J. Med. Chem. 2007, 50, 4419-4430
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Nitrated Indenoisoquinolines as Topoisomerase I Inhibitors: A Systematic Study and Optimization Andrew Morrell,† Michael Placzek,† Seth Parmley,† Smitha Antony,‡ Thomas S. Dexheimer,‡ Yves Pommier,‡ and Mark Cushman*,† Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, and the Purdue Cancer Center, Purdue UniVersity, West Lafayette, Indiana 47907, and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255 ReceiVed March 27, 2007
The biological activity of indenoisoquinoline topoisomerase I (Top1) inhibitors can be greatly enhanced depending on the choice of substituents on the aromatic rings and lactam side chain. Previously, it was discovered that a 3-nitro group and a 9-methoxy group afforded enhanced biological activity. In the present investigation, indenoisoquinoline analogues were systematically prepared using combinations of nitro groups, methoxy groups, and hydrogen atoms in an effort to understand the contribution of each group toward cytotoxicity and Top1 inhibition. Analysis of the biological results suggests that the nitro group is important for Top1 inhibition and the methoxy group improves cytotoxicity. In addition, previously identified structureactivity relationships were utilized to select favorable lactam side chain functionalities for incorporation on the aromatic skeleton of analogues in this study. As a result, this investigation has provided optimal Top1 inhibitors equipotent to camptothecin that demonstrate low nanomolar cytotoxicities toward cancer cells. Introduction DNA topoisomerases are important for the successful replication, transcription, and recombination of DNA, as well as chromatin remodeling.1-5 Thus, the proliferation and survival of eukaryotic cells are dependent on topoisomerases, which are therefore potential therapeutic targets for anticancer therapy. The cellular role of topoisomerase I is to relieve torsional strain in DNA by creating a single strand nick in the phosphodiester backbone, a process that renders the enzyme covalently ligated to the DNA until strain is relieved.1 Thus, a potential therapeutic agent could inhibit topoisomerase I (Top1) by two distinct pathways: (1) “suppressing” its ability to nick the DNA strand and (2) “poisoning” the enzyme by trapping it as a covalent complex with DNA. The identification of indenoisoquinoline 1 as a novel lead molecule for the inhibition of Top1 resulted from a COMPARE analysis of its cytotoxicity profile demonstrating a similarity to that of the known Top1 inhibitor, camptothecin (2).6 These molecules intercalate between the DNA base pairs at the site of single strand cleavage and effectively stabilize a ternary complex consisting of the drug molecule, DNA, and Top1. This mechanism of action classifies these inhibitors as Top1 poisons. Recently published X-ray crystal structures for camptothecins,7,8 indenoisoquinolines,8,9 and an indolocarbazole8 elegantly demonstrate this mechanism of action and illustrate the general features for poisoning Top1 by intercalation at the cleavage site.10 Clinical validation of Top1 as a drug target has occurred, with the camptothecin derivatives topotecan and irinotecan representing the only Top1 inhibitors currently approved by the U.S. Food and Drug Administration for anticancer therapy.11 However, the camptothecins suffer from pharmacokinetic problems, most notably the hydrolysis of their lactone ring, * To whom correspondence should be addressed. Phone: 765-494-1465. Fax: 765-494-6790. E-mail:
[email protected]. † Purdue University ‡ National Cancer Institute, NIH.
which results in a hydroxy acid product that has high affinity for human serum albumin.12-15 Indenoisoquinoline 1, unlike the camptothecins, is hydrolytically stable but alternatively suffers from intrinsically low biological activity.6 As a result, considerable research has been expended to improve the biological activity of the indenoisoquinolines.9,16-26 Novel insights have been gleaned regarding the contributions of the indenone ring, isoquinoline ring, and lactam side chain toward biological activity.20,22,27-30 Recent efforts have indicated that compounds 3-5 (Figure 1) represent novel lead compounds with potencies comparable to the potencies of camptothecins and with cytotoxicities approximately 1000 times more potent than that of the original lead molecule 1.30 In the present study, second-generation analogues were systematically prepared to examine the contributions of the nitro group and methoxy group toward Top1 inhibition and cytotoxicity. Previously observed structure-activity relationships have indicated that lactam substituents such as amino, morpholino, dimethylamino, ethanolamino, and imidazolyl afford excellent biological activity for unsubstituted, dimethoxy-substituted, and methylenedioxy-substituted indenoisoquinolines.17,20,22,24,26,29,31 Thus, it seemed prudent to select these functionalities for incorporation in the present study along with analogues possessing nitrated and methoxy-substituted aromatic rings. Figure 2 indicates previously synthesized analogues of interest to the present study.17,22,26 Previously, it has been demonstrated that indenoisoquinolines possessing a 3-nitro group display enhanced biological activity, especially when combined with a 9-methoxy group.21,30,32 This enhancement of biological activity has been hypothesized to result from favorable hydrogen-bonding interactions and electrostatic charge complementarity between Top1, the indenoisoquinolines, and the DNA base pairs in the cleavage complex.21,30 However, a systematic examination of the independent effects that these substituents may exert on the biological activity of the indenoisoquinolines has not been conducted. As a result, the current study has been designed in such a way as to improve the current level of understanding regarding the effects of the nitro and methoxy groups on the
10.1021/jm070361q CCC: $37.00 © 2007 American Chemical Society Published on Web 08/15/2007
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Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Figure 1. Representative topoisomerase I inhibitors.
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compound (27, Scheme 2). This procedure was also utilized, as previously described, for the preparation of compounds 6-13.17,22,26 With compounds 20, 24, 25, and 6 appropriately functionalized for analogue synthesis, target compounds were then prepared according to Scheme 3. The alkyl halide side chain present in compound 6 was initially converted into the corresponding alkylazide 28 by treatment with sodium azide in dimethylsulfoxide. Compound 28 was not subjected to reduction (analogous to 22) because the corresponding derivative 7 was previously prepared by an alternative method.22 Furthermore, the corresponding azido- and amino-substituted analogues derived from 20 and 26 have been previously prepared.21,30 Compounds 20, 24, and 25 were treated with morpholine in refluxing 1,4-dioxane to provide target compounds 29-31. Compounds 32-35 were prepared by treatment of 20, 24, 25, and 6 with ethanolamine in DMSO and isolated as their respective hydrochloride salts. Compounds 24 and 25 were treated with dimethylamine in refluxing 1,4-dioxane to afford target compounds 36 and 37, which were isolated as their respective hydrochloride salts. Finally, target compounds 38 and 39 were prepared by treatment of 24 and 25 with imidazole in refluxing 1,4-dioxane. Biological Results and Discussion
Figure 2. Indenoisoquinolines included in the current study.
biological activity of the indenoisoquinolines. Furthermore, the study has been conducted utilizing lactam side chains expected to confer enhanced biological activity to the analogues in an effort to provide new lead molecules for anticancer therapy. Chemistry The syntheses of advanced indenoisoquinoline analogues 20 and 22-25 are described in Scheme 1. Condensation of homophthalic anhydrides 1433 and 15 with Schiff bases 1634 and 17 provided carboxylic acids 18 and 19. The cis stereochemical relationship was established by the observed coupling constant of ∼6 Hz for the two methine protons.35 These carboxylic acids were subjected to oxidative Friedel-Crafts ring closure with thionyl chloride36 and aluminum chloride21,30 to provide indenoisoquinolines 20 and 21. Indenoisoquinoline 3 was readily available from a previous synthetic endeavor.30 It was quickly recognized that the choice of halide leaving group on the lactam side chain was critical for successful SN2 displacement with organic amines such as dimethylamine, ethanolamine, morpholine, and imidazole. Azide, however, was a suitably active nucleophile for displacement of the alkylchloride present in 21 to provide analogue 22, which was subsequently reduced to provide compound 23 upon isolation as the hydrochloride salt. Unsuccessful efforts to displace the alkyl chloride motifs present in compounds 3 and 21 using organic amines as nucleophiles prompted their conversion to the corresponding alkyl iodides 24 and 25, thereby providing advanced intermediates amenable to the preparation of the desired analogues. Subsequent experiments determined that lactam-substituted alkyl bromides, such as 20, were suitably functionalized for nucleophilic displacement to provide the desired analogues. Benz[d]indeno[1,2-b]pyran-5,11-dione 2626 was treated with the corresponding primary amine to provide the desired target
The indenoisoquinolines were examined for antiproliferative activity against the human cancer cell lines in the National Cancer Institute screen, in which the activity of each compound was evaluated with approximately 55 different cancer cell lines of diverse tumor origins.37,38 The GI50 values obtained with selected cell lines, along with the mean graph midpoint (MGM) values, are summarized in Table 1. The MGM is based on a calculation of the average GI50 for all of the cell lines tested (approximately 55) in which GI50 values below and above the test range (10-8-10-4 M) are taken as the minimum (10-8 M) and maximum (10-4 M) drug concentrations used in the screening test. For comparison purposes, the activities of the previously reported lead compound 1, camptothecin (2), and compounds 3-5 are also included in the table.6,30 The relative potencies of the compounds in the production of topoisomerase I mediated DNA cleavage are also listed in the table. These results were expressed semiquantitatively as follows: 0, no detectable activity; +, weak activity; ++, similar activity as compound 1; +++ and ++++, greater activity than compound 1; ++++, similar activity as 1 µM camptothecin. Examination of the results in Table 1 indicates that the substitution pattern at the indenoisoquinoline 3-position, 9-position, and the lactam side chain has a pronounced effect on the biological activities of the molecules. If an “active” compound is defined as one that displays an MGM value of less than 1 µM or Top1 inhibitory activity of +++ or greater, then the trends in substitution pattern can be readily evaluated. Four groups of compounds can be categorized by evaluating the 3and 9-positions: group 1, hydrogen-substituted (6-9, 27, 28, and 35); group 2, 3-position hydrogen and 9-methoxy (21-23, 31, 34, 37, and 39); group 3, 3-nitro and 9-position hydrogen (10-13, 20, 29, and 32); group 4, 3-nitro and 9-methoxy (35, 24, 30, 33, 36, and 38). These four groups all possess optimized lactam side chains and differ only in the presence or absence of a nitro or methoxy substituent at the 3- or 9-positions. The enhancing effect of a 3-nitro group and a 9-methoxy group on biological activity has previously been identified, and an electrostatic charge complementarity hypothesis has been offered to help rationalize the effects.30 However, a detailed analysis
Indenoisoquinolines as Topoisomerase I Inhibitors
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Scheme 1a
a Reagents and conditions: (a) CHCl , room temp; (b) (i) SOCl , benzene, reflux; (ii) AlCl , nitrobenzene, 100 °C; (c) NaN , DMSO, 100 °C; (d) (i) 3 2 3 3 P(OEt)3, benzene, reflux; (ii) 3 M HCl in MeOH, reflux; (e) NaI, acetone, reflux.
Scheme 2
of the extent to which each substituent modulates the biological activity of the indenoisoquinolines has not been performed. Thus, examination of these groups in the context of this study should provide insight into the importance of each substituent regarding cytotoxicity and Top1 inhibition. Investigation of the results for group 1 (3- and 9-positions substituted with hydrogen) reveals that only two compounds (7 and 35) satisfy the criterion for active compounds as defined by having an MGM value of less than 1 µM. Furthermore, four compounds (7, 8, 9, and 35) possessed Top1 inhibitory activities of +++ or greater. However, one can readily conclude from Table 1 that substituting the 3- or 9-position with hydrogen atoms is less optimal (relative to the other compounds in Table 1) for biological activity. It is noted that the compounds designed and evaluated in group 1 were not intended to be the most active compounds because this distinction was intended for group 4 on the basis of previously gleaned structure-activity relationships.21,30 However, the biological results for the compounds in group 1 do serve to provide a benchmark for evaluating the efficacy of the nitro and methoxy substituents at the 3- and 9-positions in the other groups. In contrast to group 1, the analogues classified according to group 2 (3-position hydrogen atom and 9-methoxy group) displayed improved cytotoxicity profiles. Four compounds (23, 34, 37, and 39) possessed submicromolar MGM values (a 2-fold improvement versus group 1) and four compounds (21, 22, 31, and 39) demonstrated potent Top1 inhibition (+++). Therefore, it can be concluded that the 9-methoxy substituent contributes greatly to the improvement in cellular cytotoxicity relative to the hydrogen-substituted analogues in group 1. Interestingly, only analogue 39, with its imidazolyl-substituted lactam side chain, fits the criterion established for an “active” compound in both assays. This implies that Top1 inhibition and cytotoxicity
in the NCI 55 cell screen are not always strongly correlated within the indenoisoquinoline class of Top1 inhibitors and several of these molecules may be interacting with other cellular targets leading to the observed cytotoxicity. Previously derived structure-activity relationships predicted that the group 3 analogues, with their 3-nitro substituent, would greatly outperform the group 2 analogues in the biological assays.21,30 Interestingly, the magnitude of this trend was less than predicted. In group 3, four compounds (11, 13, 29, and 32) displayed submicromolar MGM values. This was the same number of compounds as that observed for group 2, thereby indicating that the presence of the 3-nitro group did not improve the relative cytotoxic nature of the analogues to a greater extent than the 9-methoxy group. Five compounds (11, 12, 13, 29, and 32) displayed Top1 inhibition of +++ or greater. This was a slight improvement over the groups 1 and 2 analogues and suggests a role for the 3-nitro group in improving Top1 inhibition. Furthermore, group 3 demonstrated an improvement for both cytotoxicity and Top1 inhibition relative to group 1. Thus, it appears that the 3-postion nitro group and the 9-methoxy group are both independently capable of affording potent biological activity. If this speculation is correct, then preparing analogues with the 3- and 9-position both substituted with a nitro and methoxy group, respectively, should provide more potent analogues than in situations in which only one of the two substituents is present.30 Analysis of the analogues in group 4 appears to confirm this hypothesis. In group 4 all eight analogues (3, 4, 5, 24, 30, 33, 36, and 38) possessed submicromolar MGM values. Additionally, all of the compounds in group 4 displayed potent Top1 inhibition greater than or equal to +++. Furthermore, compounds 5, 30, 33, 36, and 38 were all 2-3 times more cytotoxic than camptothecin (2). Except for compound 4, all of the group 4 analogues were at least equipotent to camptothecin in their abilities to inhibit the enzyme. A comparison of the analogues in group 4 with those in group 1 provides an even more striking example of the substituents’ ability to modulate biological activity. The possession of both the nitro and methoxy substituents provided 4 times as many “active” analogues based on their MGM values. Additionally, those molecules were 201000 times more cytotoxic. Thus, when it comes to the
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Scheme 3a
a Reagents and conditions: (a) NaN , DMSO, 100 °C; (b) morpholine, 3 K2CO3, 1,4-dioxane, reflux; (c) 3 M HCl in MeOH; (d) ethanolamine, DMSO, room temp; (e) dimethylamine, K2CO3, 1,4-dioxane, reflux; (f) imidazole, K2CO3, 1,4-dioxane, reflux.
biological activity of these indenoisoquinoline analogues, a definite synergistic benefit results from possessing both a 3-nitro group and a 9-methoxy group. The results in Table 1 also provide for an alternative method of analysis by evaluating analogues with the same lactam side chain but different 3- and 9-position substituents. From this method, it follows that there are five groups represented in Table 1 that differ by their respective lactam side chains (each of which has been proven to improve the biological activity of the indenoisoquinolines)17,20,22,24,26,29,31 and four members in each group. Compounds 5 (MGM, 0.027 µM; Top1, ++++), 7 (MGM, 0.320 µM; Top1, +++), 11 (MGM, 0.143 µM; Top1, ++++), and 23 (MGM, 0.048 µM; Top1, +) all possess propylamino-substituted lactam nitrogens but have different aromatic ring substituents. Thus, evaluating the differences in the biological activities between these molecules should allow conclusions to be drawn regarding specific contributions to the biological activity by the 3- and 9-position substituents. Using
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compound 7 as the benchmark (since it possesses hydrogen atoms substituted at the 3- and 9- positions), one can clearly see that substituting a nitro group at the 3-position (analogue 11) resulted in a 2-fold increase in cytotoxicity with a concomitant 20% increase in Top1 inhibition. Substituting a methoxy group at the 9-position (analogue 23) resulted in a 7-fold increase in cytotoxicity but at the cost of Top1 inhibition. Aromatic rings with both substituents (analogue 5) resulted in a 12-fold increase in cytotoxicity and a 60% increase in Top1 inhibition. Therefore, it is possible to make the generalization that the 3-nitro group tends to improve Top1 inhibition to a greater extent than cytotoxicity and the 9-methoxy group improves cytotoxicity more than Top1 inhibition. Compounds 8 (MGM, 1.86 µM; Top1, +++), 12 (MGM, 9.77 µM; Top1, +++), 36 (MGM, 0.020 µM; Top1, ++++), and 37 (MGM, 0.300 µM; Top1, 0) all possess dimethylaminopropyl-substituted lactam nitrogens and displayed biological activities similar to the previous group. Surprisingly, the trend observed for the previous group with aminopropyl-substituted lactam nitrogens was not exactly similar to the current group, especially given the similarity of the lactam side chains. In the current group, substituting the 3-position with a nitro group was not as beneficial to the cytotoxicity (analogue 12) because it was 5 times less active than the benchmark compound 8. However, it did retain Top1 inhibition. Substituting the 9-position with a methoxy group (analogue 37) resulted in a compound that was 6 times more cytotoxic but provided a poor Top1 inhibitor. This was similar to the previously identified trend for the 9-methoxy group. Accordingly, when the 3- and 9-positions were substituted with the nitro and methoxy groups, respectively, analogue 36 demonstrated a 93-fold improvement in cytotoxicity and provided a potent Top1 inhibitor. From continuation of the analysis, compounds 27 (MGM, 15.1 µM; Top1, ++), 29 (MGM, 0.632 µM; Top1, +++), 30 (MGM, 0.014 µM; Top1, ++++), and 31 (MGM, 2.70 µM; Top1, +++) all possess morpholine-substituted lactam nitrogens. By return to a previously identified trend, substituting a nitro group at the 3-position (analogue 29) resulted in a 24fold increase in cytotoxicity relative to the benchmark compound 27. Interestingly, the 9-methoxy-substituted analogue 31 did not display the typical large increase in cytotoxicity as previously observed, although a small increase in activity did occur and good Top1 inhibition was maintained. Exactly as before, substituting both the 3- and 9-positions resulted in an exceedingly potent compound 30 that was over 1000 times more cytotoxic than 27 and was a potent Top1 inhibitor. The DNA cleavage patterns produced by camptothecin (2, lane 3), the indenoisoquinoline NSC 314622 (lane 4), and compounds 27 and 29-31 are displayed in Figure 3. The following points are apparent from inspection of the gels: (1) The potencies of the indenoisoquinolines as Top1 inhibitors are reflected in the intensities of the DNA cleavage bands. The bands produced by compounds 27 (Top1, ++), 29 (Top1, +++), and 31 (Top1, +++) are slightly weaker in comparison with 30 (Top1, ++++) and camptothecin (Top1, ++++). (2) Top1 inhibitors can be classified as Top1 suppressors, which inhibit DNA cleavage, and as Top1 poisons, which inhibit the religation reaction after DNA cleavage. Many of the Top1mediated DNA cleavages are trapped at lower compound concentrations and suppressed at higher concentrations, and therefore, the indenoisoquinolines act as Top1 poisons at lower concentrations and as Top1 suppressors at higher concentrations. The suppression could result from binding of the drug to the DNA, rendering it a poorer enzyme substrate at high drug
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Table 1. Cytotoxicities and Topoisomerase I Inhibitory Activities of Indenoisoquinoline Analogues cytotoxicity (GI50 in µM)a compd 1 2 3 4 5 6 7 8 9 10 11 12 13 20 21 22 23 24 27 28 29 30 31 32 33 34 35 36 37 38 39
lung HOP-62
colon HCT-116
CNS SF-539
melanoma UACC-62
1.3 0.01 0.295
