Azaindenoisoquinolines as Topoisomerase I Inhibitors and Potential Anticancer Agents: A Systematic Study of Structure-Activity Relationships

Abstract

A comprehensive study of a series of azaindenoisoquinoline topoisomerase I (Top1) inhibitors is reported. The synthetic pathways have been developed to prepare 7-, 8-, 9-, and 10-azaindenoisoquinolines. The present study shows that 7-azaindenoisoquinolines possess the greatest Top1 inhibitory activity and cytotoxicity. Additionally, the introduction of a methoxy group into the D-ring of 7-azaindenoisoquinolines improved their biological activities, leading to new lead molecules for further development. A series of QM calculations were performed on the model “sandwich” complexes of azaindenoisoquinolines with flanking DNA base pairs from the Drug–Top1–DNA ternary complex. The results of these calculations demonstrate how changes in two forces contributing to the π–π stacking, dispersion and charge-transfer interactions, affect the binding of the drug to the Top1–DNA cleavage complex and thus modulate the drug’s Top1 inhibitory activity.

Introduction

After the discovery of NSC 314622’s (1, Figure 1) cytotoxic and topoisomerase I (Top1) inhibitory properties, the indenoisoquinolines emerged as potential anticancer agents. Following the demonstration of similarity between the cytotoxicity profiles of 1 and the plant alkaloid Top1 inhibitor camptothecin (2) by the National Cancer Institute’s (NCI’s) COMPARE analysis, it was shown that 1 is also capable of inducing single-strand DNA breaks in the presence of Top1 at micromolar concentrations.¹ Top1 plays its vital role in cell survival and replication by unwinding supercoiled DNA, thus allowing its processing. The major step in Top1-mediated DNA relaxation is reversible single strand cleavage of DNA by Top1 that results in the formation of a covalent Top1–DNA cleavage complex (Top1-DNAcc).^{2, 3} A series of elegant crystallographic studies of Top1-DNAcc in presence of inhibitors 2 and MJ-III-65 (3) revealed that by binding between DNA base pairs at the site of cleavage, molecules like 2 and 3 cause separation of the ends of the cleaved strand, preventing DNA religation and release of active enzyme.^{4, 5} Both camptothecins and indenoisoquinolines have demonstrated similarity in their mode of action by showing the ability to intercalate their polyaromatic cores between DNA base pairs and to selectively form hydrogen bond interactions with Top1, thereby acting as interfacial inhibitors (Figure 2).⁶

Figure 1.

Representative Top1 inhibitors.

Figure 2.

Superimposition of ternary complexes of 2 (green) and 3 (blue) with Top1-DNAcc.

In comparison with 2, indenoisoquinolines offer greater chemical stability. The lactone hydrolysis of 2 at physiological pH results in loss of its biological activity.⁷ The indenoisoquinoline Top1 inhibitors express DNA cleavage cite specificity different from that of 2, in addition to slower reversibility and thus greater stability of Drug–Top1–DNA ternary complexes. These advantages of indenoisoquinolines triggered their development as anticancer agents. The structure optimization of the lead compound 1 resulted in the identification and promotion of two members of the family, LMP400 (4) and LMP776 (5), into phase I clinical study at the NCI (Figure 1).^8–10

Through the synthesis and evaluation of a large number of analogues of 1, a series of important modifications that advantageously affect the inhibitory and cytotoxic properties of the indenoisoquinolines have been identified. It was shown that replacement of lactam methyl group with omega-aminoalkyl substituents having 2–4 carbon atoms (e.g. 4 and 5) positively affects the biological activities of the drug.^{8, 11, 12} Diverse substituents on the A- and D-rings of the core indenoisoquinoline molecules have been investigated leading to drugs with greatly improved cytotoxic properties.^{11, 13} The modifications of the indenone side of the indenoisoquinolines were almost exclusively confined to electron-donating alkoxy groups, and only very limited number of electron withdrawing substituent at 9^th position of indenoisoquinoline were studied.^{14, 15} Recently, the syntheses of 7-azaindenoisoquinolines 6 and 7 have been reported.¹⁶ The replacement of benzene D-ring with a pyridine ring has afforded improved water solubility. The introduction of the pyridine motif into the indenoisoquinoline system was in accordance with our hypothesis that the increase in electron deficiency of the system would potentially provide an increase in charge-transfer interactions. A series of 8-, 9-, and 10-azaindenoisoquinolines have therefore been prepared in order to complete the systematic study of indenoisoquinolines with electron-deficient D-rings.

The desired series of 8-, 9-, and 10-azaindenoisoquinolines was prepared from the corresponding cyanomethylpyridines (Schemes 1–3), similarly to the synthesis of 7-azaindenoisoquinolines from 2-cyano-3-methylpyridine.^{16, 17} Synthesis of 8-azaindenoisoquinolines was started with preparation of the key starting material, 4-methylnicotinonitrile (12, Scheme 1).¹⁸ Cyanoacetamide (8) was condensed with ethyl acetoacetate (9) in the presence of potassium hydroxide to afford dihydroxypyridine 10. Conversion of 10 to dichloride 11 by heating with excess of phosphorus oxychloride in a sealed reaction vessel followed by hydrogenation in the presence of palladium dichloride yielded 12. Further synthesis proceeded as previously described for 7-azaindenoisoquinolines.¹⁶ In short, intermediate bromide 13 obtained by bromination of 12 with N-bromosuccinimide (NBS) was reacted with 4,5-dimethoxyhomophthalic anhydride (14)¹⁹ to produce 15 (Scheme 1). Oxidation of 15 with selenium dioxide afforded the key intermediate dioxoindenoisoquinoline 16. The synthesis of 8-azaindenoisoquinolines analogues was completed with alkylation of 16 by means of Mitsunobu reaction to prepare the dimethylaminopropyl and morpholinopropyl analogues 17 and 18, respectively.

Scheme 1^a.

^aReagents and conditions: (a) KOH, methanol, reflux, 12 h (75%); (b) POCl₃, sealed tube, 150–180 °C, 8 h (67%); (c) H₂, PdCl₂, CH₃CO₂Na, methanol, 23 °C, 14 h (93%); (d) (1) NBS, AIBN, CCl₄, reflux, 3.5 h, (2) 14, triethylamine, acetonitrile, reflux 14 h (13%); (e) SeO₂, 1,4-dioxane, reflux, 4 h (49%); (f) (1) DIAD, triphenylphosphine, 3-dimethylamino-1-propanol (for 17) or 4- (3-hydroxypropyl)morpholine (for 18), THF, 23 °C, 3 h, (2) TFA, diethyl ether, chloroform, 23 °C (17 34%, 18 35%).

Scheme 3^a.

^aReagents and conditions: (a) DMF, reflux, 3 d (31%); (b) POCl₃, reflux, 6 h (54%); (c) HCO₂NH₄, Pd/C, methanol, 23 °C, 12 h (81%); (d) (1) NBS, AIBN, 1,2-dichloroethane, reflux, 9 h, (2) 14, triethylamine, acetonitrile, reflux 2 d (31%); (e) SeO₂, 1,4-dioxane, reflux, 3 d (90%); (f) (1) DIAD, triphenylphosphine, 3-dimethylamino-1-propanol (for 37) or 4-(3-hydroxypropyl)morpholine (for 38), THF, 23 °C, 12 h, (2) TFA, diethyl ether, chloroform, 23 °C (37 22%, 38 7%).

The 3-methyl-4-cyanopyridine (23) necessary for the preparation of 9-azaindenoisoquinolines was obtained from 3,4-lutidine (19, Scheme 2). Oxidation of 19 with selenium dioxide in hot diphenyl ether yielded acid 20.²⁰ Treatment of 20 with thionyl chloride gave crude acyl chloride 21 that was added in small portions to a cold concentrated ammonium hydroxide solution in order to obtain amide 22. Dehydration of 22 in phosphorus oxychloride produced the key starting material 23. The crude bromide 24 derived from 23 was condensed with 14 to prepare 25. Oxidation of 25 to 26 with selenium dioxide, followed by derivatization through Mitsunobu reaction, yielded the final products 27 and 28 (Scheme 2).

Scheme 2^a.

^aReagents and conditions: (a) SeO₂, Ph₂O, 180 °C, 1 h (47%); (b) (1) SOCl₂, reflux, 3 h, (2) concd aq NH₃, 0–5 °C (60%); (c) POCl₃, reflux, 24 h (90%); (d) (1) NBS, AIBN, CCl₄, reflux, 2 h, (2) 14, triethylamine, acetonitrile, reflux 14 h (14%); (e) SeO₂, 1,4-dioxane, reflux, 4 h (94%); (f) (1) DIAD, triphenylphosphine, 3-dimethylamino-1-propanol (for 27) or 4-(3-hydroxypropyl)morpholine (for 28), THF, 23 °C, 3 h, (2) TFA, diethyl ether, chloroform, 23 °C (27 62%, 28 10%).

The synthesis of 10-azaindenoisoquinolines began with the preparation of 3-cyano-2-methylpyridine (33, Scheme 3). Nitrile 33 was obtained from 3-aminocrotonitrile (29) and ethyl propiolate (30) in three steps. Condensation of 29 and 30 afforded pyridone 31, which was further converted into chloropyridine 32 by treatment with phosphorus oxychloride.²¹ Compound 32 was reduced to 33 with ammonium formate in the presence of palladium on charcoal. Benzylic bromination of 33 and condensation of the intermediate bromide with 14 yielded compound 35, which contains the 10-azaindenoisoquinoline polycyclic core. Compound 36, the product of selenium dioxide oxidation of 35, was introduced into Mitsunobu reaction to derive 37 and 38.

It is interesting to note that reduction of 32 to 33 required different reaction conditions than were employed to convert 11 to 12. Instead of catalytic hydrogenation of 32, reduction with ammonium formate in the presence of palladium on carbon was required (Scheme 3). When conditions required for the reduction of 11 were applied to compound 32, the only isolated product was tetrahydropyridine 39 (Scheme 4).²² In contrast, attempted reduction of compound 11 with ammonium formate in the presence of palladium on activated carbon resulted in isolation of monochloride 40 in high yield, but pyridine 12 was not detected (Scheme 5). A similar reported reduction produced 40 in less than 10% yield in a mixture with 12 as a major component.²³ The conversion of 11 to 40 reported here might provide a pathway for preparation of similarly substituted pyridines.

Scheme 4^a.

^aReagents and conditions: (a) H₂, PdCl₂, CH₃CO₂Na, methanol, 23 °C (84%).

Scheme 5^a.

^aReagents and conditions: (a) HCO₂NH₄, Pd/C, methanol, 23 °C, 3 d (87%).

It was found in earlier SAR studies of indenoisoquinolines that the introduction of alkoxy groups in the 9^th position generally benefits the Top1 inhibitory activities and cytotoxic properties of the target compounds. Having the chloropyridine 32 in hand provided the opportunity to investigate the effect of the methoxy group on the activity of 10-azaindenoisoquinolines.

Luckily, the arrangement of substituents in the pyridine 32 is such that the displacement of chlorine with a methoxy group would produce 41, which could be converted to the 9-methoxy derivatives of 10-azaindenoisoquinolines (Scheme 6). 3-Cyano-6-methoxy-2-methylpyridine (41) was subjected to radical bromination with NBS followed by condensation with 14 (Scheme 6). The isolated azaindenoisoquinoline 43 was oxidized with selenium dioxide and the product 44 was reacted with 3-dimethylamino-1-propanol and 4-(3-hydroxypropyl)morpholine under Mitsunobu conditions to prepare 45 and 46, respectively.

Scheme 6^a.

^aReagents and conditions: (a) NaOCH₃, methanol, reflux, 1.5 h (87%); (b) (1) NBS, AIBN, 1,2-dichloroethane, reflux, 3.5 h, (2) 14, triethylamine, acetonitrile, reflux 14 h (19%); (c) SeO₂, 1,4-dioxane, reflux, 24 h (89%); (d) DIAD, triphenylphosphine, 3-dimethylamino-1-propanol (for 45) or 4-(3-hydroxypropyl)morpholine (for 46), THF, 23 °C, 3 d, (2) TFA, diethyl ether, chloroform, 23 °C (45 30%, 46 31%).

The evaluation of Top1 inhibitory activity and cytotoxicity of the azaindenoisoquinolines showed that the introduction of a methoxy group into the 9^th position of 10-azaindenoisoquinoline resulted in an increase in Top1 inhibitory activity.^{14, 15} But unfortunately, it did not improve cytotoxicity and 10-azaindenoisoquinolines 37, 45 and 46 remained largely non-toxic to cancer cell lines (Table 1). In contrast, the 7-azaindenoisoquinoline series has demonstrated the greatest cytotoxicity of all of the currently available azaindenoisoquinolines (Table 1).¹⁶ In order to investigate the effect of the 9-methoxy substituent in the 7-azaindenoisoquinoline series, analogues 55 and 56 were synthesized (Scheme 7).

Table 1.

Top1 Inhibitory and Antiproliferative Activity of Azaindenoisoquinolines.

compd	Top1 cleavagea	cytotoxicity (GI50, μM)c
		MGMb	lung HOP-62	colon HCT-116	CNS SF-539	melanoma UACC-62	ovarian OVCAR-3	renal SN12C	prostate DU-145	breast MCF7
1	++	8.5	2.8	11.5	1.7	0.56	22	26	4.8	1.9
2	++++	0.040	0.010	0.030	0.010	0.010	0.22	0.020	0.010	0.013
6	+++	4.5	3.4	1.6	4.1	13	3.6	3.2	1.7	0.44
7	++	0.30	0.30	0.22	0.29	0.10	0.37	0.52	0.31	0.052
17	++	NDd	ND	ND	ND	ND	ND	ND	ND	ND
18	++	16	6.5	2.5	40	48	24	16	24	4.1
27	+	6.5	6.5	0.62	12	>100	7.4	8.5	2.9	3.9
28	++	9.5	5.0	0.39	>100	85	12	5.9	7.6	1.9
37	++	ND	ND	ND	ND	ND	ND	ND	ND	ND
38	NTe	NT	NT	NT	NT	NT	NT	NT	NT	NT
45	+++	ND	ND	ND	ND	ND	ND	ND	ND	ND
46	+++	ND	ND	ND	ND	ND	ND	ND	ND	ND
55	+++	1.8	0.92	1.5	1.1	3.9	2.9	3.6	0.88	0.13
56	++	0.48	0.24	0.33	0.27	0.22	0.31	0.34	0.34	0.10
63	+++	0.40	0.30	0.34	0.57	0.54	0.94	0.26	0.30	0.21
64	++	3.0	4.1	2.5	3.5	1.7	6.3	2.6	3.6	0.60
65	+++	0.11	0.054	0.074	0.078	0.052	0.14	0.057	0.051	0.024
66	++++	0.085	0.051	0.050	0.035	0.040	0.11	0.043	0.040	0.020

^aThe relative Top1 inhibitory potencies of the compounds are presented as follows: 0: no detectable activity; +: weak activity; ++: similar activity as compound 1; +++ and ++++: greater activity than compound 1; ++++: similar activity as 1 μM 2.

^bMean graph midpoint (MGM) for growth inhibition of all human cancer cell lines successfully tested.

^cThe cytotoxicity GI₅₀ values listed are the concentrations corresponding to 50% growth inhibition, and are the result of single determinations.

^dGI₅₀s were not determined because the low activities revealed in the initial single-concentration testing at 10 μM did not warrant the multiple-concentration testing required for determination of GI₅₀ values.

^eNot tested.

Scheme 7^a.

^aReagents and conditions: (a) NBS, CH₃CO₂NH₄, acetonitrile, 0–23 °C, 25 min (65%); (b) Br₂, NaNO₂, aq HBr −15 °C, then 23 °C, 3 h (94%); (c) CuCN, DMF, reflux, 2 h (74%); (d) NaOCH₃, methanol, reflux, 12 h (79%); (e) (1) NBS, AIBN, 1,2-dichloroethane, reflux, 24 h, (2) 14, triethylamine, acetonitrile, reflux 24 h (21%); (f) SeO₂, 1,4-dioxane, reflux, 24 h (92%); (g) (1) DIAD, triphenylphosphine, 3-dimethylamino-1-propanol (for 55) or 4-(3-hydroxypropyl)morpholine (for 56), THF, 23 °C, 2 d, (2) HCl, methanol, chloroform, 23 °C (55 52%, 56 59%).

The key starting material required for the preparation of this series, 2-cyano-5-methoxy- 3-methylpyridine (51), was prepared in four steps from commercially available 2-amino-3-picoline (47, Scheme 7). Bromination of 47 with NBS in acetonitrile in presence of ammonium acetate yielded 2-amino-5-bromo-3-methylpyridine (48).²⁴ The amino group of 48 was replaced with bromide to provide 49, followed by further displacement with cyanide to yield pyridine 50. For this transformation sodium nitrite was added to the solution of aminopyridine 48 and bromine in concentrated hydrobromic acid at −15 °C.^25–27 Treatment of 49 with one equivalent of copper(I) cyanide in dry DMF led to the selective substitution of only the bromide in the second position of the pyridine, yielding 50.²⁸ The direct conversion of aminopyridine 48 to nitrile 50 via Sandmeyer-type reaction is prevented by the instability of the intermediate 2-pyridyldiazonium salt that is apparently capable of decomposing at relatively low temperatures prior to its transformation into nitrile 50 in the presence of copper(I) cyanide. Nucleophilic substitution of the second bromide with methoxide provided 51.

A series of transformations starting from 51 to the targets 55 and 56 were performed in a way that is similar to that described for other azaindenoisoquinolines (Scheme 7). Bromination of 51 in presence of radical initiator AIBN, followed by condensation with 14, produced 53. Oxidation of 53 to 54, and Mitsunobu reaction of 54 with 3-dimethylamino-1-propanol and 4-(3-hydroxypropyl)morpholine, led to analogues 55 and 56, respectively.

In order to further explore the potential of the 7-aza-9-methoxyindenoisoquinoline series as anticancer agents, compounds 63–66 were prepared (Scheme 8). These analogues contain a differently substituted indenoisoquinoline A-ring compared to compounds 55 and 56. Pyridine 51 was utilized in the preparation of 63–66 (Scheme 8). Bromination of 51 followed by condensation with homophthalic and 5-nitrohomophthalic anhydrides²⁹ (57 and 58) yielded azaindenoisoquinolines 59 and 60, respectively. Compounds 59 and 60 underwent oxidation to 61 and 62, respectively, followed by Mitsunobu transformation, to yield 63–66.

Scheme 8^a.

^aReagents and conditions: (a) (1) NBS, AIBN, 1,2-dichloroethane, reflux, 24 h, (2) homophthalic anhydride (57, for 59) or 5-nitrohomophthalic anhydride (58, for 60), triethylamine, acetonitrile, reflux 24 h (59 46%, 60 26%); (e) SeO₂, 1,4-dioxane, reflux, 24 h (61 76%, 62 86%); (d) DIAD, triphenylphosphine, 3-dimethylamino-1-propanol (for 63 and 65) or 4-(3-hydroxypropyl)morpholine (for 64 and 66), THF, 23 °C, 2 d (63 38%, 64 40%, 65 61%, 66 47%).

Biological Results and Discussion

All of the target compounds were tested for induction of DNA damage in Top1-mediated DNA cleavage assays.³⁰ For this purpose, a ³²P 3′-end-labeled 117-bp DNA fragment was incubated with human recombinant Top1 and increasing concentrations of the test compounds. The DNA fragments were separated on a denaturing gel (Figure 3). The Top1 inhibitory activity was assigned based on the visual inspection of the number and intensities of the DNA cleavage bands and expressed in semiquantitative fashion relative to the Top1 inhibitory activities of compounds 1 and 2: 0, no detectable activity; +, weak activity; ++, similar activity to compound 1; +++, greater activity than 1; ++++, equipotent to 2 (Table 1).

Figure 3.

Top1-mediated DNA cleavage induced by azaindenoisoquinolines. Lanes 1, 29: DNA alone. Lanes 2, 30: Top1 alone. Lanes 3, 31: Top1 + 2 (1 μM). Lane 4: Top1 + 1 (100 μM). Lanes 5–8:Top1 + 6 at 0.1 μM, 1 μM, 10 μM, 100 μM. Lanes 9–12: Top1 + 17 at 0.1 μM, 1 μM, 10 μM, 100 μM. Lanes 13–16: Top1 + 27 at 0.1 μM, 1 μM, 10 μM, 100 μM. Lanes 17–20: Top1 + 37 at 0.1 μM, 1 μM, 10 μM, 100 μM. Lanes 21–24: Top1 + 45 at 0.1 μM, 1 μM, 10 μM, 100 μM. Lanes 25–28: Top1 + 55 at 0.1 μM, 1 μM, 10 μM, 100 μM. Lanes 32–35: Top1 + 65 at 0.1 μM, 1 μM, 10 μM, 100 μM. Lanes 36–39: Top1 + 66 at 0.1 μM, 1 μM, 10 μM, 100 μM. Numbers on right and arrows show the cleavage site positions.

The antiproliferative activity of each compound was determined in the National Cancer Institute (NCI) screen.^{31, 32} Cells of approximately 60 different human cancer cell lines were incubated for 48 h with five 10-fold dilutions of the test compounds starting from 100 μM, and then treated with sulforhodamine B dye. The ratios of recorded optical densities relative to those of the control were plotted as a function of the common logarithm of the tested compound concentrations. The interpolation between the points located above and below the 50% percentage growth provided 50% growth inhibition (GI₅₀) values. The GI₅₀ and the mean graph midpoint (MGM) values of the prepared indenoisoquinolines in selected cell lines are presented in Table 1. The MGM is based on a calculation of the average GI₅₀ for all of the cell lines tested in which GI₅₀ values above and below tested range (10⁻⁴ to 10⁻⁸ M) are taken as the maximum (10⁻⁴ M) and minimum (10⁻⁸ M) drug concentrations used in the screening test. The Top1 inhibitory and cytotoxicity (MGM and GI₅₀) data for lead compound 1,^{1, 33} compound 2, and previously reported 7-azaindenoisoquinolines 6 and 7¹⁶ are included in Table 1 for comparison purposes.

The results of the biological evaluations presented in Table 1 clearly show that 7-azaindenoisoquinolines 6 and 7 are more active than their corresponding isomers, 8-, 9-, and 10-azaindenoisoquinolines, 17, 18, 27, 28, and 37. The Top1 inhibitory activities of 6 and 7 are at the +++/++ level and their cytotoxicities are in the low micromolar concentration range. Top1 inhibition is observed at only the ++/+ level for 8-, 9-, and 10-azaindenoisoquinolines. The MGM of compounds 18, 27 and 28 increased 2 to 50 times relative to the MGM of 6 and 7. The 8-azaindenoisoquinoline 17 and 10-azaindenoisoquinoline 37 were not cytotoxic enough for GI₅₀ and MGM values to be determined in the NCI testing concentration range. These results indicate that the introduction of a nitrogen atom in positions other than 7 of the indenoisoquinoline polycyclic system results in compounds with diminished activity.

The synthetic protocol developed for the preparation of the 10-azaindenoisoquinolines 37 and 38 was modified, allowing for an addition of the methoxy group to position 9, which resulted in the preparation of compounds 45 and 46 (Schemes 3 and 6). It was previously noted that introduction of a methoxy group into the position 9 of the indenoisoquinolines positively affects the inhibitory and cytotoxic activities of the target compounds.^{14, 15} 10-Aza-9-methoxyindenoisoquinolines 45 and 46 (Scheme 6) were ranked +++ in the Top1-mediated DNA cleavage assay, showing an improvement in comparison to the corresponding analogue 37 that lacks a 9-methoxy group (Top1: ++, Table 1). Unfortunately, the 10-azaindenoisoquinolines were largely non-cytotoxic in the tested cancer cell lines.

The 7-aza-9-methoxyindenoisoquinoline series was prepared (Scheme 7) in view of the positive Top1 inhibition results observed on the introduction of a 9-methoxy group in the 10-aza series. In the case of 7-azaindenoisoquinolines, there was no apparent increase in the Top1 inhibitory activity of compounds 55 and 56 over 6 and 7, respectively. However, in the case of 7-aza-9-methoxyindenoisoquinoline 55, a nearly three-fold improvement in cytotoxicity was observed relative to 7-azaindenoisoquinoline 6 (Table 1).

Having achieved the desired effect, 7-aza-9-methoxyindenoisoquinolines 63–66 were prepared that bear different substituents on the A-ring (Scheme 8). An improvement in cytotoxicity was observed for the dimethylaminopropyl analogues 63 and 65, which had MGM values of 0.40 and 0.11 μM. No loss of Top1 inhibitory activity was observed for 63 and 65 in comparison with the closely related compounds 6 and 55. In case of compound 66, there was a surprising improvement in Top1 inhibitory activity, up to ++++, making it equipotent with 2.

The MGM value obtained for compound 66 was 85 nM, making it the most active compound to date in the entire azaindenoisoquinoline series. The cleavage band intensities displayed in Figure 3 indicate that the azaindenoisoquinolines offer different DNA cleavage site specificity in comparison to 2. Most notably, the bands at positions 44, 62 and 106 become more prominent whereas bands at positions 32 and 97 are weaker than in the case of 2. The cleavage site specificity of the azaindenoisoquinoline series is consistent with that previously observed for 1, 4 and 5.^1,10 By trapping Top1-DNA cleavage complexes having different DNA cleavage sites than the camptothecins, the indenoisoquinolines and azaindenoisoquinolines target the genome differently than the camptothecins. This suggests that the cancer treatment profiles of the indenoisoquinolines might be different from the camptothecins.

In an attempt to understand the effect of introduction of nitrogen into various positions of the indenoisoquinoline D-ring on the π–π stacking interactions, a series of hypothetical azaindenoisoquinoline structures were modeled and subjected to quantum mechanical single point energy calculations. It was shown earlier that π–π stacking interactions of indenoisoquinolines with the flanking base pairs are responsible for their orientation in the ternary complex and their DNA cleavage site specificities. The results of these calculations indicate that π–π stacking interactions are the major complex stabilizing force.^34–36

The model for these calculations was derived from the X-ray crystal structure of the 3–Top1–DNA ternary complex (PDB entry 1SC7).⁵ The deoxyribose rings of the flanking base pairs and the lactam side chain of 3 were replaced with methyl groups because they are considered to be nonparticipating groups in π–π stacking interactions.³⁵ The geometry optimizations and frequency calculations at the HF/6-31G** level were performed on the indenoisoquinoline molecule, as well as A–T and G–C base pairs, using the Gaussian 09 software package.³⁷ The original complex was then replaced with the geometry optimized parts (Figure 4 and Figure 5, model A). The CH groups in the positions 7–10 of the D-ring of the inhibitor were replaced with nitrogen to produce 7-, 8-, 9- and 10-azaindenoisoquinoline models (Figure 5, models B–F). Geometry optimizations were performed on these molecules before fitting them into their corresponding complexes with the DNA base pairs.

Figure 4.

Model of the complex of the inhibitor with DNA base pairs.

Figure 5.

Modeled indenoisoquinoline-DNA complexes.

After the complexes were assembled, MP2/6-31G* single point energy calculations were performed. The basis set superposition error (BSSE) for each complex was calculated using the counterpoise correction method within the Gaussian 09 package, specifying two fragments: the first being the two flanking DNA base pairs, and the second being the intercalating indenoisoquinoline inhibitor. The π–π staking interaction energy was calculated as E_int = E_complex − E_ligand − E_bp + BSSE, where E_complex, E_ligand, and E_bp are the corresponding MP2/6-31G* calculated energies of complex, ligand and DNA base pairs at their normal distance in absence of intercalator (Table 2). The polarizable continuum model (PCM) was used at the MP2/6-31G* level of theory to estimate the effect of solvation on stabilization of the complex (E_{int, aq}, Table 2). The dispersion energy E_corr was calculated as E_corr = E_int (MP2) − E_int(HF). The energy associated with charge transfer was obtained as result of Natural Bond Orbital (NBO) analysis in Gaussian 09.³⁸ The NBO analysis was performed at the HF/6-31G** level of theory and presented as E_CT in Table 2. The calculated transfer of 0.01e can be equated to 1 kcal/mol of complex stabilization energy.³⁹

Table 2.

MP2 calculated energies for models A–F.

Model	Eint(au)	Eint(kcal/mol)	Eint, aq(kcal/mol)	Ecorr(kcal/mol)	ECT(kcal/mol)
A	−0.01108	−6.95	−15.17	−26.03	−11.50
B	−0.01273	−7.99	−15.57	−25.79	−12.58
C	−0.01452	−9.11	−16.20	−15.11	−13.57
D	−0.01663	−10.43	−17.01	−14.73	−11.99
E	−0.01459	−9.16	−18.59	−14.27	−10.05
F	−0.01305	−8.19	−17.03	−40.04	−10.91

^aE_int is derived from MP2/6-31G* single point energy calculations.

^bE_corr = E_{int(MP2/6-31G*)} − E_{int(HF/6-31G*)}.

^cThe magnitude of the charge transfer as estimated by NBO analysis at HF/6-31G** level.

Although E_corr and E_CT could not be summed directly to obtain the whole or at least a part of E_int, they could be used to evaluate the effect of various modifications on particular forces responsible for stabilization of drug–Top1–DNA ternary complexes. For example, comparison of E_corr for ‘classical’ indenoisoquinoline (model A) and 7-azaindenoisoquinoline (model B) shows that introduction of nitrogen into the 7^th position did not decrease the dispersion energy contribution to the stabilization resulting from π–π stacking. Analysis of the E_CT column on the other hand suggests that as expected the charge-transfer stabilization energy is greater by 1.08 kcal/mol for 7-azindenoisoquinoline represented by model B, in comparison to indenoisoquinoline in model A. Table 2 indicates that the dispersion interaction is decreased significantly for models C–E, but not B (relative to A). This decrease in dispersion energy contributes to the decrease of Top1 inhibitory activity and cytotoxicity. Even in the case of 8-azaindenosioquinolines (model C), despite the increase of the absolute magnitude of E_CT to almost 13.6 kcal/mol, the decrease in dispersion interactions was significant enough to decrease Top1 inhibitory activity. Comparison of the experimental results of Top1-mediated DNA cleavage for compounds 37 and 45 and calculated E_CT and E_corr values for models E and F demonstrate the importance of the methoxy group in position 9 of the indenoisoquinolines (Tables 1 and 2). This methoxy substitution drastically increases dispersion (E_corr) interaction energy while maintaining charge-transfer (E_CT) interactions. The increase in the dispersion interaction of 9-methoxy analogues could be attributed to the electron donating properties of the methoxy group and increase in electron delocalization in comparison to the analogues lacking this group. The increase of the Top1 inhibitory activity that corresponds to the addition of methoxy group is observed in the case of 10-aza- and 10-aza-9-methoxyindenoisoquinolines, 37 and 45, respectively. These findings were extrapolated to 7-azaindenoisoquinolines. The resulting 7-aza-9-methoxyindenoisoquinolines 55, 56, and 63–66 demonstrated improvement in their biological properties relative to those lacking the methoxy group in position 9.

It was previously hypothesized that a nitro group in the 3^rd position of indenoisoquinoline system is capable of forming a direct hydrogen bond with Asn722.¹⁴ Overlaying the structure of 66 with 3 in its ternary complex (PDBID: 1SC7)⁵ revealed that this interaction would be possible in addition to the contact with Arg364 formed by carbonyl oxygen of indenone moiety (Figure 6). Addition of an extra hydrogen bond to the network of contacts of the drug with the Top1-DNAcc might explain the increase of Top1 inhibitory and antiproliferative potency of 66 in comparison to other azaindenoisoquinolines such as 7 and 56.

Figure 6.

Hypothetical binding mode of 66 in complex with Top1-DNAcc. Hydrogen bonds (dashed lines) represented as distances between corresponding heavy atoms.

In conclusion, a series of 7-, 8-, 9- and 10-azaindenoisoquinolines have been prepared. The established synthetic protocols allow the preparation of these compounds from easily affordable starting materials. Azaindenoisoquinolines have been evaluated for their ability to stabilize the Top1-DNAcc and thus inhibit Top1. The ab initio calculations show that the values of the components of the π–π stacking interaction energy, i.e. dispersion forces (E_corr) and charge-transfer interactions (E_CT), significantly depend on the position of the nitrogen atom. The 7-azaindenoisoquinoline system allows for an increase of charge transfer interaction without compromising the level of dispersion interactions. Previous studies have also revealed that incorporation of nitrogen into position 7 of indenoisoquinolines allows for improvement of water solubility without compromising the Top1 inhibitory activities and cytotoxicities of the resulting 7-azaindenoisoquinolines.¹⁶ Further development of the 7-azaindenoisoquinolines presented in this study led to the discovery of 7-aza-9-methoxy-3-nitroindenoisoquinoline 66, which demonstrated high activity in the Top1-mediated DNA cleavage assay, ++++, and in cytotoxicity screening at the NCI, with an MGM of 85 nM. Further aminopropyl chain modifications and optimizatization^8,13 could be done in order to further improve the Top1 inhibitory and cytotoxic performance of compounds such as 65 and 66.

Experimental Section

General

Melting points were determined using capillary tubes with a Mel-Temp apparatus and are uncorrected. The nuclear magnetic resonance spectra (¹H and ¹³C NMR) were recorded using ARX300 300 MHz and DRX500 500 MHz Bruker NMR spectrometers. IR spectra were recorded using a Perkin-Elmer 1600 series FTIR spectrometer. Purities of all tested compounds were ≥95%, as established by combustion and/or estimated HPLC analysis. Combustion microanalyses were performed at the Purdue University Microanalysis Laboratory or Galbraith Laboratories Inc. and the reported values were within 0.4% of the calculated values. HPLC analyses were performed on a Waters 1525 binary HPLC pump/Waters 2487 dual λ absorbance detector system. For purities estimated by HPLC, the major peak accounted for ≥95% of the combined total peak area when monitored by a UV detector at 254 nm. Analytical thin-layer chromatography was carried out on Baker-flex silica gel IB2-F plates and compounds were visualized with UV light at 254 nm. Silica gel flash chromatography was performed using 230–400 mesh silica gel.

3-Cyano-2,6-dihydroxy-4-methylpyridine (10).¹⁸

Cyanoacetamide (8, 34 g, 0.40 mol) and ethyl acetoacetate (9, 52 g, 0.40 mol) were dissolved in methanol (250 mL) at room temperature, a solution of potassium hydroxide (28 g, 0.42 mol) in methanol (200 mL) was slowly added, and the resulting mixture was heated to reflux for 12 h. The reaction mixture was cooled to room temperature and the white amorphous precipitate was filtered and washed with methanol (2 × 50 mL). The solid product was redissolved in hot water. The solution was carefully acidified and the off-white precipitate was allowed to form. The precipitate was filtered and washed with water and methanol to yield 10 (46 g, 75%): mp >300 °C (dec) [lit.¹⁸ mp 315–320°C (dec)]. ¹H NMR (300 MHz, DMSO-d₆) δ 5.58 (s, 1 H), 2.22 (s, 3 H).

3-Cyano-2,6-dichloro-4-methylpyridine (11).¹⁸

3-Cyano-2,6-dihydroxy-4-methylpyridine (10, 10 g, 0.07 mol) and phosphorus oxychloride (25 mL, 0.27 mol) were sealed in a heavy-walled tube and the mixture was heated to 150–180 °C in an oil bath for 8 h. The resulting mixture was allowed to cool to room temperature and carefully quenched by pouring it into ice (200 g). The light brown precipitate was filtered, washed with water and dried to yield 11 (8.3 g, 67%): mp 114–118 °C (lit.¹⁸ mp 109–110 °C). ¹H NMR (300 MHz, DMSO-d₆) δ 6.77 (s, 1 H), 1.49 (s, 3 H).

3-Cyano-4-methylpyridine (12).¹⁸

Palladium dichloride (50 mg, 0.3 mmol) was added to a degassed solution of 11 (5.0 g, 27 mmol) and sodium acetate (4.5 g, 55 mmol) in methanol (100 mL). The resulting mixture was stirred under hydrogen (1 atm) for 14 h at room temperature. The precipitate was filtered and washed with methanol (3 × 20 mL). The combined filtrates were evaporated under reduced pressure, and chloroform (50 mL) was added to the residue. The chloroform solution was filtered through a thin pad of silica gel, washing with additional portions of chloroform. The filtrate was evaporated to dryness to provide 12 (2.9 g, 93%) as a yellow oil. ¹H NMR (300 MHz, DMSO-d₆) δ 8.77 (s, 1 H), 8.63 (s, J = 6.0 Hz, 1 H), 7.3 (d, J = 6.0 Hz, 1 H), 2.56 (s, 3 H). 3-Cyano-4-methylpyridine (12) was used further without additional purification.

8-Aza-5,6-dihydro-2,3-dimethoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (15)

3-Cyano-4-methylpyridine (12, 1.5 g, 13 mmol), NBS (3.3 g, 19 mmol) and AIBN (100 mg, 0.6 mmol) were diluted with carbon tetrachloride (60 mL) and the mixture was heated at reflux for 3.5 h. The reaction mixture was concentrated to one-half its original volume, filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was diluted with acetonitrile (60 mL), 4,5-dimethoxyhomophthalic anhydride (14, 5.6 g, 25 mmol) was added, followed by triethylamine (3.5 mL, 25 mmol), and the solution was heated at reflux for 14 h. The solution was allowed to cool to room temperature and the precipitate was filtered and washed with acetonitrile (2 × 15 mL) to provide a grey solid (500 mg, 13%): mp 270–272 °C. IR (KBr) 1633, 1611, 1593 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 12.31 (s, 1 H), 9.08 (s, 1 H), 8.48 (d, J = 6.0 Hz, 1 H), 7.61 (m, 2 H), 7.13 (s, 1 H) 3.93 (s, 5 H), 3.86 (s, 3 H); positive ESIMS m/z (rel intensity): 295 (MH⁺, 100).

8-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (16)

8-Aza-5,6-dihydro-2,3-dimethoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (15, 250 mg, 0.85 mmol) and SeO₂ (190 mg, 1.7 mmol) were diluted with 1,4-dioxane (20 mL) and the mixture was heated at reflux for 4 h. The reaction mixture was filtered while hot and the precipitate was washed with hot dioxane (3 × 10 mL). The combined filtrates were evaporated to dryness under reduced pressure. The solid residue was purified by flash column chromatography (silica gel), eluting with 5% methanol in chloroform, to obtain 16 (130 mg, 49%): mp 300–302 °C. IR (KBr) 1708, 1648, 1611, 1579 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 12.96 (s, 1 H), 8.56 (d, J = 6.2 Hz and 1.4 Hz, 1 H), 7.86–7.81 (m, 2 H), 7.59 (s, 1 H), 7.40 (t, J = 6.8 Hz, 1 H); positive ESIMS m/z (rel intensity): 309 (MH⁺, 100).

8-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-2,3-dimethoxy-5,11-dioxo-11H-indeno[ 1,2-c]isoquinoline Trifluoroacetate (17)

8-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (16, 92 mg, 0.30 mmol), 3-dimethylamino-1-propanol (0.1 mL, 0.9 mmol), and PPh₃ (240 mg, 0.92 mmol) were diluted with THF (15 mL). Diisopropyl azodicarboxylate (0.18 mL, 0.92 mmol) was added to the THF solution and the resulting mixture was stirred at room temperature for 3 h. The reaction mixture was then evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel), eluting with 10% methanol in chloroform, followed by preparative TLC (silica gel), eluting with 5% methanol in chloroform, to provide orange solid. The solid was redissolved in chloroform (5 mL) and trifluoroacetic acid (2M in diethyl ether, 1 mL) was added. The precipitate was collected by filtration and washed with ether (2 × 2 mL) to yield the product in the form of its trifluoroacetate salt (53 mg, 34%): mp 230–232 °C (dec). IR (KBr) 1690, 1612 cm⁻¹; ¹H NMR (300 MHz, CD₃OD) δ 8.68–8.60 (m, 2 H), 7.73 (d, J = 5.5 Hz, 1 H), 7.69 (s, 1 H), 7.26 (s, 1 H), 4.57 (t, J = 6.1 Hz, 2 H), 3.76 (s, 3 H), 3.70 (s, 3 H), 3.24–3.08 (m, 2 H), 2.69 (s, 6 H), 2.24–2.06 (m, 2 H); positive ion ESIMS m/z (rel intensity): 394 (MH⁺, 100); HRMS–ESI m/z: MH⁺ calcd for C₂₂H₂₃N₃O₄, 394.1767; found, 394.1769; HPLC purity: 97.44% [C-18 reverse phase, MeOH (1% CF₃COOH)/H₂O, 80:20].

8-Aza-5,6-dihydro-6-(3-(4-morpholino)propyl)-2,3-dimethoxy-5,11-dioxo-11H-indeno[ 1,2-c]isoquinoline Trifluoroacetate (18)

8-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (16, 100 mg, 0.32 mmol), 4-(3-hydroxypropyl)morpholine (94.3 mg, 0.65 mmol), and PPh₃ (170 mg, 0.65 mmol) were diluted with THF (10 mL). Diisopropyl azodicarboxylate (0.13 mL, 0.65 mmol) was added to the THF solution and the resulting mixture was stirred at room temperature for 3 h. The reaction mixture was then evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel), eluting with 10% methanol in chloroform, followed by preparative TLC (silica gel), eluting with 5% methanol in chloroform, to provide an orange solid. The solid was redissolved in chloroform (5 mL) and trifluoroacetic acid (2M in diethyl ether, 1 mL) was added. The precipitate was collected by filtration and washed with ether (2 × 2 mL) to yield the product in the form of its trifluoroacetate salt (62 mg, 35%): mp 213–214 °C. IR (KBr) 1778, 1753, 1679, 1614 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 10.15 (s, 1 H), 8.83–8.58 (m, 2 H), 7.69 (s, 1 H), 7.44 (d, J = 4.5 Hz, 1 H), 7.31 (s, 1 H), 4.69 (t, J = 5.8 Hz, 2 H), 4.02 (d, J = 12.3 Hz, 2 H), 3.92 (s, 3 H), 3.90 (s, 3 H), 3.67 (t, J = 11.5 Hz, 2 H), 3.54 (d, J = 12.1 Hz, 2 H), 3.40 (s, 2 H), 3.16 (s, 2 H), 2.32 (s, 2 H); positive ion ESIMS m/z (rel intensity): 436 (MH⁺, 100); HRMS–ESI m/z: MH⁺ calcd for C₂₄H₂₅N₃O₅, 436.1872; found, 436.1769; HPLC purity: 97.05% [C-18 reverse phase, MeOH (1% CF₃COOH)/H₂O, 90:10]; 97.36% [C-18 reverse phase, MeOH (1% CF₃COOH)/H₂O, 70:30].

3-Methyl-4-nicotinic Acid (20).²⁰

A solution of 3,4-lutidine (19, 30 g, 0.28 mol) in diphenyl ether (150 mL) was heated to 150–170 °C and selenium dioxide (62 g, 0.56 mol) was carefully added to the hot solution in small portions in the course of 1 h. The resulting mixture was heated to 180 °C for 1 h. The reaction mixture was filtered while hot and the collected precipitate was washed with boiling water (3 × 300 mL). The combined filtrates were extracted with chloroform (3 × 300 mL). The aqueous phase was evaporated to dryness and the remaining product was recrystallized from ethanol (450 mL) to obtain pure acid 20 (18 g, 47%): mp 220–222 °C (lit.⁴⁰ mp 232 °C). ¹H NMR (300 MHz, DMSO-d₆) δ 8.59 (s, 1 H), 8.04 (s, 1 H), 8.47 (d, J = 4.8, 1 H), 7.69 (d, J = 4.8, 1 H), 2.48 (s, 3 H).

3-Methyl-4-nicotinamide (22)

A solution of 3-methyl-4-nicotinic acid (20, 5.0 g, 37 mmol) in thionyl chloride (20 mL, 0.28 mol) was heated at reflux for 3 h. The thionyl chloride was evaporated. The solid acid chloride 21 was added in small portions to a concentrated ammonium hydroxide solution (300 mL) while cooling the reaction mixture to 0–5 °C. The reaction mixture was saturated with potassium carbonate, and the solution was extracted with chloroform (2 × 150 mL) and ethyl acetate (2 × 150 mL). The aqueous phase was evaporated to dryness and the resulting solid was extracted with hot ethyl acetate (3 × 150 mL). The combined extracts were evaporated to dryness to yield crude 22 (3.0 g, 60%): mp 140–142 °C. ¹H NMR (300 MHz, DMSO-d₆) δ 8.47 (s, 1 H), 8.44 (d, J = 4.9 Hz, 1 H), 7.94 (s, 1 H), 7.65 (s, 1 H), 7.29 (d, J = 4.9 Hz, 1 H), 2.32 (s, 3 H).

3-Methyl-4-cyanopyridine (23)

Phosphorus oxychloride (100 mL, 1.1 mol) was slowly added to the crude amide 22 (15 g, 0.11 mol) while cooling the mixture in an ice bath. The resulting solution was heated at reflux for 24 h. The reaction mixture was cooled to room temperature and the excess phosphorus oxychloride was removed under reduced pressure. Crushed ice (150 g) was slowly added to the oily residue and the solution was neutralized with saturated ammonium hydroxide. The crude product was extracted with chloroform (3 × 100 mL). The combined extracts were filtered through a layer of silica gel, washing with extra portions of chloroform. The filtrates were evaporated to dryness to yield 23 as colorless crystals (12 g, 90%): mp 45–47 °C (lit.⁴¹ mp 50 °C). ¹H NMR (300 MHz, CDCl₃) δ 8.66 (s, 1 H), 8.59 (d, J = 5.0 Hz, 1 H), 7.45 (d, J = 5.0 Hz, 1 H), 2.54 (s, 3 H).

9-Aza-5,6-dihydro-2,3-dimethoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (25)

3-Methylisonicotinonitrile (23, 590 mg, 5.0 mmol), NBS (1.2 g, 7.0 mmol) and AIBN (50 mg, 0.3 mmol) were diluted with carbon tetrachloride (20 mL) and the mixture was heated at reflux for 2 h. The reaction mixture was concentrated to one-half its original volume, filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was diluted with acetonitrile (25 mL), and 14 (2.2 g, 10 mmol) was added, followed by triethylamine (5 mL, 36 mmol), and the solution was heated at reflux for 14 h. The solution was allowed to cool to room temperature and the precipitate was filtered and washed with acetonitrile (50 mL) to provide a light-brown solid (200 mg, 14%): mp 306–308 °C. IR (KBr) 1639, 1613, 1592 cm⁻¹; ¹H NMR (300 MHz, DMSOd₆) δ 12.32 (s, 1 H), 8.73 (s, 1 H), 8.54 (d, J = 6.0 Hz, 1 H), 7.87 (d, J = 6.0 Hz, 1 H), 7.65 (s, 1 H), 7.23 (s, 1 H), 3.96 (s, 3 H), 3.88 (s, 3 H), 3.37 (s, 2 H); positive ESIMS m/z (rel intensity): 295 (MH⁺, 100).

9-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (26)

9-Aza-5,6-dihydro-2,3-dimethoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (25, 100 mg, 0.34 mmol) and SeO₂ (75 mg, 0.68 mmol) were diluted with 1,4-dioxane (10 mL) and the mixture was heated at reflux for 4 h. The reaction mixture was filtered while hot and the precipitate was washed with hot dioxane (3 × 10 mL). The combined filtrates were evaporated to dryness under reduced pressure. The solid residue was purified by flash column chromatography (silica gel), eluting with 5% methanol in chloroform, to obtain 26 (98 mg, 94%): mp 312–314 °C. IR (KBr) 1710, 1638, 1608 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 12.96 (s, 1 H), 8.56 (d, J = 6.2 Hz and 1.4 Hz, 1 H), 7.86-7.81 (m, 2 H), 7.59 (s, 1 H), 7.40 (t, J = 6.8 Hz, 1 H); positive ESIMS m/z (rel intensity): 309 (MH⁺, 100), negative ion ESIMS m/z (rel intensity): 307 [(M–H⁺)⁻, 100].

9-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-2,3-dimethoxy-5,11-dioxo-11H-indeno[ 1,2-c]isoquinoline Trifluoroacetate (27)

9-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (26, 92 mg, 0.3 mmol), 3-dimethylamino-1-propanol (0.1 mL, 0.9 mmol), and PPh₃ (240 mg, 0.92 mmol) were diluted with THF (15 mL). Diisopropyl azodicarboxylate (0.18 mL, 0.92 mmol) was added to the THF solution and the resulting mixture was stirred at room temperature for 3 h. The reaction mixture was then evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel), eluting with 10% methanol in chloroform, to provide a dark-orange solid. The solid was redissolved in chloroform (10 mL) and trifluoroacetic acid (2M in diethyl ether, 1 mL) was added. The precipitate was collected by filtration and washed with ether (2 × 2 mL) to yield the product in the form of its trifluoroacetate salt (94 mg, 62%): mp 220 °C (dec). IR (KBr) 1772, 1688, 1633, 1612 cm⁻¹; ¹H NMR (300 MHz, CD₃OD) δ 8.61 (d, J = 5.2 Hz, 1 H), 8.44 (s, 1 H), 7.54 (d, J = 7.0 Hz, 2 H), 7.16 (s, 1 H), 4.62 (t, J = 6.0 Hz, 2 H), 3.84 (s, 3 H), 3.81 (s, 3 H), 3.40–3.29 (m, 2 H), 2.89 (s, 6 H), 2.38–2.24 (m, 2 H); positive ion ESIMS m/z (rel intensity): 394 (MH⁺, 100) ); HRMS–ESI m/z: MH⁺ calcd for C₂₂H₂₃N₃O₄, 394.1767; found, 394.1770; HPLC purity: 96.18% [C-18 reverse phase, MeOH (1% CF₃COOH)/H₂O, 70:30]; 97.23% [C-18 reverse phase, MeOH (1% CF₃COOH)/H₂O, 80:20].

9-Aza-5,6-dihydro-6-(3-(4-morpholino)propyl)-2,3-dimethoxy-5,11-dioxo-11H-indeno[ 1,2-c]isoquinoline Trifluoroacetate (28)

9-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (26, 85 mg, 0.28 mmol), 4-(3-hydroxypropyl)morpholine (120 mg, 0.84 mmol), and PPh₃ (230 mg, 0.84 mmol) were diluted with THF (10 mL). Diisopropyl azodicarboxylate (0.17 mL, 0.84 mmol) was added to the THF solution and the resulting mixture was stirred at room temperature for 3 h. The reaction mixture was then evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel), eluting with 10% methanol in chloroform, to provide a dark-orange solid. The solid was redissolved in chloroform (2 mL) and trifluoroacetic acid (2M in diethyl ether, 1 mL) was added. The precipitate was collected by filtration and washed with ether (2 × 2 mL) to yield the product in the form of its trifluoroacetate salt (16 mg, 10%): mp 222–224 °C (dec). IR (KBr) 1772, 1712, 1677, 1635, 1612 cm⁻¹; ¹H NMR (300 MHz, CD₃OD) δ 8.73 (s, 1 H), 8.62 (s, 1 H), 8.55 (d, J = 8.3 Hz, 1 H), 8.24 (d, J = 8.0 Hz, 1 H), 7.80 (t, J = 7.6 Hz, 2 H), 7.59 (t, J = 7.7 Hz, 1 H), 4.75 (d, J = 6.4 Hz, 2 H), 3.99 (d, J = 11.5 Hz, 2 H), 3.68 (t, J = 12.7 Hz, 2 H), 3.50 (d, J = 12.2 Hz, 2 H), 3.44–3.35 (m, 2 H), 3.13–2.98 (m, 2 H), 2.36 (td, J = 11.7, 5.7 Hz, 2 H); positive ion ESIMS m/z (rel intensity): 436 (MH⁺, 100); HRMS–ESI m/z: MH⁺ calcd for C₂₀H₁₉N₃O₂, 436.1872; found, 436.1870; HPLC purity: 98.14% [C-18 reverse phase, MeOH (1% CF₃COOH)/H₂O, 70:30]; 96.84% [C-18 reverse phase, MeOH (1% CF₃COOH), 100].

2-Methyl-6-oxo-1,6-dihydropyridine-3-carbonitrile (31).²¹

3-Aminocrotonitrile (29, 2.9 g, 36 mmol) and ethyl propiolate (30, 3.0 mL, 36 mmol) were dissolved in dry DMF (17 mL). The reaction mixture was stirred for 1 h at room temperature and the mixture was heated at reflux for 3 d. The precipitate formed after cooling to room temperature was collected, washed with methanol (5 mL), ether (10 mL), and dried to yield 31 (1.5 g, 31%): mp >300 °C. ¹H NMR (300 MHz, DMSO-d₆) δ 12.43 (s, 1 H), 7.58 (d, J = 9.6 Hz, 1 H), 6.23 (d, J = 9.6 Hz, 1 H), 2.37 (s, 3 H).

6-Chloro-2-methylnicotinonitrile (32).²¹

A mixture of 31 (1.5 g, 11 mmol) and phosphorus oxychloride (9 mL, 0.1 mol) was heated at reflux for 6 h. The reaction mixture was cooled to room temperature and the excess phosphorus oxychloride was removed under reduced pressure. Ice cold water (50 mL) was added to the residue. The brown precipitate was collected and washed with ice cold water (3 × 25 mL), ether (2 × 20 mL), and dried to provide 32 as a light-brown solid (0.9 g, 54%): mp 104–105 °C (lit.²¹ mp 106–108 °C). ¹H NMR (300 MHz, CDCl₃) δ 7.83 (d, J = 8.1 Hz, 1 H), 7.29 (d, J = 8.1 Hz, 1 H), 2.77 (s, 3 H).

2-Methylnicotinonitrile (33)

6-Chloro-2-methylnicotinonitrile (32, 10 g, 66 mmol) and ammonium formate (41 g, 0.65 mol) were dissolved in methanol (250 mL), and palladium (5% on activated carbon, 3.5 g, 2.5 mol%) was added. The mixture was stirred at room temperature for 12 h, filtered through celite, and washed with methanol (3 × 50 mL). The combined filtrates were evaporated, and the yellow oily residue was subjected to flash column chromatography on silica gel, eluting with chloroform to provide 33 as an off-white solid (6.3 g, 81%): mp 55 °C (lit.⁴² mp 56–58 °C). ¹H NMR (300 MHz, CDCl₃) δ 7.80 (dd, J = 4.9, 1.6 Hz, 1 H), 7.02 (dd, J = 7.8, 1.7 Hz, 1 H), 6.37 (dd, J = 7.8, 5.0 Hz, 1 H), 1.88 (s, 3 H); positive ion ESIMS m/z (rel intensity): 119 (MH⁺, 100).

10-Aza-5,6-dihydro-2,3-dimethoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (35)

2-Methylnicotinonitrile (33, 3.3 g, 34 mmol), NBS (5.5 g, 33 mmol) and AIBN (600 mg, 4 mmol) were diluted with 1,2-dichloroethane (60 mL) and the mixture was heated at reflux for 9 h. The reaction mixture was concentrated to the half its original volume, filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was redissolved in acetonitrile (70 mL), 14 (11 g, 48 mmol) was added, followed by triethylamine (7 mL, 50 mmol), and the solution was heated at reflux for 2 d. The hot solution was filtered, and the precipitate was washed with boiling acetonitrile (2 × 25 mL) to provide a gray solid (2.8 g, 31%): mp 270–272 °C. IR (KBr) 1635, 1610, 1528, 1503 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 12.26 (s, 1 H), 8.40 (d, J = 4.9 Hz, 1 H), 8.19 (d, J = 7.7 Hz, 1 H), 7.63 (s, 1 H), 7.34 (dd, J = 7.6, 5.1 Hz, 1 H), 7.19 (s, 1 H), 3.95 (s, 3 H), 3.91 (s, 2 H), 3.86 (s, 3 H); positive ESIMS m/z (rel intensity): 295 (M⁺, 100).

10-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (36)

10-Aza-5,6-dihydro-2,3-dimethoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (35, 1.5 g, 5.1 mmol) and SeO₂ (1.13 g, 10.2 mmol) were diluted with 1,4-dioxane (50 mL) and the mixture was heated at reflux for 3 d. The reaction mixture was filtered while hot and the precipitate was extracted in a Soxhlet extractor with chloroform–methanol mixture (4:1). The extracts were evaporated to dryness to get 36 (1.4 g, 90%): mp >300 °C. IR (KBr) 1708, 1659, 1600, 1574 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 8.49 (d, J = 4.9 Hz, 1 H), 8.07 (d, J = 7.6 Hz, 1 H), 7.83 (s, 1 H), 7.56 (s, 1 H), 7.42 (dd, J = 7.4, 5.2 Hz, 1 H), 3.93 (s, 3 H), 3.87 (s, 3 H); negative ion ESIMS m/z (rel intensity): 307 [(M–H⁺)⁻, 100].

10-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-2,3-dimethoxy-5,11-dioxo-11H-indeno[ 1,2-c]isoquinoline Trifluoroacetate (37)

10-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (36, 92 mg, 0.3 mmol), 3-dimethylamino-1-propanol (0.1 mL, 0.9 mmol), and PPh₃ (240 mg, 0.92 mmol) were diluted with THF (15 mL). Diisopropyl azodicarboxylate (0.18 mL, 0.92 mmol) was added to the THF solution and the resulting mixture was stirred at room temperature for 12 h. The reaction mixture was then evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel), eluting with 10% methanol in chloroform, followed by preparative TLC (silica gel), eluting with 5% methanol in chloroform, to provide an orange solid. The solid was redissolved in chloroform (5 mL) and trifluoroacetic acid (2M in diethyl ether, 1 mL) was added. The precipitate was collected by filtration and washed with ether (2 × 2 mL) to yield the product in the form of its trifluoroacetate salt (34 mg, 22%): mp 212–214 °C (dec). ¹H NMR (300 MHz, DMSO-d₆) δ 10.60 (s, 1 H), 8.50 (dd, J = 5.1, 1.3 Hz, 1 H), 7.83 (dd, J = 7.5, 1.3 Hz, 1 H), 7.73 (s, 1 H), 7.41 (dd, J = 7.5, 5.1 Hz, 1 H), 7.32 (s, 1 H), 4.67 (t, J = 6.1 Hz, 2 H), 3.93 (s, 3 H), 3.90 (s, 3 H), 3.34–3.25 (m, 2 H), 2.81 (d, J = 4.7 Hz, 6 H), 2.38–2.28 (m, 2 H); positive ion ESIMS m/z (rel intensity): 394 (MH⁺, 100); HRMS–ESI m/z: MH⁺ calcd for C₂₂H₂₃N₃O₄, 394.1767; found, 394.1769; HPLC purity: 98.32% [C-18 reverse phase, MeOH (1% CF₃COOH)/H₂O, 80:20].

10-Aza-5,6-dihydro-6-[3-(4-morpholino)propyl]-2,3-dimethoxy-5,11-dioxo-11H-indeno[ 1,2-c]isoquinoline Trifluoroacetate (38)

10-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (36, 100 mg, 0.32 mmol), 4-(3-hydroxypropyl)morpholine (94 mg, 0.65 mmol), and PPh₃ (170 mg, 0.65 mmol) were diluted with THF (10 mL). Diisopropyl azodicarboxylate (130 mg, 0.65 mmol) was added to the THF solution and the resulting mixture was stirred at room temperature for 12 h. The reaction mixture was then evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel), eluting with 10% methanol in chloroform, followed by preparative TLC (silica gel), eluting with 5% methanol in chloroform, to provide an orange solid. The solid was redissolved in chloroform (5 mL) and trifluoroacetic acid (2M in diethyl ether, 1 mL) was added. The precipitate was collected by filtration and washed with ether (2 × 2 mL) to yield the product in the form of its trifluoroacetate salt (12 mg, 7%): mp 208–210 °C. ¹H NMR (300 MHz, DMSO-d₆) δ 11.05 (s, 1 H), 8.51 (d, J = 4.0 Hz, 1 H), 7.88 (d, J = 7.4 Hz, 1 H), 7.81 (s, 1 H), 7.44 (dd, J = 7.4, 5.1 Hz, 1 H), 7.40 (s, 1 H), 4.71 (t, J = 5.8 Hz, 2 H), 3.96 (t, J = 10.8 Hz, 8 H), 3.81 (dd, J = 20.1, 8.5 Hz, 2 H), 3.50 (d, J = 12.1 Hz, 2 H), 3.11 (dd, J = 21.2, 9.3 Hz, 4 H), 2.41–2.33 (m, 2 H); positive ion ESIMS m/z (rel intensity): 436 (MH⁺, 100); HRMS–ESI m/z: MH⁺ calcd for C₂₄H₂₅N₃O₅, 436.1872; found, 436.1769; HPLC purity: 98.28% [C-18 reverse phase, MeOH (1% CF₃COOH)/H₂O, 90:10]; 97.14% [C-18 reverse phase, MeOH(1% CF₃COOH)/H₂O, 70:30].

2-Methyl-1,4,5,6-tetrahydropyridine-3-carbonitrile (39)

A solution of 6-chloro-2-methylnicotinonitrile (32, 6.0 g, 39 mmol) and potassium acetate (7.8 g, 80 mmol) in methanol (80 mL) was degassed. Palladium dichloride (350 mg, 2.0 mmol) was added to the solution, and the reaction vessel was filled with hydrogen (1 atm.). The mixture was stirred at room temperature until no more hydrogen was consumed. The solvent was removed under reduced pressure and the oily residue was subjected to flash column chromatography on silica gel, eluting with chloroform, to provide 39 as light-brown solid (4.0 g, 84%): mp 49–52 °C. ¹H NMR (300 MHz, CDCl₃) δ 4.43 (s, 1 H), 3.18 (td, J = 6.2, 3.0 Hz, 2 H), 2.20 (t, J = 6.3 Hz, 2 H), 1.97 (s, 3 H), 1.80–1.64 (m, 2 H); ¹³C NMR (75 MHz, CDCl₃) δ 153.22, 123.85, 71.19, 41.22, 23.27, 20.75, 19.85.

3-Cyano-2-chloro-4-methylpyridine (40)

3-Cyano-2,6-dichloro-4-methylpyridine (11, 35.1 g, 0.19 mol) and ammonium formate (235 g, 3.73 mol) were added to a suspension of palladium (5% on activated carbon, 4.0 g, 0.8 mol%) in methanol (600 mL), and the mixture was stirred at room temperature for three days. The mixture was filtered through a Celite bed and the filtrate was evaporated to dryness, water (100 mL) and chloroform (100 mL) were added. The organic layer was separated, and the aqueous layer was extracted with chloroform (3 × 50 mL). The combined extracts were washed with water (50 mL), brine (100 mL), dried with sodium sulfate, and filtered through a thin pad of silica gel, washing with chloroform. The combined filtrates were evaporated to yield a light-brown solid (25.3 g, 87%): mp 104–106 °C (lit.²³ mp 105–108 °C). ¹H NMR (300 MHz, CDCl₃) δ 8.39 (d, J = 5.1 Hz, 1 H), 7.21 (d, J = 5.1 Hz, 1 H), 2.57 (s, 3 H).

6-Methoxy-2-methylnicotinonitrile (41)

Sodium methoxide (20 g, 0.4 mol) was added to a solution of 6-chloro-2-methylnicotinonitrile (32, 10 g, 66 mmol) in methanol (150 mL) and the mixture was heated at reflux for 1.5 h and cooled to room temperature. The precipitate was removed by filtration and the filtrate was concentrated to dryness. The crude solid was redissolved in chloroform and the resulting solution was filtered through a layer of silica gel, washing with extra portions of chloroform. The combined filtrates were evaporated to dryness to yield 41 (8.3 g, 87%): mp 81–82 °C (lit.²¹ mp 80–80.5 °C). ¹H NMR (300 MHz, CDCl₃) δ 7.66 (d, J = 8.5 Hz, 1H), 6.59 (d, J = 8.5 Hz, 1H), 3.94 (s, 3H), 2.62 (s, 3H).

10-Aza-5,6-dihydro-2,3,9-trimethoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (43)

6-Methoxy-2-methylnicotinonitrile (41, 2.2 g, 15 mmol), NBS (2.9 g, 16 mmol) and AIBN (100 mg, 0.6 mmol) were diluted with 1,2-dichloroethane (50 mL) and the mixture was heated at reflux for 3.5 h. The reaction mixture was concentrated to one-half its original volume, filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was diluted with acetonitrile (60 mL), and 14 (5.3 g, 24 mmol) was added, followed by triethylamine (3.5 mL, 25 mmol), and the solution was heated at reflux for 14 h. The solution was allowed to cool to room temperature and the obtained precipitate was filtered and washed with acetonitrile (2 × 15 mL) to provide an off-white solid (0.9 g, 19%): mp 284–286 °C. IR (KBr) 1648, 1614 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 12.20 (s, 1 H), 8.14 (d, J = 8.4 Hz, 1 H), 7.61 (s, 1 H), 7.10 (s, 1 H), 6.81 (d, J = 8.5 Hz, 1 H), 3.94 (s, 3 H), 3.91 (s, 3 H), 3.86 (s, 3H), 3.85 (s, 2 H); EIMS m/z: 324 (M⁺); CIMS m/z (rel intensity): 325 (MH⁺, 100).

10-Aza-5,6-dihydro-2,3,9-trimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (44)

10-Aza-5,6-dihydro-2,3,9-trimethoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (43, 0.7 g, 2.2 mmol) and SeO₂ (0.48 mg, 4.3 mmol) were diluted with 1,4-dioxane (50 mL) and the mixture heated at reflux for 24 h. The reaction mixture was filtered while hot and the precipitate was washed with hot dioxane (3 × 100 mL). The combined filtrates were evaporated to dryness under reduced pressure to obtain 44 (0.65 g, 89%): mp >350 °C. IR (KBr) 1713, 1645, 1624, 1612, 1592 cm⁻¹; NMR data has not been obtained due to poor solubility of the sample. Negative ion ESIMS m/z (rel intensity): 337 [(M–H⁺)⁻, 100].

10-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-2,3,9-trimethoxy-5,11-dioxo-11H-indeno[ 1,2-c]isoquinoline Trifluoroacetate (45)

10-Aza-5,6-dihydro-2,3,9-trimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (44, 110 mg, 0.32 mmol) was added to a stirred solution of PPh₃ (170 mg, 0.65 mmol) and diisopropyl azodicarboxylate (0.13 mL, 0.65 mmol) in tetrahydrofuran (10 mL). The mixture was stirred at room temperature until the solid material completely disappeared to form a dark-red solution. 3-Dimethylamino-1-propanol (67 mg, 0.65 mmol) was added dropwise to the resulting solution over the course of 30 min, and the reaction mixture was stirred at room temperature for 3 d. The resulting mixture was evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel), eluting with 3% methanol in chloroform, to provide a red solid. The solid was redissolved in chloroform (10 mL) and trifluoroacetic acid (2M in diethyl ether, 1 mL) was added. The precipitate was collected by filtration and washed with ether (2 × 2 mL) to yield the product in the form of its trifluoroacetate salt (52 mg, 30%): mp 250–252 °C (dec). IR (KBr) 1692, 1621, 1605 1562 cm⁻¹; ¹H NMR (500 MHz, CD₃OD) δ 7.28 (d, J = 8.1 Hz, 1 H), 7.16 (s, 1 H), 6.95 (s, 1 H), 6.54 (d, J = 8.1 Hz, 1 H), 4.57 (t, J = 5.8 Hz, 2 H), 3.94 (s, 3 H), 3.83 (s, 3 H), 3.82 (s, 3 H), 3.58–3.48 (m, 2 H), 3.07 (s, 6 H), 2.46–2.32 (m, 2 H); positive ion ESIMS m/z (rel intensity): 424 (MH⁺, 100). Anal. Calcd for C₂₅H₂₆F₃N₃O₇: C, 55.87; H, 4.88; N, 7.82. Found: C, 55.45; H, 4.62; N, 7.75.

10-Aza-5,6-dihydro-6-(3-(4-morpholino)propyl)-2,3,9-trimethoxy-5,11-dioxo-11H-indeno[ 1,2-c]isoquinoline Trifluoroacetate (46)

10-Aza-5,6-dihydro-2,3,9-trimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (44, 110 mg, 0.32 mmol) was added to a stirred solution of PPh₃ (170 mg, 0.65 mmol) and diisopropyl azodicarboxylate (0.13 mL, 0.65 mmol) in tetrahydrofuran (10 mL). The mixture was stirred at room temperature until the solid material completely disappeared to form a dark-red solution. 4-(3-Hydroxypropyl)morpholine (94 mg, 0.65 mmol) was added dropwise to the resulting solution over the course of 30 min, and the reaction mixture was stirred at room temperature for 3 d. The resulting mixture was evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel), eluting with 3% methanol in chloroform, to provide a red solid. The solid was redissolved in chloroform (10 mL) and hydrochloric acid (2M in methanol, 1 mL) was added. The precipitate was collected by filtration and washed with ether (2 × 2 mL) to yield the product in the form of its trifluoroacetate salt (61 mg, 31%): mp 237–238 °C (dec). IR (KBr) 1709, 1618, 1605, 1561 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 10.81 (s, 1 H), 7.79 (d, J = 8.2 Hz, 1 H), 7.70 (s, 1 H), 7.33 (s, 1 H), 6.86 (d, J = 8.2 Hz, 1 H), 4.68 (s, 2 H), 4.00 (s, 2 H), 3.94 (s, 3 H), 3.92 (s, 3 H), 3.90 (s, 3 H), 3.78 (t, J = 11.8 Hz, 2 H), 3.62–3.40 (m, 4 H), 3.18–3.04 (m, 2 H), 2.35 (s, 2 H).; positive ion ESIMS m/z (rel intensity): 466 (MH⁺, 100); HRMS–ESI m/z: MH⁺ calcd for C₂₅H₂₇N₃O₆, 466.1978; found, 466.1980; HPLC purity: 96.75% [C-18 reverse phase, MeOH (1% CF₃COOH)/H₂O, 70:30]; 95.27% [C-18 reverse phase, MeOH (1% CF₃COOH)].

5-Bromo-3-methylpyridin-2-amine (48).²⁴

N-Bromosuccinimide (170 g, 0.95 mol) was added to a solution of 47 (99 g, 0.92 mol) and ammonium acetate (7g, 10 mol%) in acetonitrile (500 mL). The temperature of the reaction mixture during addition was controlled with an ice bath. After the full amount of NBS was added, the ice bath was removed and the reaction mixture was stirred at room temperature for 25 min and acetonitrile was removed under reduced pressure. A mixture of ethyl acetate (1 L) and water (1 L) was added to the solid residue. The resulting mixture was stirred and the organic layer was separated. The water layer was extracted with ethyl acetate (3 × 500 mL). The combined extracts were washed with water (300 mL), saturated sodium bicarbonate solution (500 L), dried with sodium sulfate and evaporated to dryness to give a dark brown solid. The crude product was redissolved in chloroform (300 mL) and the solution was filtered through a thin pad of silica gel, eluting with chloroform. The combined filtrates were evaporated under reduced pressure to yield 48 as a light-brown solid (113 g, 65%): mp 89–90 °C (lit.⁴³ mp 91–93 °C). ¹H NMR (300 MHz, CDCl₃) δ 7.97 (d, J = 2.3 Hz, 1 H), 7.36 (d, J = 2.3 Hz, 1 H), 4.50 (s, 2 H), 2.09 (s, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 156.10, 146.29, 140.03, 118.79, 108.58, 77.72, 77.30, 76.88, 17.27.

2,5-Dibromo-3-methylpyridine (49).²⁷

5-Bromo-3-methylpyridin-2-amine (48, 69 g, 0.37 mol) was suspended in hydrobromic acid (200 mL, 48% in water) and the mixture was cooled to −15 °C. Bromine (95 g, 0.59 mol) was added dropwise to the mixture followed by addition of sodium nitrite (69 g, 1 mol) in water (100 mL). Temperature of the reaction mixture was kept below −15 °C during addition. After addition, the cooling bath was removed and the reaction mixture was stirred for 3 h. The reaction mixture was cooled to −15 °C and quenched with potassium hydroxide (112 g, 2 mol) in water (500 mL). The cooling bath was removed and the mixture was stirred for 1.5 h. The products were extracted with ethyl acetate (3 × 300 mL). The combined extracts were washed with water (2 × 200 mL), saturated aqueous sodium bicarbonate (200 mL), dried with sodium sulfate, and evaporated to dryness. The oily residue was redissolved in chloroform (100 mL), and the solution was filtered through a pad of silica gel, washing with chloroform. The combined filtrates were evaporated to provide 49 as light-yellow solid (87 g, 94%): mp 38–40 °C (lit.⁴⁴ mp 41–42 °C). ¹H NMR (300 MHz, CDCl₃) δ 8.22 (d, J = 2.4 Hz, 1 H), 7.61 (d, J = 2.4 Hz, 1 H), 2.33 (s, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 148.29, 143.05, 141.15, 137.15, 119.68, 22.06.

5-Bromo-3-methylpicolinonitrile (50)

Copper(I) cyanide (21 g, 0.24 mol) was added to a solution of 2,5-dibromo-3-methylpyridine (49, 60 g, 0.24 mol) in dry DMF (200 mL), and the mixture was heated at reflux for 2 h. After cooling to room temperature, water (1500 mL) was added to the mixture and the products were extracted with ethyl acetate (3 × 300 mL). The combined extracts were washed with water (3 × 300 mL), brine (300 mL), dried with sodium sulfate and evaporated to dryness. The brown oily residue was subjected to flash column chromatography (silica gel), eluting with chloroform, to yield white solid (35 g, 74%): mp 86–88 °C. ¹H NMR (300 MHz, CDCl₃) δ 8.55 (d, J = 1.8 Hz, 1 H), 7.84 (d, J = 1.5 Hz, 1 H), 2.53 (s, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 149.75, 140.68, 139.95, 132.25, 124.61, 115.90, 18.59; EIMS m/z 196/198 (M⁺); CIMS 197/199 (MH⁺). The ¹H NMR spectrum is consistent with previously published data.⁴⁵

5-Methoxy-3-methylpicolinonitrile (51)

5-Bromo-3-methylpicolinonitrile (50, 35 g, 0.18 mol) was added to a solution of sodium methoxide (18 g, 0.54 mol) in methanol (200 mL), and the mixture was heated at reflux for 12 h. The solution was cooled to room temperature and concentrated to one third of its volume. The concentrated solution was diluted with water (150 mL), and the products were extracted with chloroform (3 × 50 mL). The combined extracts were washed with water (2 × 50 mL), brine (50 mL), dried with sodium sulfate and filtered through a pad of silica gel, washing with chloroform, to produce 51 as an off-white solid (21 g, 79%): mp 80–81 °C. IR (film) 2225, 1645, 1589 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.06 (d, J = 2.6 Hz, 1 H), 7.03 (d, J = 2.4 Hz, 1 H), 3.83 (s, 3 H), 2.43 (s, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 157.65, 139.99, 137.68, 125.05, 120.52, 116.76, 55.84, 18.71; EIMS m/z (rel intensity): 148 (M⁺, 100); CIMS m/z (rel intensity): 149 (MH⁺, 100).

7-Aza-5,6-dihydro-2,3,9-trimethoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (53)

5-Methoxy-3-methylpicolinonitrile (51, 5.0 g, 34 mmol), NBS (6.6 g, 37 mmol) and AIBN (500 mg, 3 mmol) were diluted with 1,2-dichloroethane (50 mL) and the mixture was heated at reflux for 24 h. The reaction mixture was concentrated to one-half its original volume, filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was redissolved in acetonitrile (100 mL), 4,5-dimethoxyhomophthalic anhydride (14, 11.2 g, 50 mmol) was added, followed by triethylamine (8 mL, 58 mmol), and the solution was heated at reflux for 24 h. The hot solution was filtered, and the precipitate was washed with boiling acetonitrile (2 × 25 mL) to provide a gray solid (2.4 g, 21%): mp >260 °C. IR (KBr) 1635, 1608 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 11.94 (s, 1 H), 8.22 (d, J = 2.6 Hz, 1 H), 7.63 (s, 2 H), 7.17 (s, 1 H), 3.95 (s, 3 H), 3.89 (s, 3 H), 3.87 (s, 3 H), 3.85 (s, 2 H); positive ion ESIMS m/z (rel intensity): 265 (MH⁺, 100).

7-Aza-5,6-dihydro-2,3,9-trimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (54)

10-Aza-5,6-dihydro-2,3,9-trimethoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (53, 2.08 g, 6.4 mmol) and SeO₂ (1.42 g, 12.8 mmol) were diluted with 1,4-dioxane (100 mL) and the mixture was heated at reflux for 24 h. The reaction mixture was filtered while hot and the precipitate was washed with hot dioxane (2 × 500 mL). The combined filtrates were evaporated to dryness under reduced pressure to afford 54 (2.0 g, 92%): mp >300 °C. IR (KBr) 1705, 1662, 1615, 1602, 1563 cm⁻¹; NMR data has not been obtained due to poor solubility of the sample. Negative ion ESIMS m/z (rel intensity): 339 [(M–H⁺)⁻, 100]. The product was introduced into the next step without additional purification.

7-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-2,3,9-trimethoxy-5,11-dioxo-11H-indeno[ 1,2-c]isoquinoline Hydrochloride (55)

7-Aza-5,6-dihydro-2,3,9-trimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (54, 338 mg, 1 mmol) was added to a stirred solution of PPh₃ (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390 mg, 1.9 mmol) in tetrahydrofuran (10 mL). The mixture was stirred for 4 h at room temperature. 3-Dimethylamino-1-propanol (200 mg, 1.9 mmol) was added dropwise to the resulting solution over the course of 30 min, and the reaction mixture was stirred at room temperature for 12 h. PPh₃ (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390 mg, 1.9 mmol) were added to the reaction mixture. The mixture was stirred for 6 h, and 3-dimethylamino-1-propanol (200 mg, 1.9 mmol) was added, forming a dark red solution. The solution was stirred at room temperature for 24 h and evaporated to dryness under reduced pressure. The residue was subjected to flash column chromatography (silica gel), eluting with a gradient of 1% to 5% methanol in chloroform, to provide a red solid. The solid was redissolved in chloroform (10 mL) and hydrochloric acid (1M in methanol, 1 mL) was added. The precipitate was collected by filtration and washed with ether (2 × 2 mL) to yield the product in the form of its hydrochloride salt (238 mg, 52%): mp 245 °C (dec). IR (KBr) 3445, 1699, 1651, 1611 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 9.90 (s, 1 H), 8.21 (s, 1 H), 7.81 (s, 1 H), 7.49 (s, 1 H), 7.43 (s, 1 H), 4.78 (s, 2 H), 3.94 (s, 3 H), 3.91 (s, 3 H), 3.86 (s, 3 H), 3.15 (s, 2 H), 2.76 (s, 3 H), 2.74 (s, 3 H), 2.14 (s, 2 H); positive ion ESIMS m/z (rel intensity): 424 (MH⁺, 100); HRMS–ESI m/z: MH⁺ calcd for C₂₃H₂₅N₃O₅, 424.1822; found, 424.1869; HPLC purity: 98.61% [C-18 reverse phase, MeOH]; 97.99% [C-18 reverse phase, MeOH/H₂O, 85:15].

7-Aza-5,6-dihydro-6-[3-(4-morpholino)propyl]-2,3,9-trimethoxy-5,11-dioxo-11H-indeno[ 1,2-c]isoquinoline Hydrochloride (56)

7-Aza-5,6-dihydro-2,3,9-trimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (54, 338 mg, 1 mmol) was added to a stirred solution of PPh₃ (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390 mg, 1.9 mmol) in tetrahydrofuran (10 mL). The mixture was stirred for 4 h at room temperature. 4-(3-Hydroxypropyl)morpholine (280 mg, 1.9 mmol) was added dropwise to the resulting solution over the course of 30 min, and the reaction mixture was stirred at room temperature for 12 h. PPh₃ (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390 mg, 1.9 mmol) were added to the reaction mixture. The mixture was stirred for 6 h, and 4-(3-hydroxypropyl)-morpholine (280 mg, 1.9 mmol) was added, forming a dark red solution. The solution was stirred at room temperature for 24 h and evaporated to dryness under reduced pressure. The residue was subjected to flash column chromatography (silica gel), eluting with a gradient of 1% to 5% methanol in chloroform, to provide a red solid. The solid was redissolved in chloroform (10 mL) and hydrochloric acid (1 M in methanol, 1 mL) was added. The precipitate was collected by filtration and washed with chloroform (20 mL) and diethyl ether (10 mL) to yield the product in the form of its hydrochloride salt (276 mg, 59%): mp 260–261 °C (dec). ¹H NMR (300 MHz, DMSO-d₆) δ 8.18 (d, J = 2.6 Hz, 1 H), 7.76 (s, 1 H), 7.45 (s, 1 H), 7.38 (d, J = 2.6 Hz, 1 H), 4.76 (s, 2 H), 3.93 (s, 3 H), 3.90 (s, 3 H), 3.84 (s, 3 H), 3.75 (t, J = 11.6 Hz, 4 H), 3.20 (s, 2 H), 3.06 (s, 4 H), 2.20 (s, 2 H); positive ion ESIMS m/z (rel intensity): 466 (MH⁺, 100); HRMS–ESI m/z: MH⁺ calcd for C₂₃H₂₅N₃O₅, 466.1978; found, 466.1974; HPLC purity: 95.45% [C-18 reverse phase, MeOH]; 96.67% [C-18 reverse phase, MeOH/H₂O, 85:15].

7-Aza-5,6-dihydro-9-methoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (59)

5-Methoxy-3-methylpicolinonitrile (51, 5.0 g, 34 mmol), NBS (6.6 g, 37 mmol) and AIBN (500 mg, 3 mmol) were diluted with 1,2-dichloroethane (50 mL) and the mixture was heated at reflux for 24 h. The reaction mixture was concentrated to one-half its original volume, filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was redissolved in acetonitrile (100 mL) and homophthalic anhydride (57, 9 g, 55 mmol) was added, followed by triethylamine (8 mL, 58 mmol), and the solution was heated at reflux for 24 h. The hot solution was filtered, and the precipitate was washed with boiling acetonitrile (2 × 30 mL) to provide a gray solid (4.1 g, 46%): mp 232–233 °C. IR (KBr) 1666, 1621, 1607 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 12.11 (s, 1 H), 8.29–8.19 (m, 2 H), 7.75 (d, J = 4.1 Hz, 2 H), 7.68 (s, 1 H), 7.48 (dd, J = 8.1, 4.3 Hz, 1 H), 3.89 (s, 5 H); positive ion ESIMS m/z (rel intensity): 310 (MH⁺, 100).

7-Aza-5,6-dihydro-3-nitro-9-methoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (60)

5-Methoxy-3-methylpicolinonitrile (51, 5.0 g, 34 mmol), NBS (6.6 g, 37 mmol) and AIBN (500 mg, 3 mmol) were diluted with 1,2-dichloroethane (50 mL) and the mixture was the mixture was heated at reflux for 24 h. The reaction mixture was concentrated to one-half its original volume, filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was redissolved in acetonitrile (100 mL) and 5-nitrohomophthalic anhydride (58, 11 g, 53 mmol) was added, followed by triethylamine (8 mL, 58 mmol), and the solution was heated at reflux for 24 h. The hot solution was filtered, and the precipitate was washed with boiling acetonitrile (2 × 30 mL) to provide a gray solid (2.7 g, 26%): mp >260 °C. IR (KBr) 1690, 1616, 1559 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 8.89 (d, J = 2.5 Hz, 1 H), 8.47 (dd, J = 8.8, 2.5 Hz, 1 H), 8.30 (d, J = 2.6 Hz, 1 H), 7.91 (d, J = 8.8 Hz, 1 H), 7.72 (d, J = 2.5 Hz, 1 H), 3.94 (s, 2 H), 3.91 (s, 3 H); positive ion ESIMS m/z (rel intensity): 310 (MH⁺, 100).

7-Aza-5,6-dihydro-9-methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (61)

7-Aza-5,6-dihydro-9-methoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (59, 2.64 g, 10 mmol) and SeO₂ (2.22 g, 20 mmol) were diluted with 1,4-dioxane (120 mL) and the mixture was heated at reflux for 24 h. The reaction mixture was filtered while hot and the precipitate was washed with hot dioxane (3 × 300 mL). The combined filtrates were evaporated to dryness under reduced pressure to afford 61 (2.10 g, 76%): mp >350 °C. IR (KBr) 1717, 1689, 1618, 1574 cm⁻¹; NMR and MS data has not been obtained due to poor solubility of the sample. The product was introduced into the next step without additional purification.

7-Aza-5,6-dihydro-3-nitro-9-methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (62)

7-Aza-5,6-dihydro-3-nitro-9-methoxy-5-oxo-11H-indeno[1,2-c]isoquinoline (60, 2.2 g, 7 mmol) and SeO₂ (1.6 g, 14 mmol) were diluted with 1,4-dioxane (100 mL) and the mixture was heated at reflux for 24 h. The reaction mixture was filtered while hot and the precipitate was washed with hot dioxane (2 × 500 mL). The combined filtrates were evaporated to dryness under reduced pressure to yield 62 (1.94 g, 86%): mp >300 °C. IR (KBr) 1693, 1618, 1571 cm⁻¹; NMR data has not been obtained due to poor solubility of the sample. Negative ion ESIMS m/z (rel intensity): 322 [(M–H⁺)⁻, 100]. The product was introduced into the next step without additional purification.

7-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-9-methoxy-5,11-dioxo-11H-indeno[ 1,2-c]isoquinoline (63)

7-Aza-5,6-dihydro-9-methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (61, 278 mg, 1 mmol) was added to a stirred solution of PPh₃ (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390 mg, 1.9 mmol) in tetrahydrofuran (20 mL). The mixture was stirred at room temperature for 4 h. 3-Dimethylamino-1-propanol (200 mg, 1.9 mmol) was added dropwise to the resulting solution over the course of 15 min, and the reaction mixture was stirred at room temperature for 12 h. PPh₃ (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390 mg, 1.9 mmol) were added to the reaction mixture. The mixture was stirred for 6 h, and 3-dimethylamino-1-propanol (200 mg, 1.9 mmol) was added, forming orange solution. The solution was stirred at room temperature for 24 h and evaporated to dryness under reduced pressure. The residue was subjected to flash column chromatography (silica gel), eluting with a gradient of 1% to 5% methanol in chloroform, to provide an orange solid (138 mg, 38%): mp 190–192 °C. ¹H NMR (300 MHz, CDCl₃) δ 8.35 (d, J = 8.1 Hz, 1 H), 8.16 (d, J = 8.1 Hz, 1 H), 7.93 (d, J = 2.7 Hz, 1 H), 7.59–7.49 (m, 1 H), 7.35–7.27 (m, 1 H), 7.09 (d, J = 2.7 Hz, 1 H), 4.82–4.69 (m, 2 H), 3.81 (s, 3 H), 2.40 (t, J = 7.1 Hz, 2 H), 2.18 (s, 6 H), 1.89 (dt, J = 14.6, 7.5 Hz, 2 H); positive ion ESIMS m/z (rel intensity): 364 (MH⁺, 100); HRMS–ESI m/z: MH⁺ calcd for C₂₁H₂₁N₃O₃, 364.1661; found, 364.1663; HPLC purity: 98.39% [C-18 reverse phase, MeOH]; 98.46% [C-18 reverse phase, MeOH/H₂O, 85:15].

7-Aza-5,6-dihydro-6-(3-(4-morpholino)propyl)-9-methoxy-5,11-dioxo-11Hindeno[ 1,2-c]isoquinoline (64)

7-Aza-5,6-dihydro-9-methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (61, 278 mg, 1 mmol) was added to a stirred solution of PPh₃ (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390 mg, 1.9 mmol) in tetrahydrofuran (20 mL). The mixture was stirred at room temperature for 4 h. 4-(3-Hydroxypropyl)morpholine (280 mg, 1.9 mmol) was added dropwise to the resulting solution over the course of 15 min, and the reaction mixture was stirred at room temperature for 12 h. PPh₃ (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390 mg, 1.9 mmol) were added to the reaction mixture. The mixture was stirred for 6 h, and 4-(3-hydroxypropyl)morpholine (280 mg, 1.9 mmol) was added, forming orange solution. The solution was stirred at room temperature for 24 h and evaporated to dryness under reduced pressure. The residue was subjected to flash column chromatography (silica gel), eluting with a gradient of 1% to 5% methanol in chloroform, to provide an orange solid (163 mg, 40%): mp 218–224 °C (dec). ¹H NMR (300 MHz, DMSO-d₆) δ 8.45 (d, J = 8.2 Hz, 1 H), 8.25 (d, J = 2.5 Hz, 1 H), 8.20 (d, J = 7.8 Hz, 1 H), 7.82 (t, J = 7.7 Hz, 1 H), 7.61–7.45 (m, 2 H), 4.82 (s, 2 H), 3.92 (d, J = 12.6 Hz, 5 H), 3.74 (t, J = 11.9 Hz, 2 H), 3.39 (d, J = 12.2 Hz, 2 H), 3.21 (s, 2 H), 3.03 (d, J = 11.8 Hz, 2 H), 2.22 (s, 2 H); positive ion ESIMS m/z (rel intensity): 406 (MH⁺, 100); HRMS–ESI m/z: MH⁺ calcd for C₂₁H₂₁N₃O₃, 406.1767; found, 406.1773; HPLC purity: 97.30% [C-18 reverse phase, MeOH]; 98.60% [C-18 reverse phase, MeOH/H₂O, 85:15].

7-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-9-methoxy-3-nitro-5,11-dioxo-11H-indeno[ 1,2-c]isoquinoline (65)

7-Aza-5,6-dihydro-9-methoxy-3-nitro-5,11-dioxo-11Hindeno[ 1,2-c]isoquinoline (62, 323 mg, 1 mmol) was added to a stirred solution of PPh₃ (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390 mg, 1.9 mmol) in tetrahydrofuran (20 mL). The mixture was stirred at room temperature for 4 h. 3-Dimethylamino-1-propanol (200 mg, 1.9 mmol) was added dropwise to the resulting solution over the course of 15 min, and the reaction mixture was stirred at room temperature for 12 h. PPh₃ (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390 mg, 1.9 mmol) were added to the reaction mixture. The mixture was stirred for 6 h, and 3-dimethylamino-1-propanol (200 mg, 1.9 mmol) was added, forming orange solution. The solution was stirred at room temperature for 24 h and evaporated to dryness under reduced pressure. The residue was subjected to flash column chromatography (silica gel), eluting with a gradient of 1% to 5% methanol in chloroform, to provide a red solid (250 mg, 61%): mp 224–226 °C. ¹H NMR (300 MHz, CDCl₃) δ 9.17 (d, J = 2.2 Hz, 1 H), 8.71 (d, J = 8.8 Hz, 1 H), 8.45 (dd, J = 8.9, 2.4 Hz, 1 H), 8.21 (d, J = 2.8 Hz, 1 H), 7.41 (d, J = 2.8 Hz, 1 H), 5.12–4.88 (m, 2 H), 3.98 (d, J = 4.0 Hz, 3 H), 2.48 (t, J = 7.0 Hz, 2 H), 2.22 (d, J = 3.9 Hz, 6 H), 1.99 (dt, J = 14.3, 7.1 Hz, 2 H); positive ion ESIMS m/z (rel intensity): 409 (MH⁺, 100); HRMS–ESI m/z: MH⁺ calcd for C₂₁H₂₀N₄O₅, 409.1512; found, 409.1510l; HPLC purity: 100% [C-18 reverse phase, MeOH]; 99.03% [C-18 reverse phase, MeOH/H₂O, 85:15].

7-Aza-5,6-dihydro-6-(3-(4-morpholino)propyl)-9-methoxy-3-nitro-5,11-dioxo-11H-indeno[ 1,2-c]isoquinoline (66)

7-Aza-5,6-dihydro-9-methoxy-3-nitro-5,11-dioxo-11Hindeno[ 1,2-c]isoquinoline (62, 323 mg, 1 mmol) was added to a stirred solution of PPh₃ (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390 mg, 1.9 mmol) in tetrahydrofuran (20 mL). The mixture was stirred at room temperature for 4 h. 4-(3-Hydroxypropyl)morpholine (280 mg, 1.9 mmol) was added dropwise to the resulting solution over the course of 15 min, and the reaction mixture was stirred at room temperature for 12 h. PPh₃ (510 mg, 1.9 mmol) and diisopropyl azodicarboxylate (390 mg, 1.9 mmol) were added to the reaction mixture. The mixture was stirred for 6 h, and 4-(3-hydroxypropyl)morpholine (280 mg, 1.9 mmol) was added, forming orange solution. The solution was stirred at room temperature for 24 h and evaporated to dryness under reduced pressure. The residue was subjected to flash column chromatography (silica gel), eluting with a gradient of 1% to 5% methanol in chloroform, to provide a red solid (212 mg, 47%): mp 243–245 °C (dec). ¹H NMR (300 MHz, DMSO-d₆) δ 8.81 (s, 1 H), 8.55 (s, 2 H), 8.32 (d, J = 2.5 Hz, 1 H), 7.59 (d, J = 2.5 Hz, 1 H), 4.84 (s, 2 H), 3.96 (s, 3 H), 3.79 (s, 4 H), 3.21 (m, 6 H), 2.22 (s, 2 H); positive ion ESIMS m/z (rel intensity): 451 (MH⁺, 100); HPLC purity: 95.42% [C-18 reverse phase, MeOH]; 95.93% [C-18 reverse phase, MeOH/H₂O, 85:15].

Topoisomerase I-Mediated DNA Cleavage Reactions

Human recombinant Top1 was purified from baculovirus as previously described.⁴⁶ DNA cleavage reactions were prepared as previously reported with the exception of the DNA substrate.³⁰ Briefly, a 117-bp DNA oligonucleotide (Integrated DNA Technologies) encompassing the previously identified Top1 cleavage sites in the 161-bp fragment from pBluescript SK(−) phagemid DNA was employed. This 117-bp oligonucleotide contains a single 5′-cytosine overhang, which was 3′-end-labeled by fill-in reaction with [α-³²P]dGTP in React 2 buffer (50 mM Tris–HCl, pH 8.0, 100 mM MgCl₂, 50 mM NaCl) with 0.5 units of DNA polymerase I (Klenow fragment, New England BioLabs). Unincorporated [³²P]dGTP was removed using mini Quick Spin DNA columns (Roche, Indianapolis, IN), and the eluate containing the 3′-end-labeled DNA substrate was collected. Approximately 2 nM of radiolabeled DNA substrate was incubated with recombinant Top1 in 20 μL of reaction buffer [10 mM Tris–HCl (pH 7.5), 50 mM KCl, 5 mM MgCl₂, 0.1 mM EDTA, and 15 μg/mL BSA] at 25 °C for 20 min in the presence of various concentrations of compounds. The reactions were terminated by adding SDS (0.5% final concentration) followed by the addition of two volumes of loading dye (80% formamide, 10 mM sodium hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue). Aliquots of each reaction mixture were subjected to 20% denaturing PAGE. Gels were dried and visualized by using a phosphoimager and ImageQuant software (Molecular Dynamics). For simplicity, cleavage sites were numbered as previously described in the 161-bp fragment.⁴⁶

Quantum Mechanics Calculations

All calculations were performed in Gaussian 09. The structures of model indenoisoquinoline, azaindenoisoquinolines, A–T and G–C base pairs from models A–F were optimized at the HF/6-31G** level of theory. The single point energy calculations were performed at the MP2/6-31G* and HF/6-31G* levels of theory. The NBO analysis was performed at the HF/6-31G** level of theory.

List of abbreviations

AIBN

azobisisobutyronitrile

DIAD

diisopropyl azodicarboxylate

DMF

N,N-dimethylformamide

DMSO-d₆

dimethyl-d₆ sulfoxide

NBS

N-bromosuccinimide

TFA

trifluoroacetic acid

THF

tetrahydrofuran

Top1

topoisomerase type I

Top1-DNAcc

topoisomerase type I–DNA cleavage complex