Design, Synthesis, and Biological Evaluation of Indenoisoquinoline Rexinoids with Chemopreventive Potential

Abstract

Nuclear receptors, such as the retinoid X receptor (RXR), are proteins that regulate a myriad of cellular processes. Molecules that function as RXR agonists are of special interest for the prevention and control of carcinogenesis. The majority of these ligands possess an acidic moiety that is believed to be key for RXR activation. This communication presents the design, synthesis and biological evaluation of both acidic and non-acidic indenoisoquinolines as new RXR ligands. In addition, a comprehensive structure-activity relationship study is presented that identifies the important features of the indenoisoquinoline rexinoids. The ease of modification of the indenoisoquinoline core and the lack of the necessity of a carboxyl group for activity make them an attractive and unusual family of RXR agonists. This work establishes a structural foundation for the design of new and novel rexinoid cancer chemopreventive agents.

Introduction

Nuclear receptors are cellular proteins that control gene expression¹ and regulate cellular functions such as growth, differentiation, apoptosis and metabolism.² There are 48 nuclear receptors,³ all of which share a similar structural organization.^4–6 The preferred binding partner for one-third of all nuclear receptors is retinoid X receptor (RXR). For this reason, RXR has been called the “master partner.”^{7, 8} The RXR heterodimers can be classified into two distinct groups: permissive and non-permissive. The former group is activated by agonists of RXR or the other nuclear receptor partner, as in the case of RXR-liver X receptor (LXR) heterodimers. The latter group requires the presence of the ligand of the heterodimerization partner to be activated. This group is further divided into two subgroups: conditional, where the full response to the RXR ligand occurs in the presence of the partner’s ligand, as in the case of the RXR-retinoid acid receptor (RAR) partnership; and non-conditional, where RXR-ligands cannot activate the dimer even if an agonist of the partner receptor is present, as in the case of RXR-vitamin D receptor (VDR).⁹ RXR also has the ability to form homodimers that contain ligand-binding and DNAbinding domains.

There are three isoforms of RXR: α, which is mainly found in the kidney, liver and intestine, and is the major isotype found in the skin; β, which can be detected in nearly every tissue; and γ, which is found in the pituitary gland, brain and muscles.^10–14 Literature reports suggest that there is overlap between the functions of the three isoforms, but malfunction of RXRα has far worse consequences than those of the other two types. For example, knockout mouse studies have shown that absence of the α isoform is fatal to fetal life, produces cardiac failure, and results in ocular malformations. Inactivation of the α type has an effect similar to the one observed in vitamin A-deficient fetuses, implying that this isoform is key for retinoid signaling.¹⁵

Retinoids are natural or synthetic vitamin A derivatives. The effects of retinoids, such as 9-cis-retinoic acid (9cRA, 1, Figure 1), are modulated by two families of nuclear receptors, the retinoic acid receptor (RAR) and the retinoid X receptor (RXR). When 9cRA or other retinoids bind to RAR/RXR heterodimers, the receptor is activated and conformational changes take place. The nuclear receptor then binds to its cis-acting response element initiating transcriptional activity. This up-regulates the production of the cyclin-dependent kinase p21, which produces chemopreventive effects such as stopping the cell cycle progression of cancer cells and apoptosis.^16–18 The RAR/RXR heterodimer binds to the retinoic acid response element (RARE) and, with lesser affinity, to the retinoid X receptor response element (RXRE). The RAR/RXR heterodimer binds RXRE with higher affinity than the RXR homodimer. Therapies based on 9cRA and other retinoids have two limitations: (1) a high dose is needed to achieve a biological response;^{19, 20} and (2) a variety of side effects are observed including hypothyroidism, liver toxicity and teratogenicity.^21–25

Figure 1.

RXR ligands.

Rexinoids are RXR-specific ligands having the potential to function as cancer-preventive agents without the serious side effects associated with retinoids. One rexinoid, bexarotene (2), is used to treat cutaneous T-cell lymphoma and holds promise as a chemopreventive agent against various cancers.^26–29 The RXR binding pocket is composed of hydrophobic amino acids except for an Arg316 residue that is believed to be important for transcriptional activation. A common characteristic of RXR agonists is the presence of an acidic moiety, the conjugate base of which can bind to the positively charged Arg316.^{22, 26, 30–32} The indenoisoquinoline 3 (Figure 1) was synthesized in our laboratory and found to bind to RXRα and induce apoptosis in MCF-7 breast cancer cells in a dose-dependent manner.³³ The rexinoid activity of the lead compound 3 was discovered through its inclusion in a 5000-compound library that was screened for RXR agonist activity in an RXRE-luciferase reporter gene assay, and it proved to be the only active compound in the whole array.³³ It also inhibited HL-60 cell proliferation with an IC₅₀ of 92 nM after a 96 h incubation and caused the accumulation of these cells in the subG1 phase, from 4.5% at 13 nM to 28.7% at 250 nM.³³ These results were unexpected given the lack of an acidic substituent in this indenoisoquinoline. In order to validate the indenoisoquinolines as RXRα ligands and to understand the key features for RXR agonist activity, an extensive SAR study was undertaken.

Chemistry

Exploratory docking studies were performed on RXRα in order to facilitate the rational design of indenoisoquinoline rexinoids. Various indenoisoquinolines were docked into the 9cRA binding site using GOLD, and the top binding poses calculated for each ligand were energyminimized with SYBYL. The obtained binding poses suggest that the aminopropyl side chain of compound 3 does not interact with any key residues of the protein, and thus it might possibly be removed without loss of activity. The computational studies also suggested that the addition of an acidic substituent in the 3 position of the indenoisoquinoline core may interact with the Arg316 and Ala 327 residues as seen with other rexinoids. Thus, a working hypothesis was proposed that addition of carboxylic acid side chains at C-3 would improve activity. Several indenoisoquinolines were designed and docked inside the binding pocket of RXR (PDB: 1FBY). Figure 2 shows one of the docked compounds, indenoisoquinoline 4, inside of the RXR cavity where 9cRA (1) binds.

Figure 2.

Model of indenoisoquinoline 4 inside RXR binding pocket. The image is programmed for walleyed (relaxed) viewing.

In order to prepare indenoisoquinolines with carboxylic acid side chains of various lengths and flexibilities that could mimic the structure of 1, possible precursors such as halides were synthesized. Compounds 6–8 (Table 1) were prepared from 3-amino-6-methyl-5Hindeno[ 1,2-c]isoquinoline-5,11(6H)-dione (5) by modifying a previously reported method³³ using Sandmeyer chemistry (Scheme 1). Precursor 8 was reacted with acrylonitrile (9) or methyl acrylate (10), using Heck-coupling conditions, to provide compounds 11 and 12, which feature an ethylene linker between the functional group and the indenoisoquinoline (Scheme 2). The ester group of compound 12 was hydrolyzed to provide the acid analogue 13.

Table 1.

Screening N-Methylated Indenoisoquinolines vs RXR.

Compd	R1	IR	Compd	R1	IRa
4	CH2CO2H	<1	36	CH3	<1
6	Cl	<1	37	C(O)H	<1
7	Br	<1	39	CN	<1
8	I	<1	40	NHC(O)CO2CH3	<1
11	CH=CHCN	<1	41	NHC(O)CH2CO2CH3	<1
12	CH=CHCO2CH3	<1	42	NHC(O)(CH2)2CO2CH3	<1
13	CH=CHCO2H	<1	43	NHC(O)(CH2)3CO2CH3	<1
15	C≡CO2CH3	<1	44	NHC(O)(CH2)4CO2CH3	<1
18	(CH2)3CO2CH3	<1	45	NHC(O)CO2H	<1
19	(CH2)3CO2H	<1	46	NHC(O)CH2CO2H	<1
22	CH2CO2CH3	<1	47	NHCH2CO2CH2CH3	<1
29	CO2CH3	<1	48	NHCH2CO2H	<1
30	CO2H	<1	49	H	<1
31	CO2NH2	<1	50	NO2	<1

^aIR: Induction ratio. Testing concentration: 50 µM.

Scheme 1^a.

^aReagents and conditions: (a) NaNO₂, HX, H₂O, dioxane 0 °C; (b) CuCl, CuBr, or CuI + KI.

Scheme 2^a.

^aReagents and conditions: (a) Pd(OAc)₂, Et₃N, n-Bu₄NBr, DMF; (b) KOH, EtOH, H₂O, DMF, THF.

Precursor 8 was also used to synthesize compounds with acetylene and propyl linkers between the carboxylic acid and the indenoisoquinoline. Iodide 8 was reacted with methyl propiolate (14) using Sonogashira cross-coupling conditions to provide indenoisoquinoline 15, which was hydrolyzed to furnish 16 (Scheme 3). Indenoisoquinoline 8 was also reacted with methyl but-3-enoate (17) using Heck coupling conditions. The obtained intermediate (not shown) was reduced by catalytic hydrogenation to provide 18, which was hydrolyzed to yield acid 19.

Scheme 3^a.

^aReagents and conditions: (a) Pd(PPh₃)₂Cl₂, CuI, Cs₂CO₃, DMF; (b) KOH, MeOH, H₂O, THF; (c) Pd(OAc)₂, K₂CO₃, n-Bu₄NBr, DMF; (d) H₂, Pd-C, THF, MeOH, 50 psi.

Next, an indenoisoquinoline with a single methylene linker between the functional group and the 3-position was prepared. Indenoisoquinoline 7 was coupled with ethyl cyanoacetate (20), using tetrakis(triphenylphosphine)palladium(0) (Scheme 4). The position alpha to the carbonyl of 20 was deprotonated with sodium hydride to generate the anion coupling partner in situ.^{34, 35} The intermediate 21 was hydrolyzed and decarboxylated in the presence of sodium hydroxide in aqueous ethanol at reflux to provide the indenoisoquinoline 4.³⁶ Esterification of this indenoisoquinoline afforded the methyl ester 22.

Scheme 4^a.

^aReagents and conditions: (a) i. NaH, THF, ii. Pd(PPh₃)₄, reflux; (b) NaOH, H₂O, EtOH, reflux; (c) AcCl, MeOH, reflux.

The next challenge was to attach a carboxylic acid substituent directly to the A ring of the indenoisoquinoline core. Ethyl 3-oxo-1,3-dihydroisobenzofuran-5-carboxylate (23, Scheme 5) was prepared by modifying published procedures.^37–39 Bromination of 23 with N-bromosuccinimide yielded intermediate 24, which was hydrolyzed to precursor 25. This precursor was condensed with phthalide (26) as previously described to afford methyl 5,11- dioxo-5,11-dihydroindeno[1,2-c]isochromene-3-carboxylate (27).⁴⁰ Treatment of 27 with methylamine (28) yielded indenoisoquinoline 29, which was hydrolyzed to 6-methyl-5,11-dioxo- 6,11-dihydro-5H-indeno[1,2-c]isoquinoline-3-carboxylic acid (30). The resulting acid 30 was converted into the acid chloride derivative and treated with ammonia to provide the amide analogue 31.

Scheme 5^a.

^aReagents and conditions: (a) NBS, CCl₄, AIBN, hυ, reflux; (b) H₂O, reflux; (c) i. EtOAc, MeOH, NaOMe, reflux, ii. HCl, iii. PhH, pTsOH, reflux; (d) THF; (e) KOH, THF, MeOH, H₂O, ii. HCl, H₂O; (f) i. SOCl₂, PhH, reflux, ii. NH₃, THF.

Once the acid-containing series was finished, other indenoisoquinolines containing hydrogen bond acceptors were synthesized, such as the aldehyde 37 (Scheme 6), which might interact with the Arg316 and Ala327 residues. Hence, 5-methylhomophthalic anhydride (33), obtained from 32,^{41, 42} and imine 34 were reacted as previously published.³³ Intermediate 35 was treated with thionyl chloride, followed by aluminum chloride using Friedel-Crafts conditions to provide the methyl-substituted indenoisoquinoline 36, which was oxidized to aldehyde 37.

Scheme 6^a.

^aReagents and conditions: (a) AcCl, reflux ; (b) CHCl₃; (c)( i) SOCl₂, PhH, (ii) AlCl₃, PhNO₂, (ClCH₂)₂; (d) i. NBS, AIBN, hυ, CCl₄, reflux, ii. AgNO₃, dioxane.

Compounds bearing a cyano group were prepared to further understand the role of indenoisoquinolines bearing electron-withdrawing groups on RXR activation. Lactone 38 was prepared as before⁴³ and reacted with methylamine (28) to afford the desired product 39 (Scheme 7).

Scheme 7^a.

^aReagents and conditions: (a) THF, Et₃N.

All of the N-methyl-containing indenoisoquinolines were inactive irrespective of the substituents on the 3 position (Table 1). This was perplexing given the molecular modeling results and the literature precedents showing that RXR agonists have an acid moiety that interacts with Arg316. At this point, some other indenoisoquinolines were tested to understand these results and to determine whether compound 3^{33, 44} could actually be the sole indenoisoquinoline rexinoid. The previously synthesized compounds 40–62⁴³ were therefore tested (Tables 1 and 2). Two additional active compounds 52 and 61 were discovered, suggesting that an aminopropyl side chain, rather than an acid substituent, may be important for activity. With the isochromenone 27 at hand (Scheme 5), it was easy to explore lactam side chains beyond the methyl group to develop an SAR model. The lactone 27 was reacted with amines 63–66 to produce analogues 67–70, respectively (Scheme 8). Compound 67 was converted into the acid and ester derivatives 72 and 73 using basic and acidic conditions, respectively (Scheme 9).

Table 2.

Screening N-(3-Aminopropyl)indenoisoquinoline Derivatives of the Lead Compound 3 with Acid and Ester Side Chains vs RXR.

Compd	R1	IRa
3	NH2	>1
51	NHC(O)CO2CH3	<1
52	NHC(O)CH2CO2CH3	>1
53	NHC(O)(CH2)2CO2CH3	<1
54	NHC(O)(CH2)3CO2CH3	<1
55	NHC(O)(CH2)4CO2CH3	<1
56	NHC(O)CO2H	<1
57	NHC(O)CH2CO2H	<1
58	NHC(O)(CH2)2CO2H	<1
59	NHC(O)(CH2)3CO2H	<1
60	NHC(O)(CH2)4CO2H	<1
61	NHCH2CO2H	>1
62	NHCH2CO2CH2CH3	<1

^aIR: Induction ratio. Testing concentration: 50 µM.

Scheme 8^a.

^aReagents and conditions: (a) i. CHCl₃, THF, Et₃N; ii. 3 M HCl in Et₂O, CHCl₃ (68–70 only); iii. K₂CO₃, H₂O (69 only).

Scheme 9^a.

^aReagents and conditions: (a) HCl, ethyl ether, CHCl₃; (b) NaOH, THF, EtOH; (c) HCl, CHCl₃.

At this point of the investigation, several literature reports on RXR agonists appeared that disputed the necessity of the binding of an acidic moiety on the ligand to the Arg316 residue of RXR. Kakuta et al. suggested that a carboxylic acid moiety may not be key for RXR activation.⁴⁵ The authors synthesized compounds without a carboxylic acid moiety, such as the tetrazole 74 (Figure 3), which activate RXRα heterodimers by interacting with the RXR component. The following year, the cocrystal structure of the sesquiterpene rexinoid agonist bigelovin (75) inside the RXR binding pocket was published.⁴⁶ This compound activates RXRα heterodimers by binding to the RXR component but does not activate RXR homodimers. The authors of this work suggested that hydrophobic interactions may rule RXR activation. The anticancer natural products honokiol (76) and magnolol (77) are also among the few recently reported RXRα agonists lacking a carboxylic acid moiety.⁴⁷ The crystal structure of 77 in complex with RXR has been published. An interaction between Asn306 and the hydroxyl group of 77 is observed in the crystal structure.⁴⁸ These recent publications motivated the further exploration of the indenoisoquinolines because the template 3 is novel for an activator of RXRα homodimers, it lacks a carboxylic acid, and it is distinct from compounds 74–77.

Figure 3.

RXR agonists lacking carboxylic acid side chain.

The next aim of the project was to define the optimal length of the lactam side chain in indenoisoquinolines lacking a carboxylic acid group. Compounds 78–82⁴⁹ (Figure 4), which were previously synthesized, were tested as RXRE activators. The results indicated that 3 methylene units provided the optimal length for activation as seen in Table 3. This suggested the synthesis of compounds with various substituents on the aminopropyl side chain and on the 3- position of the indenoisoquinoline scaffold to develop an SAR model.

Figure 4.

Indenoisoquinolines bearing side chains of different lengths.

Table 3.

Induction of RXR Activity by Indenoisoquinolines with N-Aminoalkyl Side Chains of Different Lengths.

Compd	n	IRa	EC50 (µM)b	% Cell Survival	cLogPc
78	2	8.2	15.9	30.7	−0.16
79	3	11.4	10.2	52.5	0.11
80	4	9.9	11.3	53.8	0.38
81	5	4.9	12.2	40.7	0.88
82	6	6.7	16.4	24.9	1.39

^aInduction ratio at a testing concentration of 50 µM.

^bEC₅₀: concentration to achieve half the maximum activity.

^ccLogP: miLogP from Molinspiration LogP Calculator.

The synthesis of indenoisoquinoline 7 (Scheme 1) was cumbersome and therefore an alternative synthesis was designed, starting from 6-bromo-3-hydroxyisobenzofuran-1(3H)-one (83, Scheme 10),⁵⁰ which reacted with phthalide (26) to produce isochromenone 84. This compound reacted with methylamine (28) to afford the indenoisoquinoline 7. Once the isochromenone 84 was at hand, it was reacted with the Boc-protected amine 63 to yield indenoisoquinoline 85 (Scheme 11). This compound was functionalized by Heck coupling with methyl acrylate (10) to provide compound 86, which was converted to the ester derivative 87.

Scheme 10^a.

^aReagents and conditions: (a) (i) EtOAc, MeOH, NaOMe, reflux, (ii) HCl, (iii) PhH, pTsOH, reflux, (iv) Ac ₂O, reflux; (b) THF, CH₃NH₂ (28).

Scheme 11^a.

^aReagents and conditions: (a) THF, CHCl₃, Et₃N; (b) Pd(OAc)₂, PPh₃, DMF; (c) HCl, CHCl₃.

Additionally, the bromo-lactone 84 was reacted with various amines 64–66, and 88–90 to provide indenoisoquinolines 91–96 (Scheme 12). The ester derivative 96 was hydrolyzed to the carboxylate 97. The aim of these reactions was to study the effect of a bromo substituent combined with different side chains on RXRE activation.

Scheme 12^a.

^aReagents and conditions: (a) CHCl₃, THF; (b) LiOH, THF, H₂O.

The next goal was to investigate the activities of indenoisoquinolines without substituents on the A ring. A set of analogues was prepared from the unsubstituted isochromenone 98 (Scheme 13).⁴⁰ This compound was reacted with various amines to provide analogues 102–103, 105–106, and 108–109. The quaternary ammonium compound 104 was prepared by methylation of 103 to understand the effect that a positively charged nitrogen, lacking hydrogen bonding capabilities, has on activity. Amide 107 was prepared from the acid derivative 106 through the acid chloride intermediate.

Scheme 13^a.

^aReagents and conditions: (a) THF, CHCl₃; (b) THF, MeI; (c) (i) SOCl₂, (ii) NH₃, THF.

The nitro group is a bioisostere of a carboxylic acid. In order to determine if nitrosubstituted compounds can work as RXR agonists and to study the effect of various side chains on the activity, lactone 110 was converted into various analogues 113–121 as shown in Scheme 14 by reaction with different amines. Compound 113 was reduced to the aniline analogue 121 under catalytic hydrogenation conditions (Scheme 14). The acetal 118 was converted to the aldehyde derivative 119 under acidic conditions.

Scheme 14^a.

^aReagents and conditions: (a) CHCl₃, THF, Et₃N; (b) i. SOCl₂, ii NH₃, THF; (c) HCl, MeOH; (d) HCl, H₂O; (e) H₂ (50 psi), Pd-C, THF.

Indenoisoquinoline 124 (Scheme 15), which lacks a ketone at C-11, was prepared to evaluate the effect that the ketone has on activity. Briefly, isochromenone 110 was subjected to catalytic hydrogenation to yield intermediate 122. Treatment of 122 with the mono Bocprotected diamine 63 unexpectedly produced 123. Removal of the protecting group under acidic conditions provided compound 124.

Scheme 15^a.

^aReagents and conditions: (a) H₂, Pd-C, THF, MeOH, CHCl₃; (b) CHCl₃, reflux; (c) HCl, CHCl₃, MeOH, TFA.

Compounds 125–161^{33, 44, 49} have been previously synthesized and the RXR activities of some of them have been reported elsewhere. These analogues have been included in Table 7 for comparison to explain and expand the present SAR study.

Table 7.

Maximum IR, EC₅₀, cLogP, Volume and pK_a of Selected Indenoisoquinolines

Compd	Max IRa	EC50 (µM)b	% Cell Survival	cLogPc	Vol	pKad
3	39.19	4.4	52.1	−0.838	299.9	9.5
52	12.47	>44.5	85.0	−0.911		9.5
61	23.03	8.4	61.2	−0.661		9.5
62	NAe	NA	22.2	0.182		9.5
70	25.9	19.46	21.7	0.457		8.5
72	NA	NA	83.7	−0.549		9.5
73	NA	NA	55.0	0.257		9.5
79	57.53	8.66	50.8	0.11		9.5
87	NA	NA	90.4	0.674		9.5
92	NA	NA	100.0	3.462		6.1
93	11.75	18.44	48.5	1.095		8.5
95	32.93	10.72	31.3	0.995		10.1
97	NA	NA	98.0	0.756		NA
102	NA	NA	100.0	3.121		6.7
103	43.94	9.6	52.5	0.31		8.5
104	NA	NA	99.4	−0.508		NA
105	106.00	9.22	51.2	0.21		10.1
106	NA	NA		−0.029		NA
107	NA	NA		2.17		NA
113	NA	NA	83.7	0.245		8.5
121	34.35	7.0	53.7	−0.638		8.5
124	73.15	10.01	49.3	−0.474		9.5
125	NA	NA	33.1	0.145		10.1
126	59.03	15.2	53.0	−0.738	300.7	10.1
129	7.14	>20.7	54.8	0.041		8.5
131	18.75	>34.7	69.2	−0.695	337.3	9.5
132	NA	NA	90.6	2.002		NA
135	NA	NA	32.1	0.045	314.1	9.5
142	NA	NA	85.9	1.859		NA
149	NA	NA	30.6	0.188	331.2	9.5
150	22.91	4.22	45.9	−0.057		9.5
151	NA	NA		2.807		NA
160	NA	NA	23.8	−1.101		9.5
161	25.02	4.84	46.8	0.143	315.1	9.5
162	3.15	0.04	84.3	5.366

^aMax IR: the maximum induction ratio observed when compounds are tested at variable concentrations.

^bEC₅₀: concentration to achieve half the maximum activity.

^ccLogP: miLogP from Molinspiration LogP Calculator.

^dThe numbers listed are the calculated pK_a values of the conjugate acids of the bases.

^eNA: Max IR not determined because IR < 2.

Biological Results

The biological data enabled a detailed and thorough SAR analysis. All of the N-methyl-substituted indenoisoquinolines containing an acidic moiety, such as 4, 13, 19, 30, 45, etc, were inactive regardless of the length of the acidic side chain (Table 1). N-Methylated compounds containing other electronegative substituents such as esters, nitriles, or aldehydes, as exemplified by 11, 12, 31, 37, and 39, were also inactive. The halide- (6–8) or methyl-substituted (36) indenoisoquinolines were not active either. Table 2 shows the preliminary screening results for acid- and ester-containing indenoisoquinolines bearing an aminopropyl side chain. Most of these compounds were also inactive. Compounds 52, with an ester moiety, and 61, which contains a carboxylic acid moiety, were the only exceptions, with maximum induction ratios (IR) of 12.47 and 23.03 respectively, and EC₅₀ (the concentration that achieves one-half the maximum IR) values of >44.5 and 8.4 µM (Tables 2 and 7).

With the notable exception of the carboxylic acid 61 containing an N-glycinyl side chain, the results did not support the initial hypothesis that incorporation of carboxylic acid-containing moieties onto the structure of indenoisoquinoline 3 would yield potent RXR agonists, but instead suggested a novel mechanism of action for RXR agonism that does not include interactions between hydrogen bond acceptors, such as carboxylates, and Arg316. However, several of the N-(3-aminopropyl) compounds were active as RXR inducers. Therefore, further experiments were carried out to find the optimal chain length for RXRE activation. Compound 79 presented the highest induction ratio of the evaluated compounds, 78–82, with a value of 11.4 at the testing concentration of 50 µM (Table 3). It also presented the lowest EC₅₀ value for the tested compounds at 10.2 µM (Table 3). Therefore, the propyl side chain was selected for further development and modified in order to understand the key structural features of these novel rexinoids. Replacement of the side-chain amino group with methoxy, methyl, chloride, or azide groups led to inactive compounds, such as 108, 109, 133, 134, 136–139 (Table 4). Likewise, acetylation of the side chain amine as in 144 significantly reduced the IR as shown in our previous publication (induction was achieved only at high concentrations of the sample).⁴⁴ Attachment of a Boc group (141) inactivated the compound. Moreover, exchanging the amino functionality with a hydroxyl group on the propyl side chain rendered the compounds inactive as seen with 94, 120, 130, 132, 142, 145 and 151 (Table 4). This result is interesting given the ability of the hydroxyl group to form hydrogen bonds. The mono or dimethylation of the amine did not eliminate the activity as seen in Table 7 for compounds 93 (IR = 11.75), 95 (32.93), 103 (43.94), 105 (106.00), 121 (34.35), and 126 (59.03). Other analogues bearing basic nitrogens on the side chain, such as morpholine and imidazole, were prepared. However, all of the compounds containing these substituents were inactive, as exemplified by 68, 69, 91, 92, 102, 127, and 128 (Table 4).

Table 4.

Screening Derivatives of N-Ethylindenoisoquinoline vs RXR.

Compd	R1	R2	IRa	Compd	R1	R2	IRa
68	CO2CH3	bCH2Imid	<1	121	NH2	CH2N(CH3)2	>1
69	CO2CH3	bCH2Morph	<1	125	NO2	CH2NHCH3	<1
70	CO2CH3	CH2N(CH3)2	>1	126	NH2	CH2NHCH3	>1
72	CO2H	CH2NH2	<1	127	CN	bCH2Imid	<1
73	CO2CH3	CH2NH2	<1	128	CN	bCH2Morph	<1
87	CH=CHCO2CH3	CH2NH2	<1	129	CN	CH2N(CH3)2	>1
91	Br	bCH2Imid	<1	130	NH2	CH2OH	<1
92	Br	bCH2Morph	<1	131	NHAc	CH2NH2	>1
93	Br	CH2N(CH3)2	>1	132	NHAc	CH2OH	<1
94	Br	CH2OH	<1	133	NO2	CH2Cl	<1
95	Br	CH2NHCH3	>1	134	NO2	CH2N3	<1
96	Br	CO2CH3	<1	135	NO2	CH2NH2	<1
97	Br	CO2H	<1	136	NH2	CH2Cl	<1
102	H	bCH2Imid	<1	137	NHAc	CH2Cl	<1
103	H	CH2N(CH3)2	>1	138	NHAc	CH2N3	<1
104	H	CH2N+(CH3)3	<1	139	NO2	N3	<1
105	H	CH2NHCH3	>1	140	NO2	CH2NHBoc	<1
106	H	CO2H	<1	141	NH2	CH2NHBoc	<1
107	H	CONH2	<1	142	NH2	CH2OH	<1
108	H	CH2OCH3	<1	143	NO2	CH2NHAc	<1
109	H	CH2CH3	<1	144	NH2	CH2NHAc	>1
113	NO2	CH2N(CH3)2	<1	145	NO2	CH2OH	<1
114	NO2	CO2H	<1	146	NO2	CH2Br	<1
115	NO2	CONH2	<1	147	NHCH3	CH2NHBoc	<1
116	NO2	CH2OCH3	<1	148	N(CH3)2	CH2NHBoc	<1
117	NO2	CH(OCH2CH3)2	<1	149	N(CH3)2	CH2NH2	<1
118	NO2	CH(OCH3)2	<1	150	NHCH3	CH2NH2	>1
119	NO2	C(O)H	<1	151	H	CH2OH	<1
120	NO2	(CH2)2OH	<1

^aIR: Induction ratio. Testing concentration: 50 µM.

^b"Imid" = N-imidazolyl; "Morph" = N morpholinyl.

Various conclusions can be drawn regarding the effect of other substituents in the 3 position of the indenoisoquinoline core. Monomethylation of the aniline nitrogen (150, Table 4) and acetylation (131) decreases the IR value to one-half of that observed with the lead compound 3 (Table 7). Moreover, induction was achieved only at concentrations higher than 34 µM as also seen in our previous publication.⁴⁴ Addition of two methyl groups rendered the analogue inactive (149) even in the presence of an aminopropyl side chain. Compounds containing nitro, cyano, carboxylic acid, and ester groups were evaluated. The nitro group was detrimental for activity regardless of the presence of an aminopropyl side chain as observed in 113, 125, 135, or 143 (Table 4). Other substituents were added to the propyl side chain of nitro-containing indenoisoquinolines but none was able to produce activity as observed with 114–120 (Table 4). The same lack of activity was observed with the esters, carboxylic acids, and nitriles such as in 68, 69, 72, 87, 127, and 128. Only one ester-substituted indenoisoquinoline 70 was active, with an IR of 25.9 and an EC₅₀ of 19.46 µM (Table 7). Also, one nitrile-containing indenoisoquinoline, compound 129, was active with an IR of 7.14 and an EC₅₀ higher than 20.7 µM. The aniline amino group can be replaced with bromine and hydrogen atoms to produce active compounds as seen with 93, 95, 103, and 105 (Table 7). Once again, only compounds containing basic amines on the side chain were active. Figure 5 shows the dose-response curves for some of the active compounds. The curves are bell-shaped rather than sigmoidal because the compounds are cytotoxic at higher concentrations.

Figure 5.

Dose-response curves of active indenoisoquinolines.

As reported previously, reduction of the ketone group of the C ring to an alcohol provided inactive indenoisoquinolines such as 152–156 (Table 5).⁴⁴ Compound 124, which lacks C-ring oxygenation, displays good activity, proving that the ketone may be removed with retention, or even as in this case, improvement of the RXR induction ratio. Moreover, it has previously been shown that the ketone group of the lead compound 3 is metabolized to an alcohol, which renders the compound inactive.⁴⁴ Therefore, compounds such as 124 may be viable alternatives to indenoisoquinoline 3.

Table 5.

Screening Indenoisoquinolines Lacking a C-11 Carbonyl vs RXR.

Compd	R1	R2	R3	IRa
124	NH2	NH2	H	>1
152	NH2	NH2	OH	<1
153	NH2	OH	OH	<1
154	NHCH3	NHBoc	OH	<1
155	NHCH3	NH2	OH	<1
156	NH2	NHCH3	OH	<1

^aIR: Induction ratio. Testing concentration: 50 µM.

Compounds containing other substituents on the indenoisoquinoline core were also tested. All of these additional compounds were inactive with the exception of 161 (Table 6). Compound 161 had an IR value of 25.02, which is lower than that observed for compound 79 (maximum IR 57.53, Table 7). Overall, the results indicate that addition of methoxy groups to the A or D rings has a detrimental effect on the activity.

Table 6.

Screening Variously Substituted Indenoisoquinolines vs RXR.

Compd	R1	R2	R3	R4	R5	IRa
157	H	CN	N(CH3)2	OCH2O		<1
158	H	N(CH3)2	NH2	H	OCH3	<1
159	OCH3	OCH3	NH2	H	OCH3	<1
160	OCH3	OCH3	NH2	H	F	<1
161	H	H	NH2	H	OCH3	>1

^aIR: Induction ratio. Testing concentration: 50 µM.

In order to gain further insight into possible determinants of the biological activities, the molecular volumes, cLogP values, and pK_b values of selected compounds were calculated and are shown in Table 7. The binding pocket of RXR is L-shaped and has a volume of 489 ų.⁴⁶ The docking results demonstrate that the indenoisoquinolines fit the cavity space. Still, the molecular volumes of some indenoisoquinolines were calculated to further understand the effect that sterics may play in the biological activity. As seen in Table 7, the larger indenoisoquinolines 131 and 149 have volumes of 337.3 ų and 331.3 ų. The larger compound, 131, occupies 69% of the space. Therefore, the differences in activities among the indenoisoquinolines are not due to any of them being too large to fit into the pocket. The calculated partition coefficients, or cLogP values, of various compounds were estimated using Molinspiration software,⁵¹ while the basicity was calculated using the Ace Organic pK_a calculator.⁵² As seen in Table 7, the active compounds tend to have lower cLogP values than the inactive analogues. Compounds with a cLogP values higher than 1.100, such as 102, 132, 142, and 151 were inactive. However, this is not the only criterion for activity. For example, the inactive acid derivative 106 has a low cLogP (−0.029). The same can be seen with the ester- or nitro-containing compounds 73, 125 or 135. The control bexarotene analogue 162 (Figure 6) had a lower IR than any of the tested compounds with a value of 3.15. The EC₅₀ of 162 was also significantly lower with a value of 0.04 µM.

Figure 6.

Bexarotene analogue LG100268.

The quaternary ammonium compound 104 was prepared in order to investigate the possibility that an electrostatic interaction is responsible for RXR activation. Even though this compound possesses a cLogP of −0.508 and a positively-charged nitrogen, it is inactive. To confirm that the activity is not related to the polarity of the side chain, the two acids 97 and 106 were prepared and found to be inactive. The addition of hydrogen-bonding groups such as alcohols (142 or 151) or amides (107) also provided inactive compounds. Basic aliphatic amines tend to have high activity as seen with compounds 3, 79, 103, 105, 121, and 126 (Table 7).

It is uncertain whether GOLD⁵³ is the best docking program for this investigation. It is known that for a given system one docking program may perform better than another.^54–56 Therefore, a new docking study using both GOLD and AutoDock Vina⁵⁷ was carried out to identify potential interactions between the aminopropyl side chain and the protein and to compare the obtained binding poses. The centroid of bigelovin⁴⁶ (75, Figure 3), a molecule which lacks an acidic group, bound in another RXR crystal structure (PDB: 3OZJ) was used as a reference. Irrespective of the docking program, only one potential hydrogen bonding interaction between the aminopropyl side chain of indenoisoquinoline 105 and the carbonyl group of Cys432 was found as depicted in Figure 7. However, there are several hydrophobic interactions between the ligand and the protein as in the case of bigelovin; for example, Phe313 and Ile310 are near the indenoisoquinoline core.

Figure 7.

GOLD (orange) and AutoDock (green) binding poses of compound 105 inside holo RXR (PDB: 3OZJ). Docking was performed using the bigelovin (75) centroid. The image is programmed for walleyed (relaxed) viewing.

Past efforts in computer-aided drug design of RXR agonists have mostly used the holo form of the protein.^7,31,58–61 This protocol assumes ligand binding to the protein once conformational changes have taken place. However, it has been proposed that upon ligand binding, and not before, RXR changes its conformation and traps the ligand inside the cavity.^62–65 It cannot be assumed that all ligands will induce the same conformational change in RXR (although, so far, the published holo RXR crystal structures present similar morphology). Therefore, the same ligand 105 was docked inside the apo-RXR structure, which was obtained by removing the ligand from the crystal structure (PDB: 1G5Y) of the all trans-retinoic acid- RXRα complex. Since all trans-retinoic acid is non-activating, the protein exists in the apo conformation that is present before activating ligand binding. This could potentially indicate a set of initial interactions between a ligand and the apoprotein that occur before the activating conformational change. The top binding poses obtained by each docking program, GOLD and AutoDock Vina, were identical. After minimization of the system, two hydrogen bond interactions between the aminopropyl side chain of 105 and Gln275 and Leu309 were found with distances of 2.6 Å and 3.5 Å, respectively. Also, the amide carbonyl is within hydrogen bonding distance, 2.8 Å, of Ala 327 (Figure 8). This interaction as well as hydrophobic interactions with the pocket are hypothesized to cause receptor activation. After binding, a conformational change takes place and the ligand is transported to a more hydrophobic environment with fewer hydrogen bonds to the protein.

Figure 8.

Hypothetical model of compound 105 inside apo RXR (PDB: 1G5Y). The image is programmed for walleyed (relaxed) viewing.

RXRα Binding

As mentioned in a previous publication, the RXRE-luciferase reporter gene assay not only assesses RXR transcriptional activation but also the binding of the ligand to RXR because trans-activation will not take place in the absence of ligand binding.³³ The binding to RXRα was further confirmed using a novel ultrafiltration affinity technique developed in our lab, and the results are detailed in Table 8 and Figure 9.⁶⁶ The ranking obtained with the ultrafiltration affinity assay was 105 > 103 > 121 > 3 > 52 > 62, which agrees with the transcriptional activation results. An analogue of bexarotene (2), LG100268 (162, Figure 6), served as a positive control.⁶⁷ Compound 162 binds to RXR with higher affinity than 2.⁶⁷

Table 8.

RXR Ultracentrifugation Binding Affinity of Indenoisoquinolines

Compd	Max IRa	Binding Ratio (Binding:Control)b	Binding Affinity Rankc
105	106.00	7.6	1
103	43.94	2.6	2
121	34.35	2.5	3
3	39.19	1.9	4
52	12.47	1.7	5
62d	NAe	-	6

^aMax IR: highest point on the titration curve. IR: Induction ratio. Testing concentration: 50 µM.

^bBinding Ratio (Binding:Control): ligand peak area for the experiment to that of the control.

^cCompounds ranked as a function of Binding Ratio.

^dTested, but no specific binding was detected.

^eNA: Max IR not determined because IR <2.

Figure 9.

Ultrafiltration LC-MS testing of postive control LG100268 (162) and compounds 105, 103, 121, 3, and 52 for binding to RXRα. Binding of the test compound to RXRα produced peak enhancement (solid line) relative to the incubation using denatured RXRα (dashed lines). Numbers at right indicate precursor and product ion m/z from tandem mass spectrometry with selected reaction monitoring.

VDR Binding

The aim of this research was to evaluate the performance of indenoisoquinolines as RXR agonists. The thorough study of the selectivity of these compounds against other receptors is outside the scope of this report. However, in a preliminary investigation the ultracentration affinity technique was used to study binding to the vitamin D receptor (VDR). This nuclear receptor is a common partner for RXR and it has been proposed that binding to both receptors produces a synergistic activation effect.^{68, 69} All of the active indenoisoquinolines were screened using the technique but only some of them display binding (Table 9 and Figure 10). Some of the RXR agonists, such as 103 and 105 (Table 4), were inactive in this assay. However, other RXR agonsists, such as 81 and 82, were inactive vs VDR. Additionally, the lead compound 3 was assayed for competitive binding against the known VDR ligand curcumin⁷⁰ at equimolar concentration. Compound 3 did not compete with curcumin for binding to VDR. These results suggest that there is limited overlap between the RXR and VDR agonists, and that some of the compounds could be combined with VDR agonists for increased transcriptional activation activity.

Table 9.

VDR Ultrafiltration Binding Affinity of Indenoisoquinolines

Compd	Positive VDR signal	Ratio to 82	Percentage of 82
82	9397620	1.0000	100.0000
81	7877048	0.8382	83.8196
131	4875144	0.5066	50.6585
79	4013179	0.4270	42.7042
80	3320824	0.3534	35.3369
126	2155086	0.2226	22.2551
121	2011978	0.2141	21.4094
3	1903415	0.1727	17.2679
124	1422175	0.1513	15.1334
62	152467	0.0162	1.6224

Figure 10.

VDR competitive ultracentrifugation assay.

Conclusions

A series of indenoisoquinolines with homologous carboxylic acid-containing substituents at C-3 were synthesized in order to evaluate the hypothesis that the RXR agonist activity of the lead compound 3 could be improved through bonding of the carboxylate of the ligand to the Arg316 side chain guanidinium moiety of the receptor. This idea proved to be non-productive, since with the exception of the glycinyl derivative 61, all of the C-3 carboxyl-containing analogues were inactive as rexinoids, regardless of the length or conformational mobility of the side chain. In fact, the presence of an N-6 alkylamine substituent proved to be a key structural determinant for activity, and the reason for this was not readily apparent from molecular modeling based on the hypothetical structures derived from docking the ligands in the 9-cis-retinoic acid binding site (Figure 2). The unusual lack of a requirement for a carboxylic acid for rexinoid activity in this series of compounds led to the consideration of alternative binding modes, resulting in the realization that more attractive binding models that were more consistent with the biological results could be derived from docking the indenoisoquinolines in the bigelovin binding site.

The important conclusions derived from analysis of the structure-activity relationships in this series include the following:

1. A 3-aminopropyl substituent at N-6 of the indenoisoquinoline is required for maximum activity.

2. In almost all cases attachment of side chains to C-3 with terminal carboxyl groups led to inactive compounds.

3. Active substituents at C-3 include a primary amine, methylamino, halogen, or in some cases a methyl ester.

4. Reduction of the C-11 ketone to an alcohol is inactivating, but replacement of the ketone of the lead compound 3 with a methylene led to the second most active compounds in the series (124, maximum IR 73.15).

5. Methylation of the side chain amino group of the lead compound 3 led to an improvement in potency (126, maximum IR 59.03).

6. Removal of the C-3 amino group from the lead compound 3 increased the potency (79, maximum IR 57.53).

7. Removal of the C-3 amino group of the lead compound 3 and methylation of the side chain amino group produced the most active compound in the series (105, maximum IR 106.00).

The key changes in the structure of the lead compound 3 that led to increases in potency are summarized the Figure 11.

Figure 11.

Summary of key SAR trends resulting in potency enhancements of the lead compound 3.

RXR activation by the indenoisoquinoline rexinoids that lack a carboxylic acid group does not seem to be linked to Arg316 binding. Instead, they should be categorized with bigelovin (75), homokiol (76), and magnolol (77), a small but growing class of rexinoids that lack a carboxylic acid group.

Experimental Section

NMR spectra were obtained at 300 or 500 (¹H) and 75 or 125 (¹³C) MHz using CDCl₃ or DMSO-d₆ as solvents with a Bruker ARX300 (QNP probe) or Bruker DX-2 500 (BBO probe) spectrometers. Silica gel column chromatography was performed with 230–400 mesh silica gel. Melting points were determined using capillary tubes with a Mel-Temp apparatus and are uncorrected. IR spectra were obtained as films on salt plates using CH₂Cl₂ or CHCl₃ as solvents or as KBr pellets, using a Perkin-Elmer 1600 series FTIR spectrometer. HPLC analyses were performed on a Waters 1525 binary HPLC pump/Waters 2487 dual λ absorbance detector system using a 5 µm C₁₈ reversed phase column. Mass spectral analyses were performed at the Purdue University Campus-wide Mass Spectrometry Center. The mass spectrometric studies were performed using ESIMS on a FinniganMAT LCQ Classic mass spectrometer, EI/CIMS on a Hewlett-Packard Engine or GCQ FinniganMAT mass spectrometer, or APCIMS on an Agilent 6320 ion trap mass spectrometer. All yields refer to isolated compounds. Unless otherwise stated, chemicals and solvents were of reagent grade and used as obtained from commercial sources without further purification. The purities were estimated by HPLC, and the major peak accounted for ≥95% of the combined total peak area when monitored by a UV detector at 254 nm.

Docking using GOLD

Molecular modeling studies were carried out using GOLD.⁵³ Compounds were docked inside the binding site of the X-ray structure of RXRα (PDB: 1FBY, 3OZJ or 1GY5). To prepare the protein, the co-crystallized ligand and water molecules were removed. Hydrogens were added as needed. The calculated centroids of 9cRA (1FBY, x = 15. 249, y = 28.495, z = 48.261), bexarotene (3OZJ, x = 15.702, y = 13.870, z = 18.037) or all-trans-retinoic acid (1G5Y, x = 53.972, y = 31.197, z = 29.896) were used as reference. The default settings with a population size of 100 were used for 1FBY. The 7–8 times speed-up default setting was used with a population size of 1000. The hydrogen bond parameter was modified to 4.0 Å. The pose with the highest GOLD score was minimized using SYBYL software. The ligand was surrounded by a sphere of radius 6.0 Å (1FBY) or 8.0 Å (3OZJ or 1GY5) and the subset was minimized by the conjugate gradient method using the MMFF94s force field and MMFF94 charges until a convergence of 0.05 kcal/(mol·Å) was achieved. The study was validated by docking 9cRA and comparing the obtained binding pose vs the crystal structure.

A second docking study was conducted with the 3OZJ structure using the centroid: x = 15.393, y = 13.965, z = 18.079. This centroid was obtained from the downloaded pdb file of the protein, which lacks hydrogen. After the centroid was determined hydrogens were added to the protein structure. The docking and minimizations were performed as previously done with 3OZJ.

Docking using AutoDock Vina

Alternatively, the ligand structures were docked using AutoDock Vina.⁵⁷ The preparation of the ligands and protein structures (3OZJ), as well as the docking simulation, were performed with an integrated computer-aided drug design platform⁷¹ based on Pymol.⁷² A ligand library was created that contained the compounds of interest in PDBQT format. To prepare the protein, the co-crystallized ligand and water molecules were removed. The protein was submitted to the Pymol-based platform to generate the required PDBQT format. To perform the docking simulation, the visualization tool in the Pymol-based platform was used to define the searching space that is represented as a rectangular box. The searching space was carefully adjusted such that the co-crystallized ligand of 3OZJ was centered in the middle of the box and all of the important binding site residues (Cys269, Asn306, Phe313, Arg316, Ile324, Ile326, Ile345, Cys432, Phe438, etc.) were included inside the box. The final searching box had dimensions of 25Å×25Å×25Å. The maximum energy difference between the best binding mode and the worst one displayed was set to be 5 kcal/mol. A maximum of 10 docking modes were output for further analysis. The study was validated by docking a bexarotene analogue⁷³ and comparing the obtained binding pose vs the crystal structure.

2-(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)acetic Acid (4)

Compound 21 (360 mg, 1.00 mmol) was dissolved in ethanol (15 mL). A solution of sodium hydroxide (100 mg) in water (15 mL) was added and the reaction mixture heated at reflux, with stirring, for 10 h. The reaction mixture was allowed to reach room temperature and concentrated hydrochloric acid (2 mL) was added. A light brown solid precipitated. The solid was washed with water (15 mL), dried, washed with diethyl ether (10 mL) and dichloromethane-hexanes (4:1, 20 mL), and dried. The product was obtained as an orange solid (287 mg, 89%): mp 283–285 °C. IR (film) 2953, 1753, 1693, 1663, 1619, 1579, 1544, 1511, 1433, 1317, 1054, 898, 763 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 12.43 (s, 1 H), 8.44 (d, J = 8.3 Hz, 1 H), 8.08 (s, 1 H), 7.86 (d, J = 7.5 Hz, 1 H), 7.70 (d, J = 8.3 Hz, 1 H), 7.54-7.45 (m, 3 H), 3.95 (s, 3 H), 3.73 (s, 2 H); EIMS m/z (rel intensity) 319 (M⁺, 44), 274 [(M - CO₂H)⁺, 100]; HREIMS calcd for C₁₉H₁₃NO₄ 319.0845 (M⁺), found 319.0840 (M⁺); HPLC purity: 99.48% (C₁₈ reversed phase, 1% TFA in MeOH-H₂O, 90:10).

3-Chloro-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (6)

3-Amino-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (5, 129 mg, 0.46 mmol) was dissolved in water (5 mL) and dioxane (10 mL). Concentrated hydrochloric acid (0.5 mL) was added and the reaction mixture was cooled to 0 °C. A solution of sodium nitrite (42 mg, 0.60 mmol) in water (5 mL) was added dropwise. The reaction mixture was stirred for 30 min at 0 °C. A solution of copper(I) chloride (97.5 mg, 1 mmol) in water (10 mL) and hydrochloric acid (1 mL) was added slowly while the temperature was maintained at 0 °C. The reaction mixture was stirred at 0 °C for 30 min and then heated at reflux for 6 h. The solution was cooled down to room temperature and extracted with chloroform (3 × 200 mL). The organic extracts were combined and concentrated in vacuo. The product was purified by silica gel column chromatography, eluting with dichloromethane. The product 6 was obtained as a light red solid (17 mg, 12%): mp 281–283 °C. IR (film) 3078, 2942, 1690, 1670, 1657, 1606, 1573, 1541, 1499, 1314, 1195, 885, 835, 711 cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 8.61 (d, J = 8.1 Hz, 1 H), 8.30 (s, 1 H), 7.69-7.61 (m, 3 H), 7.46-7.41 (m, 2 H), 4.06 (s, 3 H); ¹³C NMR (125 MHz, DMSO-d₆) δ 190.1, 162.4, 156.0, 137.4, 134.9, 134.2, 133.1, 133.0, 131.2, 130.4, 127.9, 125.0, 124.4, 123.4, 122.8, 107.8, 32.5; ESIMS m/z (rel intensity) 295 (MH⁺, 100); HRESIMS m/z calcd for C₁₇H₁₀O₂Cl 295.0400 (MH⁺), found 295.0397 (MH⁺); HPLC purity: 95.08% (C₁₈ reversed phase, MeOH, 100).

3-Bromo-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (7)

Method 1. 3- Amino-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (5, 957 mg, 3.46 mmol) was dissolved in water (15 mL). Hydrobromic acid (48% in water, 2.1 mL) was added, the reaction mixture was cooled to 0 °C, and a solution of sodium nitrite (310 mg, 4.49 mmol) in water (15 mL) was added slowly. The reaction mixture was stirred for 0.5 h at 0 °C. This solution was added dropwise to a solution of copper(I) bromide (1.08 g, 7.53 mmol) in water (10 mL) and dioxane (10 mL) that was maintained at 0 °C. The reaction mixture was stirred at 0 °C for 0.5 h and then stirred at room temperature for 16 h. The solution was cooled down to room temperature and extracted with chloroform (3 × 200 mL). The organic extracts were combined and concentrated in vacuo. The product was purified by silica gel column chromatography, eluting with chloroform. The title compound 7 was obtained as an orange solid (605 mg, 52%). Method 2. 3-Bromoindeno[1,2-c]isochromene-5,11-dione (84, 653 mg, 2 mmol) was dissolved in tetrahydrofuran (10 mL). A solution of methylamine (28, 0.5 mL, 33% in ethanol) in chloroform (5 mL) was added and the reaction mixture was stirred at room temperature for 6 h and then heated at reflux for 1 h. The solvent was removed under vacuum. The product was obtained as a red solid (380 mg, 100%): mp 265–267 °C. IR (film) 3066, 2917, 1693, 1657, 1603, 1571, 1539, 1498, 1453, 1432, 1381, 1312, 1196, 1082, 882, 845, 833, 719, 691 cm⁻¹, ¹H NMR (500 MHz, CDCl₃) δ 8.54 (d, J = 8.6 Hz, 1 H), 8.47 (d, J = 2.0 Hz, 1 H), 7.78 (dd, J = 8.6 Hz, J = 2.1 Hz, 1 H), 7.65 (d, J = 6.7 Hz, 1 H), 7.63 (d, J = 7.0 Hz, 1 H), 7.43 (m, 2 H), 4.05 (s, 3 H); ¹³C NMR (125 MHz, CDCl₃) δ 190.0, 162.2, 156.1, 137.4, 136.9, 134.9, 133.1, 131.2, 131.0, 130.7, 128.5, 125.1, 124.6, 123.4, 122.9, 120.8, 107.8, 33.1; EIMS m/z (rel intensity) 339, (M⁺, 100); HREIMS m/z calcd for C₁₇H₁₀NO₂Br 338.9895 (M⁺), found 338.9892 (M⁺); HPLC purity: 95.10% (C₁₈ reversed phase, MeOH-H₂O, 90:10); 95.72% (C₁₈ reversed phase, MeOH, 100).

3-Iodo-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (8)

3-Amino-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (5, 1.12 g, 4.06 mmol) was dissolved in hydroiodic acid (55%, 5 mL), dioxane (10 mL) and dimethylsulfoxide (1 mL). The reaction mixture was placed in a calcium chloride ice bath at −20 ± 5 °C. This bath was placed inside a container filled with dry ice and acetone at −78 °C. A solution of sodium nitrite (1.15 g, 16.7 mmol) in water (10 mL) was added slowly. The reaction mixture was stirred for 30 min at −20 ± 5 °C. Potassium iodide (3.20 g, 19.3 mmol) and copper (I) iodide (3.60 g, 18.9 mmol) were partially dissolved in hydroiodic acid (10 mL). This mixture was added dropwise to the reaction mixture. The reaction mixture was allowed to warm to room temperature and stirred for 8 h. The reaction mixture was diluted with water (30 mL) and chloroform (30 mL), and filtered. The aqueous layer was extracted with chloroform (3 × 50 mL). The organic extracts were combined, washed with water (50 mL) and brine (50 mL), and concentrated under vacuum. The compound was purified by silica gel column chromatography, eluting with dichloromethane. The product 8 was obtained as a red solid (813 mg, 51.7 %): mp 263–265 °C. IR (KBr) 3065, 1690, 1664, 1602, 1571, 1536, 1497, 1426, 1310 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) d 8.67 (d, J = 1.8 Hz, 1 H), 8.39 (d, J = 8.5 Hz, 1 H), 7.98 (dd, J = 8.4, 2.0 Hz, 1 H), 7.65 (m, 2 H), 7.43 (m, 2 H), 4.05 (s, 3 H); EIMS m/z (rel intensity) 387 (M⁺, 100); HREIMS calcd for C₁₇H₁₀NO₂I 386.9756 (M⁺), found 386.9754 (M⁺); HPLC purity: 97.57% (C₁₈ reversed phase, MeOH/H₂O, 90:10); 97.66 (C₁₈ reversed phase, MeOH, 100).

(E)-3-(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3- yl)acrylonitrile (11)

3-Iodo-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (8, 303 mg, 0.78 mmol), acrylonitrile (9, 100 mL, 1.56 mmol), palladium (II) acetate (18 mg, 0.08 mmol), tetrabutylammonium bromide (10 mg), and triethylamine (220 mL, 1.56 mmol) were dissolved in dimethylformamide (6 mL). The reaction mixture was purged with argon and heated in a closed flask at 100 °C for 14 h. The reaction mixture was filtered through celite, and the celite washed with chloroform (100 mL). The organic phases were combined, and chloroform was removed under vacuum to give a dimethylformamide solution. The product was precipitated with ether and purified by silica gel column chromatography, eluting with chloroform. The desired compound was obtained as a red solid (79 mg, 33%): mp > 350 °C. IR (film) 3065, 2915, 1698, 1666, 1517, 1433, 1197, 1047, 987, 821, 758, 700, 666 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 8.46 (d, J = 8.3 Hz, 1 H), 8.34 (s, 1 H), 8.06 (d, J = 8.5 Hz, 1 H), 7.90 (d, J = 7.5 Hz, 1 H), 7.76 (d, J = 16.7 Hz, 1 H), 7.58-7.46 (m, 3 H), 6.55 (d, J = 16.7 Hz, 1 H), 3.95 (s, 3 H); EIMS m/z (rel intensity) 312 (M⁺, 100); CIMS m/z (rel intensity) 313 (MH⁺, 100); HREIMS m/z calcd for C₂₀H₁₂N₂O₂ 312.0899 (M⁺), found, 312.0900 (M⁺); HPLC purity: 98.44% (C₁₈ reversed phase, MeOH-H₂O, 85:15), 98.35% (C₁₈ reversed phase, MeOH-H₂O, 90:10).

(E)-Methyl 3-(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c] isoquinolin-3-yl)acrylate (12)

3-Iodo-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (8, 703 mg, 1.81 mmol), methyl acrylate (10, 650 mg, 7.85 mmol), triethylamine (1.80 mg, 17.8 mmol), tetrabutylammonium bromide (10 mg), and palladium (II) acetate (40 mg, 0.18 mmol) were dissolved in DMF (10 mL). The reaction mixture was purged with argon for 20 min. The reaction vessel was closed, placed in an oil bath and stirred at 105 °C for 16 h. The reaction mixture was filtered through celite and the celite was washed with chloroform (100 mL). The organic phase was washed with 1% hydrochloric acid (50 mL), water (3 × 40 mL) and brine (50 mL). The product was purified by silica gel column chromatography, eluting with chloroform-hexane, 9:1. The product was obtained as an orange solid (345 mg, 77.2%): mp 282–284 °C. IR (film) 3075, 2950, 1720, 1705, 1663, 1611, 1516, 1435, 1200, 977, 840 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.68 (d, J = 8.5 Hz, 1 H), 8.47 (s, 1 H), 7.78-7.64 (m, 4 H), 7.46-7.43 (m, 2 H), 6.55 (d, J = 16 Hz, 1 H), 4.08 (s, 3 H), 3.83 (s, 3 H); EIMS m/z (rel intensity): 345 (M⁺, 97), 314 (M⁺- OCH₃, 100); HREIMS m/z calcd for C₂₁H₁₅NO₄ 345.1001 (M⁺), found, 345.0997 (M⁺); HPLC purity: 98.51% (C₁₈ reversed phase, MeOH-H₂O, 95:5), 98.48% (C₁₈ reversed phase, MeOH-H₂O, 90:10).

(E)-3-(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)acrylic Acid (13)

(E)-Methyl 3-(6-methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3- yl)acrylate (12, 103 mg, 0.298 mmol) was dissolved in a mixture of tetrahydrofuran (5 mL), dimethylformamide (5 mL) and ethanol (5 mL). Potassium hydroxide (168 mg, 3.00 mmol) was dissolved in water (5 mL), and the obtained solution was added to the indenoisoquinoline reaction mixture. The reaction mixture was heated at reflux for 24 h and then quenched with saturated aqueous ammonium chloride (20 mL). The reaction mixture was filtered and the obtained solid was washed with water (15 mL) and dried. The filtered liquid was extracted with chloroform (20 mL) and the water layer discarded. The solid obtained from the filtration was added to the chloroform solution. Methanol (10 mL) and chloroform (10 mL) were added to dissolve the residue, and the solution was heated, filtered, and placed in the freezer at −20 °C overnight. The precipitate was filtered. This solid was placed in a vial containing water (3 mL), sonicated and filtered to give the product as a red solid (46 mg, 47%): mp 317–319 °C. Starting material (41 mg) was also recovered. IR (KBr) 3421, 3062, 3027, 1708, 1687, 1675, 1608, 1533, 1516, 1433, 1318, 1294, 1198, 899, 839, 762, 546 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 12.4 (s, 1 H), 8.46 (d, J = 8.4 Hz, 1 H), 8.30 (s, 1 H), 8.12 (d, J = 8.2 Hz, 1 H), 7.90 (d, J = 7.5 Hz, 1 H), 7.65 (d, J = 16.0 Hz, 1 H), 7.55-7.43 (m, 3 H), 6.58 (d, J = 16.0 Hz, 1 H), 3.95 (s, 1 H); ESIMS m/z (rel intensity) 332 (MH⁺, 100); HRESIMS m/z calcd for C₂₀H₁₃NO₄ 332.0923 (MH⁺), found 332.0926 (MH⁺); HPLC purity: 97.43% (C₁₈ reversed phase, 1% TFA in MeOH-H₂O, 97:3).

Methyl 3-(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)propiolate (15)

3-Iodo-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (8, 340 mg, 0.88 mmol), methyl propiolate (14, 306 mg, 3.64 mmol), cesium carbonate (572 mg, 1.76 mmol), dichloro-bis(triphenylphosphine)palladium(II) (36 mg, 0.05 mmol), and copper(I) iodide (19 mg, 0.1 mmol) were dissolved in DMF (4 mL). The reaction mixture was purged with argon for 20 min. The reaction vessel was closed, placed in an oil bath and stirred at 90 °C for 12 h. The reaction mixture was filtered through celite, and the celite was washed with chloroform (60 mL). The organic phase was washed with 1% hydrochloric acid (25 mL), water (3 × 30 mL) and brine (30 mL). The product was purified by silica gel column chromatography, eluting with chloroform, to afford a brown compound that was pure by TLC. The product was purified for a second time, eluting with ethyl acetate-hexane, 2:1. The product was obtained as a dark yellow solid (117 mg, 38.7%): mp 232–234 °C. IR (film) 3018, 2953, 2222, 1710, 1664, 1577, 1533, 1432, 1198, 1160, 903, 843, 761, 721 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.64 (d, J = 8.3 Hz, 1 H), 8.55 (d, J = 1.4 Hz, 1 H), 7.81 (dd, J = 1.6 Hz, J = 8.9 Hz, 1 H), 7.67 (m, 2 H), 7.45 (m, 2 H), 4.06 (s, 3 H), 3.86 (s, 3 H); ¹³C NMR (300 MHz, CDCl₃) δ 189.6, 162.0, 157.2, 154.2, 136.9, 136.7, 134.8, 133.8, 133.2, 131.6, 123.5, 123.4, 123.2, 122.8, 117.6, 107.4, 85.4, 81.3, 52.8, 32.2; ESIMS m/z (rel intensity) 344 (MH⁺, 100); HRESIMS m/z calcd for C₂₁H₁₃NO₄ 344.0923 (MH⁺), found, 344.0928 (MH⁺); HPLC purity: 96.50% (C₁₈ reversed phase, MeOH-H₂O, 85:15), 97.06% (C₁₈ reversed phase, MeOH-H₂O, 90:10).

3-(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)propiolic Acid (16)

Methyl 3-(6-methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3- yl)propiolate (15, 80 mg, 0.23 mmol) was dissolved in methanol (5 mL) and tetrahydrofuran (5 mL). A solution of potassium hydroxide (100 mg, 1.78 mmol) in water (2 mL) was added, and the reaction mixture heated at reflux for 1 h and at room temperature for 3 h. Concentrated hydrochloric acid (1 mL) was added, and a precipitated formed. The reaction mixture was filtered and the residue washed with diethyl ether-hexanes (1:1, 10 mL). The product was obtained as an orange solid (19 mg, 25%): mp >350 °C. IR (KBr) 2215, 1697, 1668, 1608, 1577, 1533, 1510, 1434, 1388, 1317, 1198, 922, 846, 761 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 8.50 (d, J = 8.3 Hz, 1 H), 8.25 (s, 1 H), 7.94-7.89 (m, 2H), 7.57-7.50 (m, 3 H), 3.95 (s, 3 H); ¹³C NMR (300 MHz, CDCl₃) δ 189.6, 162.0, 157.2, 154.2, 136.9, 136.7, 134.8, 133.8, 133.2, 131.6, 123.5, 123.4, 123.2, 122.8, 117.6, 107.4, 85.4, 81.3, 52.8, 32.2; ESIMS m/z (rel intensity) 330 (MH⁺, 100); HRESIMS m/z calcd for C₂₀H₁₁NO₄ 330.0766 (MH⁺), found, 330.0772 (MH⁺).

Methyl 5-(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)butanoate (18)

3-Iodo-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (8, 217 mg, 0.56 mmol) was dissolved in dimethylformamide (10 mL). The flask was purged with argon, and tetrabutylammonium bromide (10 mg) and potassium carbonate (219 mg, 1.58 mmol) were added. Palladium(II) acetate (23 mg) and methyl but-3-enoate (17, 203 mg, 2.03 mmol) were added, and the reaction mixture was heated at 90 °C for 12 h. Water (100 mL) was added, and the reaction mixture was filtered. The obtained residue was washed with water (100 mL). The aqueous extracts were combined and extracted with chloroform (4 × 15 mL). The organic extracts were combined, washed with water (3 × 50 mL), aqueous hydrochloric acid (1 mL of concentrated acid in 50 mL water, twice), saturated aqueous lithium chloride (50 mL) and brine (50 mL) and dried over sodium sulfate. The solvent was removed in vacuo, and the residue combined with the solid obtained in the filtration step. The compound was purified by silica gel column chromatography, eluting with chloroform-methanol, 50:1. The obtained solid was dissolved in THF (10 mL) and methanol (50 mL), Pd-C (10 mg) was added, and the reaction mixture placed under hydrogen at 50 psi for 8 h. The solvent was removed under vacuum and the compound was purified by silica gel column chromatography, eluting with chloroformmethanol, 50:1. The title compound was obtained as an orange solid (91 mg, 43%): mp 163–165 °C. IR (film) 2951, 1737, 1698, 1661, 1579, 1515, 1434, 1317, 1197, 763 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 8.57 (d, J = 8.2 Hz, 1 H), 8.13 (s, 1 H), 7.63-759 (m, 2 H), 7.55 (d, J = 8.4 Hz, 1 H), 7.42-7.37 (m, 2 H), 4.05 (s, 3 H), 3.67 (s, 3 H), 2.77 (t, J = 8.0 Hz, 2 H), 2.36 (t, J = 7.4 Hz, 2 H), 2.02 (t, J = 7.7 Hz, 2 H); ¹³C NMR (CDCl₃, 125 MHz) δ 190.1, 173.6, 162.9, 155.1, 140.5, 137.4, 134.7, 134.3, 132.8, 130.6, 129.9, 127.3, 123.3, 123.1, 122.8, 122.4, 108.1, 51.5, 34.9, 33.2, 32.0, 26.1; ESIMS m/z (rel intensity) 362 (MH⁺, 88); HRESIMS calcd for C₂₂H₁₉NO₄ 362.1392 (MH⁺), found 362.1395 (MH⁺); HPLC purity: 95.09% (C₁₈ reversed phase, MeOH-H₂O, 95:5), 95.12% (C₁₈ reversed phase, MeOH, 100).

5-(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)butanoic Acid (19)

Methyl 5-(6-methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3- yl)pentanoate (18, 69 mg, 0.18 mmol) was dissolved in tetrahydrofuran (5 mL) and methanol (5 mL). A solution of potassium hydroxide (110 mg) in water (5 mL) was added, and the reaction mixture was stirred at room temperature for 12 h. Concentrated hydrochloric acid (1 mL) was added and the reaction mixture diluted with water (30 mL). The aqueous phase was extracted with chloroform (3 × 20 mL). The organic phase was dried with sodium sulfate and the solvent removed in vacuo. The residue was precipitated from chloroform-hexane. The desired compound was obtained as an orange solid (34 mg, 51%): mp 212–214 °C. IR (film) 3368, 2918, 2849, 1738, 1703, 1689, 1644, 1579, 1543, 1514, 1431, 1338, 1275, 1195, 897, 782, 763, 665 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 8.61 (d, J = 8.2 Hz, 1 H), 8.17 (s, 1 H), 7.66-7.60 (m, 2 H), 7.55 (d, J = 8.5 Hz, 1 H), 7.47-7.39 (m, 2 H), 4.07 (s, 3 H), 2.81 (t, J = 7.4 Hz, 2 H), 2.39 (t, J = 7.3 Hz, 2 H), 2.04 (t, J = 7.2 Hz, 2 H); ¹³C NMR (CDCl₃-MeOH-d₄, 125 MHz) δ 190.1, 163.6, 155.5, 140.9, 137.5, 134.7, 134.6, 133.1, 130.8, 130.1, 127.3, 123.3, 123.1, 122.7, 108.4, 34.8, 31.1, 26.1; ESIMS m/z (rel intensity) 348 (MH⁺, 100); HRESIMS calcd for C₂₁H₁₇NO₄ 348.1236 (MH⁺), found 348.1240 (MH⁺); HPLC purity: 97.14% (C₁₈ reversed phase, 1% TFA MeOH-H₂O, 85:15), 95.18% (C₁₈ reversed phase, 1% TFA MeOH-H₂O, 80:20).

Ethyl 2-Cyano-2-(6-methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)acetate (21)

Ethyl cyanoacetate (20, 360 mg, 4.29 mmol) was dissolved in dry tetrahydrofuran (40 mL) under an argon atmosphere at 0 °C. Sodium hydride (150 mg, 6.25 mmol) was added immediately, and the reaction mixture was stirred while keeping the temperature constant. 3-Bromo-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (7, 1.12 g, 3.30 mmol) was dissolved in dry tetrahydrofuran (30 mL), and the solution added to the reaction mixture. Tetrahydrofuran (10 mL) was used to wash the flask in order to transfer any leftovers of compound 7 and added to the reaction mixture, which was heated at reflux for 10 h. The reaction mixture was cooled to room temperature, ethyl acetate (50 mL) was added and the solvent was removed under vacuum. The residue was purified by silica gel column chromatography, eluting with chloroform-methanol, 100:1. The product was obtained as a red solid (331 mg, 40%): mp 180 °C (dec). ¹H NMR (300 MHz, CDCl₃) δ 8.74 (d, J = 8.5 Hz, 1 H), 8.41 (d, J = 2.0 Hz, 1 H), 7.80 (dd, J = 8.5 Hz, J = 2.0 Hz, 1 H), 7.70-7.63 (m, 2 H), 7.48-7.46 (m, 2 H), 4.84 (s, 1 H), 4.28 (dd, J = 7.1 Hz, J = 6.8 Hz, 2 H), 4.08 (s, 3 H), 1.31 (t, J = 7.1 Hz, 3 H).

Methyl 2-(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)acetate (22)

2-(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)acetic acid (4, 65 mg, 0.2 mmol) was dissolved in methanol (5 mL). The solution was cooled to 0 °C, and thionyl chloride (0.5 mL) was slowly added. The reaction mixture was stirred for 3 h at room temperature. A cold solution prepared from thionyl chloride (0.5 mL) and methanol (5 mL) was added and the reaction mixture heated at reflux for 3 h. The solvent was removed under vacuum and the residue was dissolved in chloroform (15 mL). The organic phase was washed with water and brine (20 mL each) and dried over anhydrous sodium sulfate. The solvent was removed under vacuum and the residue washed with hexanes. The product was obtained as red solid (59 mg, 87%): mp 192–194 °C. IR (film) 2953, 1736, 1696, 1664, 1580, 1545, 1516, 1434, 1318, 1197, 1017, 898, 763 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.54 (d, J = 8.3 Hz, 1 H), 8.15 (s, 1 H), 7.61-7.51 (m, 3 H), 7.37-7.31 (m, 2 H), 3.97 (s, 3 H), 3.72 (s, 2 H), 3.71 (s, 3 H); ESIMS m/z (rel intensity) 334 (MH⁺, 100); HRESIMS calcd for C₂₀H₁₅NO₄ 334.1079 (MH⁺), found 334.1077 (MH⁺); HPLC purity: 94.95% (C₁₈ reversed phase, MeOH-H₂O, 95:5).

Ethyl 1-Bromo-3-oxo-1,3-dihydroisobenzofuran-5-carboxylate (24)

Ethyl 3-oxo-1,3- dihydroisobenzofuran-5-carboxylate (23, 2.10 g, 10.2 mmol), N-bromosuccinimide (2.00 g, 11.23 mmol) and AIBN (100 mg) were mixed together in a round-bottomed flask containing carbon tetrachloride (100 mL). The reaction mixture was purged with argon and irradiated with a 200 W light. The reaction mixture was gently heated at reflux for 16 h. After the reaction mixture reached room temperature, it was diluted with chloroform (100 mL) and washed with a solution of potassium hydroxide (1.5 g) in water (100 mL) and brine (50 mL). The solvent was removed under vacuum and the residue purified by silica gel column chromatography, eluting with ethyl acetate-hexane, 1:1. The product was obtained as a brown oil (2.29 g, 79.0%). IR (film) 3101, 2984, 2940, 2908, 1774, 1721, 1626, 1444, 1370, 1292, 1253, 1205, 1100, 929, 760, 720, 691 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.57 (s, 1 H), 8.70 (dd, J = 7.9 Hz, J = 1.2 Hz, 1 H), 7.72 (d, J = 8.1 Hz, 1 H), 7.43 (s, 1 H), 4.45 (dd, J = 7.1 Hz, J = 7.0 Hz, 2 H), 1.43 (t, J = 7.1 Hz, 3 H); ESIMS m/z (rel intensity) 205 [(M + H – HBr) ⁺, 100].

Ethyl 1-Hydroxy-3-oxo-1,3-dihydroisobenzofuran-5-carboxylate (25)

Ethyl 1-bromo- 3-oxo-1,3-dihydroisobenzofuran-5-carboxylate (24, 10.48 g, 36.67 mmol) was dissolved in hot water (150 mL). The reaction mixture was heated at reflux for 16 h, cooled down to 4 °C, and the water was filtered off in order to remove solid particles. Concentrated hydrochloric acid was added to the aqueous filtrate, which was extracted with ethyl acetate (2 × 100 mL). The organic solvent was removed under vacuum. The product was obtained as a brown oil (8.01 g, 98%). IR (film) 3405, 2984, 1721, 1626, 1445, 1370, 1254, 1204, 1100, 1016, 931, 759, 720, 645 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.45 (s, 1 H), 8.36 (d, J = 7.8 Hz, 1 H), 7.70 (d, J = 8.1 Hz, 1 H), 6.68 (s, 1 H), 4.39 (q, J = 7.1 Hz, 2 H), 1.41 (t, J = 7.1 Hz, 3 H); ESIMS m/z (rel intensity) 223 (MH⁺, 100).

Methyl 5,11-Dioxo-5,11-dihydroindeno[1,2-c]isochromene-3-carboxylate (27)

Ethyl 1-hydroxy-3-oxo-1,3-dihydroisobenzofuran-5-carboxylate (25, 3.32 g, 14.94 mmol) and phthalide (26, 1.69 g, 12.61 mmol) were dissolved in ethyl acetate (30 mL). A solution of sodium (1.61 g) in methanol (60 mL) was added, and the reaction mixture was heated at reflux, with stirring, for 12 h. Concentrated hydrochloric acid (15 mL) was added until a yellow color appeared and the pH of the solution was 1. Ethanol (10 mL) and benzene (10 mL) were added and the solvents were removed under vacuum. para-Toluenesulfonic acid (50 mg) and benzene (100 mL) were added to the flask, which was connected to a Dean-Stark trap, and the reaction mixture was heated at reflux for 16 h. The reaction mixture was diluted with chloroform (100 mL) and methanol (20 mL), heated and filtered. The solvent was removed under vacuum giving an orange solid that was precipitated from chloroform to provide compound 27 as an orange solid (689 mg, 17.9%): mp 233–235 °C. IR (film) 2923, 2852, 1763, 1726, 1712, 1646, 1616, 1544, 1503, 1457, 1438, 1385, 1282, 1201, 875, 759, 685 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.95 (s, 1 H), 8.42 (s, 2 H), 7.63 (d, J = 6.8 Hz, 1 H), 7.53-7.46 (m, 3 H), 3.98 (s, 3 H); EIMS m/z (rel intensity) 306 (M⁺, 100).

Methyl 6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinoline-3-carboxylate (29)

Methyl 5,11-dioxo-5,11-dihydroindeno[1,2-c]isochromene-3-carboxylate (27, 463 mg, 1.51 mmol) was dissolved in THF (40 mL). A solution of methylamine (28) in ethanol (33%, 1 mL) was slowly added and the reaction mixture stirred for 13 h at room temperature. Hexanes (20 mL) were added, and the reaction mixture was placed in the freezer at −20 °C for 2 h. The liquid was filtered off, and the remaining residue was re-dissolved in hot chloroform (40 mL). Hexanes (10 mL) were added, and the reaction mixture was heated until reflux (~ 30 sec) and placed inside the freezer overnight. The desired compound was obtained as an orange solid (314 mg, 65%): mp 291–293 °C. IR (KBr) 2955, 1763, 1725, 1615, 1504, 1282, 1201, 1097, 875, 760, 685 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 9.00 (s, 1 H), 8.70 (d, J = 8.5 Hz, 1 H), 8.30 (dd, J = 8.5 Hz, J = 1.6 Hz, 1 H), 7.70 (d, J = 7.4 Hz, 1 H), 7.66 (d, J = 6.7 Hz, 1 H), 7.48-7.44 (m, 2 H), 4.08 (s, 3 H), 3.97 (s, 3 H); ¹³C NMR (125 MHz, DMSO-d₆) δ 189.9, 166.1, 162.9, 157.7, 137.1, 135.3, 135.1, 133.9, 133.1, 131.6, 130.7, 128.3, 123.4, 123.2, 122.8, 107.7, 52.2, 32.3; ESIMS m/z (rel intensity) 320 (MH⁺, 100); HRESIMS m/z calcd for C₁₉H₁₃N₂O₄ 320.0923 (MH⁺), found, 320.0920 (MH⁺); HPLC purity: 96.88% (C₁₈ reversed phase, MeOH-H₂O, 95-5), 97.58% (C₁₈ reversed phase, MeOH, 100).

6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinoline-3-carboxylic Acid (30)

Methyl 6-methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinoline-3-carboxylate (29, 109 mg, 0.34 mmol) was dissolved in THF (5 mL) and methanol (5 mL). A solution of potassium hydroxide (102 mg, 1.82 mmol) in water (5 mL) was added, and the reaction mixture was stirred at room temperature for 36 h. The solvent was removed under vacuum, concentrated hydrochloric acid (1 mL) and water (10 mL) were added, and the solution was placed inside the refrigerator at 4 °C for 1 h. The water was filtered off, and the residue washed first with diethyl ether (5 mL) and then with chloroform-hexane, 1:1 (10 mL). The desired compound was obtained as an orange solid (79 mg, 76%): mp 358–360 °C. IR (KBr) 3445, 2799, 1672, 1612, 1579, 1541, 1514, 1436, 1290, 1197, 895, 762, 575, 527 cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 13.15 (br s, 1 H), 8.66 (d, J = 1.5 Hz, 1 H), 8.50 (d, J = 8.5 Hz, 1 H), 8.19 (dd, J = 8.5 Hz, J = 1.6 Hz, 1 H), 7.90 (d, J = 7.4 Hz, 1 H), 7.66 (d, J = 6.7 Hz, 1 H), 7.54-7.44 (m, 2 H), 3.93 (s, 3 H); ¹³C NMR (125 MHz, DMSO-d₆) δ 189.9, 166.7, 162.6, 159.1, 137.1, 135.1, 134.7, 134.1, 132.1, 130.2, 128.8, 125.2, 123.0 (2 C), 122.5, 106.5, 32.2; negative ion ESIMS m/z (rel intensity) 304 [(M-H⁺)⁻, 100]; HRESIMS m/z calcd for C₁₈H₁₁NO₄ 306.0766 (MH⁺), found, 306.0761 (MH⁺); HPLC purity: 97.21% (C₁₈ reversed phase, MeOH-H₂O-TFA, 90-10-1), 96.11% (C₁₈ reversed phase, 1% TFA MeOH-H₂O, 95-5).

6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinoline-3-carboxamide (31)

Compound 30 (60 mg, 0.19 mmol) was dissolved in benzene (10 mL) at 10 °C. Thionyl chloride (0.5 mL) was added, and the reaction mixture was stirred for 2 h at room temperature. Another portion of thionyl chloride (0.5 mL) was added, and the reaction mixture was heated at reflux for 1 h and cooled to 10 °C. A solution of ammonia in tetrahydrofuran (0.4 M, 3 mL) was added and the reaction mixture was stirred for 3 h at room temperature. Chloroform (10 mL) was added and the organic phase washed with saturated aqueous potassium carbonate (2 × 10 mL), water (10 mL) and brine (10 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under vacuum. The obtained residue was washed with diethyl ether-hexane (1:1, 10 mL). The title compound was obtained as an orange solid (50 mg, 83%): mp 338–340 °C. IR (film) 3428, 2918, 1713, 1693, 1669, 1621, 1578, 1518, 1319, 1200, 891, 759 cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 8.71 (d, J = 1.8 Hz, 1 H), 8.52 (d, J = 8.4 Hz, 1 H), 8.22-8.20 (m, 2 H), 7.93 (d, J = 7.3 Hz, 1 H) 7.57-7.47 (m, 4 H), 3.99 (s, 3 H); ¹³C NMR (125 MHz, DMSO-d₆) δ 190.0, 167.1, 162.7, 158.5, 137.2, 134.6, 134.0, 133.9, 132.8, 131.9, 125.0, 122.9, 122.6, 122.5, 106.5, 32.6; EIMS m/z (rel intensity) 304 (M⁺, 100); HREIMS calcd for C₁₈H₁₂N₂O₃ 304.0848 (M⁺), found 304.0846 (M⁺); HPLC purity: 95.87 % (C₁₈ reversed phase, MeOH-H₂O, 90:10), 97.40 % (C₁₈ reversed phase, MeOH-H₂O, 95:5).

5-Methylhomophthalic Anhydride (33)

2-(Carboxymethyl)-5-methylbenzoic acid⁴² 32, 3.30 g, 17.0 mmol) was dissolved in acetyl chloride (25 mL) and the reation mixture was heated at reflux for 3 h. The solvent was removed under vacuum. Toluene (10 mL) was added and the residue sonicated for 5 min to break most of the solid particles. The toluene was removed under vacuum to give the desired compound as a white solid (3.10 g, 100%): mp 212 °C (dec). IR (KBr) 2962, 1793, 1740, 1705, 1424, 1293, 1040, 765, 659 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.00 (s, 1 H), 7.49 (d, J = 7.6 Hz, 1 H), 7.22 (d, J = 7.8 Hz, 1 H), 4.08 (s, 2 H), 2.43 (s, 3 H); CIMS m/z (rel intensity) 177 (MH⁺, 81).

3,6-Dimethyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (36)

5-methylhomophthalic anydride (33, 2.99 g, 16.4 mmol) was dissolved in chloroform (35 mL). A solution of N-benzylidenemethanamine (34, 2.17 g, 18.2 mmol) in chloroform (15 mL) was slowly added and the reaction mixture stirred for 3 h at room temperature. Hexanes (20 mL) were added and the reaction mixture placed inside the refigerator at ~ 4 °C for 2 h. A precipitated was formed. The solvents were filtered off and the obtained solid washed with a mixture of dichloromethane-hexanes, 1:1 (20 mL). This provided compound 35 (2.07 g) as a mixture of cis and trans acids (1.0:1.6). 2,7-Dimethyl-1-oxo-3-phenyl-1,2,3,4- tetrahydroisoquinoline-4-carboxylic acid (35, 2.07 g, 7.01 mmol), thionyl chloride (10 mL) and benzene (90 mL) were placed in a round-bottomed flask. The reaction mixture was heated at reflux for 3 h until complete dissolution of the precipitate. The solvent was removed under vacuum and cold nitrobenzene (40 mL) and cold 1,2-dichloroethane (20 mL) were added. The reaction mixture was placed inside an ice bath and anhydrous aluminum chloride (1.70 g, 12.7 mmol) was added. The reaction mixture was heated at reflux for 3 h. Ice-water (200 mL) was added, and the reaction mixture extracted with chloroform (80 mL). The organic phase was washed with aqueous hydrochloric acid (5 mL of concentrated HCl in 45 mL of water), water (100 mL), saturated sodium bicarbonate (100 mL), water (100 mL) and brine (100 mL). The reaction mixture was concentrated, hexanes were added and the flask was placed in the freezer for 4 h. The solvent was filtered off and the obtained residue was purified by silica gel column chromatography, eluting with hexane-ethyl acetate, 4:1. The desired compound was obtained as a red solid (275 mg, 14.3%): mp 260–262 °C. IR (KBr) 1687, 1650, 1575, 1544, 1511, 1449, 1429, 1314, 1193, 892, 829, 758, 715 cm ⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.48 (d, J = 8.2 Hz, 1 H), 8.06 (s, 1 H), 7.56 (d, J = 7.4 Hz, 1 H), 7.54 (d, J = 7.0 Hz, 1 H), 7.49 (d, J = 8.3 Hz, 1 H), 7.35 (t, J = 7.6 Hz, 1 H), 7.33 (t, J = 7.0 Hz, 1 H), 3.98 (s, 3 H), 2.44 (s, 3 H); ¹³C NMR (125 MHz, CDCl₃) δ 190.3, 163.2, 155.0, 137.7, 137.2, 135.1, 135.0, 132.8, 130.6, 129.6, 127.8, 123.2, 122.4, 108.4, 32.1, 21.4; ESIMS m/z (rel intensity) 276 (MH⁺, 100); HRESIMS m/z calcd for C₁₈H₁₃NO₂ 276.1025 (MH⁺), found 276.1028 (MH⁺); HPLC purity: 96.05% (C₁₈ reversed phase, MeOH-H₂O, 90:10), 97.77% (C₁₈ reversed phase, MeOH, 100).

6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinoline-3-carbaldehyde (37)

3,6-Dimethyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (36, 218 mg, 0.79 mmol) was dissolved in carbon tetrachloride (35 mL). NBS (178 mg, 1 mmol) and AIBN (50 mg) were added and the reaction mixture was stirred at reflux for 8 h while being irradiated with a 200W lamp. The reaction mixture was cooled to room temperature, diluted with chloroform (100 mL) and washed with water, aqueous sodium bicarbonate, water and brine (30 mL each). The solvent was removed in vacuo and the residue purified by silica gel column chromatography, eluting with dichloromethane. The solid obtained after evaporation of the solvent (175 mg) was a mixture of monobrominated and dibrominated products that were extremely difficult to separate. Additionally, compound 36 (30 mg, 0.01 mmol) was recovered. The mixture of compounds was dissolved in dioxane (20 mL) and water (5 mL). Silver nitrate (80 mg, 0.47 mmol) was added and the reaction mixture heated at reflux for 7 h. The silver bromide residue was separated by filtration and the filter paper washed with chloroform (10 mL). Chloroform (15 mL) was added and the organic phase washed with water (15 mL) and brine (50 mL). The solvent was removed under vacuum and the residue was purified by silica gel column chromatography, eluting with chloroform. The product 37 was obtained as an orange solid (10 mg, 5% over two steps): mp 279–281 °C. IR (KBr) 2929, 1761, 1699, 1672, 1611, 1539, 1435, 1317, 1265, 1196, 1112, 845, 737, 665 cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 10.1 (s, 1 H), 8.78 (m, 2 H), 8.19 (dd, J = 8.5 Hz, J = 1.6 Hz, 1 H), 7.73 (d, J = 6.9 Hz, 1 H), 7.68 (d, J = 8.0 Hz, 1 H), 7.50-7.47 (m, 2 H), 4.09 (s, 3 H), 2.07 (m, 2 H); ¹³C NMR (125 MHz, DMSO-d₆) δ 191.1, 189.9, 162.9, 158.4, 136.9, 136.6, 135.1, 134.6, 133.3, 133.2, 131.9, 131.7, 124.1, 123.6, 123.4, 123.0, 107.7, 32.3; EIMS m/z (rel intensity) 289 (M⁺, 100); HREIMS calcd. for C₁₈H₁₁NO₃ 289.0739 (M⁺), found 289.0744 (M⁺); HPLC purity: 95.00% (C₁₈ reversed phase, MeOH-H₂O, 95:5).

3-Cyano-5,11-dihydro-6-methyl-5,11-dioxoindeno[1,2-c]isoquinoline (39)

5,11-Dioxo-5,11-dihydroindeno[1,2-c]isochromene-3-carbonitrile⁴³ (38, 310 mg, 1.13 mmol) was dissolved in tetrahydrofuran (30 mL). Methylamine hydrochloride (205 mg, 3.06 mmol) was added and the reaction mixture was stirred at 0 °C. Triethylamine (0.3 mL) was immediately added and the reaction mixture stirred for 12 h at room temperature and then heated at reflux for 1 h. Initially, the mixture turned red and after about 2 h, an orange precipitate formed. The reaction mixture was diluted with chloroform (50 mL) and washed with water (4 × 70 mL) and brine (100 mL). The compound was purified by silica gel column chromatography, eluting with chloroform-acetone, 20:1. After evaporation of the solvent, the compound was precipitated with dichloromethane to remove some trace impurities. The desired compound was obtained as a dark-orange solid (263 mg, 81.3%): mp 252–254 °C. IR (KBr) 3081, 2226, 1702, 1667, 1611, 1578, 1511, 1433, 1315, 1195, 1051, 894, 843, 722 cm ⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 8.76 (d, J = 8.5 Hz, 1 H), 8.63 (d, J = 1.1 Hz, 1 H), 7.87 (dd, J = 8.4 Hz, J = 1.6 Hz, 1 H), 7.73 (d, J = 7.1 Hz, 1 H), 7.69 (d, J = 6.8 Hz, 1 H), 7.51-7.49 (m, 2 H), 4.09 (s, 3 H); ¹³C NMR (125 MHz, DMSO-d₆) δ 189.5, 162.0, 158.3, 136.8, 135.4, 134.9, 133.6, 133.4, 132.0, 124.3, 123.7, 123.5, 122.9, 118.2, 110.0, 107.2, 105.2, 32.5; ESIMS m/z (rel intensity) 287 (MH⁺, 100); HRESIMS m/z calcd for C₁₈H₁₀N₂O₂ 287.0821 (MH⁺), found, 287.0825; HPLC purity: 95.17% (C₁₈ reversed phase, MeOH-H₂O, 90:10), 95.60% (C₁₈ reversed phase, MeOH-H₂O, 95:5).

General Procedure for the Synthesis of Indenoisoquinolines 67–70

Methyl 5,11- dioxo-5,11-dihydroindeno[1,2-c]isochromene-3-carboxylate (27, 101 mg, 0.33 mmol) was dissolved in tetrahydrofuran (15 mL). A solution of the desired N-substituted 1,3- propylenediamine (63–66, 1.00–1.02 mmol) in chloroform (5 mL), triethylamine (3 drops) and molecular sieves was added and the reaction mixture was stirred for 24 h at room temperature. Water (40 mL) was added, followed by chloroform (25 mL), and the organic layer was separated. The aqueous layer was extracted with chloroform (25 mL) and the organic layers combined and washed with water (3 × 50 mL), brine (40 mL), and dried over anhydrous sodium sulfate. The solvent was removed under vacuum. For compounds 68–70, the residue was dissolved in chloroform (15 mL) and hydrochloric acid (0.5 mL, 3 M solution in diethyl ether) was added to the solution. The solvent was filtered off.

Methyl 6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-6,11-dihydro-5Hindeno[1,2-c]isoquinoline-3-carboxylate (67)

The title compound was obtained as a red oil (109 mg, 70.0%): mp 172–174 °C. IR (film) 3373, 2976, 1714, 1674, 1614, 1512, 1455, 1437, 1366, 1279, 1242, 1169, 959, 858, 762 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 8.98 (d, J = 1.7 Hz, 1 H), 8.73 (d, J = 8.5 Hz, 1 H), 8.32 (dd, J = 1.9 Hz, J = 8.6 Hz, 1 H), 7.64 (d, J = 1.6 Hz, 1 H), 7.58 (d, J = 6.6 Hz, 1 H), 7.49-7.43 (m, 2 H), 5.40 (br s, 1 H), 4.63 (t, J = 7.3 Hz, 2 H), 3.96 (s, 3 H), 3.27 (q, J = 6.0 Hz, 2 H), 2.10 (br t, 2 H), 1.43 (s, 9 H); EIMS m/z (rel intensity) 467 (M⁺, 22).

Methyl 6-(3-(1H-Imidazol-1-yl)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinoline-3-carboxylate Hydrochloride (68)

The title compound was obtained as a dark yellow solid (93 mg, 63%): mp 244–246 °C. IR (KBr) 2950, 1720, 1667, 1614, 1577, 1542, 1509, 1432, 1282, 1242, 1197, 1094, 762 cm⁻¹, ¹H NMR (300 MHz, DMSO-d₆) δ 9.10 (s, 1 H), 8.76 (d, J = 1.7 Hz, 1 H), 8.66 (d, J = 8.5 Hz, 1 H), 8.33 (dd, J = 8.6 Hz, J = 1.9 Hz, 1 H), 7.82 (s, 1 H), 7.74 (d, J = 7.0 Hz, 1 H), 7.67-7.57 (m, 4 H), 4.58 (t, J = 6.8 Hz, 2 H), 4.58 (t, J = 7.1 Hz, 2 H), 2.40 (p, J = 6.6 Hz, 2 H); EIMS m/z (rel intensity) 413 (M⁺, 9), 305 ([M - C₆H₈N₂]⁺, 100); HREIMS m/z calcd for C₂₄H₁₉N₃O₄ 413.1376, found, 413.1378; HPLC purity: 95.28% (C₁₈ reversed phase, 1% TFA in MeOH-H₂O, 90:10).

Methyl 6-(3-Morpholinopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinoline-3-carboxylate (69)

Potassium carbonate (100 mg) in water (5 mL) was added and the compound extracted with chloroform (3 × 15 mL). The organic phases were combined, washed with brine (15 mL) and dried over anhydrous sodium sulfate. The title compound was obtained as a pale orange solid (61 mg, 43%): mp 251–253 °C. IR (film) 2939, 2835, 1698, 1647, 1599, 1489, 1431, 1316, 1273, 1197, 1107, 1039, 966, 787, 760, 694 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 9.00 (d, J = 1.6 Hz, 1 H), 8.76 (d, J = 8.5 Hz, 1 H), 8.32 (dd, J = 8.5 Hz, J = 1.8 Hz, 1 H), 7.83-7.80 (m, 1 H), 7.70-7.66 (m, 1 H), 7.48-7.44 (m, 2 H), 4.64 (t, J = 7.6 Hz, 2 H), 3.96 (s, 3 H), 3.71 (t, J = 4.4 Hz, 4 H), 2.59 (t, J = 6.3 Hz, 2 H), 2.50 (br s, 4 H), 2.07 (p, J = 8.7 Hz, 2 H); ESIMS m/z (rel intensity) 433 (MH⁺, 100), 455 (MH⁺, 99); HRESIMS calcd for C₂₅H₂₄N₂O₅ 433.1763 (MH⁺), found 433.1759 (MH⁺); HPLC purity: 99.34% (C₁₈ reversed phase, MeOH-H₂O, 90:10).

Methyl 6-(3-(Dimethylamino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinoline-3-carboxylate Hydrochloride (70)

The product was obtained as an orange solid (67 mg, 48%): mp 230–232 °C. IR (KBr) 3426, 2952, 1751, 1718, 1667, 1614, 1542, 1437, 1284, 1243, 1095, 763, 697 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 9.70 (br s, 1 H) 8.77 (d, J = 1.5 Hz, 1 H), 8.67 (d, J = 8.5 Hz, 1 H), 8.33 (dd, J = 8.4 Hz, J = 1.9 Hz, 1 H), 7.85 (d, J = 7.4 Hz, 1 H), 7.66-7.52 (m, 3 H), 4.58 (t, J = 6.7 Hz, 2 H), 2.76 (br s, 8 H, overlap CH₂ + N(CH₃)₂), 2.22 (m, 2 H); EIMS m/z (rel intensity) 390 (M⁺, 21); HRESIMS calcd for C₂₃H₂₂N₂O₄ 391.1585 (MH⁺), found 391.1581 (MH⁺); HPLC purity: 99.53% (C₁₈ reversed phase, 1% TFA in MeOH-H₂O, 90:10).

6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinoline-3-carboxylic Acid (71)

Compound 67 (285 mg, 0.61 mmol) was dissolved in tetrahydrofuran (5 mL) and ethanol (5 mL). A solution of sodium hydroxide (100 mg) in water (2 mL) was added and the reaction mixture stirred for 4 h at room temperature, and then heated at reflux for 1 h. The solvent was removed and a solution of hydrochloric acid in methanol (11 mL, obtained from 1 mL acetyl chloride in 10 mL of methanol) was added. Silica gel (~ 2 g) was added, the solvent was removed under vacuum and the compound run through a short column of silica gel, eluting with chloroform followed by chloroform-methanol-acetic acid, 20:1:0.5. The solvent was removed and the residue dissolved in warm chloroform-methanol, 5:1 (60 mL). The organic phase was diluted with water (50 mL) and concentrated hydrochloric acid (1 mL) was added. The organic phase was separated, the aqueous phase extracted with chloroform (20 mL) and the organic phases combined. The solvent was removed and the product was obtained as an orange solid (260 mg, 95.0%): mp 265 °C (dec). IR (KBr) 3374, 2977, 1714, 1675, 1614, 1579, 1512, 1437, 1279, 1243, 1170, 1097, 1021, 858, 763, 695 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 8.93 (d, J = 1.3 Hz, 1 H), 8.65 (d, J = 8.4 Hz, 1 H), 8.25 (dd, J = 1.7 Hz, J = 8.5 Hz, 1 H), 7.59-7.55 (m, 2 H), 7.43-7.37 (m, 2 H), 4.53 (t, J = 7.3 Hz, 2 H), 3.18 (t, J = 6.0 Hz, 2 H), 1.92 (br p, 2 H), 1.37 (s, 9 H); ESIMS m/z (rel intensity) 363 (MH⁺, 100).

6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinoline-3-carboxylic Acid Hydrochloride (72)

Compound 71 (102 mg, 0.23 mmol) was suspended in tetrahydrofuran (5 mL) and benzene (5 mL). A solution of hydrochloric acid in methanol (2 mL, prepared from 0.5 mL of acetyl chloride in 3 mL of methanol) was added and the reaction mixture heated at reflux for 1 h. Hydrochloric acid (2 mL, 2 M solution in diethyl ether) was added, and the reaction mixture was heated at reflux for another hour and then overnight at room temperature. The residue was filtered and washed with chloroform. The product was obtained as an orange solid (81 mg, 93%): mp >350 °C. IR (KBr) 2962, 1693, 1613, 1541, 1509, 1435, 1293, 902, 856, 767 cm⁻¹; ¹H NMR (DMSO-d₆, 500 MHz) δ 8.71 (d, J = 1.2 Hz, 1 H), 8.60 (d, J = 8.4 Hz, 1 H), 8.26 (d, J = 8.1 Hz, 1 H), 7.85-7.82 (m, 4 H), 7.60-7.59 (m, 2 H), 7.32 (m, 1 H), 4.53 (br t, 2 H), 2.96 (br t, 2 H), 2.09 (br p, 2 H); EIMS m/z (rel intensity) 348 (M⁺, 100); HRESIMS calcd for C₂₀H₁₈N₂O₄ 349.1188 (MH⁺), found 349.1182 (MH⁺); HPLC purity: 95.15% (C₁₈ reversed phase, MeOH-H₂O, 90:10), 98.48% (C₁₈ reversed phase, MeOH, 100).

Methyl 6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinoline-3-carboxylate Hydrochloride (73)

Compound 67 (103 mg, 0.21 mmol) was suspended in chloroform (15 mL). A solution of hydrochloric acid in diethyl ether (3 M, 4 mL) was added and the reaction mixture was heated at reflux for 2 h. An orange precipitate formed, the liquid filtered off and the solid washed with diethyl ether (10 mL). The residue was dissolved in water (15 mL) and the aqueous solution extracted with chloroform (3 × 10 mL). The organic extracts were combined, washed with brine (20 mL), and dried over anhydrous sodium sulfate. The solvent was removed in vacuo and the residue dissolved in chloroform. Hydrochloric acid (2 M in diethyl ether, 1 mL) was added and the reaction mixture heated at reflux for 0.5 h. The solvent was filtered off and the product was obtained as an orange solid (119 mg, 65.1%): mp 192–194 °C. IR (KBr) 3421, 2954, 1702, 1666, 1614, 1541, 1435, 1283, 1243, 1197, 1096, 1040, 856, 763 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 9.00 (d, J = 1.5 Hz, 1 H), 8.76 (d, J = 8.4 Hz, 1 H), 8.32 (dd, J = 1.9 Hz, J = 8.5 Hz, 1 H), 7.82 (d, J = 6.9 Hz, 1 H), 7.67 (d, J = 6.6 Hz, 1 H), 7.49-7.44 (m, 2 H), 4.67 (t, J = 7.3 Hz, 2 H), 3.96 (s, 3 H), 2.92 (br t, 2 H), 2.04 (p, J = 7.1 Hz, 1 H); ESIMS m/z (rel intensity) 363 (MH⁺, 100); HRESIMS calcd for C₂₁H₁₈N₂O₄ 363.1345 (MH⁺), found 363.1348 (MH⁺); HPLC purity: 95.92% (C₁₈ reversed phase, 1% TFA in MeOH-H₂O, 85:15), 98.65% (C₁₈ reversed phase, 1% TFA in MeOH-H₂O, 90:10).

3-Bromoindeno[1,2-c]isochromene-5,11-dione (84)

6-Bromo-3-hydroxyisobenzofuran-1(3H)-one (83, 6.81 g, 29.2 mmol) and phthalide (26, 3.90 g, 29.2 mmol) were dissolved in ethyl acetate (35 mL). A solution prepared by adding sodium (3.50 g) to methanol (75 mL) was added and the reaction mixture heated at reflux, with stirring, for 16 h. Concentrated hydrochloric acid (25 mL) was added until a yellow color appeared and the pH of the solution was 1. Ethanol (10 mL) and benzene (10 mL) were added and the solvents were removed under vacuum. para-Toluenesulfonic acid (100 mg) and benzene (100 mL) were added to the flask, which was connected to a Dean-Stark trap and the reaction mixture was heated at reflux for 6 h. Acetic anhydride (10 mL) was added and the reaction mixture was heated at reflux for an additional 10 h. Ethyl acetate (50 mL) and water (150 mL) were added and an orange solid precipitated (4.71 g of the title compound). The organic phase was washed with water and brine (100 mL each) and the solvent removed under vacuum. The residue was dissolved in hot chloroform and placed inside the freezer at −20 °C, and additional pure product (2.31 g) was provided. Compound 84 was obtained as an orange solid (7.02 g, 74%): mp 230–232 °C. IR (film) 2942, 2893, 1759, 1705, 1646, 1604, 1489, 1454, 1379, 1265, 1193, 1153, 1040, 863, 787, 758, 736, 687 cm⁻¹; ¹H NMR (300 MHz, CDCl₃ + MeOH-d₄) δ 8.33 (s, 1 H), 8.16 (d, J = 8.5 Hz, 1 H), 7.82 (d, J = 8.6 Hz, 1 H), 7.52 (d, J = 6.6 Hz, 1 H), 7.45-7.28 (m, 3 H); EIMS m/z (rel intensity) 326 (M⁺, 100), 328 (M⁺, 100); HREIMS calcd for C₁₆H₇BrO₃ 325.9579 (M⁺), found 325.9577 (M⁺).

tert-Butyl (3-(3-Bromo-5,11-dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)propyl)carbamate (85)

Methyl 3-bromoindeno[1,2-c]isochromene-5,11-dione (84, 600 mg, 1.84 mmol) was dissolved in tetrahydrofuran (25 mL) and chloroform (25 mL). Triethylamine (3 drops) was added, and tert-butyl (3-aminopropyl)carbamate (63, 823 mg, 4.72 mmol) was slowly added and the reaction mixture heated at reflux for 2 h. The organic phase was washed with a solution of concentrated hydrochloric acid (1 mL) in water (50 mL), water (50 mL) and brine (50 mL), and dried over anhydrous sodium sulfate. The solvent was removed under vacuum and the compound purified by silica gel column chromatography, eluting with chloroform. The product was obtained as a purple solid (611 mg, 68.8%): mp 203–205 °C. IR (film) 3378, 2973, 1699, 1656, 1601, 1569, 1537, 1501, 1429, 1365, 1242, 1167, 833, 764, 724 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.53 (d, J = 8.7 Hz, 1 H), 8.44 (d, J = 1.3 Hz, 1 H), 7.87 (dd, J = 2.1 Hz, J = 8.7 Hz, 1 H), 7.59-7.52 (m, 2 H), 745-7.38 (m, 2 H), 5.36 (br t, 1 H), 4.58 (t, J = 6.8 Hz, 2 H), 3.26 (q, J = 6.0 Hz, 2 H), 208 (p, J = 6.4 Hz, 2 H), 1.46 (s, 9 H); ESIMS m/z (rel intensity) 434 (MH⁺, 100), 436 (MH⁺, 99); HRESIMS calcd for C₂₂H₁₆N₃O₂Br 434.0504 (MH⁺), found 434.0508 (MH⁺).

(E)-Methyl 3-(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)acrylate (86)

tert-Butyl (3-(3-bromo-5,11-dioxo-5Hindeno[ 1,2-c]isoquinolin-6(11H)-yl)propyl)carbamate (85, 600 mg, 1.24 mmol) was dissolved in anhydrous dimethylformamide (10 mL). The solution was purged with argon while palladium(II) acetate (24 mg), triphenylphosphine (20 mg) and triethylamine (0.5 mL) were added sequentially. After 5 min, methyl acrylate (10, 0.35 mL, 3.65 mmol) was added and the reaction mixture was heated at reflux for 8 h. A second portion of methyl acrylate (10, 0.35 mL, 3.65 mmol) was added and the reaction mixture was heated for another 8 h. The mixture was cooled to room temperature, water (25 mL) was added and the precipitate was filtered. The residue was dissolved in warm chloroform-tetrahydrofuran (1:1, 30 mL) and allowed to reach room temperature. A mixture of diethyl ether-hexanes (1:1, 10 mL) was added and the flask placed inside the freezer at −20 °C. The solvent was decanted and the solid purified by silica gel column chromatography, eluting with chloroform. The desired compound was obtained as a red oil (421 mg, 69.6%). IR (film) 2951, 1735, 1664, 1609, 1577, 1536, 1435, 1270, 1168, 841 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.71 (d, J = 8.5 Hz, 1 H), 8.45 (s, 1 H), 7.87 (dd, J = 1.6 Hz, J = 8.5 Hz, 1 H), 7.77 (d, J = 16.1 Hz, 1 H), 7.68-7.42 (m, 4 H), 6.55 (d, J = 16.0 Hz, 1 H), 5.36 (br t, 1 H), 4.62 (t, J = 6.7 Hz, 2 H), 3.83 (s, 3 H), 3.27 (q, J = 5.7 Hz, 2 H), 2.09 (p, J = 6.3 Hz, 2 H), 1.46 (s, 9 H); ESIMS m/z (rel intensity) 511 (MNa⁺, 60).

(E)-Methyl 3-(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)acrylate Hydrochloride (87)

E)-Methyl 3-(6-(3-((tert-butoxycarbonyl) amino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3- yl)acrylate (86, 103 mg, 0.21 mmol) was dissolved in chloroform (10 mL). Hydrochloric acid (3 M in diethyl ether, 5 mL) was added and the reaction mixture heated at reflux for 1 h. Chloroform (10 mL) was added and the reaction mixture was heated for another hour. An orange solid precipitated. The solvent was decanted by filtration and the solid washed with diethyl ether (10 mL). The product was obtained as an orange solid (76 mg, 86%): mp 178–180 °C. IR (KBr) 3446, 2951, 1705, 1650, 1610, 1537, 1432, 1283, 1198, 905, 841, 763 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.53 (d, J = 8.7 Hz, 1 H), 8.38 (s, 1 H), 8.22 (d, J = 7.6 Hz, 1 H), 7.79-7.59 (m 3 H), 7.30-7.10 (m, 3 H), 6.71 (d, J = 16.0 Hz, 1 H), 4.55 (t, J = 6.1 Hz, 2 H), 3.70 (s, 3 H), 3.52 (br s, 2 H), 2.08 (br s, 2 H); ESIMS m/z (rel intensity) 389 (MH⁺, 62); HRESIMS calcd for C₂₃H₂₀N₂O₄ 389.1501 (MH⁺), found 389.1507 (MH⁺); HPLC purity: 96.79% (C₁₈ reversed phase, MeOH-H₂O, 90:10), 95.75% (C₁₈ reversed phase, MeOH-H₂O, 95:5).M

General Procedure for the Synthesis of Indenoisoquinolines 91–93

3- Bromoindeno[1,2-c]isochromene-5,11-dione (84, 100–103 mg, 0.31–0.33 mmol) was dissolved in tetrahydrofuran (5 mL) and chloroform (10 mL). A solution of the desired N-substituted 1,3- propylenediamine (64–66, 1.01–1.04 mmol) in chloroform (5 mL), triethylamine (3 drops), and molecular sieves was added and the reaction mixture was stirred for 24 h at room temperature. The molecular sieves were removed by filtration and the filter paper washed with chloroform-tetrahydrofuran (1:1) until no more solid was seen on the filter paper. The solvent was concentrated under vacuum to about 15 mL. A mixture of diethyl ether and hexane (1:4, 10 mL) was added to the reaction mixture, which was placed inside the freezer at −20 °C. A precipitate formed, the solvent was removed by filtration, and the solid washed with hexanes-dichloromethane (1:1, 20 mL), and dried.

3-Bromo-6-(3-(1H-imidazol-1-yl)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinoline (91)

The title compound was obtained as a dark yellow solid (89 mg, 64%): mp 224 °C (dec). IR (film) 2933, 1697, 1666, 1602, 1573, 1539, 1497, 1427, 1264, 1081, 832, 784, 762, 665 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.57 (d, J = 8.7 Hz, 1 H), 8.47 (d, J = 1.3 Hz, 1 H), 7.80 (dd, J = 2.1 Hz, J = 8.7 Hz, 1 H), 7.63-7.59 (m, 2 H), 7.40-7.28 (m, 2 H), 7.20 (s, 1 H), 7.08 (s, 1 H), 6.66 (d, J = 7.4 Hz, 1 H), 4.55 (t, J = 7.6 Hz, 2 H), 4.25 (t, J = 6.3 Hz, 2 H), 2.40 (m, 2 H); ESIMS m/z (rel intensity) 434 (MH⁺, 100), 436 (MH⁺, 99); HRESIMS calcd for C₂₂H₁₆N₃O₂Br 434.0504 (MH⁺), found 434.0508 (MH⁺); HPLC purity: 94.99% (C₁₈ reversed phase, MeOH-H₂O, 90:10).

3-Bromo-6-(3-morpholinopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinoline (92)

The title compound was obtained as a pale orange solid (70 mg, 51%): mp 209–211 °C. IR (film) 1697, 1654, 1539, 1498, 1425, 1313, 1276, 1111, 843, 762 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.59 (d, J = 8.6 Hz, 1 H), 8.47 (d, J = 2.1 Hz, 1 H), 7.82-7.76 (m, 2 H), 7.66-7.63 (m, 1 H), 7.46-7.42 (m, 2 H), 4.62 (t, J = 7.8 Hz, 2 H), 3.71 (t, J = 4.5 Hz, 4 H), 2.57 (t, J = 6.3 Hz, 2 H), 2.51 (m, 4 H), 2.05 (m, 2 H); ESIMS m/z (rel intensity) 453 (MH⁺, 100), 455 (MH⁺, 99); HRESIMS calcd for C₂₃H₂₁N₂O₃Br 453.0814 (MH⁺), found 453.0815 (MH⁺); HPLC purity: 97.75% (C₁₈ reversed phase, MeOH-H₂O, 90:10).

3-Bromo-6-(3-(dimethylamino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinoline (93)

The title compound was obtained as an orange solid (74 mg, 58%): mp 160–162 °C. IR (film) 3378, 2962, 2836, 1699, 1649, 1601, 1537, 1492, 1365, 1316, 1265, 1166, 1040, 875, 833, 785 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.57 (d, J = 8.6 Hz, 1 H), 8.46 (d, J = 1.3 Hz, 1 H), 7.78 (d, J = 7.2 Hz, 1 H), 8.62 (d, J = 8.6 Hz, 1 H), 7.47-7.39 (m, 2 H), 4.57 (t, J = 7.9 Hz, 2 H), 2.49 (t, J = 6.5 Hz, 2 H), 2.30 (s, 6 H), 2.03 (p, J = 8.6 Hz, 2 H); ESIMS m/z (rel intensity) 413 (MH⁺, 100), 411 (MH⁺, 99); HRESIMS calcd for C₂₁H₁₉N₂O₂Br 411.0708 (MH⁺), found 411.0706 (MH⁺); HPLC purity: 99.44% (C₁₈ reversed phase, MeOH-H₂O, 90:10).

3-Bromo-5,6-dihydro-6(3-hydroxypropyl)-5,11-dioxo-indeno[1,2-c]isoquinoline (94)

Isochromenone 84 (84 mg, 0.26 mmol) was dissolved in chloroform (35 mL). 3-Aminopropan-1-ol (88, 70 mg, 0.93 mmol) was added and the reaction mixture was stirred and heated to reflux for 16 h. The solvent was removed under vacuum and the residue was washed with diethyl ether (5 mL) to provide 94 as a red solid (97 mg, 97%): mp 200–202 °C. IR (KBr) 3524, 3072, 2953, 1694, 1651, 1498 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.60 (d, J = 8.7 Hz, 1 H), 8.50 (d, J = 2.1 Hz, 1 H), 7.82 (dd, J = 8.7 Hz, J = 2.1 Hz, 1 H), 7.72 (d, J = 7.2 Hz, 1 H), 7.64 (dd, J = 6.9 Hz, J = 1.5 Hz, 1 H), 7.44 (m, 2 H), 4.69 (t, J = 6.6 Hz, 2 H), 3.73 (m, 2 H), 2.99 (t, J = 5.1 Hz, 1 H), 2.11 (p, J = 5.7 Hz, 2 H); ESIMS m/z (rel. intensity) 384 (MH⁺, 100); HRESIMS calcd for C₁₉H₁₄NO₃Br 384.0235 (MH⁺), found 384.0238 (MH⁺); HPLC purity: 97.86% (C₁₈ reversed phase, MeOH, 100); 97.89% (C₁₈ reversed phase, MeOH-H₂O, 95:5).

3-Bromo-5,6-dihydro-6-(3-N-methylaminopropyl)-5,11-dioxo-indeno[1,2-c]isoquinoline (95)

Isochromenone 84 (82 mg, 0.25 mmol) was dissolved in chloroform (25 mL). N-Methylpropane-1,3-diamine (89, 71 mg, 0.81 mmol) was added to the reaction mixture, which was stirred and heated to reflux for 16 h. The solvent was removed under vacuum and the residue was washed with diethyl ether (5 mL) to provide 95 as an orange solid (101 mg, 100%): mp 97–100 °C. IR (KBr) 3425, 1662, 1540, 1494, 1316 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.58 (d, J = 8.6 Hz, 1 H), 8.46 (d, J = 2.1 Hz, 1 H), 7.80 (dd, J = 8.9 Hz, J = 2.1 Hz, 1 H), 7.73 (d, J = 7.2 Hz, 1 H), 7.63 (dd, J = 6.6 Hz, J = 1.8 Hz, 1 H), 7.43 (m, 2 H), 4.61 (t, J =7.5 Hz, 2 H), 2.79 (t, J = 6.3 Hz, 2 H), 2.06 (p, J = 6.9 Hz, 2 H); ESIMS m/z (rel. intensity) 412 (MH⁺, 99); HRESIMS calcd for C₂₀H₁₇N₂O₂Br 412.0184 (MH⁺), found: 412.0181 (MH⁺); HPLC purity: 97.31% (C₁₈ reversed phase, MeOH-H₂O, 90:10); 100% (C₁₈ reversed phase, MeOH-H₂O, 80:20).

3-Bromo-5,6-dihydro-6(2’-methoxycarbonylethyl)-5,11-dioxo-11H-indeno[1,2-c]isoquinolone (96)

Isochromenone 84 (82 mg, 0.25 mmol) was dissolved in chloroform (25 mL). ß-Alanine methyl ester hydrochloride (90, 112 mg, 0.25 mmol) was added and the reaction mixture heated at reflux for 16 h. The solvent was removed under vacuum and the residue was washed with diethyl ether (5 mL) to provide 96 as a red solid (100 mg, 97%): mp 180–183 °C. IR (KBr) 1730, 1693, 1662, 1497, 1313, 1049 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.58 (d, J = 8.7 Hz, 1 H), 8.47 (d, J = 2.2 Hz, 1 H), 7.81 (dd, J = 8.7 Hz, J = 2.2 Hz, 1 H), 7.64 (m, J = 7.4 Hz, 2 H), 7.47 (m, 2 H), 4.81 (m, 2 H), 3.78 (s, 3 H), 2.95 (m, 2 H); ESIMS m/z (rel. intensity) 412 (MH⁺, 99); HRESIMS calcd for C₂₀H₁₄NO₄Br 412.0184, found: 412.0181.

Lithium 6(2-Carboxyethyl)-3-bromo-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (97)

Compound 96 (76 mg, 0.23 mmol) was dissolved in tetrahydrofuran (10 mL) and water (10 mL). Lithium hydroxide monohydrate (10 mg, 0.10 mmol) was added and the reaction mixture was allowed to stir at room temperature for 48 h. The aqueous phase was washed with ethyl acetate (5 mL) and chloroform (5 mL). The aqueous portion was concentrated to yield 97 (58 mg, 62%) as an orange solid: mp 305 °C (dec). IR (KBr) 2590, 1706, 1646, 1582, 1540, 1386 cm⁻¹; ¹H NMR (300 MHz, CD₃OD) δ 8.51 (d, J = 8.7 Hz, 1 H), 8.35 (d, J = 2.1 Hz, 1 H), 7.92 (d, J = 7.5 Hz, 1 H), 7.82 (dd, J = 8.7 Hz, J = 2.1 Hz, 2 H), 7.53 (m, 2 H), 7.43 (t, J = 7.4 Hz, 2 H), 4.74 (m, 2 H), 2.73 (m, 2 H); ESIMS m/z (rel. intensity) 420 [(M-Li+H)+Na⁺, 100]; HRESIMS calcd for C₁₉H₁₂BrNNaO₄ 419.9847 [(M-Li+H)+Na⁺)], found 419.9853 [(MLi+ H)+Na⁺)]; HPLC purity: 99.58% (C₁₈ reversed phase, MeOH-H₂O, 90-10), 99.26% (C₁₈ reversed phase, MeOH-H₂O, 85-15).

Compounds 102 (5,6-Dihydro-6-(3-imidazolyl-1-propyl)-5,11-dioxo-11H-indeno[1,2- c]isoquinoline) and 103 (5,6-Dihydro-6-(3-(dimethylamino)-1-propyl)-5,11-dioxo-11Hindeno[ 1,2-c]isoquinoline)⁷⁴ were prepared according to published procedures.

6-(3-(Trimethylammonium)propyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione Iodide (104)

6-(3-(Dimethylamino)propyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (103, 67 mg, 0.20 mmol) was dissolved in anhydrous tetrahydrofuran (15 mL). Methyl iodide (0.5 mL) was added and the reaction mixture stirred at room temperature for 3 d. Another portion of methyl iodide (0.5 mL) was added and the reaction mixture was heated at reflux for 6 h. Hexanes (15 mL) were added and the reaction mixture was placed inside the freezer at −20 °C for 12 h. The solid was filtered off and the residue washed with dichloromethane-hexane (20 mL). The title compound was obtained as an orange solid (79 mg, 81%): mp 197–199 °C. IR (KBr) 3303, 1704, 1660, 1606, 1501, 1424, 1318, 1308, 1190, 958, 762 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ 8.58 (d, J = 8.0 Hz, 1 H), 8.23 (d, J = 8.1 Hz, 1 H), 7.85-7.76 (m, 2 H), 7.61-7.55 (m, 4 H), 4.55 (d, J = 6.8 Hz, 2 H), 3.55 (m, 2 H), 3.03 (s, 9 H), 2.25 (m, 2 H); ESIMS m/z (rel intensity) 347 (MH⁺, 96); HRESIMS m/z calcd for C₂₂H₂₂N₂O₂ 347.1760 (MH⁺), found 347.1763 (MH⁺); HPLC purity 96.72% (C₁₈ reversed phase, MeOH-H₂O, 90:10); 96.83% (C₁₈ reversed phase, MeOH, 100).

6-(3-(Methylamino)propyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (105)

Indeno[1,2-c]isochromene-5,11-dione (98, 102 mg, 0.41 mmol) was dissolved in chloroform (8 mL) and tetrahydrofuran (8 mL). A solution of N-methyl-1,3-diaminopropane (89, 97 mg, 1.10 mmol) in chloroform (2 mL) was slowly added to the first solution. The reaction mixture was stirred at reflux for 2 h. The solvent was removed in vacuo and the residue purified by silica gel column chromatography, eluting with dichloromethane. The product was obtained a red solid (108 mg, 82.8%): mp 151–153 °C. IR (film) 3061, 2937, 2845, 1697, 1661, 1610, 1550, 1503, 1428, 1318, 1264, 1195, 965, 756 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.69 (d, J = 8.1 Hz, 1 H), 8.33 (d, J = 7.9 Hz, 1 H), 7.63-7.38 (m, 7 H), 4.62 (t, J = 7.4 Hz, 2 H), 2.79 (t, J = 6.5 Hz, 2 H), 2.48 (s, 3 H), 2.08 (m, 2 H); ¹³C NMR (300 MHz, CDCl₃) δ 162.8, 148.9, 148.8, 136.7, 135.6, 129.5, 127.3, 126.4, 125.9, 125.4, 125.0, 122.4, 121.8, 121.7, 110.0, 71.9, 59.3, 33.1; ESIMS m/z (rel intensity) 319 (MH⁺, 100); HRESIMS m/z calcd for C₂₁H₂₀N₂O₂ 319.1447 (MH⁺), found 319.1444 (MH⁺); HPLC purity 99.53% (C₁₈ reversed phase, MeOH, 100).

3-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)propanoic Acid (106).⁷⁴

Indeno[1,2-c]isochromene-5,11-dione (98, 0.25 g, 1.00 mmol) was dissolved in THF-CHCl₃- DMF (20:20:5 mL) and triethylamine (0.303 g, 3.0 mmol) followed by β-alanine (99, 0.269 g, 3.02 mmol) were added at room temperature. The mixture was heated at reflux for 12 h. The solvent was removed under vacuum and purified by silica gel flash column chromatography (CHCl₃-MeOH, 90:10) to afford compound 106 (0.350 g, 87.2%) as a yellow solid: mp 241–243 °C. IR (KBr) 3347, 1734, 1652, 1632, 1543, 1507, 1356, 1154, 768, 759, 655 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 8.58 (d, J = 9.2 Hz, 1 H), 8.19 (d, J = 9.2 Hz, 1 H), 7.55 (m, 2 H), 7.34 (d, J = 7.1 Hz, 1 H), 7.29 (m, 3 H), 4.68 (m, 2 H), 2.80 (m, 2 H); ¹³C NMR (CDCl₃ + DMSO-d₆, 75 MHz) δ 172.0, 162.8, 136.4, 134.5, 133.5, 133.3, 131.9, 130.7, 127.9, 126.8, 123.0, 122.9, 122.8, 122.2, 40.5, 33.0; EIMS m/z (rel intensity) 319 (M⁺, 100); HPLC purity 98.98% (C₁₈ reversed phase, MeOH-H₂O, 85:15).

3-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)propanamide (107)

Thionyl chloride (4 mL) was added to 3-(5,11-dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)propanoic acid (106, 0.250 g, 0.782 mmol) at room temperature and the mixture was stirred for 2 h. The excess thionyl chloride was removed under vacuum. The crude acid chloride was treated with a solution of ammonia in tetrahydrofuran (0.4 M, 3 mL) at room temperature for 2 h. The reaction mixture was washed with water (2×50 mL) and extracted with ethyl acetate (2×60 mL). The combined organic layers were concentrated and purified by silica gel column chromatography, eluting with chloroform-methanol, 9.5:0.5. The desired compound 107 was obtained as a yellowish-orange solid (0.120 g, 50%): mp 270–271 °C. IR (KBr) 3584, 3418, 1651, 1638, 1551, 1504, 1317, 760, 666 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 8.70 (d, J = 8.1 Hz, 1 H), 8.31 (d, J = 8.1 Hz, 1 H), 7.86 (d, J = 7.5 Hz, 1 H), 7.30 (t, J = 7.5 Hz, 1 H), 7.48 (d, J = 7.5 Hz, 1 H), 7.38 (m, 2 H), 7.24 (m, 1 H), 4.83 (m, 2 H), 2.86 (m, 2 H); EIMS m/z (rel intensity) 318 (M⁺, 100); HREIMS m/z calcd for C₁₉H₁₄N₂O₃ 318.0999 (M⁺), found 318.0992 (M⁺); HPLC purity 96.59% (C₁₈ reversed phase, MeOH-H₂O, 85:15).

6-(3-Methoxypropyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (108)

Indeno[1,2-c]isochromene-5,11-dione (98, 390 mg, 1.57 mmol) was dissolved in chloroform (10 mL). A solution of 3-methoxy-1-propylamine (100, 300 mg, 3.30 mmol) in tetrahydrofuran (5 mL) was slowly added to the solution. The reaction mixture was heated at reflux for 2 h. The reaction mixture was allowed to cool to room temperature, diluted with chloroform (50 mL) and washed with water (2 × 50 mL) and brine (50 mL). The residue was purified by silica gel column chromatography, eluting with chloroform. The product was obtained as a red solid (331 mg, 66.0 %): mp 142–144 °C. IR (film) 3065, 1650, 1570, 1531, 1454, 1384, 1305, 1271, 1191, 908, 842 cm ⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 8.48 (d, J = 8.1 Hz, 1 H), 8.14 (d, J = 8.0 Hz, 1 H), 7.79 (d, J = 7.5 Hz, 1 H), 7.74 (t, J = 7.6 Hz, 1 H), 7.53-7.43 (m, 4 H), 4.47 (t, J = 7.8 Hz, 2 H), 3.49 (t, J = 5.8 Hz, 2 H), 3.25 (s, 3 H), 1.98 (dd, J = 6.1 Hz, J = 3.0 Hz, 2 H); ¹³C NMR (75 MHz, DMSO-d₆) δ 190.3, 162.7, 156.6, 136.8, 134.7, 134.3, 134.2, 132.2, 131.6, 128.4, 127.4, 124.1, 123.1, 123.0, 122.9, 107.2, 69.7, 58.6, 42.6, 29.2; ESIMS m/z (rel intensity) 320 (MH⁺, 100); HPLC purity: 98.04% (C₁₈ reversed phase, MeOH-H₂O, 95:5), 99.92% (C₁₈ reversed phase, MeOH-H₂O, 90:10).

6-Butyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (109)

Indeno[1,2-c]isochromene-5,11-dione (98, 300 mg, 1.21 mmol) was dissolved in tetrahydrofuran (20 mL). A solution of n-butylamine (101, 152 mg, 2.08 mmol) in chloroform (5 mL) was added to the flask and the reaction mixture heated at reflux for 3 h. The solvent was removed in vacuo and the solid purified by silica gel column chromatography, eluting with ethyl acetate-hexane, 8:1. The product was obtained as an orange solid (321 mg, 87.6%); mp 141–143 °C (lit⁷⁵ 157–158 °C). IR (film) 2958, 2932, 2872, 1693, 1659, 1576, 1503, 1426, 1319, 1194, 1071, 955, 755 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.71 (d, J = 8.1 Hz, 1 H), 8.35 (d, J = 8.2 Hz, 1 H), 7.72 (td, J = 1.2 Hz, J = 7.7 Hz, 1 H), 7.64 (dd, J = 1.4 Hz, J = 6.2 Hz, 1 H), 7.48-7.39 (m, 4 H), 4.52 (t, J = 7.9 Hz, 2 H), 1.89 (m, 2 H), 1.58 (m, 2 H), 1.05 (t, J = 7.3 Hz, 6 H); ¹³C NMR (75 MHz, CDCl₃) δ 189.7, 162.7, 155.1, 136.6, 134.7, 133.3, 132.8, 131.7, 130.5, 128.0, 126.6, 123.1, 123.0, 122.6, 122.1, 107.8, 44.3, 31.1, 20.0, 13.7; ESIMS m/z (rel intensity) 304 (MH⁺, 100); HRESIMS m/z calcd for C₂₀H₁₇NO₂ 304.1338 (MH⁺), found 304.1334 (MH⁺); HPLC purity 99.74% (C₁₈ reversed phase, MeOH-H₂O, 95:5); 99.81% (C₁₈ reversed phase, MeOH).

6-(3-(Dimethylamino)propyl)-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (113)⁴⁰

3-(Dimethylamino)propylamine (66, 0.105 g, 1.023 mmol) was dissolved in chloroform (5 mL) and the solution was added to a solution of 110 (0.308 g, 1.05 mmol) in chloroform (5 mL). The reaction was stirred for 3 h at room temperature, during which time the solution turned red. The mixture was heated at reflux for 1 h and the solution turned orange. The solvent was removed under vacuum and the solid purified by silica gel column chromatography, eluting with chloroform-methanol, 50:1. The product was obtained as a yellow solid (96 mg, 55%): mp 227 °C (dec). IR (film) 2916, 1699, 1658, 1590, 1567, 1356, 1223, 1145, 665 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 8.89 (d, J = 2.5 Hz, 1 H), 8.73 (d, J = 9.0 Hz, 1 H), 8.59 (dd, J = 9.0, J = 2.5 Hz, 1 H), 8.00 (d, J = 7.18 Hz, 1 H), 7.67-7.59 (m, 3 H), 4.57 (t, J = 6.9 Hz, 2 H), 2.45 (t, J = 6.5 Hz, 2 H), 2.19 (s, 6 H), 1.95 (m, 2 H); ¹³C NMR (125 MHz, DMSO-d₆) δ 189.6, 162.0, 157.9, 136.3, 135.3, 135.1, 135.0, 133.6, 133.5, 131.8, 124.4, 123.7, 123.5, 123.0, 118.2, 109.9, 107.3, 56.6, 45.6, 43.8, 29.9; ESIMS m/z (rel intensity) 378 (MH⁺, 100); HRESIMS m/z calcd for C₂₁H₁₉N₃O₄ 378.1454, found, 378.1452; HPLC purity: 97.37% (C₁₈ reversed phase, MeOH-H₂O, 95:5), 97.37% (C₁₈ reversed phase, MeOH, 100).

3-(3-Nitro-5,11-dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)propanoic Acid (114)

3-Nitroindeno[1,2-c]isochromene-5,11-dione (110, 0.3 g, 1.02 mmol) was dissolved in a mixture of THF-CHCl₃-DMF (20:20:5, 45 mL). Triethylamine (0.310 g, 3.07 mmol) followed by β-alanine (99, 0.273 g, 3.07 mmol) were added to the reaction mixture at room temperature. The mixture was heated at reflux for 12 h. The solvent was removed under vacuum and the product purified by silica gel flash column chromatography (CHCl₃-MeOH 8.5:1.5) to afford compound 114 (0.180 g, 48.3%) as a yellow solid: mp 306–308 °C. IR (KBr) 3367, 1712, 1668, 1612, 1557, 1507, 1333, 1194, 766, 748 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ 8.86 (d, J = 2.2 Hz, 1 H), 8.75 (d, J = 9.2 Hz, 1 H), 8.50 (m, 1 H), 8.05 (d, J = 9.2 Hz, 1 H), 7.58 (m, 3 H), 4.68 (m, 2 H), 2.70 (m, 2 H); EIMS m/z (rel intensity) 364 (M⁺, 100); HREIMS m/z calcd for C₁₉H₁₂N₂O₆ 364.0690 (M⁺), found 364.0692 (M⁺); HPLC purity 96.71% (C₁₈ reversed phase, MeOH, 100).

3-(3-Nitro-5,11-dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)propanamide (115)

Thionyl chloride (2 mL) was added to 3-(3-nitro-5,11-dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)propanoic acid (114, 0.125 g, 0.391 mmol) at room temperature and the mixture was stirred for 2 h before the excess thionyl chloride was removed on a rotary evaporator. The crude acid chloride was treated with 0.4 M NH₃ in THF (3 mL) at room temperature for 2 h. The reaction mixture was washed with water (2×25 mL) and extracted with ethyl acetate (2×40 mL). The combined organic layer was concentrated and purified by silica gel flash column chromatography (chloroform-methanol, 9.5:0.5) to afford compound 115 (0.050 g, 40%) as a yellow solid: mp 283–285 °C. IR (KBr) 3583, 1707, 1658, 1613, 1558, 1504, 1425, 1336, 1261, 665 cm⁻¹; ¹H NMR (CD₃OD + DMSO-d₆, 300 MHz) 9.02 (s, 1 H), 8.93 (d, J = 6.7 Hz, 1 H), 8.61 (m, 1 H), 8.10 (d, J = 7.0 Hz, 1 H), 7.69 (m, 3 H), 4.85 (m, 2 H), 2.86 (m, 2 H); EIMS m/z (rel intensity) 363 (M⁺, 100); HREIMS m/z calcd for C₁₉H₁₃N₃O₅Na 386.0753 (MNa⁺), found 386.0755 (MNa⁺); HPLC purity 97.82% (C₁₈ reversed phase, MeOH-H₂O, 85:15).

6-(3-Methoxypropyl)-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (116)

3-Nitroindeno[1,2-c]isochromene-5,11-dione (110, 150 mg, 0.51 mmol) was dissolved in chloroform (8 mL) and tetrahydrofuran (8 mL). A solution of 3-methoxy-1-propylamine (100, 219 mg, 2.30 mmol) in tetrahydrofuran (1 mL) was slowly added to the first solution. Magnesium sulfate (approximately 100 mg) was added to the flask. The reaction mixture was stirred at room temperature for 12 h. The solid was filtered off, the solvent was removed in vacuo and the residue purified by silica gel column chromatography, eluting with chloroform. The product was obtained as a red solid (111 mg, 0.30 mmol, 60.0%): mp 222–224 °C. IR (film) 3103, 2981, 2927, 2823, 1698, 1663, 1614, 1499, 1426, 1334, 1199, 991, 861, 769, 749 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 9.19 (d, J = 2.3 Hz, 1 H), 8.85 (d, J = 9.0 Hz, 1 H), 8.48 (dd, J = 2.5 Hz, J = 8.8 Hz, 1 H), 8.00 (d, J = 7.21 Hz, 1 H), 7.70 (m, 1 H), 7.53-7.49 (m, 2 H), 4.68 (t, J = 7.2 Hz, 2 H), 3.63 (t, J = 5.4 Hz, 2 H), 3.42 (s, 3 H), 2.20 (m, 2 H); ¹³C NMR (300 MHz, CDCl₃) δ 189.6, 162.4, 158.6, 145.7, 136.7, 136.0, 135.0, 133.9, 132.0, 127.6, 124.8, 124.6, 124.4, 123.5, 123.0, 69.8, 59.1, 43.4, 29.2; ESIMS m/z (rel intensity) 395 (MH⁺, 60), 333 [(MH-CH₃OH)⁺, 100]; HRESIMS m/z calcd for C₂₀H₁₆N₂O₆ 365.1137 (MH⁺), found 365.1134 (MH⁺); HPLC purity 95.89% (C₁₈ reversed phase, MeOH, 100).

6-(3,3-Diethoxypropyl)-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (117)

3-Nitroindeno[1,2-c]isochromene-5,11-dione (110, 180 mg, 0.60 mmol) was dissolved chloroform (10 mL) and tetrahydrofuran (11 mL). A solution of 1-amino-3,3-diethoxypropane (111, 312 mg, 2.11 mmol) in tetrahydrofuran (1 mL) was added dropwise. Magnesium sulfate (approximately 100 mg) was added to the flask. The reaction mixture was stirred at room temperature for 12 h. The solid was filtered off, the solvent was removed in vacuo and the residue purified by silica gel column chromatography, eluting with dichloromethane-hexane, 95:5. The product was obtained as an orange solid (171 mg, 66.1%): mp 191–193 °C. IR (film) 2973, 1776, 1708, 1672, 1612, 1557, 1431, 1328, 1275, 1118, 1060, 665 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 9.18 (d, J = 2.3 Hz, 1 H), 8.83 (d, J = 8.9 Hz, 1 H), 8.46 (dd, J = 2.2 Hz, J = 8.9 Hz, 1 H), 7.67 (m, 1 H), 7.72 (dd, J = 1.1 Hz, J = 7.9 Hz, 1 H), 7.50-7.47 (m, 2 H), 4.74 (m, 1 H), 4.67 (t, J = 8.2 Hz, 2 H), 3.81 (m, 2 H), 3.60 (m, 2 H), 2.26 (m, 2 H), 1.28 (t, J = 7.2 Hz, 6 H); APCIMS m/z (rel intensity) 445 (MNa⁺, 100); HPLC purity: 97.46 % (C₁₈ reversed phase, MeOH, 100).

6-(3,3-Dimethoxypropyl)-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (118)

6-(3,3-Diethoxypropyl)-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (117, 26 mg, 61.6 mmol) was dissolved in methanolic hydrochloric acid (1 mL, 10% solution in methanol). The reaction mixture was stirred for 2 h at room temperature. The solvent was removed and the residue purified by silica gel column chromatography, eluting with dichloromethane. The title compound was obtained as an orange solid (17 mg, 78%): mp 204–206 °C. IR (film) 3099, 2984, 2956, 2935, 2836, 1698, 1670, 1611, 1558, 1507, 1429, 1338, 1330, 1117, 1048, 924, 862 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 9.20 (d, J = 2.4 Hz, 1 H), 8.87 (d, J = 9.0 Hz, 1 H), 8.49 (dd, J = 2.5 Hz, J = 8.9 Hz, 1 H), 7.94 (d, J = 6.7 Hz, 1 H), 7.71 (dd, J = 1.7 Hz, J = 6.6 Hz, 1 H), 7.54-7.49 (m, 2 H), 4.65 (m, 3 H), 3.45 (s, 6 H), 2.24 (m, 2 H); APCIMS m/z (rel intensity) 395 (MH⁺, 100); HPLC purity: 95.43 % (C₁₈ reversed phase, MeOH, 100).

3-(3-Nitro-5,11-dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)propanal (119)

6-(3,3-Dimethoxypropyl)-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (118, 102 mg, 0.26 mmol) was dissolved in tetrahydrofuran ( 10 mL) and cooled to 0 °C. Concentrated hydrochloric acid (0.5 mL) was added and the reaction mixture stirred for 3 h while letting the temperature to rise to room temperature. The solvent was removed in vacuo and the compound purified by preparative silica gel TLC, eluting with chloroform-methanol, 20:1. Compound 119 was obtained as an orange solid (37 mg, 41%): mp >350 °C. IR (film) 2914, 2849, 1709, 1672, 1612, 1557, 1500, 1338, 1078, 837, 769, 747 cm ⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 9.96 (s, 1 H); 9.20 (d, J = 2.3 Hz, 1 H); 8.87 (d, J = 9.0 Hz, 1 H); 8.51 (dd, J = 2.3 Hz, J = 9.0 Hz, 1 H); 7.74 (m, 1 H); 7.56-7.51 (m, 3 H); 4.88 (t, J = 7.6 Hz, 2 H); 3.18 (t, J = 7.8 Hz, 2 H); HPLC purity 97.89 % (C₁₈ reversed phase, MeOH, 100).

6-(4-Hydroxybutyl)-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (120)

3-Nitroindeno[1,2-c]isochromene-5,11-dione (110, 120 mg, 0.41 mmol) was dissolved in chloroform (25 mL). 4-Amino-1-butanol (112, 100 mg, 1.12 mmol) was slowly added to the solution. The reaction mixture was heated at reflux for 2 h. The reaction mixture was allowed to cool to room temperature, diluted with chloroform (25 mL) and washed with water (30 mL) and brine (30 mL). The product was purified by silica gel column chromatography, eluting with chloroform-methanol, 98:2. The product was obtained as a yellow solid (109 mg, 72.8%): mp 187–189 °C. IR (film) 3696, 1712, 1673, 1557, 1424, 1327, 1185, 1066, 843 cm ⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 9.18 (d, J = 2.4 Hz, 1 H), 8.85 (d, J = 8.9 Hz, 1 H), 8.47 (dd, J = 8.9 Hz, J = 2.4 Hz, 1 H), 7.70 (t, J = 8.2 Hz, 1 H), 7.54 (m, 2 H), 4.62 (t, J = 7.7 Hz, 2 H), 3.82 (s, 2 H), 3.70 (br s, 1 H), 2.06 (m, 2 H), 1.85 (m, 2 H); ESIMS m/z (rel intensity) 365 (MH⁺, 100), 320 (25); HPLC purity 97.25% (C₁₈ reversed phase, MeOH-H₂O, 95:5), 99.27% (C₁₈ reversed phase, MeOH-H₂O, 90:10).

3-Amino-6-(3-(dimethylamino)propyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (121)

Compound 113 (0.2 g, 0.55 mmol) was dissolved in THF (50 mL) and methanol (10 mL) and subjected to catalytic hydrogenation in the presence of 10% Pd-C (30 mg) at 45 psi for 15 h. The catalyst was filtered off and the solvent removed. The residue was purified by silica gel column chromatography, eluting with chloroform-methanol, 9.2:0.8, to give the product 121 (0.1 g, 45%) as brown solid: mp 222–224 °C. IR (KBr) 3343, 1691, 1644, 1615, 1578, 1516, 1317, 1274, 1194, 665 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 8.28 (d, J = 7.5 Hz, 1 H), 7.68 (m, 1 H), 7.45 (m, 2 H), 7.31 (d, J = 7.8 Hz, 1 H), 7.25 (m, 1 H), 7.08 (dd, J = 4.5, 7.8 Hz, 1 H), 5.80 (br s, 1 H), 4.52 (m, 2 H), 3.20 (m, 2 H), 2.13 (m, 2 H); ESIMS m/z (rel intensity) 348 (MH⁺, 100); HRESIMS m/z calcd for C₂₁H₂₂N₃O₂ 348.1712 (MH⁺), found 348.1720 (MH⁺); HPLC purity 95.54% (C₁₈ reversed phase, MeOH-H₂O, 70:30)

3-Amino-11-hydroxyindeno[1,2-c]isochromen-5(11H)-one (122)

Nitro compound 110 (0.5 g, 1.11 mmol) was dissolved in a mixture of THF-CHCl₃-MeOH (30 mL of each), and then 10% Pd/C (30 mg) was added and the mixture was hydrogenated on a Parr apparatus at 30 psi for 15 h. The reaction mixture was filtered, concentrated and purified by silica gel flash column chromatography (2% MeOH in CHCl₃) to provide 122 as a yellowish solid (0.20 g, 44%): mp 147–148 °C. ¹H NMR (Acetone-d₆ + MeOH-d₄, 300 MHz) δ 7.10 (d, J = 6.9 Hz, 1 H), 7.55 (d, J = 6.9 Hz, 1 H), 7.46 (d, J = 2.4 Hz, 1 H), 7.37 (m, 3 H), 7.28 (dd, J = 2.4, 6.9 Hz, 1 H), 5.54 (s, 1 H); ¹³C NMR (DMSO-d₆, 75 MHz) δ 149.0, 145.7, 143.6, 134.5, 128.4, 127.0, 124.5, 124.3, 123.9, 122.1, 120.7, 119.5, 117.0, 111.6, 70.8.

tert-Butyl (3-(3-Amino-5-oxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)propyl)carbamate (123)

tert-Butyl (3-aminopropyl) carbamate (63, 0.656 g, 3.77 mmol) was added to a solution of 3-amino-11-hydroxyindeno[1,2-c]isochromen-5(11H)-one (122, 0.2 g, 0.754 mmol) in CHCl₃ (100 mL) and the mixture heated at reflux for 15 h. The solvent was removed under vacuum and the residue purified by silica gel column chromatography, eluting with chloroform-methanol, 20:1. The product was obtained as a yellowish solid (0.10 g, 33%): mp 176–178 °C. ¹H NMR (CDCl₃, 300 MHz) δ 7.68 (m, 2 H), 7.55 (m, 2 H), 7.35 (t, J = 5.6 Hz, 1 H), 7.27 (d, J = 5.5 Hz, 1 H), 6.98 (dd, J = 2.2, 5.5 Hz, 1 H), 5.76 (t, J = 3.2 Hz, 1 H), 4.63 (m, 2 H), 3.99 (br s, 2 H), 3.80 (s, 2 H), 3.10 (m, 2 H), 2.05 (m, 2 H), 1.44 (s, 9 H); ESIMS m/z (rel intensity) 406 (MH⁺, 100).

3-Amino-6-(3-aminopropyl)-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-5-one (124)

Compound 123 (0.05 g, 0.12 mmol) was dissolved in chloroform (15 mL) and then HCl in methanol (1.25 M, 2 mL) was added. The mixture was stirred at room temperature for 2 h before adding TFA (0.5 mL) as the reaction did not proceed in the presence of HCl. The reaction mixture stirred at room temperature for 8 h, and then the solvents were removed on a rotary evaporator and the product precipitated from chloroform as a light blue solid (0.025 g, 72%): mp 245–247 °C. IR (KBr) 3351, 1688, 1630, 1577, 1516, 1458, 1366, 1175, 761, 655 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.99 (m, 4 H), 7.78 (d, J = 6.5 Hz, 1 H), 7.66 (d, J = 6.5 Hz, 1 H), 7.32 (m, 3 H), 4.63 (t, J = 2.3 Hz, 2 H), 3.94 (s, 2 H), 2.89 (m, 2 H), 2.10 (m, 2 H); ESIMS m/z (rel intensity) 306 (MH⁺, 100); HRESIMS m/z calcd for C₁₉H₂₀N₃O 306.1606 (MH⁺), found 306.1610 (MH⁺); HPLC purity 96.83% (C₁₈ reversed phase, MeOH-H₂O, 50:50).

RXRα Ultrafiltration Affinity Assay

In preparation for ultrafiltration screening, 85 µL binding buffer consisting of 50 mM Tris-HCl (pH 7.5), 50 mM KCl, and 1 mM EDTA, 5 µL of a sample solution in DMSO and 10 µL of RXRα (obtained from Michael Schimerlik, Oregon State University, Corvalis, OR) (10 µM in binding buffer), were mixed and incubated for 2 h at room temperature. The final concentration of sample solution in the incubation was 10 µM. LG100268 (CVChem, Cary, NC) was used as a positive control in each batch of incubation. After incubation, each mixture was filtered through a Microcon (Millipore, Bedford, MA) YM-10 centrifugal filter containing a regenerated cellulose ultrafiltration membrane with a 10,000 molecular weight cutoff by centrifugation at 13,000 g for 10 min at 4 °C. The RXRα-ligand complexes were washed three times with 150 µL aliquots of 30 mM ammonium acetate (pH 7.5) for 15 min at 4 °C to remove the unbound compounds. The washed RXRα-ligand solution (~10 µL) was transferred to a second filter where it was treated with 200 µL of 90% methanol in deionized water to disrupt the receptor-ligand complex. The released ligands were then isolated from the denatured protein by ultrafiltration. The solvent in the ultrafiltrate was evaporated under vacuum, and the ligands were reconstituted in 50 µL of methanol/water (50:50, v/v) containing 200 nM ketoconazole as internal standard for analysis using LC-MS-MS as described below. For comparison, control analysis was carried out that was identical except for the use of denatured RXRα. Denatured RXRα was prepared by boiling the receptor at 60–70 °C for 15 min.

Aliquots (5 µL each) of each reconstituted ultrafiltrate were analyzed using UHPLC-MS-MS on a Shimadzu (Kyoto, Japan) Nexera UHPLC system interfaced with a Shimadzu LCMS-8040 triple quadrupole mass spectrometer. Analytes were separated on a Shimadzu Shim-pack XR-ODS III UHPLC column (2.0×50 mm, 1.6 µm) using a 2.5 min linear gradient from 10–100% acetonitrile in 0.1% aqueous formic acid with equilibration at 10% acetonitrile for 1.5 min. The flow rate was 0.5 mL/min. Mass spectrometer source parameters were as follows: DL temperature 300 °C, spray voltage 3500 V, nebulizing gas flow 3 L/min, and drying gas flow 20 L/min. LG100268 was detected using negative ion electrospray, collision-induced dissociation and selected reaction monitoring (SRM) by recording the signal for the transition of the deprotonated molecule of m/z 362 to the most abundant fragment ion of m/z 318. Other analytes were detected using positive ion electrospray. The SRM transitions of m/z 531 to m/z 489, m/z 320 to m/z 303, m/z 319 to m/z 288, m/z 333 to m/z 288, m/z 348 to m/z 303, and m/z 420 to m/z 403 were monitored for ketoconazole, 3, 103, 105, 121, and 62, respectively.

VRD Ultrafiltration Affinity Assay

The VDR (ProteinOne, Bethesda, MD) (4.7 µL) in storage buffer was incubated in a total volume of 20 µL with 15.3 µL of the working ligand solution. Incubations were carried out in the absence of light for 30 min at 37 °C with shaking at 300 rpm. The entire incubated volume was transferred to a Millipore membrane apparatus and subjected to 5 min of temperature controlled (4 °C) ultracentrifugation at 12,000 × g. Each membrane was rinsed with 200 µL of ice cold buffer (50 mM ammonium acetate, 0.1 mM NaCl at pH 8.0) with centrifugation at 12,000 × g for 15 min. Inversion of the apparatus and subsequent centrifugation at 3000 × g allowed for the collection of the ultrafiltrate. The ligand-bound protein complex was dissociated with the addition of 300 µL of MeOH containing 0.5 µL of 100 µM genistein (Sigma-Aldrich, St. Louis, MO). A steady stream of nitrogen (N₂) provided by an in-house generator was used to dry each ultrafiltrate. The initial LC solvent system (20 µL) was added to reconstitute each sample.

LC-MS analysis of the ligands trapped during the ultrafiltration process utilized a Shimadzu (Columbia, MD) LC20ADXR enhanced pressure tolerance HPLC system (max. 9,600 psi) coupled with a Shimadzu ion trap time-of-flight (ITTOF) mass analyzer. A universal LC-MS method for all sample types was developed allowing for an uncomplicated batch setup and data processing workflow of large sample sets. A Shimadzu 2.0 × 50 mm, 1.6 µm column was utilized with the solvent system consisted of water/0.1% formic acid channel A and acetonitrile channel B at 500 µL/min employing a linear gradient, with the following conditions: isocratic hold at 25% A for 0.2 min, 25–100% B in 1 min, isocratic 100% B for 0.8 min, equilibrate at initial condition for 1 min. A total run time of 3 min proved to be sufficient to elute all compounds and eliminate run-to-run carryover. Electrospray mass spectra were acquired in both positive and negative polarities. Operating parameters were defined as follows: Vcap = +4500 V/−3500 V, heating block and CDL set to 200 °C, N₂ drying gas flow 1.5 L/min. A total of four events with two assigned for each polarity were employed to cover the desired mass range m/z 100–1500 with a total cycle time of 500 ms. Positive control experiments for the were performed with the bile salt lithocholic acid (LCA) (Sigma-Aldrich, St. Louis, MO) and curcumin (CUR) (Sigma-Aldrich, St. Louis, MO). Control analyses can be carried out using either denatured hVDR, which was prepared by boiling in water for 10 min, or in the absence of protein when access to protein is limited.

RXRE-Luciferase Reporter Gene Assay (RXRE Assay)

The translucent reporter vector encoding firefly luciferase gene under the control of RXRE (5'-AGGTCACAGGTCACAGGTCACAGGTCACAGGTCA-3') (pRXRE) was purchased from Panomics (Fremont, CA). pBABE-puro vector encoding the cDNA for human RXRα (phRXRα) was purchased from Addgene Inc. (Cambridge, MA). Renilla reniformis luciferase vector (pRL) and Dual-Luciferase® Reporter Assay System were purchased from Promega (Madison, WI).

The RXRE assay was performed as previously described.³³ Briefly, COS-1 cells were transiently co-transfected with 100 ng of pRXRE, 50 ng of phRXRα and 3 ng of pRL in each well by using LipofectamineTM 2000 for 24 hours followed by the incubation with compounds for additional 12 hours. The RXRE transcriptional activities were calculated after the measurement of firefly and Renilla luciferase activities.

Abbreviations Used

9cRA

9-cis-retinoic acid

APCIMS

atmospheric pressure chemical ionization mass spectrometry

BBO

multinuclear broadband observe

EC₅₀

half maximal effective concentration, the concentration needed to induce a response that is equal to half the maximum response

EIMS

electron impact mass spectrometry

ESIMS

electrospray ionization mass spectrometry

IR

induction ratio

LC-MS/MS

high performance liquid chromatography-tandem mass spectrometry

QNP

quattro nucleus probe

p21

cyclin-dependent kinase inhibitor 1

RXR

retinoid X receptor

RXRE

retinoid X receptor response element

SAR

structure-activity relationships.