Dimethoxybenzo[i]phenanthridine-12-carboxylic acid derivatives and 6H-dibenzo[c,h][2,6]naphthyridin-5-ones with potent topoisomerase I-targeting activity and cytotoxicity

The exceptional TOP1-targeting activity and antitumor activity of ARC-111, 1, prompted studies on similarly substituted benzo[i]phenanthridine-12-carboxylic ester and amide derivatives. These studies were extended to include 6-substituted 8,9-dimethoxy-2,3-methylenedioxy-dibenzo[c,h][2,6]naphthyridin-5-ones, which represent reversed lactam analogues of 1. Several of these analogues retained the potent TOP1-targeting activity and cytotoxicity observed for ARC-111.     

5H-Dibenzo[c,h]1,6-naphthyridin-6-ones: novel topoisomerase I-targeting anticancer agents with potent cytotoxic activity

5H-Dibenzo[c,h]1,6-naphthyridine-6-ones can exhibit potent antitumor activity. The effect of varied substituents at the 5-position of 5H-8,9-dimethoxy-2,3-methylenedioxydibenzo[c,h]1,6-naphthyridine on relative cytotoxicity and topoisomerase I-targeting activity was evaluated. Potent TOP-1-targeting activity is observed when the 5-position is substituted with either a 2-(N,N-dimethylamino)ethyl group, as in 3a, or a 2-(pyrrolidin-1-yl)ethyl substituent, 3c. In contrast, the addition of a beta-methyl group or a beta-hydroxymethyl group to compound 3a, as in 3b and 3j, results in a loss of significant TOP1-targeting activity. While the presence of a 3-(N,N-dimethylamino)propyl substituent at the 5-position or a methyl(2-tetrahydrofuranyl) group allows for retention of TOP1-targeting activity, the 2-(4-methyl-1-piperazinyl)ethyl analogue, 3d, did not exhibit significant activity. Replacement of the N,N-dimethylamino group of 3a with either C(2)H(5) or OH, as in 3f and 3h, respectively, also had a negative impact on both cytotoxicity and TOP1-targeting activity. Treatment of 3a with LAH gave the 5,6-dihydrodibenzo[c,h]naphthyridine, 4a. This dihydro derivative has approximately 2/3 the potency of 3a as a TOP1-targeting agent. Compounds 3a, 3b, 3h, 3i, and 4a were evaluated for antitumor activity in the human tumor xenograft model using athymic nude mice. The non-estrogen responsive breast tumor cell line, MDA-MB-435, was used in these assays. Compound 3a proved to be effective in regressing tumor growth in vivo when administered either by ip injection or orally 3x week at a dose of 2.0mg/kg. Compound 4a when administered orally 5x weekly at a dose of 40 mg/kg also suppressed tumor growth.     

Nitro and amino substitution within the A-ring of 5H-8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)dibenzo[c,h][1,6]naphthyridin-6-ones: influence on topoisomerase I-targeting activity and cytotoxicity

Recently, 5H-8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-one, 1, was identified as a TOP1-targeting agent with pronounced antitumor activity. In the present study, the effect on activity of substituting a single nitro or amino group in the A-ring in lieu of the methylenedioxy moiety of 1 was evaluated. The presence of either a nitro or amino substituent at the 4-position had a pronounced adverse affect on both TOP1-targeting activity and cytotoxicity. To a lesser extent, derivatives with a nitro or amino substituent at the 1-position were also less active than 1. Replacement of the methylenedioxy moiety of 1 with either a nitro or amino substituent at either the 2- and 3-position did result in analogues with potent TOP1-targeting activity and cytotoxicity.     

Substituted dibenzo[c,h]cinnolines: topoisomerase I-targeting anticancer agents

Several substituted dibenzo[c,h]cinnolines were synthesized and evaluated for their potential to target topoisomerase I and for their relative cytotoxic activity. Select benzo[i]phenanthridines are capable of stabilizing the cleavable complex formed with topoisomerase I and DNA. This study was initiated to examine whether dibenzo[c,h]cinnolines, which are in essence aza analogues of benzo[i]phenanthridines, possess similar pharmacological properties. 2,3-Dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine is one of the more potent benzo[i]phenanthridine derivatives in regard to topoisomerase I-targeting activity and cytotoxicity. The structure-activity relationship observed with these substituted dibenzo[c,h]cinnolines parallels that observed for benzo[i]phenanthridine derivatives. Compared to similarly substituted benzo[i]phenanthridines, the dibenzo[c,h]cinnoline analogues exhibit more potent topoisomerase I-targeting activity and cytotoxicity. The relative IC(50) values obtained in assessing the cytotoxicity of 2,3-dimethoxy-8,9-methylenedioxydibenzo[c,h]cinnoline and 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine in the human lymphoblastma cell line, RPMI8402, are 70 and 400 nM, respectively. In tumor cell lines selected for resistance to camptothecin and known to express mutant topoisomerase I, benzo[i]phenanthridine derivatives were not cross-resistant. In contrast, similarly substituted dibenzo[c,h]cinnolines with significant topoisomerase I-targeting activity did exhibit cross-resistance in these camptothecin-resistant cell lines. The cytotoxicity of these dibenzo[c,h]cinnolines was not diminished in cells overexpressing the efflux transporter, MDR1. These data indicate that substituted dibenzo[c,h]cinnolines can exhibit potent topoisomerase I-targeting activity and are capable of overcoming the multi-drug resistance associated with this efflux transporter.     

11H-Isoquino[4,3-c]cinnolin-12-ones; novel anticancer agents with potent topoisomerase I-targeting activity and cytotoxicity

Recent studies have identified 2,3-dimethoxy-8,9-methylenedioxy-11-[(2-dimethylamino)ethyl]-11H-isoquino[4,3-c]cinnolin-12-one (1a) as a novel topoisomerase I-targeting agent with potent cytotoxic activity. The effect of varied substituents at the 11-position of 2,3-dimethoxy-8,9-methylenedioxy-11H-isoquino[4,3-c]cinnolin-12-ones on topoisomerase I-targeting activity and cytotoxicity was evaluated. Potent TOP1-targeting activity was observed when the 11-position was substituted with either a 2-(N,N-dimethylamino)ethyl, a 2-(N,N-diethylamino)ethyl, a n-butyl, or a 2-(pyrrolidin-1-yl)ethyl group. The addition of a beta-methyl group to 1a provided an analogue with dramatically reduced TOP1-targeting activity and cytotoxicity. Analogues of 1a wherein the 2-(N,N-dimethylamino)ethyl group was replaced with a (2-tetrahydrofuranyl)methyl, a 2-(piperidin-1-yl)ethyl, or a 2-(4-methylpiperazin-1-yl)ethyl substituent exhibited decreased activity as TOP1-targeting agents. Replacement of the dimethoxy groups of 1a with hydrogen atoms resulted in an analogue with significantly decreased TOP1-targeting activity and cytotoxicity. Removal of both the vicinal dimethoxyl groups and the methylenedioxy moiety resulted in a complete loss of TOP1-targeting activity. The presence of a 9-nitro substituent in place of the 8,9-methylenedioxy group of 1a resulted in a decrease in relative TOP1-targeting activity and cytotoxicity. Compounds 1a and the 11-n-butyl analogue 1d were evaluated for antitumor activity in the human tumor xenograft model using athymic nude mice. The non-estrogen responsive breast tumor cell line MDA-MB-435 was used in these assays. At dose levels that approached its maximum tolerated dose, 1a proved to be effective in inhibiting tumor growth in vivo when administered orally or by ip injection.     

8,9-methylenedioxybenzo[i]phenanthridines: topoisomerase I-targeting activity and cytotoxicity

Substituted benzo[i]phenanthridines that have incorporated within their structure an 8,9-methylenedioxy group can exhibit topoisomerase I-targeting activity. Structure-activity studies were performed to examine the influence of saturation at the 11,12-positions of several substituted 8,9-methylenedioxybenzo[i]phenanthridines. The activities of these dihydro analogues were compared to those of their unsaturated analogues. In addition, the influence of varying substituents at the 2- and 3-positions within the A-ring of these 8,9-methylenedioxybenzo[i]phenanthridines on their relative potency as topoisomerase I-targeting agents and cell proliferation as determined using the MTT assay was investigated. 2,3-Dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine and its 11,12-dihydro derivative were among the more potent analogues evaluated with regard to topoisomerase I-targeting activity and cytotoxicity.     

Synthesis of N-substituted 5-[2-(N-alkylamino)ethyl]dibenzo[c,h][1,6]naphthyridines as novel topoisomerase I-targeting antitumor agents

Several N-alkyl and N,N-dialkyl 5H-8,9-dimethoxy-5-(2-aminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-ones have been identified as topoisomerase I-targeting agents with potent antitumor activity. In the present study, the impact on biological activity of substitution of a trifluoromethyl, cyano, aminocarbonyl, or ethynyl group on a N-methyl substituent of N,N-dimethyl-, N-methyl-N-ethyl-, and N-methyl-N-isopropyl 5H-8,9-dimethoxy-5-(2-aminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-ones was assessed.     

Facile formation of hydrophilic derivatives of 5H-8,9-dimethoxy-5-[2-(N,N-dimethylamino)ethyl]-2,3-methylenedioxydibenzo[c,h] [1,6]naphthyridin-6-one (ARC-111) and its 12-aza analog via quaternary ammonium intermediates

Several new TOP1-targeting agents were prepared using as intermediates the N,N,N-trimethyl quaternary ammonium salts of either ARC-111 or its 12-aza analog (ARC-31), 3 and 4, respectively. Direct displacement of the quaternary ammonium group with water, imidazole, alkylethylenediamines, or polyhydroxylated alkylamines provides a convenient means for furthering the structure-activity relationships associated with these non-camptothecin TOP1-targeting agents.     

SAR and evaluation of novel 5H-benzo[c][1,8]naphthyridin-6-one analogs as Aurora kinase inhibitors

Several potent Aurora kinase inhibitors derived from 5H-benzo[c][1,8]naphthyridin-6-one scaffold were identified. A crystal structure of Aurora kinase A in complex with an initial hit revealed a binding mode of the inhibitor within the ATP binding site and provided insight for structure-guided compound optimization. Subsequent SAR campaign provided a potent and selective pan Aurora inhibitor, which demonstrated potent target modulation and antiproliferative effects in the pancreatic cell line, MIAPaCa-2. Furthermore, this compound inhibited phosphorylation of histone H3 (pHH3) in mouse bone morrow upon oral administration, which is consistent with inhibition of Aurora kinase B activity.     

5H-8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)dibenzo[c,h][1,6]naphthyridin-6-ones and related compounds as TOP1-targeting agents: influence of structure on the ternary cleavable complex formation

In this paper, we present our results from a docking study of the title compounds with the DNA/topoisomerase I complex based on the recently published X-ray crystal structure of the topotecan/DNA/topoisomerase I ternary cleavable complex (Staker, B.L., et al. PNAS 2002, 99, 15387) using the Autodock program. Simple intermolecular docking energies (E(dock)) correlate well with in vitro DNA cleavage data suggesting that the binding mode from the crystal structure is a reasonable binding mode for these compounds.     

Design, synthesis and anticancer activity of novel dihydrobenzofuro[4,5-b][1,8]naphthyridin-6-one derivatives

On the basis of the chemical structures of psorospermin with a xanthone template and acronycine derivatives with an acridone template, rac-1 and rac-2 constructed on an 1,2-dihydrobenzofuro[4,5-b][1,8]naphthyridin-6(11H)-one scaffold were designed and synthesized as potential anticancer agents. Their anticancer activities were evaluated against five human cancer cell lines. Rac-2 showed similar anticancer activity to doxorubicin and rac-1 exhibited even higher anticancer activity against LNCaP (IC(50)=0.14 μM), DU145 (IC(50)=0.15 μM), PC3 (IC(50)=0.30 μM) and MCF-7 (IC(50)=0.26 μM) cancer lines than doxorubicin and rac-2. Also, rac-1 revealed very potent anticancer activity (IC(50)=0.15 μM) against MCF-7/ADR cell (doxorubicin-resistant breast cancer cell) lines and induced G2/M phase arrest of the cell cycle in MCF-7/ADR cells.     

Thiopyrano[2,3-e]indol-2-ones: angelicin heteroanalogues with potent photoantiproliferative activity

A new class of compounds, the thiopyrano[2,3-e]indol-2-ones, bioisosters of the angular furocoumarin angelicin, was synthesized with the aim of obtaining new photochemotherapeutic agents. In particular 7,8-dimethyl-thiopyranoindolone 6c s showed a remarkable phototoxicity and a great dose UVA dependence reaching IC(50) values at submicromolar level. This latter photoinduced a massive apoptosis and a remarkable photodamage to lipids and proteins. Although it did not intercalate DNA, it was able to cause photooxidation of DNA bases.     

Synthesis,anti-thymidine phosphorylase activity and molecular docking of 5-thioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-ones

In our lead finding program, a series of 5-thioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-ones and their 5-thio-alkyl derivatives were designed and synthesized which contained different substituents at ortho-position of 2-phenyl ring attached to the fused ring structure. The preliminary pharmacological evaluation demonstrated that the synthesized compounds exhibited a varying degree of inhibitory activity towards thymidine phosphorylase (TP), comparable to reference compound, 7-Deazaxanthine (7-DX, 2) (IC₅₀ value = 42.63 μM). The study also inferred that the ortho-substituted group at the phenyl ring and 5-thio-alkyl moiety imparted steric hindrance effects in the binding site of the enzyme, leading to a reduced inhibitory response. In addition, compound 3a was identified as a mixed-type inhibitor of TP. Moreover, computational docking study was performed to illustrate the important structural information on the plausible ligand-enzyme binding interactions.     

Pyrrolo[2,1-d][1,2,3,5]tetrazine-4(3H)-ones,a new class of azolotetrazines with potent antitumor activity

Pyrrolo[2,1-d][1,2,3,5]tetrazinones 10a-o, compounds that hold the deaza skeleton of the antitumor drug temozolomide, were prepared by reaction of 2-diazopyrroles 9 and isocyanates. Such a synthetic route represents, among those leading to azolotetrazinones reported so far, the only possible one since attempts to cyclize to the title ring system 2-amino-1-carbamoylpyrroles 11 or the mono substituted 2-triazenopyrrole 12 failed. Compounds 10 were screened at the National Cancer Institute (NCI) for their activity against a panel of about 60 human tumor cell lines. Most of them possess remarkable antineoplastic activity having GI(50) values in the low micromolar or sub-micromolar range and reaching, in the case of compound 10d, nanomolar concentrations. The most sensitive cell lines were MDA-N and MDA-MB-435 of the breast sub-panel, and SR, K-562, HL60 (TB) and CCRF-CEM of the leukaemia sub-panel. SAR evaluation and COMPARE computations indicate, for compounds 10, a mechanism of action different from that of temozolomide.     

7-(4H-1,2,4-Triazol-3-yl)benzo[c][2,6]naphthyridines: a novel class of Pim kinase inhibitors with potent cell antiproliferative activity

A novel class of pan-Pim kinase inhibitors was designed by modifying the CK2 inhibitor CX-4945. Introduction of a triazole or secondary amide functionality on the C-7 position and 2'-halogenoanilines on C-5 resulted in potent inhibitors of the Pim-1 and Pim-2 isoforms, with many analogs active at single digit nanomolar concentrations. The molecules inhibited the phosphorylation at Serine 112 of the apoptosis effector BAD, and had potent antiproliferative effects on the AML cell line MV-4-11 (IC(50) <30 nM). This work delivers an excellent lead-optimization platform for Pim targeting anticancer therapies.     

Chemical modification of apomorphine to discover sigma ligands: 6H-dibenzo[b,d]pyran and carbazole analogues.

It seems that many sigma ligands have been designed from known sigma ligands. We focused on a difference in structural flexibility between haloperidol and apomorphine, and studied chemical modification of apomorphine, a compound with high affinity for dopamine D2 receptors but not for sigma receptors, for discovery of sigma ligands. The first modification yielded good results with 6H-dibenzo[b,d]pyran analogues with weak affinity for sigma receptors but not D2 receptors. Furthermore, carbazole analogues, compounds designed from 6H-dibenzo[b,d]pyran analogues, potentially acted at sigma receptors with high selectivity. This paper describes the design, synthesis and sigma/D2 selectivity of 6H-dibenzo[b,d]pyran and carbazole analogues.     

Design and synthesis of poly(ADP-ribose)polymerase-1 (PARP-1) inhibitors. Part 3: In vitro evaluation of 1,3,4,5-tetrahydro-benzo[c][1,6]- and [c][1,7]-naphthyridin-6-ones

The 1,3,4,5-tetrahydro-benzo[c][1,6]- and [c][1,7]-napthyridin-6-ones are presented as a potent class of PARP-1 inhibitors. Derivatives of these partially saturated aza-5[H]-phenanthridin-6-ones were designed and synthesized with tertiary amines for salt formation, thus enhancing aqueous solubility, iv formulation and their potential use in acute ischemic injuries (i.e., myocardial ischemia and stroke). We found that partial saturation of the C-ring results in derivatives that are several times more potent than the aromatic C-ring derivatives. The general synthetic routes are presented herein as well as thorough in vitro potencies and SAR discussion for selected derivatives.     

Anticancer thiopyrano[2,3-d][1,3]thiazol-2-ones with norbornane moiety. Synthesis, cytotoxicity, physico-chemical properties, and computational studies

A series of novel 9-substituted-3,7-dithia-5-azatetracyclo[9.2.1.0(2,10).0(4,8)]tetradecen-4(8)-ones-6 have been synthesized by a stereoselective hetero-Diels-Alder reaction of 5-ylidene-4-thioxo-2-thiazolidone derivatives with norbornene-2. All the compounds have been evaluated for antitumor activity in in vitro human tumor cell lines, and 10 of them possessed significant and selective cytotoxicity (MGM logGI50 approximately -4.17 to -4.98, for individual cell lines logGI50 up to -8). COMPARE analyses of differential growth inhibition patterns of compounds at the GI50 level showed high correlations with some of the antitubulin agents. The lipophilicity of the compounds was studied by RP-TLC and found to correlate well with calculated logP values. Docking and structure-activity relationship studies produced seven QSAR models with 2 or 3 variables, with correlation coefficients r2>0.9 and leave-one-out cross-validation correlation coefficients, q2>0.8.     

Lamellarin-inspired potent topoisomerase I inhibitors with the unprecedented benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one scaffold

A new class of topoisomerase I inhibitors containing the unprecedented benzo[g][1]benzopyrano[4,3-b]indol-6(13H)-one (abbreviated as BBPI) ring system have been developed based on structure-activity relationship studies of the cytotoxic marine alkaloid lamellarin D. The pentacyclic BBPI scaffold was constructed from N-tert-butoxycarbonylpyrrole by sequential and regioselective functionalization of the pyrrole core using directed lithiation, conventional electrophilic substitution, and palladium-catalyzed cross-coupling reactions. Further N-alkylation of the scaffold followed by selective deprotection of the O-isopropyl group produced a range of N-substituted BBPI derivatives. The BBPIs thus prepared exhibited potent topoisomerase I inhibitory activity in DNA relaxation assays. The activities of BBPIs were higher than those of lamellarin D and camptothecin; they showed potent and selective antiproliferative activity in the panel of 39 human cancer cell lines established by Japanese Foundation for Cancer Research. COMPARE analyses indicated that the inhibition patterns of the BBPIs correlated well with those of the known topoisomerase I inhibitors such as SN-38 and TAS-103. The water-soluble valine ester derivative exhibited antitumor activity in vivo against murine colon carcinoma colon 26. The activity was comparable to that of the approved anticancer agent irinotecan.     

Synthesis of benzo[3,4]azepino[1,2-b]isoquinolin-9-ones from 3-arylisoquinolines via ring closing metathesis and evaluation of topoisomerase I inhibitory activity, cytotoxicity and docking study

Benzo[3,4]azepino[1,2-b]isoquinolinones were designed and developed as constraint forms of 3-arylisquinolines with an aim to inhibit topoisomerase I (topo I). Ring closing metathesis (RCM) of 3-arylisoquinolines with suitable diene moiety provided seven membered azepine rings of benzoazepinoisoquinolinones. Spectral analyses of these heterocyclic compounds demonstrated that the methylene protons of the azepine rings are nonequivalent. The shielding environment experienced by these geminal hydrogens differs unusually by 2.21ppm. As expected, benzoazepinoisoquinolinones displayed potent cytotoxicity. However, cytotoxic effects of the compounds were not related to topo I inhibition which is explained by non-planar conformation of the rigid compounds incapable of intercalating between DNA base pairs. In contrast, flexible 3-arylisoquinoline 8d attains active conformation at drug target site to exhibit topo I inhibition identical to cytotoxic alkaloid, camptothecin (CPT).