 [M]+ 395.0732; found 395.0732.

Disobutyl 5-(Trifluoromethoxy)-1H-indole-2,3-dicarboxylate(5a): Dark brown solid (32.2 mg, 40 %). M.p. 119.3–121.8 °C. 1H NMR(400 MHz, CDCl3): δ = 9.41 (br. s, 1 H), 7.94 (s, 1 H), 7.44 (d, J =8.9 Hz, 1 H), 7.23 (dd, J = 8.9, 1.5 Hz, 1 H), 4.20–4.18 (m, 4 H), 2.15–2.07 (m, 2 H), 1.06–1.02 (m, 12 H) ppm. 13C NMR (100 MHz, CDCl3):δ = 163.7, 161.0, 144.8 (q, JC,F = 1.8 Hz), 132.8, 130.2, 127.1, 120.6(q, JC,F = 255.3 Hz), 120.0, 114.9, 112.9, 112.4, 72.2, 71.2, 27.9, 27.8,19.2, 19.0 ppm. IR (KBr): ν̃ = 3286, 2964, 2877, 1743, 1727, 1698,1536, 1458, 1363, 1268, 1184, 1154, 1063, 986, 740, 704 cm–1. HRMS(quadrupole, EI): calcd. for C19H22F3NO5 [M]+ 401.1450; found401.1447.

Diisopropyl 5-(Trifluoromethoxy)-1H-indole-2,3-dicarboxylate(5b): Dark brown solid (31.5 mg, 42 %). M.p. 110.1–113.0 °C. 1H NMR(400 MHz, CDCl3): δ = 9.56 (br. s, 1 H), 7.91 (s, 1 H), 7.43 (dd, J =8.9, 0.3 Hz, 1 H), 7.23–7.20 (m, 1 H), 5.36–5.28 (m, 2 H), 1.45–1.40(m, 12 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 163.0, 160.1, 144.6(q, JC,F = 1.9 Hz), 132.7, 130.3, 127.0, 120.6 (q, JC,F = 255.6 Hz), 119.8,114.7, 112.8, 112.6, 70.2, 68.5, 21.9, 21.7 ppm. IR (KBr): ν̃ =3286, 3066, 2982, 2363, 1741, 1698, 1363, 1256, 1157, 1108, 1062,741 cm–1. HRMS (quadrupole, EI): calcd. for C17H18F3NO5 [M]+

373.1137; found 373.1134.

Ethyl 3-Methyl-5-(trifluoromethoxy)-1H-indole-2-carboxylate(5c): Dark brown solid (20.4 mg, 35 %). M.p. 161.4–163.1 °C. 1H NMR(400 MHz, CDCl3): δ = 8.88 (br. s, 1 H), 7.49 (s, 1 H), 7.35 (d, J =8.8 Hz, 1 H), 7.18 (d, J = 9.1 Hz, 1 H), 4.43 (q, J = 7.1 Hz, 2 H), 2.58(s, 3 H), 1.43 (t, J = 7.1 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3):δ = 162.3, 143.0 (q, JC,F = 1.9 Hz), 133.9, 128.6, 125.2, 120.7 (q, JC,F =254.2 Hz), 120.1, 119.7, 113.0, 112.5, 60.9, 14.4, 9.9 ppm. IR (KBr):ν̃ = 3316, 3056, 2989, 2927, 2364, 1737, 1673, 1546, 1367, 1262,1169, 1124, 867, 741 cm–1. HRMS (quadrupole, EI): calcd. forC13H12F3NO3 [M]+ 287.0769; found 287.0769.

Ethyl 3-Phenyl-5-(trifluoromethoxy)-1H-indole-2-carboxylate(5d): Dark brown solid (16.6 mg, 24 %). M.p. 155.5–157.1 °C. 1H NMR(400 MHz, CDCl3): δ = 9.19 (br. s, 1 H), 7.53–7.38 (m, 7 H), 7.23 (d,J = 8.9 Hz, 1 H), 4.33–4.27 (m, 2 H), 1.23 (t, J = 7.1 Hz, 3 H) ppm.13C NMR (100 MHz, CDCl3): δ = 161.6, 133.8, 132.7, 130.4, 128.0,127.9, 127.5, 124.5, 124.3, 120.6 (q, JC,F = 254.3 Hz), 120.0, 113.9,112.6, 61.1, 13.9 ppm. IR (KBr): ν̃ = 3328, 3188, 2358, 1741, 1682,1602, 1537, 1496, 1362, 1266, 1185, 1156, 1077, 1021, 903, 763 cm–1.HRMS (quadrupole, EI): calcd. for C18H14F3NO3 [M]+ 349.0926; found349.0923.

Ethyl 6-Ethyl-3-(trifluoromethyl)-1H-indole-2-carboxylate (5e):White solid (20.5 mg, 36 %). M.p. 124.7–126.5 °C. 1H NMR (400 MHz,CDCl3): δ = 9.47 (br. s, 1 H), 7.81 (d, J = 8.5 Hz, 1 H), 7.26 (s, 1 H),7.13 (dd, J = 8.5, 1.4 Hz, 1 H), 4.47 (q, J = 7.1 Hz, 2 H), 2.77 (q, J =7.6 Hz, 2 H), 1.44 (t, J = 7.1 Hz, 3 H), 1.29 (t, J = 7.5 Hz, 3 H) ppm.13C NMR (100 MHz, CDCl3): δ = 160.5, 142.9, 135.0, 125.2 (q, JC,F =4.3 Hz), 123.9 (q, JC,F = 266.3 Hz), 123.8, 123.1 (q, JC,F = 1.9 Hz),121.6 (q, JC,F = 2.9 Hz), 110.2, 109.9 (q, JC,F = 37.1 Hz), 62.0, 29.1,15.6, 14.0 ppm. IR (KBr): ν̃ = 3325, 2976, 1739, 1695, 1538, 1479,1453, 1362, 1331, 1289, 1267, 1199, 1117, 1013, 871, 815, 781 cm–1.HRMS (quadrupole, EI): calcd. for C14H14F3NO2 [M]+ 285.0977; found285.0979.


(6-Ethyl-1H-indole-2,3-diyl)bis(phenylmethanone) (5f): Dark yel-low solid (45.3 mg, 64 %). M.p. 177.8–179.9 °C. 1H NMR (400 MHz,CDCl3): δ = 9.83 (br. s, 1 H), 7.83 (d, J = 8.3 Hz, 1 H), 7.44–7.41 (m,2 H), 7.37–7.30 (m, 5 H), 7.17–7.12 (m, 5 H), 5.79 (q, J = 7.5 Hz, 2 H),1.31 (t, J = 7.5 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 192.7,189.0, 143.6, 140.7, 138.9, 136.1, 134.9, 132.4, 132.0, 129.0, 128.9,128.1, 127.9, 125.6, 124.0, 122.3, 120.8, 110.3, 29.3, 15.7 ppm. IR(KBr): ν̃ = 3292, 3058, 2923, 2853, 2366, 1961, 1739, 1633, 1596,1576, 1519, 1498, 1447, 1418, 1327, 1261, 1222, 1175, 1142, 1002,935, 885, 819 cm–1. HRMS (quadrupole, EI): calcd. for C24H19NO2[M]+ 353.1416; found 353.1417.

Experimental Procedure for the Synthesis of Pyridazinoindole-1,4-dione and Characterization Data: Dibutyl 5-(trifluoro-methoxy)-1H-indole-2,3-dicarboxylate (3c; 80.3 mg, 0.2 mmol,100 mol-%) and EtOH (1 mL) were placed into an oven-dried sealedtube, and then NH2NH2·H2O (50 % in H2O; 100.1 mg, 1 mmol,500 mol-%) was added at room temperature. The reaction mixturewas stirred at 100 °C for 12 h. The resulting mixture was then cooledand diluted with H2O (2 mL), treated with diluted NaOH (1 mL), andwarmed at 50 °C for 10 min. Then acetone (2 mL) and HCl (35 %aq.; 0.5 mL) were added. The resulting brown precipitate was col-lected by filtration and washed with water to give 6a (45.9 mg,80 %).

8-(Trifluoromethoxy)-2,3-dihydro-1H-pyridazino[4,5-b]indole-1,4(5H)-dione (6a): Pale brown solid (45.9 mg, 80 %). M.p. > 300 °C(decomposition). 1H NMR (400 MHz, [D6]DMSO): δ = 12.83 (s, 1 H),11.72 (br. s, 2 H), 7.96 (s, 1 H), 7.69 (d, J = 9.2 Hz, 1 H), 7.45 (d, J =8.8 Hz, 1 H) ppm. 13C NMR (100 MHz, [D6]DMSO): δ = 155.5, 149.5,143.1 (q, JC,F = 2.2 Hz), 136.7, 133.2 (q, JC,F = 1.7 Hz), 122.2, 120.4(q, JC,F = 253.8 Hz), 120.1, 114.5, 113.5, 111.8 ppm. IR (KBr): ν̃ =3327, 2924, 2356, 2340, 1680, 1398, 1263, 1159, 742 cm–1. HRMS(quadrupole, EI): calcd. for C11H6F3N3O3 [M]+ 285.0361; found285.0363.

Cancer-Cell Growth Inhibition Assay (MTT Assay): Human pros-tate adenocarcinoma cells (LNCaP), human endometrial adenocarci-noma cells (Ishikawa), and human ovarian carcinoma cells (SKOV3)were grown in DMEM medium (Dulbecco's modified Eagle medium)supplemented with penicillin/streptomycin (1 %), and fetal bovineserum (10 %) (all from Life Technologies, Grand Island, NY). Cellswere seeded in 96-well plates (3 × 103 cells/well) containing growthmedium (100 μL) for 24 h. The medium was then removed, andfresh medium (100 μL) containing individual compounds at differ-ent concentrations was added to each well. The plates were incu-bated at 37 °C for 48 h. After 24 h of culture, the cells were supple-mented with test compounds dissolved in DMSO (1 μL; less than0.025 % in each preparation). After 24 h of incubation, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 100 μL] wasadded to each well. After 4 h of incubation at 37 °C, the supernatantwas aspirated, and the formazan crystals were dissolved in DMSO(100 μL) at 37 °C for 10 min with gentle agitation. The absorbanceper well was measured at 540 nm using a VERSA max MicroplateReader (Molecular Devices Corp., USA). The IC50 is defined as thecompound concentration required for the inhibition of cell prolifer-ation by 50 % in comparison with cells treated with the maximumamount of DMSO (0.025 %), considered as 100 % viability.

Experimental Procedure for the Detection of ROS Generation:The formation of intracellular ROS was assayed by the fluorescenceemission of 2′,7′-dichlorofluorescin diacetate (DCFDA). This pene-trates into the cytoplasm of the cell, where it is hydrolysed by intra-cellular esterase and oxidized to form 2′,7′-dichlorofluorescein (DCF)by ROS. For the assay, Caki-1 cells were seeded in a cover-glass-bottomed dish at a density of 2 × 104 cells. The Caki-1 cells were

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pretreated, irradiated, washed twice with Dulbecco's phosphate-buffered saline (DPBS), and loaded with DCFDA solution (10 mM)dissolved in Dulbecco's modified Eagle medium (DMEM; withoutphenol red) at 37 °C in the dark for 30 min. The cells were subse-quently washed twice with DPBS to remove extracellular dye. Thequantification of the fluorescence intensity was carried out using aGuava EasyCyte Plus flow cytometer.

Acknowledgments

This work was supported by a National Research Foundation ofKorea (NRF) grant funded by the Korea government (MSIP) (nos.2016R1A4A1011189 and 2017R1A2B2004786).

Keywords: Cancer · Azo compounds · C-H activation ·Indoles · Nitrogen heterocycles · Alkenes

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Received: July 17, 2017

synthesis and anticancer evaluation of 2,3‐disubstituted indoles...

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