A series of [1,2,4]triazino[4,3-a]indoles was prepared in good yield by reacting 2-diazo-3-ethoxycarbonylindole with methylene active compounds. Derivatives of the title ring system were tested against a panel of 60 human tumor cell lines, and showed inhibitory activity against a wide range of cancer cell lines at micromolar concentration

ALMERICO, Anna Maria

BARRAJA, Paola

CIRRINCIONE, Girolamo

DATTOLO, Gaetano

DIANA, Patrizia

LAURIA, Antonino

MONTALBANO, Alessandra

English

Archivio istituzionale della ricerca - Università di Palermo

Abstract. A series of [1,2,4]triazino[4,3-a]indoles was preparedin good yield by reacting 2-diazo-3-ethoxycarbonylindole withmethylene active compounds. Derivatives of the title ring systemwere tested against a panel of 60 human tumor cell lines, andshowed inhibitory activity against a wide range of cancer cell linesat micromolar concentration. As part of our ongoing program directed towards the design,synthesis and evaluation of novel compounds of biologicalinterest containing the pyrrole and the indole moieties, we havereported the synthesis of 2-diazopyrroles (1) and 2-diazoindoles(2) which are versatile key intermediates for the synthesis ofbiologically active compounds. In fact we synthesized 2-triazenopyrroles and 2-triazenoindoles by reaction of thecorresponding diazo compounds with secondary amines. Suchcompounds can be considered deaza analogues of dacarbazine,a triazenoimidazole derivative used in therapy as the singlemost active drug available for the treatment of malignantmelanoma (3). Biological screenings of both classes ofderivatives gave quite interesting results, especially those of thepyrrole series. In fact one of them displayed cytotoxicity againstleukemia, lymphoma and carcinoma cell lines at micromolarlevel (3.9-21.2) (4), while those of the indole series were lessactive with GI50 in the range 23-99 ÌM (5).By reacting 2-diazopyrroles with aryl or alkyl isocyanates,we also obtained several derivatives of the new ring systempyrrolo[2,1-d][1,2,3,5]tetrazine (6), a deaza analogue of theantitumor drug temozolomide, today on the market underthe trade name TEMODAL® and used against malignantmelanoma, mycosis fungoides and brain tumors (7). Thesynthesised pyrrolotetrazinones, tested in vitro against apanel of 60 human cancer cell lines, showed potentantiproliferative activity in the low micromolar orsubmicromolar range, some of them reaching GI50 up tonanomolar concentrations (8). Furthermore, upon reaction with the sodium salts of ‚-diketones, ‚-carbonitriles, ‚-dinitriles and ‚-ketoesters, 2-diazopyrroles afforded derivatives of the new ring systempyrrolo[2,1-c][1,2,4] triazine that inhibited the growth of awide range of human cancer cell lines at micromolarconcentration (9). The title heterocyclic system contains the1,2,4-triazine moiety, to which a wide range of biologicalproperties such as antibacterial, antibiotic, antiviral andantitumor have been ascribed (10). In particular,pyrazolo[5,1-c][1,2,4]triazine derivatives exhibitedantineoplastic activity against the mouse sarcoma 180 andmethylcolanthrene-induced rat sarcoma (11).Encouraged by these results and considering the greatversatility of 2-diazoindoles, we thought it was interesting toverify whether the condensation of the 1,2,4-triazine moietywith the indole system increases the biological activity.In this paper, we report a simple synthesis of the[1,2,4]triazino[4,3-a]indole system based on the reaction of2-diazoindole of type 1, obtained by diazotization of 2-aminoindoles according to a method recently reported, (2)with methylene active compounds. The reactions werecarried out in absolute ethanol at room temperature for 24hours, with the sodium salts of type 2a-d prepared in situwith sodium ethoxide in absolute ethanol. The primarycoupling products 3a-d, which are analogous with similarcompounds in the azole series (12) and exist in the hydrazoform, were not isolated as they spontaneously cyclize to thedesired [1,2,4]triazino[4,3-a]indoles in good yields (72-80%)(Figure 1). The structure of [1,2,4]triazino[4,3-a]indoles 4a-d was confirmed by IR and NMR data as well as elementalanalysis. An evaluation of the spectroscopic data led to the3775An account of this work was presented at the Italian-Hungarian-Polish Joint Meeting on Medicinal Chemistry. Giardini Naxos(Italy) Sept 28-Oct 1, 1999. Abstract p. 106.Correspondence to: Prof. Girolamo Cirrincione, DipartimentoFarmacochimico Tossicologico e Biologico, Via Archirafi 32, 90123Palermo, Italy. Tel: +390916161606, Fax: +390916169999, e-mailgcirrinc@unipa.itKey Words: 2-Diazoindoles, triazinoindoles, antiproliferativeactivity.ANTICANCER RESEARCH 24: 3775-3780 (2004)Synthesis and Antiproliferative Activityof [1,2,4]Triazino[4,3-a]indolesPAOLA BARRAJA, PATRIZIA DIANA, ANTONINO LAURIA, ALESSANDRA MONTALBANO, ANNA MARIA ALMERICO, GAETANO DATTOLO and GIROLAMO CIRRINCIONEDipartimento Farmacochimico Tossicologico e Biologico, Università di Palermo, Palermo 90123, Italy0250-7005/2004 $2.00+.40ANTICANCER RESEARCH 24: 3775-3780 (2004)3776Figure 1. Synthetic pathway for derivatives 4.Figure 2. Hydrogen bondings in compounds 4.conclusion that compounds 4a,c,d exist in the imino form.Such a form is stabilized by the hydrogen bonding betweenthe hydrogen on the N-1 and the ethoxycarbonylfunctionality on position 10 of the triazinoindole ringsystem. In the case of compounds 4c and 4d, an additionalhydrogen bonding of the imino hydrogen with the adjacentcarboxamido and ethoxycarbonyl moieties respectively,further stabilizes the compounds in such a form (Figure 2).On the other hand, in the azolo[1,2,4]triazine series, it hasalready been observed that hydrogen bonding determinedthe tautomer populations of the bicyclic systems (13).In the case of compound 4b, the presence of the twohydrogen bonds, analogous to those present in compounds4c and 4d, stabilizes the hydrate form which can be isolated,giving rise to a full aromatic system.Materials and MethodsChemistry. All melting points were taken on a Buchi-Tottolicapillary apparatus and are uncorrected; IR spectra weredetermined in bromoform with a Jasco FT/IR 5300spectrophotometer; 1H and 13C NMR spectra were measured at200 and 50.3 MHz, respectively, in DMSO-d6 (TMS as internalreference), using a Bruker AC-E series 200 MHz spectrometer.Column chromatography was performed with a BIOTAGEFLASH40i chromatography module (prepacked polyethylenecartridge system). For all new compounds, elemental analyses werewithin ± 0.4% of theoretical values.Preparation of 2-diazo-3-ethoxycarbonylindole (1). According to theprocedure described previously (2), to a stirred solution of 2-amino-3-ethoxycarbonylindole (10 mmol) in acetic acid (80%, 10 mL) at0ÆC, a solution of sodium nitrite (10 mmol) in the minimumamount of water (2 mL) was added under a nitrogen atmosphereand in the dark. The reaction mixture was stirred for 3 hours andneutralised with an aqueous solution of sodium carbonate (10%),keeping the temperature at 0ÆC. The brown solid separated wasfiltered, dried in the dessicator under vacuum and in the dark. Thecrude product was quickly washed with cyclohexane to give onewhich furnished an IR spectrum identical to an authentic sample.General method for the preparation of [1,2,4]triazino[4,3-a]indoles 4a-d. To a solution of active methylene compounds 2a-d (2 mmol) inanhydrous ethanol (50 mL), a solution of sodium ethoxide (2 mL,1N) in anhydrous ethanol (10 mL) was added dropwise. The reactionmixture was stirred at 0ÆC for 30 minutes then a solution of 2-diazo-3-ethoxycarbonylindole 1 (2 mmol) in anhydrous ethanol was addeddropwise with stirring at 0ÆC. The mixture was stirred at roomtemperature for an additional 24 hours, after which the solvent wasremoved under reduced pressure, the residue taken up with water,filtered and air dried. The crude [1,2,4]triazino[4,3-a]indoles werepurified by chromatography using dichloromethane: ethyl acetate(9:1) as eluant, and recrystallized from ethanol.Ethyl 3-cyano-4-imino-1,4-dihydro[1,2,4]triazino[4,3-a]indole-10-carboxylate (4a), (yield 80%), mp >320ÆC; ir: 3296 (NH), 3209(NH), 2229 (CN), 1675 (CO) cm-1; 1H nmr (ppm): 1.39 (3H, t,J=6.8 Hz, CH3), 4.41 (2H, q, J=6.8 Hz, CH2), 7.37 (1H, t, J=7.5Hz, H-7), 7.48 (1H, t, J=7.5 Hz, H-8), 8.10 (1H, d, J=7.5 Hz, H-6),8.73 (1H, d, J=7.5 Hz, H-9), 8.80 (1H, vbs, C=NH), 11.50 (1H, vbs,NH); 13C nmr (ppm): 14.1 (q), 59.8 (t), 90.3 (s), 113.9 (s), 116.5(d), 119.7 (d), 123.0 (d), 123.4 (s), 125.4 (d), 126.1 (s), 129.3 (s),138.3 (s), 143.9 (s), 163.1 (s).Ethyl 3-acetyl-4-hydroxy-4-methyl-1,4-dihydro[1,2,4]triazino[4,3-a]indole-10-carboxylate (4b), (yield 72%), mp 174-176ÆC; ir: 3449(OH), 3284 (NH), 1671 (CO), 1614 (CO) cm-1; 1H nmr (ppm): 1.27(3H, t, J=7.0 Hz, CH3), 2.02 (3H, s, CH3), 2.34 (3H, s, CH3), 4.26(2H, q, J=7.0 Hz, CH2), 7.05 (1H, t, J=7.8 Hz, H-7), 7.12 (1H, t,J=7.8 Hz, H-8), 7.37 (1H, s, OH), 7.69 (1H, d, J=7.8 Hz, H-6),7.83 (1H, d, J=7.8 Hz, H-9), 11.70 (1H, s, NH);13C nmr (ppm):14.7 (q), 25.1 (q), 26.6 (q), 59.0 (t), 78.8 (s), 85.0 (s), 113.6 (d),119.7 (d), 121.2 (d), 122.4 (d), 126.0 (s), 130.4 (s), 137.1 (s), 140.1(s), 163.9 (s), 194.7 (s).Ethyl 3-(aminocarbonyl)-4-imino-1,4-dihydro[1,2,4]triazino[4,3-a]indole-10-carboxylate (4c), (yield 80%), mp >320ÆC; ir: 3477(NH), 3257-3165 (NH and NH2), 1676 (CO), 1607 (CO) cm-1; 1Hnmr (ppm): 1.51 (3H, t, J=7.7 Hz, CH3), 4.51 (2H, q, J=7.7 Hz,CH2), 7.45 (1H, t, J=7.7 Hz, H-7), 7.58 (1H, t, J=7.7 Hz, H-8),7.90 (1H, bs, NH), 8.05 (1H, bs, NH), 8.25 (1H, d, J=7.7 Hz, H-6),8.90 (1H, d, J=7.7 Hz, H-9), 10.70 (1H, vbs, C=NH), 12.01 (1H,vbs, NH); 13C nmr (ppm): 14.1 (q), 59.0 (t), 116.3 (d), 119.1 (d),121.7 (d), 124.1 (s), 124.7 (d), 126.4 (s), 128.9 (s), 139.9 (s), 144.8(2xs), 163.0 (s), 166.0 (s).Diethyl 4-imino-1,4-dihydro[1,2,4]triazino[4,3-a]indole-3,10-dicarboxylate(4d), (yield 75%), mp >320ÆC; ir: 3341 (NH), 3301 (NH), 1697 (CO),1674 (CO) cm-1; 1H nmr (ppm): 1.31 (3H, t, J=6.5 Hz, CH3), 1.34 (3H,t, J=7.4 Hz, CH3), 4.31 (2H, q, J=6.5 Hz, CH2), 4.38 (2H, q, J=7.4Hz, CH2), 7.30 (1H, t, J=8.3 Hz, H-7), 7.42 (1H, t, J=8.3 Hz, H-8),8.08 (1H, d, J=8.3 Hz, H-6), 8.87 (1H, d, J=8.3 Hz, H-9), 9.40 (1H,vbs, C=NH), 10.00 (1H, vbs, NH);13C nmr (ppm): 14.0 (q), 14.6 (q),59.4 (t), 61.2 (t), 116.8 (s), 117.2 (d), 119.3 (d), 122.2 (d), 124.2 (s),124.8 (d), 126.4 (s), 130.2 (s), 145.7 (2 x s), 163.0 (s), 163.8 (s).Results and DiscussionThe triazinoindoles 4c and 4d were selected by NCI andtheir cytotoxicity was evaluated in in vitro disease-orientedantitumor screenings (14) against a panel of 60 humantumor cell lines derived from leukemia, non-small lungcancer, colon cancer, CNS cancer, melanoma, ovariancancer, renal cancer, prostate cancer and breast cancer. Thetest compounds were evaluated using five concentrations at10-fold dilutions, the highest being 10-4 M and the others10-5, 10-6, 10-7 and 10-8 M. The results obtained are shownin Table I, taking into consideration the growth inhibitorypower (GI50).Most of the susceptible tumors were generally inhibitedat micromolar concentration, expecially for the triazine 4dbearing an ethoxycarbonyl functionality in position 10,which generally resulted more active than 4c bearing acarbamoyl group in the same position. Compound 4d gavethe best result in the leukemia and melanoma subpanels,with GI50 values in the low micromolar range, 1.12-6.91 andBarraja et al: Synthesis and Antiproliferative Activity of [1,2,4]Triazino[4,3-a]indoles37772.09-5.97, respectively. The most sensitive cell lines were K-562 (1.12 ÌM) and RPMI-8226 (1.54 ÌM) of the formersubpanel; UACC-257 (2.09 ÌM) and MALME-3M (2.10ÌM) belong to the latter subpanel. Moreover, in someovarian cancer cell lines, IGROV1, OVCAR-3, OVCAR-5,OVCAR-8 and CNS cancer cell lines, SF539, SNB19 andU251, good values of GI50 were observed: 1.12-6.03 ÌM and4.93-5.66 ÌM, respectively. Compound 4c showedremarkable inhibitory activity only in the case of the CNSsubpanel, with the exception of the SF268 cell line, showingGI50 in the range 1.17-3.31 ÌM, and the ovarian cancer cellline IGROV1 with GI50 1.33 ÌM. In the other cell linescompound 4c, with some exceptions when the potency wasat the same level as compound 4d, was one magnitude orderless active and, in the case of the leukemia and in most ofthe colon cancer cell lines, not active at all.In order to discern the mechanism of action ofpyrrolotriazines, we performed COMPARE computations(15) on the NCI screening data, using [1,2,4]triazino[4,3-a]indoles 4c,d as seed compounds. Both had a PearsonCorrelation Coefficient <0.5 (data not shown) against the"Standard Agents" database, suggesting that they probably actwith a different mechanism from those of the Standard Agentsand make this class of compounds worthy of considerableattention. It is our intention to undertake studies directed atelucidating their biochemical mechanism of action.ANTICANCER RESEARCH 24: 3775-3780 (2004)3778Table I. Inhibition of in vitro tumor cell growth by triazinoindoles 4c,dGI50 (ÌM)a,b GI50 (ÌM)a,bCell lines 4c 4d Cell lines 4c 4dLeukemia MelanomaCCRF-CEM  >100 2.22 LOX IMVI 28.3 5.96HL-60(TB) >100 2.15 MALME-3M 70.9 2.10K-562  >100 1.12 M14 >100 5.97MOLT-4 >100 4.40 SK-MEL-2 20.5 5.15RPMI-8226 >100 1.54 SK-MEL-28 >100 5.85SR >100 6.91 SK-MEL-5 74.3 5.53Non-small cell lung UACC-257 99.7 2.09A549/ATCC >100 10.5 UACC-62 19.9 2.42EKVX 18.0 25.8 Ovarian cancerHOP-62 17.3 68.8 IGROV1 1.33 1.12NCI-H23 16.7 5.94 OVCAR-3 >100 5.20NCI-H322M 62.4 14.5 OVCAR-4 >100 >100NCI-H460 50.3 8.13 OVCAR-5 16.1 5.16NCI-H522 28.8 6.21 OVCAR-8 13.9 6.03Colon cancer SK-OV-3 21.9 67.0COLO 205 >100 10.2 Renal cancerHCC-2998 >100 34.0 786-0 21.0 4.85HCT-116 31.1 29.3 A498 6.07 7.19HCT-15 79.0 7.66 ACHN 32.8 7.49HT29 >100 22.7 CAKI-1 16.1 >100KM12 >100 4.43 RXF 393 >100 6.66SW-620 >100 8.26 SN12C 11.3 7.90CNS cancer  TK-10 74.3 7.87SF-268 16.0 19.3 UO-31 59.9 5.35SF-295 3.31 26.9 Breast cancerSF-539 1.62 5.66 MCF7 17.6 13.1SNB-19 1.93 4.93 NCI/ADR-RES 35.3 3.62SNB-75 1.17 11.0 MDA-MB231/ATCC 18.7 19.8U251 1.52 5.41 HS 578T 4.36 7.16Prostate cancer MDA-MB-435 4.36 5.04PC-3 59.9 53.5 MDA-N >100 4.35DU-145 >100 37.4 BT-549 22.1 13.7T-47D 43.0 30.0aData obtained from NCI’s in vitro disease-oriented tumor cells screen. bGI50 is the molar concentration causing 50% growth inhibition of tumor cells. Compounds with GI50 >100 ÌM are considered inactive. AcknowledgementsThis work was in part financially supported by Ministerodell’Istruzione dell'Università e della Ricerca, Italy.References1 Cirrincione G, Almerico AM, Diana P, Grimaudo S, Barraja P,Mingoia F, Dattolo G and Aiello E: 2-Diazopyrroles: synthesisand antileukemic activity. Il Farmaco 51: 275-277, 1996.2 Barraja P, Diana P, Lauria A, Almerico AM, Dattolo G andCirrincione G: 2-Diazo-2H-indoles. Helv Chim Acta 84: 2212-2219, 2001. 3 a) Lucas US and Huang AT: Clinical management ofmelanoma. In: Development in Oncology; Siegler HF ed;Martinus Nijhoff: The Hague 5: 382, 1982; b) Viviani S,Bonadonna G, Santoro A, Bonfante V, Zanin M, Devizzi L,Soncini F and Valagussa P: Alternating versus hybrid MOPPand ABVD combinations in advanced Hodgkin’s disease: tenyear results. 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Part22. Synthesis and reactions of imidazo[5,1-c][1,2,4]triazines. JChem Soc Perkin I: 1424-32, 1981.14 a) Grever MR Sherpatz SA and Chabner BA: The NationalCancer Institute: cancer drug discovery and developmentprogram. Semin Oncol 19: 622-638, 1992; b) Monks A, ScudieroD, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C,Langely J, Cronise P, Vaigro-Woiff A, Gray-Goodrich M,Campbell H, Mayo J and Boyd MR: Feasibility of a high-fluxanticancer drug screen using a diverse panel cultured humantunor cell lines. J Natl Cancer Inst 83: 757-766, 1991; c) BoydNR, Paull KD and Rubinstein LV: Data display and analysisstrategies for the new NCI disease-oriented in-vitro anti-tumordrug screen. In: Cytotoxic Anticancer Drugs: Models andConcepts for Drug Discovery and Development; Valeriote FA,Corbett TH and Baker LH eds., Kluwer Academic Publ.,Norwell, Mass., 11-34, 1992.15 Paull K, Shoemaker R, Hodes L, Monks A, Scudiero D,Rubistein L, Plowman J and Boyd MR: Display and analysis ofpatterns of differential activity of drugs against human tumorcell lines: development of mean graph and COMPAREalgorithm. J Natl Cancer Inst 81: 1088-1092, 1989.Received March 17, 2004Revised September 6, 2004Accepted October 13, 2004Barraja et al: Synthesis and Antiproliferative Activity of [1,2,4]Triazino[4,3-a]indoles3779

Synthesis and Antiproliferative Activity of [1,2,4]triazino[4,3-a]indoles

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Synthesis and Antiproliferative Activity of [1,2,4]triazino[4,3-a]indoles

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