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Abstract

Acridine-based compounds possess anticancer activities by intercalating to DNA. Although they have chemotherapeutic potential, acridine-based compounds are not used to treat cancer. In this study, 2,N¹⁰-acridone derivatives are designed and synthesized based on acridone, a ketone derivative of acridine. Herein, acridone is functionalized with alkyl side chains containing terminal nitrogen-based moieties at the N¹⁰-position and substituted at the C2-position. The products are evaluated for in vitro cytotoxicity against four cancer cell lines: Molt-3, HepG2, A549, and HuCCA-1. The derivative bearing two butyl piperidine side chains at the C2- and N¹⁰-positions is the most active, with IC₅₀ values ranging from 2.96 to 9.46 µM. Molecular modeling studies supported the binding of the derivatives to DNA by intercalation, thereby confirming the observed cytotoxic effects.

Keywords

acridone cytotoxicity intercalating agent synthesis

Introduction

Cancer is a class of disease characterized by an abnormal growth of tissues that can metastasize. In 2018, the World Health Organization (WHO)¹ reported approximately 9.6 million deaths from cancers worldwide. Undeniably, cancer is a serious health concern globally. The development of chemotherapeutics faces challenging obstacles including the development of potent small molecule drugs against mutant forms of protein targets with high selectivity and tolerable adverse effects. Thus, research to develop new anticancer drugs with the above-mentioned properties is highly desirable. Existing anticancer chemotherapeutic agents exert different mechanisms of action, for example, kinase inhibition, inhibition of DNA transcription activity, manipulation of tubulin dynamics, and DNA-targeting compounds. DNA is one of the most viable and successful targets for anticancer chemotherapy as seen by the approval of many DNA-targeting anticancer drugs,² for example, bleomycin, doxorubicin, mephalan, and cyclophosphamide (Figure 1).

Figure 1.

Structures of some DNA-targeting anticancer drugs.

Herein, we have developed DNA-targeting anticancer agents that can interfere with the DNA base-pairing system through intermolecular interactions. This mechanism causes conformational changes in the DNA structure and often disrupts the recognition of DNA-binding proteins such as DNA polymerases, transcription factors, and topoisomerases.³ Furthermore, intercalation is one of the most important mechanisms of action exhibited by DNA-targeting drugs, which are classified as DNA-intercalating agents. The mechanism of action relies on the flat planar structure of the drug, which can be inserted between DNA base pairs. The stability of the complex is considerably influenced by van der Waal’s forces, hydrophobic interactions, π–π stacking interactions, and electrostatic forces.⁴ Various well-known compounds, such as ethidium bromide, daunomycin, and quinacrine, interact with DNA through intercalation (Figure 2).

Figure 2.

Chemical structures of known DNA-intercalating agents: ethidium bromide, daunomycin, and quinacrine.

Acridone (1) is a flat planar heterocyclic scaffold consisting of three ortho-fused aromatic rings with a keto group and a nitrogen atom at the 9- and 10-positions, respectively. The flat planar shape allows acridone to intercalate between DNA bases disrupting the intermolecular bonding networks of the DNA molecule. Hence, acridone-based compounds are regarded to have chemotherapeutic potential. Numerous naturally occurring acridone alkaloids have been isolated and studied for their biological activities, including antiproliferative,^5–7 antiviral,^8,9 and antiparasitic¹⁰ activities. Some well-recognized naturally occurring cytotoxic acridone-based compounds include acronycine and glyfoline (Figure 3). These compounds have been studied and structurally modified to improve their potency and solubility for clinical applications as anticancer drug candidates.^11–13

Figure 3.

Chemical structures of acridone (1) and the naturally occurring acridone-based cytotoxic compounds acronycine and glyfoline.

Recognizing the potential of acridone-based compounds as DNA-intercalating chemotherapeutic agents, we focused our investigations on developing qualitative structure-activity relationships (SARs) by means of synthetic structural modifications and experimentally testing for the cytotoxic activities of the derivatives in vitro against four cancer cell lines: acute lymphoblastic leukemia (Molt-3), hepatocarcinoma (HepG2), lung carcinoma (A549), and cholangiocarcinoma (HuCCA-1). Inspired by the structure of quinacrine, we introduced various basic side chains to the core structure to enhance the electrostatic binding of acridone to the DNA target. The structures of the derivatives comprise an acridone core scaffold, which can form π–π interactions with the DNA base pair and the basic side chain at the C2-terminus, creating an electrostatic interaction with the negative phosphate backbone of DNA. Furthermore, molecular modeling was employed to understand the basic binding conformations of the compounds and interactions with the DNA target.

Results and discussion

Synthesis and cytotoxicity of N¹⁰-substituted acridones

Acridone (1) was shown to be almost inactive against all the four tested cell lines, despite its planar structure (data from our preliminary cytotoxicity screening). This finding may partly be due to its structural simplicity and lack of functionalities that can allow extensive interactions with DNA. We modified the SARs between acridone and the target molecules by adding alkyl amines with varying chain lengths and different terminal nitrogen-based moieties to the N¹⁰-position of the acridone tricyclic core via a two-step reaction, as shown in Scheme 1.

Scheme 1.

(a) NaH, DMF, dibromoalkane, 80 °C, 0.5 h; (b) amine, CH₃CN, rt, 24 h; (c) MeI, CH₂Cl₂, rt, 18 h; (d) NaOH, DMSO, pyrrole, rt, 2 h; (e) NaN₃ (0.5 M) in DMSO, rt, 1 h; (f) NaH, DMF, PrI (for 2i), or BuBr (for 3i), 80 °C, 0.5 h.

Commercially available acridone (1) was used as the starting material for the N-alkylations (Scheme 1). A strong base was required for deprotonation to achieve N-alkylation due to the weak basic nature of the nitrogen in acridone. Using sodium hydride as the base, acridone could be alkylated with different dibromoalkanes in dimethylformamide at 80 °C to afford the corresponding N¹⁰-bromoalkyl acridones (2–5) in acceptable yields. An O-alkylated compound was formed as a by-product accompanied with the N-alkylated product but was unstable and readily transformed into acridone (1) under mild acidic conditions. The number of methylenes in the alkyl chain affected the alkylation reaction. Under the same conditions, alkylation reactions with butyl to hexyl chain spacers were accomplished with acceptable yields. Other undesired side products were obtained from the alkylation of acridone with dibromopropane, whereas alkylation with dibromoethane did not occur. The resulting bromoalkyl acridones 2–5 underwent nucleophilic substitution with various nitrogen-containing moieties to yield N¹⁰-substituted acridones bearing an alkyl side chain with a nitrogen atom at the terminal position. The first set of acridone derivatives was synthesized with varying alkyl chain lengths and terminal nitrogen-based moieties as side chains (Scheme 1). The synthesized compounds were evaluated for their cytotoxic activities and the results are summarized in Table 1.

Table 1.

Cytotoxic activities and the Moriguchi octanol-water partition coefficient (MlogP) values of N¹⁰-substituted acridone derivatives.

Compound	R	n	IC₅₀ (µM)^a				ΔG (kcal mol⁻¹)	MlogP
Compound	R	n	Molt-3	HepG2	A549	HuCCA-1	ΔG (kcal mol⁻¹)	MlogP
Acridone		0	Inactive^b	15.5 ± 6.7	Inactive	Inactive	−6.6	2.44
2a		3	11.1 ± 3.9	17.2 ± 4.6	43.3 ± 3.9	61.6 ± 12.2	−7.3	3.14
3a		4	14.8 ± 2.5	14.2 ± 3.3	38.9 ± 5.1	56.9 ± 2.8	−7.4	3.36
4a		5	14.6 ± 2.5	12.0 ± 3.6	41.1 ± 10.6	58.5 ± 0.6	−7.5	3.58
5a		6	19.9 ± 1.2	14.9 ± 5.7	44.2 ± 7.0	64.0 ± 1.4	−7.0	3.79
2b		3	43.9 ± 4.7	Inactive	Inactive	Inactive	−7.3	2.11
3b		4	22.8 ± 0.2	38.7 ± 5.2	67.0 ± 6.3	103.1 ± 2.7	−7.5	2.34
4b		5	16.6 ± 2.6	42.9 ± 0.0	77.1 ± 8.1	97.1 ± 1.4	−7.2	2.55
5b		6	18.1 ± 0.6	27.5 ± 5.9	57.0 ± 0.6	93.1 ± 3.1	−7.2	2.77
2c		3	14.9 ± 3.6	16.3 ± 3.5	40.9 ± 5.3	63.1 ± 5.5	−7.0	2.92
3c		4	16.9 ± 3.0	16.7 ± 3.6	38.1 ± 6.6	55.9 ± 6.6	−6.9	3.14
4c		5	14.8 ± 3.6	19.8 ± 4.5	52.1 ± 6.8	68.3 ± 5.7	−6.8	3.36
5c		6	16.9 ± 3.1	18.2 ± 4.4	49.3 ± 1.8	60.1 ± 7.7	−6.8	3.58
2d		3	19.2 ± 2.4	Inactive	Inactive	Inactive	−7.0	2.77
3d		4	26.5 ± 5.2	69.6 ± 0.0	Inactive	Inactive	−6.7	2.99
4d		5	25.7 ± 5.1	53.0 ± 6.4	Inactive	Inactive	−6.8	3.21
5d		6	23.8 ± 0.3	65.9 ± 11.7	Inactive	Inactive	−6.9	3.42
2e		3	17.2 ± 6.3	11.6 ± 1.3	33.1 ± 3.4	69.5 ± 2.1	−6.3	3.31
3e		4	21.0 ± 0.9	14.0 ± 2.7	42.7 ± 5.1	74.2 ± 16.2	−6.3	3.54
4e		5	21.8 ± 0.6	17.5 ± 5.2	49.9 ± 3.0	68.4 ± 8.4	−6.5	3.75
5e		6	23.5 ± 3.7	17.5 ± 7.0	54.0 ± 1.2	80.0 ± 1.8	−6.1	3.97
2f		3	14.1 ± 0.2	15.6 ± 2.7	35.7 ± 4.7	50.0 ± 6.8	−6.2	2.85
3f		4	15.6 ± 3.7	21.0 ± 5.2	64.0 ± 1.9	87.8 ± 13.2	−6.8	3.09
4f		5	13.6 ± 1.2	20.6 ± 5.0	61.7 ± 1.2	73.7 ± 7.8	−6.6	3.31
5f		6	12.2 ± 3.1	18.6 ± 6.2	51.7 ± 2.8	62.9 ± 3.3	−6.4	3.54
2g		3	100.8 ± 6.6	Inactive	Inactive	Inactive	−6.7	−0.60
3g		4	38.7 ± 9.6	Inactive	Inactive	Inactive	−6.8	−0.37
4g		5	80.2 ± 29.9	Inactive	Inactive	Inactive	−6.7	−0.15
5g		6	inactive	Inactive	Inactive	Inactive	−6.7	0.07
2h	N₃	3	27.2 ± 4.5	98.9 ± 10.4	Inactive	Inactive	−6.9	2.25
3h		4	34.1 ± 11.1	79.6 ± 9.4	157.5 ± 9.7	Inactive	−6.6	2.49
4h		5	33.5 ± 11.8	83.9 ± 13.2	150.3 ± 9.2	150.3 ± 18.5	−6.8	2.73
5h		6	24.9 ± 5.4	63.3 ± 12.6	135.9 ± 11.0	Inactive	−6.7	2.96
2i	H	3	31.8 ± 0.5	80.2 ± 14.2	192.8 ± 5.4	173.0 ± 1.5	−6.2	3.21
3i	H	4	44.2 ± 2.6	95.6 ± 6.9	Inactive	Inactive	−6.1	3.45
Doxorubicin	−	−	ND^c	0.4 ± 0.2	0.2 ± 0.0	0.9 ± 0.7	−9.2	−0.82

Results are expressed as mean ± standard error; average of three independent experiments.

IC₅₀ ⩾ 50 µg mL⁻¹.

Not determined.

Table 1 shows that the introduction of the side chains, particularly tertiary amine, to the N¹⁰-position of the parent acridone (1) led to cytotoxicity against the tested cell lines. Acridones with alkyl piperidine 2a–5a, alkyl morpholine 2b–5b, alkyl pyrrolidine 2c–5c, alkyl diethylamine 2e–5e, and alkyl dimethylamine 2f–5f demonstrated improved cytotoxicity, whereas acridones with alkyl pyrrole 2d–5d, alkyl azide 2h–5h, and alkyl ammonium salts 2g–5g were inactive or displayed modest activity. These results are presumably due to their poor ability to interact with DNA. Varying the chain length from three to six carbons resulted in slight differences in the cytotoxicities. Among the tested cell lines, Molt-3 was the most sensitive against alkyl piperidines 2a–5a, alkyl pyrrolidines 2c–5c, alkyl diethylamines 2e–5e, and alkyl dimethylamines 2f–5f, with comparable IC₅₀ values ranging from 11 to 23 µM. For the HepG2 cell line, acridones bearing alkyl piperidine 2a–5a, alkyl pyrrolidine 2c–5c, and alkyl diethylamine 2e–5e units displayed cytotoxicities within 12−20 µM, which were comparable with that of unsubstituted acridone (1). For the A549 cell line, acridones containing alkyl piperidine 2a–5a exhibited the highest improvement in cytotoxicity, with IC₅₀ values ranging from 39 to 44 µM. For the HuCCA-1 cell line, acridones with alkyl piperidine 2a–5a and alkyl pyrrolidine 2c–5c substituents showed the highest improvement in cytotoxicity, with comparable IC₅₀ values ranging from 56 to 68 µM. Hence, introducing an alkyl piperidine at N¹⁰ of acridone significantly improved its cytotoxicity compared with alkyl morpholine. Alkyl piperidines (pK_a = 11.22) had a higher level of positive-charged forms at physiological pH compared to alkyl morpholines (pK_a = 8.36) because of the higher basicity of the former. The high extent of electrostatic interactions imposed by alkyl piperidine derivatives contributed to the lower IC₅₀ values compared with those of alkyl morpholines. The slightly higher pK_a values of alkyl pyrrolidines (e.g. pK_a 11.27) compared with those of alkyl piperidines (e.g. pK_a 11.22) resulted in slightly lower cytotoxicities. This phenomenon could be due to the higher level of positive-charged forms of alkyl pyrrolidine acridones, as reflected in the lower Moriguchi partition coefficients (MlogP), thereby preventing them from entering into the cell. It is noted that MlogP was calculated with nitrogen atoms negatively contributing to the parameter.¹⁴ Despite the low value of MlogP calculated from doxorubicin caused by the negative contribution of the nitrogen and an oxygen atoms, this drug is actually carried into cells by organic anion transporting peptide 1A/1B (OATP1A/1B).¹⁵ Among alkyl piperidine derivatives 2a–5a, butyl and pentyl piperidines (3a and 4a, respectively) were equally effective against the tested cell lines. However, the cytotoxicity was significantly compromised after lengthening the alkyl chain by adding six carbons. This finding could be due to the higher degree of freedom of the acridone moiety to escape from base-pair stacking.

Introducing a butyl piperidine to the N¹⁰ of acridone led to the highest cytotoxicity for compound 3a. This finding encouraged us to improve the cytotoxicity of 3a. The addition of a butyl piperidine side chain to the aromatic ring of 3a was aimed toward enhancing the cytotoxicity by increasing the binding interaction to the negative groove of DNA. Evidence suggests that conjugating an amino acid to compounds of interest can improve their bioavailability in tissues.¹⁶ Therefore, acridone 3a was conjugated to an amino acid in an effort to enhance the cytotoxicity. A carboxyl group was used to connect the butyl piperidine side chain or amino acid to the aromatic ring of acridone 3a. Hence, acridone-2-carboxylic acid was prepared and used as a precursor for the synthesis of 2,N¹⁰-substituted acridones.

Synthesis and cytotoxicity of 2,N¹⁰-substituted acridones

The precursor, acridone-2-carboxylic acid (9), was prepared via two steps (Scheme 2) to obtain key intermediates for synthesis of 2,N¹⁰-substituted compounds. The Ullmann condensation of 2-bromobenzoic acid (6) with 4-aminobenzoic acid (7) in the presence of copper and potassium carbonate in refluxing N,N-dimethylformamide yielded diphenylamine-2,4′-dicarboxylic acid (8).¹⁷ Treatment of 8 with concentrated sulfuric acid formed the desired acridone-2-carboxylic acid (9). A butyl piperidine side chain was incorporated into 9 through the previously described method. The use of NaH and 1,4-dibromobutane led to the generation of N,O- and O-alkylated products (10a and 10b, respectively). Stirring the resulting bromobutyl acridone 10a with piperidine in acetonitrile at room temperature yielded acridone 11a containing two butyl piperidine side chains. Reaction under the same conditions with 10b provided 11b. The basic hydrolysis of 11a afforded acridone 12 with a free carboxylic acid. Simple methyl ester 13 was prepared to investigate the cytotoxicity. The conjugation of 12 with an amino acid was aimed toward enhancing the cytotoxicity. The amino acid, L-valine benzyl ester, was conjugated to acridone 12 by using 1-hydroxybenzotriazole (HOBt) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) to obtain acridone 14 in good yield. The benzyl ester group of 14 was removed by hydrogenation to form acridone 15. A benzyl amide protecting group was incorporated to yield acridone 16. The simple benzyl amide of 12 (compound 17) was also prepared to investigate its cytotoxicity. Compounds 11a and 12–16 were subjected to cytotoxic testing. The corresponding molecules of these compounds without the N¹⁰-butyl piperidine side chain were also prepared by following similar procedures and were also subjected to cytotoxic testing (compounds 18, 19, 20, and 21). The IC₅₀ values of the substituted acridone derivatives and those of 3a are summarized in Table 2.

Scheme 2.

(a) Cu, K₂CO₃, DMF, reflux, 4 h; (b) Conc. H₂SO₄, 100 °C, 4 h; (c) NaH, DMF, dibromobutane, 80 °C, 1 h; (d) piperidine, CH₃CN, rt, 24 h; (e) 5% NaOH, EtOH, reflux, 1 h; (f) SOCl₂, reflux, 3 h then cold MeOH; (g) amino acid or amine, HOBt, EDCI, DIPEA, DMF, rt, 24 h; (h) H₂, 10% Pd/C, MeOH, rt, 24 h.

Table 2.

Cytotoxic activities and MlogP values of the acridone derivatives substituted at C-2 and/or the N¹⁰-position.

Compound	R	R′	IC₅₀ (µM)^a				ΔG (kcal mol⁻¹)	MlogP
Compound	R	R′	Molt-3	HepG2	A549	HuCCA-1	ΔG (kcal mol⁻¹)	MlogP
3a		H	14.8 ± 2.5	14.2 ± 3.3	38.9 ± 5.1	56.9 ± 2.8	−7.4	3.36
11a			3.3 ± 0.5	3.0 ± 0.5	5.2 ± 0.5	9.5 ± 0.3	−7.8	4.00
12		COOH	Inactive^b	Inactive	Inactive	Inactive	−7.7	2.98
13		COOMe	3.6 ± 0.4	4.3 ± 1.5	10.8 ± 1.9	25.6 ± 2.5	−7.8	3.20
14			6.1 ± 0.2	7.0 ± 0.5	8.9 ± 0.9	9.4 ± 0.3	−7.9	3.99
15			Inactive	Inactive	Inactive	Inactive	−6.7	2.81
16			9.6 ± 1.9	35.3 ± 3.5	19.3 ± 3.2	20.9 ± 3.3	−7.8	3.59
17			8.2 ± 0.6	2.0 ± 0.2	7.5 ± 1.1	9.9 ± 3.3	−8.7	3.84
9	H	COOH	Inactive	Inactive	Inactive	Inactive	−7.0	2.06
11b	H		12.4 ± 0.7	18.9 ± 7.5	20.2 ± 2.0	25.9 ± 1.8	−7.5	2.98
18	H	COOMe	43.9 ± 12.2	5.9 ± 3.4	11.1 ± 0.4	12.1 ± 2.8	−6.9	2.32
19	H		6.9 ± 1.0	13.8 ± 3.5	16.7 ± 3.1	22.5 ± 1.3	−8.3	3.40
20	H		Inactive	Inactive	Inactive	Inactive	−7.6	2.05
21	H		Inactive	Inactive	105.7 ± 11.5	Inactive	−8.3	3.15
Doxorubicin	−	−	ND^c	0.7 ± 0.1	0.1 ± 0.0	0.5 ± 0.4	−9.2	−0.82
3′-desamino-3′-(2-methoxy-4-morpholinyl)-doxorubicin			ND	ND	ND	ND	−9.4	ND

Results are expressed as mean ± standard error; average of three independent experiments.

IC₅₀ ⩾ 50 µg mL⁻¹.

Not determined.

Considering C2-derivatized 3a, the presence of a polar carboxyl moiety at C2 in compound 12 led to it being inactive in cytotoxicity studies. The inactivity of 12 could be due to the high hydrophilicity, as shown by the low partition coefficient (MlogP), thereby prohibiting cellular uptake. Decreasing the polarity of the compounds might increase their cytotoxicity. Hence, the carboxyl group of 12 was converted into a butyl piperidine ester (compound 11a), a methyl ester (compound 13), and a benzyl amide (compound 17), which greatly improved their activities. The conjugation of 12 with L-valine amino acid led to compound 15, in which the presence of the carboxyl group resulted in inactivity. Therefore, the uptake of acridone derivatives was not facilitated by amino acid transporting proteins but by passive diffusion. To confirm the above argument, the free acid was protected as a benzyl ester (compound 14) and a benzyl amide (compound 17), resulting in the recovery of the activity. When the R group was the same, for example, compound 11a, with the N¹⁰-butyl piperidine side chain, exhibited higher cytotoxic activity compared to 11b without the N¹⁰-attached group. This finding indicates the crucial role of the N¹⁰-butyl piperidine side chain in the cytotoxicity of acridone. Introducing a butyl piperidine ester, a methyl ester, a benzyl amide, a L-valine benzyl ester, and a L-valine benzyl amide at the C2-position of 3a increased the cytotoxicity. Compound 11a, which was obtained from the addition of butyl piperidine ester at the C2-position of 3a, exhibited the highest improvement in cytotoxicity against the tested cell lines. The cytotoxicity of 11a was improved by 4.5-, 4.8-, 7.5- and 6-fold against Molt-3, HepG2, A549, and HuCCA-1, respectively, compared with 3a.

Molecular modeling of 2,N¹⁰-substituted acridones

Molecular modeling was conducted using the AutoDock Vina 1.1.2 software suite¹⁸ to elucidate the binding modes and binding energies of the acridone derivatives. A double-stranded DNA crystal structure was obtained from the Protein Data Bank (PDB code: 2DES)^19,20 and was used as the docking scaffold. The native co-crystallized ligand, 3′-desamino-3′-(2-methoxy-4-morpholinyl)-doxorubicin, was removed and then docked again to the DNA crystal structure. The docked conformation and the co-crystallized conformation were superimposed (Figure S1 in the Supplementary Information) and showed similar orientations, which means that our method can be used further for all the derivatives.

All the derivatives were docked to the DNA crystal structure and the binding energies measured as ΔG values are shown in Tables 1 and 2. The docking results revealed that the positive control, doxorubicin, had the lowest ΔG = –9.2 kcal mol⁻¹, suggesting that this compound binds very tightly, as reflected in the very low value of IC₅₀ (<1 µM). The ΔG values of the N¹⁰-substituted acridone derivatives tended to be more positive with ΔG values ranging between −6.1 and −7.5 kcal mol⁻¹ compared to the more cytotoxic C2,N¹⁰-substituted acridone derivatives with ΔG values ranging between −6.7 and −9.1 kcal mol⁻¹, in congruence with the cytotoxicity results. Depicted in Figure 4(a), the tricyclic acridone core is inserted and stabilized by π–π stacking with the base pairs in DNA, thereby perturbing DNA base-pairing interactions. The N¹⁰-alkyl side chains (Figures 4(a) and (b)) interact with the DNA minor groove, most probably through van der Waal’s interactions, and the nitrogen-based moieties could potentially form electrostatic attractions with the phosphate backbones. For the C2,N¹⁰-substituted acridone derivatives (Figure 4(b)), the C2-substituted ester is predicted to interact with the DNA major groove.

Figure 4.

Binding conformation of N¹⁰-substituted acridone derivative 3a (a) and C2,N¹⁰-substituted acridone derivative 11a (b). The DNA fragment is represented as sticks, and ligands are represented as balls and sticks. The DNA surface has been rendered. Blue, red, and gray surfaces represent partial positive, negative, and neutral charges, respectively. Green solid lines are π–π stacking interactions and red dotted lines depict hydrogen bonding.

Conclusion

The introduction of two butyl piperidine side chains at the C2- and N¹⁰-positions of acridone (compound 11a) could enhance the cytotoxicity of acridine-based compounds. The optimal pK_a (piperidinyl moiety) improved the ability of the derivative to bind to the DNA phosphate backbone and enter the cell compared with the other derivatives, that is, morpholine-, pyrrolidine-, pyrrole-, 3° and 4° amines and azide-substituted compounds. The substitution of the butyl piperidine side chain at the C2-position could provide tight binding of the compound to DNA, as supported by the increased binding energy from the computational calculations. The incorporation of two butyl piperidines at the C2- and N¹⁰-positions of acridone did not affect the cellular uptake of the compound. In addition, the positive-charged piperidinium moieties could electrostatically bind to the DNA phosphate backbone, facilitating the tight binding DNA. As a result, compound 11a showed the greatest enhanced DNA intercalation.

Experimental

Chemistry

¹H NMR and ¹³C NMR spectra were recorded on Bruker AVANCE 400 (400 MHz for ¹H and 100 MHz for ¹³C) and Bruker AVANCE 300 (300 MHz for ¹H and 75 MHz for ¹³C) spectrometers. Chemical shifts are reported as δ values in ppm relative to tetramethylsilane. Mass spectra were obtained on a Finnigan Polaris GCQ mass spectrometer, while accurate masses (HRMS) were obtained using a Bruker Micro TOF in APCI or ESI positive mode. Infrared (IR) spectra were recorded in terms of cm⁻¹ on a Perkin Elmer Spectrum One FTIR spectrometer. Melting points were determined on an SMP3 melting point apparatus. Column chromatography analysis was performed on Merck silica gel 60 (70–230 mesh).

General procedure for the preparation of bromoalkyl acridones 2−5

A solution of acridone (1 mmol) in N,N-dimethylformamide (10 mL) was added to a stirred solution of sodium hydride (3 mmol) in N,N-dimethylformamide (10 mL). The mixture was heated at 80 °C for 15 min, treated with the appropriate dibromoalkane (6 mmol) and heated at 80 °C for an additional 0.5 h. The reaction mixture was cooled, water was added, and the mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and water, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

10-(3′-N-Bromopropyl)acridone ( 2 ):²¹ (32%) yellow solid, m.p. 113.5 °C−114.5 °C; FTIR, ν_max (cm⁻¹): 1631, 1596, 1489, 1461, 1377, 1177, 752, 673; ¹H NMR (400 MHz, CDCl₃): δ 8.49 (dd, J = 8.0, 1.6 Hz, 2H), 7.68−7.63 (m, 2H), 7.48 (d, J = 8.7 Hz, 2H), 7.22 (t, J = 7.5 Hz, 2H), 4.48 (t, J = 7.7 Hz, 2H), 3.56 (t, J = 6.0 Hz, 2H), 2.35−2.43 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 177.9, 141.6 (2C), 134.1 (2C), 128.1 (2C), 122.5 (2C), 121.4 (2C), 114.2 (2C), 44.5, 30.2, 29.4; MS (EI): m/z (relative intensity): 315 (M⁺, 20), 208 (100), 180 (26), 152 (13); HRMS (APCI): m/z calcd for C₁₆H₁₅NOBr [M + H]⁺: 316.0332; found: 316.0339.

10-(4′-N-Bromobutyl)acridone ( 3 ):²² (57%) yellow solid, m.p. 145.5 °C−146.5 °C; FTIR, ν_max (cm⁻¹): 1631, 1601, 1492, 1461, 1264, 1176, 732, 704; ¹H NMR (300 MHz, CDCl₃): δ 8.56 (dd, J = 8.0, 1.2 Hz, 2H), 7.75−7.68 (m, 2H), 7.47 (d, J = 8.7 Hz, 2H), 7.28 (t, J = 7.7 Hz, 2H), 4.34 (t, J = 7.7 Hz, 2H), 3.52 (t, J = 6.5 Hz, 2H), 2.05−2.15 (m, 4H); ¹³C NMR (75 MHz, CDCl₃): δ 177.9, 141.6 (2C), 134.0 (2C), 128.0 (2C), 122.4 (2C), 121.3 (2C), 114.4 (2C), 45.1, 32.7, 29.7, 25.7; MS (EI): m/z (relative intensity): 329 (M⁺, 23), 208 (100), 180 (21); HRMS (APCI): m/z calcd for C₁₇H₁₇NOBr [M + H]⁺: 330.0488; found: 330.0491.

10-(5′-N-Bromopentyl)acridone ( 4 ): (75%) yellow solid, m.p. 114.5 °C−115.0 °C; FTIR, ν_max (cm⁻¹): 1632, 1597, 1490, 1460, 1377, 1290, 1264, 1176, 753, 673; ¹H NMR (300 MHz, CDCl₃): δ 8.58 (dd, J = 8.0, 1.4 Hz, 2H), 7.78−7.68 (m, 2H), 7.47 (d, J = 8.7 Hz, 2H), 7.29 (t, J = 7.6 Hz, 2H), 4.35 (t, J = 8.2 Hz, 2H), 3.47 (t, J = 6.5 Hz, 2H), 2.05−1.91 (m, 4H), 1.78−1.68 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 177.9, 141.6 (2C), 133.9 (2C), 128.0 (2C), 122.5 (2C), 121.3 (2C), 114.4 (2C), 45.8, 33.3, 32.2, 26.3, 25.4; MS (EI): m/z (relative intensity): 343 (M⁺, 27), 208 (100), 180 (17); HRMS (APCI): m/z calcd for C₁₈H₁₉NOBr [M + H]⁺: 344.0644; found: 344.0647.

2.2.4 10-(6′-N-Bromohexyl)acridone ( 5 ): (70%) yellow solid, m.p. 103.0 °C−104.0 °C; FTIR, ν_max (cm⁻¹): 1632, 1597, 1490, 1460, 1377, 1290, 1264, 1176, 753, 673; ¹H NMR (300 MHz, CDCl₃): δ 8.59 (dd, J = 8.0, 1.7 Hz, 2H), 7.74−7.67 (m, 2H), 7.50 (d, J = 8.7 Hz, 2H), 7.31 (t, J = 7.6 Hz, 2H), 4.36 (t, J = 8.3 Hz, 2H), 3.46 (t, J = 6.6 Hz, 2H), 2.05−1.90 (m, 4H), 1.75−1.55 (m, 4H); ¹³C NMR (75 MHz, CDCl₃): δ 177.9, 141.7 (2C), 133.9 (2C), 128.0 (2C), 122.4 (2C), 121.2 (2C), 114.4 (2C), 45.9, 33.5, 32.5, 27.8, 27.0, 26.1; MS (EI): m/z (relative intensity): 357 (M⁺, 33), 208 (100), 195 (19), 180 (13); HRMS (APCI): m/z calcd for C₁₉H₂₁NOBr [M + H]⁺: 358.0801; found: 358.0795.

General procedure for the preparation of N¹⁰-substituted acridones

Preparation of compounds 2a–5a, 2b–5b, 2c–5c, 2e–5e, and 2f–5f

Amine (5 mmol) was added to a stirred solution of bromoalkyl acridone (1 mmol) in acetonitrile (25 mL). The reaction mixture was stirred at room temperature for 24 h and concentrated under reduced pressure. The residue was treated with water and extracted with dichloromethane. The combined organic layer was washed with saturated aqueous sodium hydrogen carbonate and water, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

Preparation of compounds 2d–5d

Pyrrole (3 mmol) was added to a mixture of sodium hydroxide (9 mmol) in dimethyl sulfoxide (5 mL) and the mixture was stirred at room temperature for 30 min. After cooling in an ice bath, the mixture was treated with a solution of bromoalkyl acridone (1 mmol) in dimethyl sulfoxide (15 mL) and then stirred at room temperature for 2 h. Water was added and the mixture was extracted with ethyl acetate. The combined organic layer was washed with water, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

Preparation of compounds 2g–5g

A solution of 2f–5f (1 mmol) in dichloromethane (10 mL) was treated with iodomethane (5 mmol) and the mixture was stirred under argon at room temperature for 12 h. The precipitate was collected, washed with dichloromethane, and dried to yield trimethylammonium salts 2g–5g.

Preparation of compounds 2h–5h

A solution of bromoalkyl acridone (1 mmol) in dimethyl sulfoxide (3 mL) was treated with sodium azide (0.5 M) in dimethyl sulfoxide (3 mL, 1.5 mmol) and the mixture was stirred at room temperature for 3 h. Water was added and the mixture was extracted with ethyl acetate. The combined organic layer was washed with water, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

Preparation of compounds 2i and 3i

A solution of acridone (1 mmol) in N,N-dimethylformamide (10 mL) was added to a stirred suspension of sodium hydride (3 mmol) in N,N-dimethylformamide (10 mL). The mixture was heated at 80 °C for 15 min, treated with propyl iodide (for 2i) or 1-bromobutane (for 3i) (5 mmol), and heated at 80 °C for an additional 0.5 h. The reaction mixture was cooled and then water was added and the mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and water, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography.

10-(3′-N-Piperidinopropyl)acridone ( 2a ):²³ (67%) yellow solid, m.p. 135.0 °C−136.0 °C; FTIR, ν_max (cm⁻¹): 1634, 1598, 1489, 1462, 1377, 1262, 1177, 753, 674; ¹H NMR (300 MHz, CDCl₃): δ 8.58 (dd, J = 8.0, 1.5 Hz, 2H), 7.80−7.60 (m, 4H), 7.29 (t, J = 7.6 Hz, 2H), 4.47 (t, J = 8.4 Hz, 2H), 2.55−2.40 (m, 6H), 2.10−2.00 (m, 2H), 1.75−1.60 (m, 4H), 1.60−1.45 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 178.0, 141.9 (2C), 133.8 (2C), 127.9 (2C), 122.4 (2C), 121.1 (2C), 114.8 (2C), 55.7, 54.8 (2C), 44.1, 26.1 (2C), 24.8, 24.4; MS (EI): m/z (relative intensity): 320 (M⁺, 8), 208 (4), 180 (4), 124 (10), 98 (100); HRMS (APCI): m/z calcd for C₂₁H₂₅N₂O [M + H]⁺: 321.1961; found: 321.1954.

10-(4′-N-Piperidinobutyl)acridone ( 3a ):²³ (96%) yellow solid, m.p. 115.5 °C−116.0 °C; FTIR, ν_max (cm⁻¹): 1633, 1598, 1490, 1461, 1377, 1264, 1177, 752, 732, 673; ¹H NMR (400 MHz, CDCl₃): δ 8.58 (dd, J = 8.0, 1.2 Hz, 2H), 7.74−7.60 (m, 4H), 7.31−7.25 (m, 2H), 4.37 (t, J = 7.6 Hz, 2H), 2.50−2.40 (m, 6H), 2.00−1.91 (m, 2H), 1.80−1.70 (m, 2H), 1.70−1.60 (m, 4H), 1.60−1.55 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 178.0, 141.7 (2C), 133.8 (2C), 127.8 (2C), 122.4 (2C), 121.1 (2C), 114.8 (2C), 57.9, 54.6 (2C), 45.9, 26.1 (2C), 24.6, 24.5, 23.6; MS (EI): m/z (relative intensity): 334 (M⁺, 44), 208 (10), 138 (21), 98 (100); HRMS (APCI): m/z calcd for C₂₂H₂₇N₂O [M + H]⁺: 335.2118; found: 335.2125.

10-(5′-N-Piperidinopentyl)acridone ( 4a ): (77%) yellow solid, m.p. 121.5 °C−122.5 °C; FTIR, ν_max (cm⁻¹): 1632, 1597, 1490, 1460, 1376, 1290, 1262, 1176, 753, 673; ¹H NMR (300 MHz, CDCl₃): δ 8.57 (dd, J = 8.1, 1.5 Hz, 2H), 7.74−7.68 (m, 2H), 7.47 (d, J = 8.7 Hz, 2H), 7.28 (t, J = 7.5 Hz, 2H), 4.32 (t, J = 8.4 Hz, 2H), 2.38−2.31 (m, 6H), 2.00−1.89 (m, 2H), 1.70−1.44 (m, 10H); ¹³C NMR (75 MHz, CDCl₃): δ 177.9, 141.7 (2C), 133.8 (2C), 127.9 (2C), 122.4 (2C), 121.1 (2C), 114.5 (2C), 59.2, 54.7 (2C), 46.1, 27.0, 26.7, 26.0 (2C), 25.0, 24.4; MS (EI): m/z (relative intensity): 348 (M⁺, 30), 208 (13), 154 (14), 98 (100); HRMS (APCI): m/z calcd for C₂₃H₂₉N₂O [M + H]⁺: 349.2274; found: 349.2281.

10-(6′-N-Piperidinohexyl)acridone ( 5a ): (77.5%) yellow solid, m.p. 81.0 °C−82.0 °C; FTIR, ν_max (cm⁻¹): 1631, 1598, 1492, 1460, 1377, 1289, 1263, 1178, 756, 674; ¹H NMR (400 MHz, CDCl₃): δ 8.59 (dd, J = 8.0, 1.6 Hz, 2H), 7.78−7.73 (m, 2H), 7.50 (d, J = 8.8 Hz, 2H), 7.30 (t, J = 7.2 Hz, 2H), 4.36 (t, J = 8.0 Hz, 2H), 3.00−2.77 (m, 6H), 2.00−1.75 (m, 8H), 1.66−1.40 (m, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 177.9, 141.7 (2C), 134.0 (2C), 128.0 (2C), 122.5 (2C), 121.2 (2C), 114.5 (2C), 57.9, 53.7 (2C), 45.8, 27.1, 26.8, 26.4, 24.5, 23.5 (2C), 22.7; MS (EI): m/z (relative intensity): 362 (M⁺, 5), 208 (12), 149 (25), 98 (98), 86 (100); HRMS (APCI): m/z calcd for C₂₄H₃₁N₂O [M + H]⁺: 363.2431; found: 363.2438.

10-(3′-N-Morpholinopropyl)acridone ( 2b ):²³ (78.5%) yellow solid, m.p. 114.0 °C−114.5 °C; FTIR, ν_max (cm⁻¹): 1633, 1598, 1491, 1462, 1378, 1290, 1263, 1178, 1115, 754, 674; ¹H NMR (300 MHz, CDCl₃): δ 8.59 (dd, J = 8.0, 1.2 Hz, 2H), 7.75−7.62 (m, 4H), 7.29 (t, J = 7.4 Hz, 2H), 4.49 (t, J = 7.5 Hz, 2H), 3.80 (t, J = 4.5 Hz, 4H), 2.55−2.47 (m, 6H), 2.12−2.02 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 178.0, 141.8 (2C), 133.8 (2C), 128.0 (2C), 122.4 (2C), 121.2 (2C), 114.6 (2C), 67.0 (2C), 55.4, 53.8 (2C), 43.7, 24.3; MS (EI): m/z (relative intensity): 322 (M⁺, 6), 279 (8), 222 (12), 208 (10), 180 (9), 100 (100); HRMS (APCI): m/z calcd for C₂₀H₂₃N₂O₂ [M + H]⁺: 323.1754; found: 323.1750.

10-(4′-N-Morpholinobutyl)acridone ( 3b ):²³ (73%) yellow solid, m.p. 143.5 °C−144.5 °C; FTIR, ν_max (cm⁻¹): 1632, 1600, 1493, 1462, 1378, 1265, 1116, 731, 703; ¹H NMR (400 MHz, CDCl₃): δ 8.57 (dd, J = 8.0, 1.6 Hz, 2H), 7.74−7.69 (m, 2H), 7.58 (d, J = 8.4 Hz, 2H), 7.30−7.26 (m, 2H), 4.37 (t, J = 8.3 Hz, 2H), 3.78 (t, J = 4.8 Hz, 4H), 2.55−2.47 (m, 6H), 2.02−1.93 (m, 2H), 1.82−1.73 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 177.9, 141.7 (2C), 133.8 (2C), 127.9 (2C), 122.4 (2C), 121.2 (2C), 114.6 (2C), 67.0 (2C), 57.6, 53.7 (2C), 45.8, 24.4, 23.2; MS (EI): m/z (relative intensity): 336 (M⁺, 10), 293 (14), 222 (9), 208 (23), 180 (15), 142 (14), 100 (100); HRMS (APCI): m/z calcd for C₂₁H₂₅N₂O₂ [M + H]⁺: 337.1911; found: 337.1908.

10-(5′-N-Morpholinopentyl)acridone ( 4b ): (75%) yellow solid, m.p. 116.5 °C−117.0 °C; FTIR, ν_max (cm⁻¹): 1633, 1597, 1490, 1460, 1376, 1290, 1261, 1176, 1116, 753, 673; ¹H NMR (400 MHz, CDCl₃): δ 8.58 (dd, J = 8.0, 1.6 Hz, 2H), 7.75−7.70 (m, 2H), 7.48 (d, J = 8.7 Hz, 2H), 7.29 (t, J = 7.6 Hz, 2H), 4.34 (t, J = 8.2 Hz, 2H), 3.74 (t, J = 4.6 Hz, 4H), 2.50−2.37 (m, 6H), 2.02−1.91 (m, 2H), 1.70−1.54 (m, 4H); ¹³C NMR (100 MHz, CDCl₃): δ 177.9, 141.7 (2C), 133.8 (2C), 128.0 (2C), 122.5 (2C), 121.2 (2C), 114.4 (2C), 66.9 (2C), 58.7, 53.8 (2C), 46.0, 27.0, 26.2, 24.7; MS (EI): m/z (relative intensity): 350 (M⁺, 4), 332 (9), 319 (15), 307 (19), 264 (20), 222 (18), 208 (41), 180 (20), 100 (100); HRMS (APCI): m/z calcd for C₂₂H₂₇N₂O₂ [M + H]⁺: 351.2067; found: 351.2065.

10-(6′-N-Morpholinohexyl)acridone ( 5b ): (77%) yellow solid, m.p. 95.5 °C−96.0 °C; FTIR, ν_max (cm⁻¹): 1634, 1598, 1490, 1460, 1376, 1290, 1262, 1176, 1116, 753, 673; ¹H NMR (400 MHz, CDCl₃): δ 8.58 (dd, J = 8.0, 1.5 Hz, 2H), 7.74−7.69 (m, 2H), 7.48 (d, J = 8.7 Hz, 2H), 7.31−7.26 (m, 2H), 4.32 (t, J = 8.2 Hz, 2H), 3.73 (t, J = 4.6 Hz, 4H), 2.47–2.42 (m, 4H), 2.36 (t, J = 7.3 Hz, 2H), 1.98−1.89 (m, 2H), 1.62−1.52 (m, 4H), 1.50−1.43 (m, 2H); ¹³C NMR (100 MHz, CDCl_3): δ 177.9, 141.7 (2C), 133.8 (2C), 127.9 (2C), 122.4 (2C), 121.1 (2C), 114.5 (2C), 66.9 (2C), 58.9, 53.8 (2C), 46.0, 27.2, 27.1, 26.8, 26.5; MS (EI): m/z (relative intensity): 364 (M⁺, 4) 346 (23), 333 (37), 321 (32), 277 (16), 222 (18), 208 (43), 100 (100); HRMS (APCI): m/z calcd for C₂₃H₂₉N₂O₂ [M + H]⁺: 365.2224; found: 365.2225.

10-(3′-N-Pyrrolidinopropyl)acridone ( 2c ): (69%) yellow solid, m.p. 70.5 °C−71.5 °C; FTIR, ν_max (cm⁻¹): 1633, 1598, 1490, 1461, 1377, 1290, 1263, 1177, 753, 674; ¹H NMR (300 MHz, CDCl₃): δ 8.58 (dd, J = 8.0, 1.2 Hz, 2H), 7.77−7.68 (m, 2H), 7.64 (d, J = 8.7 Hz, 2H), 7.29 (t, J = 7.5 Hz, 2H), 4.50 (t, J = 7.7 Hz, 2H), 2.75−2.62 (m, 6H), 2.22−2.11 (m, 2H), 1.92−1.85 (m, 4H); ¹³C NMR (75 MHz, CDCl₃): δ 178.0, 141.8 (2C), 133.9 (2C), 127.9 (2C), 122.4 (2C), 121.2 (2C), 114.7 (2C), 54.2 (2C), 52.9, 43.9, 26.4, 23.5 (2C); MS (EI): m/z (relative intensity): 306 (M⁺, 13), 208 (6), 180 (6), 110 (7), 84 (100); HRMS (APCI): m/z calcd for C₂₀H₂₃N₂O [M + H]⁺: 307.1805; found: 307.1819.

10-(4′-N-Pyrrolidinobutyl)acridone ( 3c ): (93%) yellow solid, m.p. 108.5 °C−109.5 °C; FTIR, ν_max (cm⁻¹): 1633, 1597, 1490, 1461, 1378, 1264, 1177, 752, 673; ¹H NMR (400 MHz, CDCl₃): δ 8.57 (dd, J = 8.0, 1.6 Hz, 2H), 7.73−7.68 (m, 2H), 7.59 (d, J = 8.7 Hz, 2H), 7.30−7.25 (m, 2H), 4.37 (t, J = 8.3 Hz, 2H), 2.62−2.53 (m, 6H), 2.03−1.94 (m, 2H), 1.86−1.73 (m, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 177.9, 141.7 (2C), 133.8 (2C), 127.9 (2C), 122.4 (2C), 121.1 (2C), 114.7 (2C), 55.3, 54.1 (2C), 45.9, 25.7, 24.9, 23.5 (2C) ppm; MS (EI): m/z (relative intensity): 320 (M⁺, 25), 208 (9), 180 (11), 124 (21), 84 (100); HRMS (APCI): m/z calcd for C₂₁H₂₅N₂O [M + H]⁺: 321.1961; found: 321.1962.

10-(5′-N-Pyrrolidinopentyl)acridone ( 4c ): (73%) yellow solid, m.p. 113.0 °C−114.0 °C; FTIR, ν_max (cm⁻¹): 1633, 1597, 1490, 1460, 1377, 1290, 1263, 1176, 753, 673; ¹H NMR (400 MHz, CDCl₃): δ 8.58 (dd, J = 8.0, 1.4 Hz, 2H), 7.75−7.70 (m, 2H), 7.49 (d, J = 8.7 Hz, 2H), 7.29 (t, J = 7.4 Hz, 2H), 4.34 (t, J = 8.2 Hz, 2H), 2.58−2.50 (m, 6H), 2.01−1.91 (m, 2H), 1.85−1.78 (m, 4H), 1.74−1.56 (m, 4H); ¹³C NMR (100 MHz, CDCl₃): δ 177.9, 141.8 (2C), 133.8 (2C), 128.0 (2C), 122.5 (2C), 121.2 (2C), 114.5 (2C), 56.3, 54.3 (2C), 46.1, 28.6, 27.1, 25.0, 23.4 (2C); MS (EI): m/z (relative intensity): 334 (M⁺, 13), 208 (11), 180 (8), 140 (17), 84 (100); HRMS (APCI): m/z calcd for C₂₂H₂₇N₂O [M + H]⁺: 335.2118; found: 335.2106.

10-(6′-N-Pyrrolidinohexyl)acridone ( 5c ): (71%) yellow solid, m.p. 76.0 °C−77.0 °C; FTIR, ν_max (cm⁻¹): 1630, 1596, 1491, 1460, 1377, 1263, 1177, 755, 674; ¹H NMR (300 MHz, CDCl₃): δ 8.58 (dd, J = 8.0, 1.5 Hz, 2H), 7.77−7.72 (m, 2H), 7.50 (d, J = 8.7 Hz, 2H), 7.30 (t, J = 7.5 Hz, 2H), 4.34 (t, J = 8.0 Hz, 2H), 3.08 (br s, 4H), 2.88 (t, J = 7.9 Hz, 2H), 2.10−1.81 (m, 8H) 1.63−1.45 (m, 4H); ¹³C NMR (75 MHz, CDCl₃): δ 177.9, 141.7 (2C), 134.0 (2C), 127.9 (2C), 122.4 (2C), 121.2 (2C), 114.5 (2C), 55.6, 53.7 (2C), 45.8, 27.0, 26.8, 26.5, 26.4, 23.3 (2C); MS (EI): m/z (relative intensity): 348 (M⁺, 13), 208 (8), 180 (9), 110 (11), 84 (100); HRMS (APCI): m/z calcd for C₂₃H₂₉N₂O [M + H]⁺: 394.2274; found: 349.2283.

10-(3′-(N-Pyrrole)propyl)acridone ( 2d ): (78%) yellow solid, m.p. 154.5 °C−155.5 °C; FTIR, ν_max (cm⁻¹): 1632, 1598, 1491, 1461, 1291, 1264, 1177, 732, 673; ¹H NMR (300 MHz, CDCl₃): δ 8.54 (dd, J = 8.0, 1.6 Hz, 2H), 7.68−7.60 (m, 2H), 7.26 (t, J = 7.0 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 6.78 (t, J = 2.0 Hz, 2H), 6.30 (t, J = 2.1 Hz, 2H), 4.25 (t, J = 8.1 Hz, 2H), 4.15 (t, J = 6.2 Hz, 2H), 2.40−2.26 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 177.9, 141.4 (2C), 134.0 (2C), 128.0 (2C), 122.4 (2C), 121.3 (2C), 120.8 (2C), 114.0 (2C), 109.1 (2C), 46.8, 43.1, 28.9; MS (EI): m/z (relative intensity): 302 (M⁺, 41), 222 (62), 208 (35), 180 (20), 152 (13), 81 (100); HRMS (APCI): m/z calcd for C₂₀H₁₉N₂O [M + H]⁺: 303.1492; found: 303.1494.

10-(4′-(N-Pyrrole)butyl)acridone ( 3d ): (66%) yellow solid, m.p. 145.0 °C−146.0 °C; FTIR, ν_max (cm⁻¹): 1633, 1603, 1594, 1491, 1458, 1377, 1172, 752, 726, 673; ¹H NMR (300 MHz, CDCl₃): δ 8.56 (d, J = 8.0, 1.6 Hz, 2H), 7.72−7.65 (m, 2H), 7.35 (d, J = 8.7 Hz, 2H), 7.30−7.22 (m, 2H), 6.69 (t, J = 2.1 Hz, 2H), 6.19 (t, J = 2.1 Hz, 2H), 4.25 (t, J = 8.2 Hz, 2H), 4.00 (t, J = 6.8 Hz, 2H), 2.08−1.84 (m, 4H); ¹³C NMR (75 MHz, CDCl₃): δ 177.9, 141.6 (2C), 133.9 (2C), 128.0 (2C), 122.4 (2C), 121.3 (2C), 120.4 (2C), 114.4 (2C), 108.5 (2C), 49.0, 45.6, 28.6, 24.4; MS (EI): m/z (relative intensity): 316 (M⁺, 40), 222 (23), 208 (100), 180 (31), 152 (16), 122 (49), 80 (46); HRMS (APCI): m/z calcd for C₂₁H₂₁N₂O [M + H]⁺: 317.1648; found: 317.1640.

10-(5′-(N-Pyrrole)pentyl)acridone ( 4d ): (63%) yellow solid, m.p. 109.0 °C−110.0 °C; FTIR, ν_max (cm⁻¹): 1630, 1597, 1492, 1458, 1375, 1279, 1174, 749, 720, 699; ¹H NMR (300 MHz, CDCl₃): δ 8.58 (dd, J = 8.0, 1.6 Hz, 2H), 7.74−7.68 (m, 2H), 7.43 (d, J = 8.7 Hz, 2H), 7.29 (t, J = 7.2 Hz, 2H), 6.67 (t, J = 2.0 Hz, 2H), 6.18 (t, J = 2.0 Hz, 2H), 4.29 (t, J = 8.2 Hz, 2H), 3.94 (t, J = 7.0 Hz, 2H), 1.96−1.87 (m, 4H), 1.57−1.47 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 177.9, 141.7 (2C), 133.8 (2C), 128.0 (2C), 122.5 (2C), 121.2 (2C), 120.4 (2C), 114.4 (2C), 108.1 (2C), 49.2, 45.8, 31.2, 26.8, 24.1; MS (EI): m/z (relative intensity); 330 (M⁺, 35), 236 (26), 222 (46), 208 (100), 195 (17), 180 (40), 149 (27), 80 (43); HRMS (APCI): m/z calcd for C₂₂H₂₃N₂O [M + H]⁺: 331.1805; found: 331.1804.

10-(6′-(N-Pyrrole)hexyl)acridone ( 5d ): (70%) yellow solid, m.p. 82.0 °C−83.0 °C; FTIR, ν_max (cm⁻¹): 1632, 1597, 1490, 1460, 1376, 1289, 1262, 1176, 753, 723, 673; ¹H NMR (300 MHz, CDCl₃): δ 8.56 (dd, J = 8.0, 1.5 Hz, 2H), 7.73−7.76 (m, 2H), 7.42 (d, J = 8.7 Hz, 2H), 7.27 (t, J = 7.7 Hz, 2H), 6.65 (t, J = 2.1 Hz, 2H), 6.15 (t, J = 2.1 Hz, 2H), 4.27 (t, J = 8.1 Hz, 2H), 3.90 (t, J = 7.0 Hz, 2H), 1.94−1.77 (m, 4H), 1.58−1.36 (m, 4H); ¹³C NMR (75 MHz, CDCl₃): δ 177.9, 141.6 (2C), 133.9 (2C), 128.0 (2C), 122.4 (2C), 121.2 (2C), 120.4 (2C), 114.4 (2C), 108.0 (2C), 49.4, 45.9, 31.5, 27.1, 26.6, 26.5; MS (EI): m/z (relative intensity): 344 (M⁺, 80), 264 (36), 222 (50), 208 (100), 196 (36), 180 (40), 80 (38); HRMS (APCI): m/z calcd for C₂₃H₂₅N₂O [M + H]⁺: 345.1961; found: 345.1952.

10-(3′-(N-Diethylamino)propyl)acridone ( 2e ):²⁴ (69%) yellow solid, m.p. 100.5 °C−101.0 °C; FTIR, ν_max (cm⁻¹): 1633, 1598, 1490, 1461, 1376, 1291, 1265, 1178, 752, 673; ¹H NMR (300 MHz, CDCl₃): δ 8.58 (dd, J = 8.0, 1.5 Hz, 2H), 7.75−7.62 (m, 4H), 7.28 (t, J = 7.8 Hz, 2H), 4.45 (t, J = 8.0 Hz, 2H), 2.70−2.60 (m, 6H), 2.10−2.00 (m, 2H), 1.13 (t, J = 7.1 Hz, 6H); ¹³C NMR (75 MHz, CDCl₃): δ 178.0, 141.8 (2C), 133.8 (2C), 127.9 (2C), 122.4 (2C), 121.1 (2C), 114.7 (2C), 50.2, 46.9 (2C), 44.3, 25.4, 11.7 (2C); MS (EI): m/z (relative intensity): 308 (M⁺, 9), 208 (7), 180 (6), 166 (9), 86 (100); HRMS (APCI): m/z calcd for C₂₀H₂₅N₂O [M + H]⁺: 309.1961; found: 309.1966.

10-(4′-(N-Diethylamino)butyl)acridone ( 3e ):²⁴ (94%) yellow solid, m.p. 63.0 °C−64.0 °C; FTIR, ν_max (cm⁻¹): 1633, 1598, 1490, 1461, 1378, 1290, 1264, 1177, 752, 674; ¹H NMR (400 MHz, CDCl₃): δ 8.57 (dd, J = 8.0, 1.5 Hz, 2H), 7.74−7.69 (m, 2H), 7.58 (d, J = 8.7 Hz, 2H), 7.28 (t, J = 7.5 Hz, 2H), 4.36 (t, J = 8.3 Hz, 2H), 2.67−2.57 (m, 6H), 2.00−1.90 (m, 2H), 1.82−1.72 (m, 2H), 1.10 (t, J = 7.2 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 178.0, 141.7 (2C), 133.8 (2C), 127.9 (2C), 122.4 (2C), 121.1 (2C), 114.7 (2C), 52.1, 46.6, 46.0 (2C), 24.8, 24.1, 11.3 (2C); MS (EI): m/z (relative intensity): 322 (M⁺, 19), 208 (14), 180 (9), 112 (13), 86 (100); HRMS (APCI): m/z calcd for C₂₁H₂₇N₂O [M + H]⁺: 323.2118; found: 323.2107.

10-(5′-(N-Diethylamino)pentyl)acridone ( 4e ):²⁴ (85%) yellow solid, m.p. 61.5 °C−62.5 °C; FTIR, ν_max (cm⁻¹): 1630, 1597, 1492, 1459, 1378, 1290, 1263, 1178, 751, 671; ¹H NMR (300 MHz, CDCl₃): δ 8.55 (d, J = 8.1 Hz, 2H), 7.70 (t, J = 7.0 Hz, 2H), 7.46 (d, J = 8.7 Hz, 2H), 7.26 (t, J = 7.5 Hz, 2H), 4.29 (t, J = 8.1 Hz, 2H), 2.60−2.45 (m, 6H), 2.00−1.85 (m, 2H), 1.70−1.50 (m, 4H), 1.05 (t, J = 7.2 Hz, 6H); ¹³C NMR (75 MHz, CDCl₃): δ 177.9, 141.7 (2C), 133.9 (2C), 127.9 (2C), 122.4 (2C), 121.1 (2C), 114.6 (2C), 52.6, 47.0, 46.1 (2C), 27.0, 26.9, 24.9, 11.6 (2C); MS (EI): m/z (relative intensity): 336 (M⁺, 3), 208 (5), 180 (4), 126 (6), 86 (100); HRMS (APCI): m/z calcd for C₂₂H₂₉N₂O [M + H]⁺: 337.2274; found: 337.2278.

10-(6′-(N-Diethylamino)hexyl)acridone ( 5e ):²⁴ (79%) yellow solid, m.p. 150.0 °C−151.0 °C; FTIR, ν_max (cm⁻¹): 1632, 1597, 1490, 1460, 1376, 1289, 1262, 1176, 754, 674; ¹H NMR (400 MHz, CDCl₃): δ 8.56 (dd, J = 8.0, 1.6 Hz, 2H), 7.75−7.69 (m, 2H), 7.48 (d, J = 8.7 Hz, 2H), 7.28 (t, J = 7.7 Hz, 2H), 4.32 (t, J = 8.1 Hz, 2H), 2.81 (q, J = 7.2 Hz, 4H), 2.71−2.66 (m, 2H), 1.96−1.87 (m, 2H), 1.75−1.65 (m, 2H), 1.63−1.54 (m, 2H), 1.50−1.42 (m, 2H), 1.20 (t, J = 7.2 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 177.8, 141.6 (2C), 133.9 (2C), 127.8 (2C), 122.3 (2C), 121.1 (2C), 114.5 (2C), 52.0, 46.6 (2C), 45.9, 27.0 (2C), 26.5, 25.4, 10.1 (2C); MS (EI): m/z (relative intensity): 350 (M⁺, 23), 335 (9), 321 (9), 208 (23), 180 (13), 86 (100); HRMS (APCI): m/z calcd for C₂₃H₃₁N₂O [M + H]⁺: 351.2431; found: 351.2430.

10-(3′-(N-Dimethylamino)propyl)acridone ( 2f ):²⁵ (78%) yellow solid, m.p. 86.0 °C−86.5 °C; FTIR, ν_max (cm⁻¹): 1634, 1597, 1490, 1461, 1377, 1291, 1261, 1178, 753, 674; ¹H NMR (400 MHz, CDCl₃): δ 8.58 (dd, J = 8.0, 1.6 Hz, 2H), 7.75−7.69 (m, 2H), 7.62 (d, J = 8.7 Hz, 2H), 7.29 (t, J = 7.6 Hz, 2H), 4.46 (t, J = 7.7 Hz, 2H), 2.48 (t, J = 6.6 Hz, 2H), 2.33 (s, 6H), 2.11−2.03 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 178.0, 141.8 (2C), 133.9 (2C), 127.9 (2C), 122.4 (2C), 121.2 (2C), 114.7 (2C), 56.4, 45.5 (2C), 43.9, 25.4; MS (EI): m/z (relative intensity): 280 (M⁺, 100), 236 (46), 222 (51), 209 (43), 180 (31) 166 (40), 152 (28), 58 (74); HRMS (APCI): m/z calcd for C₁₈H₂₁N₂O [M + H]⁺: 281.1648; found: 281.1642.

10-(4′-(N-Dimethylamino)butyl)acridone ( 3f ): (95%) yellow solid, m.p. 96.5 °C−97.5 °C; FTIR, ν_max (cm⁻¹): 1632, 1596, 1490, 1460, 1378, 1290, 1264, 1177, 1043, 753, 674; ¹H NMR (400 MHz, CDCl₃): δ 8.57 (dd, J = 8.0, 1.5 Hz, 2H) 7.72 (m, 2H), 7.55 (d, J = 8.7 Hz, 2H), 7.28 (t, J = 7.2 Hz, 2H), 4.37 (t, J = 8.3 Hz, 2H), 2.44 (t, J = 7.2 Hz, 2H), 2.31 (s, 6H), 2.01−1.91 (m, 2H), 1.80−1.71 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 177.9, 141.7 (2C), 133.8 (2C), 127.9 (2C), 122.4 (2C), 121.1 (2C), 114.6 (2C), 58.8, 45.9, 45.3 (2C), 24.9, 24.6; MS (EI): m/z (relative intensity): 294 (M⁺, 5), 222 (3), 208 (3), 180 (3), 100 (4), 71 (4), 58 (100); HRMS (APCI): m/z calcd for C₁₉H₂₃N₂O [M + H]⁺: 295.1805; found: 295.1797.

10-(5′-(N-Dimethylamino)pentyl)acridone ( 4f ): (71%) yellow solid, m.p. 65.0 °C−65.5 °C; FTIR, ν_max (cm⁻¹): 1632, 1591, 1492, 1459, 1376, 1261, 1181, 1037, 754, 673; ¹H NMR (400 MHz, CDCl₃): δ 8.58 (dd, J = 8.0, 1.4 Hz, 2H), 7.73 (m, 2H), 7.49 (d, J = 8.7 Hz, 2H), 7.29 (t, J = 7.8 Hz, 2H), 4.34 (t, J = 8.1 Hz, 2H), 2.36 (t, J = 6.6 Hz, 2H), 2.28 (s, 6H), 2.01−1.91 (m, 2H), 1.68−1.55 (m, 4H); ¹³C NMR (100 MHz, CDCl₃): δ 177.9, 141.7 (2C), 133.8 (2C), 128.0 (2C), 122.5 (2C), 121.1 (2C), 114.4 (2C), 59.4, 46.0, 45.4 (2C), 27.3, 27.0, 24.7; MS (EI): m/z (relative intensity): 308 (M⁺, 4), 208 (3), 180 (3), 149 (4), 114 (11), 58 (100); HRMS (APCI): m/z calcd for C₂₀H₂₅N₂O [M + H]⁺: 309.1961; found: 309.1958.

10-(6′-(N-Dimethylamino)hexyl)acridone ( 5f ): (95%) yellow solid, m.p. 102.5 °C−103.0 °C; FTIR, ν_max (cm⁻¹): 1629, 1594, 1494, 1461, 1380, 1291, 1265, 1179, 748, 670; ¹H NMR (400 MHz, CDCl₃): δ 8.58 (dd, J = 8.0, 1.6 Hz, 2H), 7.72 (m, 2H), 7.48 (d, J = 8.7 Hz, 2H), 7.29 (t, J = 7.4 Hz, 2H), 4.33 (t, J = 8.0 Hz, 2H), 2.35−2.30 (m, 2H), 2.26 (s, 6H), 2.00−1.90 (m, 2H), 1.63−1.44 (m, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 177.9, 141.7 (2C), 133.8 (2C), 128.0 (2C), 122.5 (2C), 121.1 (2C), 114.5 (2C), 59.5, 46.1, 45.3 (2C), 27.5, 27.1 (2C), 26.8; MS (EI): m/z (relative intensity): 322 (M⁺, 5), 208 (4), 180 (4), 167 (6), 149 (19), 58 (100); HRMS (APCI): m/z calcd for C₂₁H₂₇N₂O [M + H]⁺: 323.2118; found: 323.2113.

10-(3′-(N-Trimethylammonium)propyl)acridone ( 2g ): (99% from 2f) yellow solid, m.p. 266.0 °C−267.0 °C; FTIR, ν_max (cm⁻¹): 1608, 1590, 1497, 1460, 1267, 1179, 1020, 759, 673; ¹H NMR (300 MHz, DMSO-d₆): δ 8.37 (d, J = 7.9 Hz, 2H), 8.00−7.80 (m, 4H), 7.38 (t, J = 7.4 Hz, 2H), 4.51 (t, J = 7.7 Hz, 2H), 3.70 (t, J = 7.8 Hz, 2H), 3.12 (s, 9H), 2.30−2.15 (m, 2H); ¹³C NMR (75 MHz, DMSO-d₆): δ 176.5, 141.3 (2C), 134.4 (2C), 126.9 (2C), 122.6 (2C), 121.5 (2C), 115.9 (2C), 62.3, 52.5 (3C), 42.2, 20.7; MS (EI): m/z (relative intensity): 280 ([M – CH₃]⁺, 7), 208 (7), 142 (17), 127 (7), 58 (100); HRMS (ESI): m/z calcd for C₁₉H₂₃N₂O [M]⁺:295.1805; found: 295.1796.

10-(4′-(N-Trimethylammonium)butyl)acridone ( 3g ): (87% from 3f) yellow solid, m.p. 260.0 °C−261.0 °C; FTIR, ν_max (cm⁻¹): 1627, 1607, 1594, 1492, 1457, 1261, 1174, 754, 676; ¹H NMR (400 MHz, DMSO-d₆): δ 8.35 (dd, J = 8.1, 1.3 Hz, 2H), 7.89−7.81 (m, 4H), 7.35 (t, J = 6.6 Hz, 2H), 4.51 (t, J = 8.2 Hz, 2H), 3.48−3.38 (m, 2H), 3.43 (s, 9H), 2.07−1.97 (m, 2H), 1.84−1.75 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 176.9, 141.8 (2C), 134.7 (2C), 127.3 (2C), 122.1 (2C), 121.8 (2C), 116.2 (2C), 65.5, 52.9 (3C), 45.2, 24.1, 19.9; MS (EI): m/z (relative intensity): 294 ([M – CH₃]⁺, 4), 208 (6), 142 (13), 69 (36), 58 (100); HRMS (APCI): m/z calcd for C₂₀H₂₅N₂O [M]⁺: 309.1961; found: 309.1951.

10-(5′-(N-Trimethylammonium)pentyl)acridone ( 4g ): (61% from 4f) yellow solid, m.p. 263.0 °C−263.5 °C; FTIR, ν_max (cm⁻¹): 1627, 1607, 1596, 1494, 1459, 1378, 1265, 1178, 756, 674; ¹H NMR (400 MHz, DMSO-d₆): δ 8.35 (d, J = 7.8 Hz, 2H), 7.85−7.81 (m, 4H), 7.36−7.30 (m, 2H), 4.50 (f, J = 7.8 Hz, 2H), 3.36−3.34 (m, 11H), 1.85−1.46 (m, 2H), 1.72−1.63 (m, 2H), 1.60−1.51 (m, 2H), 1.41−1.32 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 176.9, 141.9 (2C), 134.7 (2C), 127.2 (2C), 122.1 (2C), 121.7 (2C), 116.4 (2C), 65.6, 52.7 (3C), 45.3, 26.9, 23.2, 22.5; MS (EI): m/z (relative intensity): 296 ([M – 27]⁺, 12), 268 (9), 185 (9), 69 (63), 55 (100); HRMS (APCI): m/z calcd for C₂₁H₂₇N₂O [M]⁺: 323.2118; found: 323.2109.

10-(6′-(N-Trimethylammonium)hexyl)acridone ( 5g ): (99% from 5f) yellow solid, m.p. 103.0 °C−104.0 °C; FTIR, ν_max (cm⁻¹): 1627, 1607, 1596, 1494, 1459, 1378, 1265, 1178, 756, 674; ¹H NMR (400 MHz, DMSO-d₆): δ 8.35 (d, J = 7.8 Hz, 2H), 7.85−7.81 (m, 4H), 7.36−7.30 (m, 2H), 4.48 (t, J = 7.8 Hz, 2H), 3.36−3.34 (m, 11H), 1.85−1.46 (m, 2H), 1.72−1.63 (m, 2H), 1.60−1.51 (m, 2H), 1.41−1.32 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 176.9, 141.9 (2C), 134.7 (2C), 127.2 (2C), 122.1 (2C), 121.7 (2C), 116.3 (2C), 65.8, 52.7 (3C), 45.5, 27.1, 26.1, 25.9, 22.4; MS (EI): m/z (relative intensity): 322 ([M – CH₃]⁺, 4), 208 (9), 180 (4), 142 (13), 58 (100); HRMS (APCI): m/z calcd for C₂₂H₂₉N₂O [M]⁺: 337.2274; found: 337.2265.

10-(3′-N-Azidopropyl)acridone ( 2h ): (89%) yellow solid, m.p. 78.5 °C−79.5 °C; FTIR, ν_max (cm⁻¹): 2094, 1632, 1595, 1490, 1461, 1377, 1290, 1261, 1177, 751, 673; ¹H NMR (300 MHz, CDCl₃): δ 8.55 (d, J = 7.8, 2H), 7.75−7.69 (m, 2H), 7.50 (d, J = 8.7 Hz, 2H), 7.31−7.24 (m, 2H), 4.44 (t, J = 7.8 Hz, 2H), 3.58 (t, J = 6.0 Hz, 2H), 2.19−2.10 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 177.8, 141.6 (2C), 134.0 (2C), 128.1 (2C), 122.5 (2C), 121.4 (2C), 114.2 (2C), 48.9, 43.0, 26.6; MS (EI): m/z (relative intensity): 278 ([M]⁺, 59), 250 (13), 208 (100), 195 (34), 180 (53), 166 (28); HRMS (APCI): m/z calcd for C₁₆H₁₅N₄O [M + H]⁺: 279.1240; found: 279.1238.

10-(4′-N-Azidobutyl)acridone ( 3h ): (95%) yellow solid, m.p. 112.5 °C−113.5 °C; FTIR, ν_max (cm⁻¹): 2094, 1632, 1596, 1491, 1460, 1379, 1290, 1263, 1175, 753, 673; ¹H NMR (400 MHz, CDCl₃): δ 8.58 (dd, J = 8.0, 1.6 Hz, 2H), 7.76−7.71 (m, 2H), 7.48 (d, J = 8.7 Hz, 2H), 7.32−7.27 (m, 2H), 4.38 (t, J = 8.1 Hz, 2H), 3.45 (t, J = 6.6 Hz, 2H), 2.08−1.90 (m, 2H), 1.88−1.80 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 177.9, 141.6 (2C), 134.0 (2C) 128.0 (2C), 122.4 (2C), 121.3 (2C), 114.3 (2C), 51.0, 45.4, 26.2, 24.5; MS (EI): m/z (relative intensity): 292 ([M]⁺, 66), 220 (59), 208 (100), 195 (20), 180 (43), 152 (23); HRMS (APCI): m/z calcd for C₁₇H₁₇N₄O [M + H]⁺: 293.1397; found: 293.1387.

10-(5′-N-Azidopentyl)acridone ( 4h ): (72%) yellow solid, m.p. 79.0 °C−80.0 °C; FTIR, ν_max (cm⁻¹): 2085, 1634, 1594, 1489, 1458, 1377, 1263, 1171, 755, 672; ¹H NMR (400 MHz, CDCl₃): δ 8.58 (t, J = 7.9 Hz, 2H), 7.73 (t, J = 8.2 Hz, 2H), 7.47 (d, J = 8.7 Hz, 2H), 7.29 (t, J = 7.4 Hz, 2H), 4.34 (t, J = 7.6 Hz, 2H), 3.36 (t, J = 6.3 Hz, 2H), 2.00−1.90 (m, 2H), 1.78−1.60 (m, 4H); ¹³C NMR (100 MHz, CDCl₃): δ 177.8, 141.7 (2C), 133.9 (2C), 128.0 (2C), 122.5 (2C), 121.2 (2C), 114.3 (2C), 51.2, 45.8, 28.7, 26.8, 24.1; MS (EI): m/z (relative intensity): 306 ([M]⁺, 27), 208 (100), 196 (44), 180 (44), 166 (19), 152 (22); HRMS (APCI): m/z calcd for C₁₈H₁₉N₄O [M + H]⁺: 307.1553; found: 307.1545.

10-(6′-N-Azidohexyl)acridone ( 5h ): (96%) yellow solid, m.p. 77.0 °C−78.0 °C; FTIR, ν_max (cm⁻¹): 2093, 1634, 1598, 1491, 1460, 1376, 1290, 1262, 1176, 754, 673; ¹H NMR (300 MHz, CDCl₃): δ 8.58 (dd, J = 8.0, 1.5 Hz, 2H), 7.75−7.70 (m, 2H), 7.47 (d, J = 8.7 Hz, 2H), 7.29 (t, J = 7.8 Hz, 2H), 4.33 (t, J = 8.1 Hz, 2H), 3.32 (t, J = 6.5 Hz, 2H), 2.00−1.90 (m, 2H), 1.70−1.50 (m, 6H); ¹³C NMR (75 MHz, CDCl₃): δ 177.9, 141.7 (2C), 133.9 (2C), 128.0 (2C), 122.4 (2C), 121.2 (2C), 114.4 (2C), 51.3, 45.9, 28.8, 27.1, 26.5, 26.4; MS (EI): m/z (relative intensity): 320 ([M]⁺, 78), 292 (15), 208 (98), 180 (100), 166 (54), 152 (60), 140 (47); HRMS (APCI): m/z calcd for C₁₉H₂₁N₄O [M + H]⁺: 321.1710; found: 321.1699.

10-(N-propyl)acridone ( 2i ):²⁶ (74%) yellow solid, m.p. 130.0 °C−131.0 °C; FTIR, ν_max (cm⁻¹): 1634, 1597, 1490, 1458, 1377, 1290, 1263, 1174, 747, 670; ¹H NMR (400 MHz, CDCl₃): δ 8.59 (dd, J = 8.0, 1.6 Hz, 2H), 7.75−7.70 (m, 2H), 7.50 (d, J = 8.7 Hz, 2H), 7.31−7.26 (m, 2H), 4.31 (t, J = 8.3 Hz, 2H), 2.03−1.93 (m, 2H), 1.17 (t, J = 7.5 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 178.0, 141.8 (2C), 133.8 (2C), 127.9 (2C), 122.4 (2C), 121.2 (2C), 114.5 (2C), 47.7, 20.5, 11.1; MS (EI): m/z (relative intensity): 237 ([M]⁺, 54), 208 (100), 180 (13), 166 (8), 152 (8), 140 (5); HRMS (APCI): m/z calcd for C₁₆H₁₆NO [M + H]⁺: 238.1226; found: 238.1231.

10-(N-butyl)acridone ( 3i ):²⁶ (70%) yellow solid, m.p. 98.5 °C−99.0 °C; FTIR, ν_max (cm⁻¹): 1633, 1596, 1489, 1459, 1375, 1290, 1259, 1175, 751, 671; ¹H NMR (400 MHz, CDCl₃): δ 8.59 (dd, J = 8.0, 1.7 Hz, 2H), 7.75−7.70 (m, 2H), 7.50 (d, J = 8.7 Hz, 2H), 7.31−7.26 (m, 2H), 4.34 (t, J = 8.3 Hz, 2H), 1.97−1.88 (m, 2H), 1.65−1.55 (m, 2H), 1.09 (t, J = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 178.0, 141.7 (2C), 133.8 (2C), 128.0 (2C), 122.4 (2C), 121.1 (2C), 114.5 (2C), 45.9, 29.2, 20.2, 13.8; MS (EI): m/z (relative intensity): 251 ([M]⁺, 41), 208 (100), 180 (21), 166 (9), 152 (8), 140 (6); HRMS (APCI): m/z calcd for C₁₇H₁₈NO [M + H]⁺: 252.1383; found: 252.1377.

Diphenylamine-2,4′-dicarboxylic acid ( 8 ):²⁷ A mixture of 2-bromobenzoic acid (6) (2.0 g, 9.95 mmol), 4-aminobenzoic acid (7) (1.37 g, 9.95 mmol), potassium carbonate (1.38 g, 9.95 mmol) and copper powder (60 mg, 3% w/w) in N,N-dimethylformamide (10 mL) was refluxed for 4 h. The mixture was cooled and slowly added to aqueous HCl (1:1) (50 mL). The precipitate was collected, washed with hot water, and dried to provide the title compound.

9(10H)-Acridone-2-carboxylic acid ( 9 ):²⁷ Diphenylamine-2,4′-dicarboxylic acid (8) (2.57 g, 10.0 mmol) in concentrated H₂SO₄ (5 mL) was heated at 100 °C for 4 h. The reaction mixture was cooled and poured into ice water. The precipitate was filtered and washed with hot water. The solid was redissolved in 5% aqueous sodium hydroxide and filtered. The filtrate was diluted with an equal volume of ethanol and acidified with acetic acid. The precipitate was filtered off, washed with water, and dried to yield 9 (1.70 g, 71% from 2-bromobenzoic acid). m.p. > 300 °C; FTIR, ν_max (cm⁻¹): 3387, 1634, 1595, 1576, 1399, 1136; ¹H NMR (300 MHz, DMSO-d₆): δ 12.16 (s, 1H), 8.85 (s, 1H), 8.22 (t, J = 8.3 Hz, 2H), 7.76 (t, J = 7.5 Hz, 1H), 7.59 (d, J = 8.5 Hz, 2H), 7.30 (t, J = 7.5 Hz, 1H); ¹³C NMR (75 MHz, DMSO-d₆): δ 176.9, 167.2, 143.3, 140.9, 133.9, 133.4, 128.6, 126.1, 124.0, 121.8, 120.9, 119.7, 117.7, 117.6; MS (EI): m/z (relative intensity): 239 ([M]⁺, 100), 222 (32), 195 (24), 139 (28), 97 (45), 83 (44); HRMS (ESI): m/z calcd for C₁₄H₁₀NO₃ [M + H]⁺: 240.0655; found: 240.0652.

10-(4′-N-Bromobutyl)acridone-2-(4-bromobutyl)carboxylate ( 10a ) and 9(10H)-Acridone-2-(4-bromobutyl)carboxylate ( 10b ): A solution of 9 (500 mg, 2.09 mmol) in N,N-dimethylformamide (25 mL) was added to a stirred suspension of sodium hydride (250 mg, 6.25 mmol) in N,N-dimethylformamide (10 mL). The mixture was heated at 80 °C for 15 min, treated with 1,4-dibromobutane (1.5 mL, 12.71 mmol) and heated at 80 °C for an additional 1 h. The reaction mixture was cooled and water was added and the mixture was extracted with ethyl acetate. The combined organic layer was washed with brine and water, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to provide 10a (245 mg, 23%) and 10b (226 mg, 29%).

10a : Yellow solid, m.p. 107.5 °C−108.0 °C; FTIR, ν_max (cm⁻¹): 1716, 1639, 1610, 1596, 1482, 1297, 1246, 1139, 754; ¹H NMR (300 MHz, CDCl₃): δ 9.16 (s, 1H), 8.84 (d, J = 7.9 Hz, 1H), 8.31 (d, J = 8.9 Hz, 1H), 7.76 (t, J = 8.0 Hz, 1H), 7.51 (d, J = 9.0 Hz, 2H), 7.34 (t, J = 7.6 Hz, 1H), 4.45−4.35 (m, 4H), 3.60−3.50 (m, 4H), 2.20−1.90 (m, 8H); ¹³C NMR (75 MHz, CDCl₃): δ 177.5, 165.8, 144.2, 141.5, 134.4, 134.3, 130.5, 128.1, 122.9, 122.8, 122.3, 121.6, 114.7, 114.6, 64.1, 45.5, 33.2, 32.5, 29.5, 29.3, 27.4, 25.6; MS (EI): m/z (relative intensity): 507 ([M]⁺, 36), 386 (98), 356 (12), 306 (11), 252 (100), 224 (15), 165 (16); HRMS (ESI): m/z calcd for C₂₂H₂₄Br₂NO₃ (M + H)⁺: 508.0117; found: 508.0125.

10b : Yellow solid, m.p. 257.5 °C−258.5 °C; FTIR, ν_max (cm⁻¹): 1713, 1634, 1593, 1562, 1526, 1478, 1293, 1243, 1130, 756, 676; ¹H NMR (400 MHz, DMSO-d₆): δ 12.08 (s, 1H), 8.84 (d, J = 2.0 Hz, 1H), 8.25−8.20 (m, 2H), 7.81−7.76 (m, 1H), 7.63−7.56 (m, 2H), 7.31 (t, J = 7.4 Hz, 1H), 4.35 (t, J = 6.4 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 2.00−1.86 (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆): δ 176.7, 165.3, 143.7, 140.8, 134.1, 133.1, 128.6, 126.1, 122.1, 122.0, 120.9, 119.7, 117.9, 117.7, 63.9, 34.7, 29.1, 27.1; MS (EI): m/z (relative intensity): 373 ([M]⁺, 44), 239 (96), 222 (100), 194 (42), 166 (23), 139 (29); HRMS (ESI): m/z calcd for C₁₈H₁₇BrNO₃ [M + H]⁺: 374.0386; found: 374.0389.

10-(4′-N-Piperidinobutyl)acridone-20[(N-piperidino)butyl]carboxylate ( 11a ): A solution of 10a (1 mmol) and piperidine (5 mmol) in acetonitrile was stirred at room temperature for 24 h. The reaction mixture was then concentrated under reduced pressure, the residue was treated with water and then extracted with dichloromethane. The combined organic layer was washed with saturated aqueous sodium hydrogen carbonate and water, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to yield 11a in 97% yield as a yellow solid: m.p. 104.5 °C−105.5 °C; FTIR, ν_max (cm⁻¹): 1703, 1639, 1602, 1479, 1251, 1137, 758; ¹H NMR (300 MHz, acetone-d₆): δ 9.04 (s, 1H), 8.41 (d, J = 7.8 Hz, 1H), 8.26 (d, J = 8.7 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.94 (d, J = 7.2 Hz, 1H), 7.80 (t, J = 7.6 Hz, 2H), 7.34 (t, J = 7.3 Hz, 1H), 4.52 (t, J = 8.2 Hz, 2H), 4.37 (t, J = 6.6 Hz, 2H), 2.50−2.30 (m, 12H), 2.10−1.35 (m, 20H); ¹³C NMR (75 MHz, acetone-d₆): δ 177.5, 166.2, 145.4, 142.7, 135.1, 134.4, 130.2, 128.0, 123.6 (2C), 122.8, 122.4, 117.0 (2C), 65.6, 59.4, 58.1, 55.3 (2C), 55.2 (2C), 46.8, 27.6, 27.0 (2C), 26.9 (2C), 25.4, 25.3, 24.8, 24.2, 23.6; MS (EI): m/z (relative intensity): 368 (6), 284 (6), 185 (14), 129 (47), 98 (93), 83 (91), 55 (100); HRMS (APCI): m/z calcd for C₃₂H₄₄N₃O₃ [M + H]⁺: 518.3377; found: 518.3379.

9(10H)-Acridone-2-[(N-piperidino)butyl]carboxylate ( 11b ): A solution of 10b (1 mmol) and piperidine (5 mmol) in N,N-dimethylformamide was stirred at room temperature for 24 h. Water was added to the mixture and the solvent was removed under reduced pressure. The precipitate was collected, washed with water, and then dichloromethane to give 11b in 92% yield as a yellow solid: m.p. 143.5 °C−144.5 °C; FTIR, ν_max (cm⁻¹): 1711, 1627, 1594, 1575, 1525, 1475, 1275, 1241, 763; ¹H NMR (300 MHz, DMSO-d₆): δ 12.17 (s, 1H), 8.84 (d, J = 2.1 Hz, 1H), 8.25−8.19 (m, 2H), 7.81−7.75 (m, 1H), 7.65−7.58 (m, 2H), 7.33 (t, J = 7.2 Hz, 1H), 4.35−4.31 (m, 2H), 2.65−2.55 (m, 6H), 1.80−1.42 (m, 10H); ¹³C NMR (75 MHz, DMSO-d₆): δ 176.7, 165.3, 143.7, 140.8, 134.1, 133.0, 128.6, 126.1, 122.1, 121.9, 120.9, 119.6, 117.9, 117.7, 64.4, 57.1, 53.3 (2C), 26.1 (2C), 24.4, 23.1, 21.9; MS (EI): m/z (relative intensity): 378 ([M]⁺, 23), 284 (2), 252 (10), 138 (24), 98 (100); HRMS (APCI): m/z calcd for C₂₃H₂₇N₂O₃ [M + H]⁺:379.2016; found: 379.2024.

10-(4′-N-Piperidinobutyl)acridone-2-carboxylic acid ( 12 ): A stirred solution of 11a (248 mg, 0.48 mmol) in ethanol (5 mL) was treated with 2% aqueous NaOH (5 mL) and refluxed for 1 h. The reaction mixture was cooled and concentrated under reduced pressure to remove the solvent. Water was added to the residue and the mixture was neutralized with 5 M HCl. The yellow precipitate was filtered and washed with water to give 12 (180 mg, 99%) as a yellow solid: m.p. 136.0 °C−137.0 °C; FTIR, ν_max (cm⁻¹): 3369, 1614, 1593, 1557, 1484, 1376, 1268, 781, 756; ¹H NMR (300 MHz, DMSO-d₆): δ 8.89 (s, 1H), 8.34 (d, J = 7.6 Hz, 1H), 8.24 (d, J = 8.6 Hz, 1H), 8.00–7.80 (m, 3H), 7.40 (t, J = 6.0 Hz, 1H), 4.55–4.45 (m, 2H), 3.20−2.90 (m, 6H), 2.10−1.90 (m, 2H), 1.90−1.70 (m, 6H), 1.60−1.45 (m, 2H); ¹³C NMR (75 MHz, DMSO-d₆): δ 176.4, 166.7, 143.9, 141.3, 134.7, 134.0, 129.0, 126.8, 123.3, 122.2, 122.0, 120.8, 116.4, 116.3, 55.5, 52.0 (2C), 45.2, 24.2, 22.4 (2C), 21.6, 20.4; MS (EI): m/z (relative intensity): 378 ([M]⁺, 23), 252 (10), 138 (24), 98 (100); HRMS (APCI): m/z calcd for C₂₃H₂₇N₂O₃ [M + H]⁺: 379.2016; found: 379.2015.

10-(4′-N-Piperidinobutyl)acridone-2-methylcarboxylate ( 13 ): Compound 12 (74 mg, 0.20 mmol) in thionyl chloride (1 mL) was refluxed for 3 h. The reaction mixture was cooled and added dropwise to cold methanol. The solution was then treated with water and extracted with ethyl acetate. The combined organic layer was washed with saturated aqueous sodium hydrogen carbonate and water, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to yield 13 (63 mg, 82%) as a yellow solid: m.p. 126.5 °C−127.5 °C; FTIR, ν_max (cm⁻¹): 1717, 1643, 1608, 1481, 1297, 1250, 766; ¹H NMR (300 MHz, CDCl₃): δ 9.18 (s, 1H), 8.54 (d, J = 8.1 Hz, 1H), 8.29 (d, J = 8.7 Hz, 1H), 7.74 (t, J = 7.2 Hz, 1H), 7.64 (d, J = 8.7 Hz, 2H), 7.32 (t, J = 7.2 Hz, 1H), 4.41 (t, J = 8.0 Hz, 2H), 3.96 (s, 3H), 2.60−2.40 (m, 6H), 2.05−1.60 (m, 8H), 1.60−1.45 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 177.7, 166.5, 144.4, 141.7, 134.3, 134.1, 130.5, 128.0, 122.8, 122.7, 122.1, 121.6, 115.1, 115.0, 57.7, 54.5 (2C), 52.1, 46.2, 25.7 (2C), 24.6, 24.2, 23.2; MS (EI): m/z (relative intensity): 392 ([M]⁺, 21), 167 (3), 149 (9), 138 (19), 98 (100); HRMS (ESI): m/z calcd for C₂₄H₂₉N₂O₃ [M + H]⁺: 393.2173; found: 393.2177.

N-(10-(4′-N-Piperidinobutyl)acridone-2-carbonyl)-L-valine benzyl ester ( 14 ): A solution of 12 (100 mg, 0.26 mmol), L-valine benzyl ester hydrochloride (64.5 mg, 0.26 mmol), HOBt (53.6 mg, 0.40 mmol), and EDCI (76 mg, 0.40 mmol) in N,N-dimethylformamide (10 mL) was stirred at −10 °C for 10 min. The solution was then treated with DIPEA (0.14 mL, 0.78 mmol) and stirred at −10 °C to room temperature for 24 h. Water was added and the mixture was extracted with ethyl acetate. The combined organic layer was washed with saturated aqueous ammonium chloride, brine, saturated aqueous sodium hydrogen carbonate, and water. The organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to yield 14 (70%) as a yellow solid: m.p. 89.0 °C−90.0 °C; FTIR, ν_max (cm⁻¹): 2934, 1739, 1637, 1608, 1482, 1466, 1264, 1181, 1155, 757; ¹H NMR (400 MHz, CDCl₃): δ 8.92 (d, J = 2.4 Hz, 1H), 8.60 (dd, J = 8.0, 1.2 Hz, 1H), 8.34 (dd, J = 9.0, 2.0 Hz, 1H), 7.81−7.70 (m, 3H), 7.40−7.32 (m, 5H), 6.93 (d, J = 8.4 Hz, 1H), 5.26−5.18 (m, 2H), 4.86 (t, J = 5.2 Hz, 1H), 4.48−4.43 (m, 2H), 2.50−2.45 (m, 4H), 2.35−2.30 (m, 1H), 2.02−1.96 (m, 2H), 1.82−1.75 (m, 2H), 1.70−1.63 (m, 6H), 1.55−1.45 (m, 2H), 1.02 (t, J = 7.2 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 177.7, 171.9, 166.3, 143.6, 141.6, 135.3, 134.3, 133.4, 128.6(2C), 128.4 (3C), 127.9, 126.3, 125.8, 122.6, 122.0, 121.2, 115.5, 115.1, 67.0, 57.7, 57.6, 54.4 (2C), 46.1, 31.6, 25.5 (2C), 24.6, 24.0, 23.0, 19.1, 18.0; MS (EI): m/z (relative intensity): 567 ([M]⁺, 13), 415 (3), 376 (3), 207 (9), 138 (41), 98 (100); HRMS (ESI): m/z calcd for C₃₅H₄₂N₃O₄ [M + H]⁺: 568.3170; found: 568.3161.

N-(10-(4′-N-Piperidinobutyl)acridone-2-carbonyl)-L-valine ( 15 ): To a solution of 14 (30 mg, 0.05 mmol) in methanol (3 mL) was added 10% Pd/C (4 mg, 10% w/w) and the suspension was stirred under a H₂ atmosphere at room temperature for 24 h. The reaction was filtered through Celite and eluting with methanol. The filtrate was concentrated under reduced pressure to yield 15 (80%); FTIR, ν_max (cm⁻¹): 1632, 1603, 1593, 1481, 1463, 1369, 1260, 761; ¹H NMR (300 MHz, DMSO-d₆): δ 8.88 (s, 1H), 8.65−8.55 (m, 1H), 8.33 (d, J = 7.2 Hz, 1H), 8.24 (d, J = 8.3 Hz, 1H), 7.92 (d, J = 8.7 Hz, 2H), 7.90−7.70 (m, 1H), 7.40−7.30 (m, 1H), 4.55−4.40 (m, 2H), 4.28 (t, J = 7.1 Hz, 1H), 2.55−2.35 (m, 6H), 2.30−2.15 (m, 1H), 1.85−1.40 (m, 10H), 1.00−0.90 (m, 6H); ¹³C NMR (75 MHz, DMSO-d₆): δ 176.5, 173.4, 165.6, 143.0, 141.4, 134.4, 132.9, 126.8, 126.7, 126.5, 121.8 (2C), 120.7, 116.2, 116.0, 58.9, 56.8, 53.6 (2C), 45.2, 29.8, 25.2 (2C), 24.0, 23.8, 22.2, 19.5, 18.9; MS (EI): m/z (relative intensity): 477 ([M]⁺, 1), 415 (16), 373 (61), 282 (39), 250 (28), 138 (27), 98 (100), 91 (76); HRMS (ESI): m/z calcd for C₂₈H₃₆N₃O₄ [M + H]⁺: 478.2700; found: 478.2693.

N-(10-(4′-N-Piperidinobutyl)acridone-2-carbonyl)-L-valine benzyl amide ( 16 ): A procedure analogous to the preparation of 14 and starting from 15 and benzyl amine was conducted to obtain compound 16 in 60% yield as a yellow solid: FTIR, ν_max (cm⁻¹): 3284, 2931, 1633, 1607, 1481, 1257, 757; ¹H NMR (400 MHz, CDCl₃): δ 8.89 (d, J = 2.4 Hz, 1H), 8.55 (d, J = 8.0 Hz, 1H), 8.23 (dd, J = 9.0, 2.4 Hz, 1H), 7.78−7.67 (m, 3H), 7.35−7.23 (m, 5H), 7.11 (d, J = 8.4 Hz, 1H), 6.59 (br s, 1H), 4.52−4.39 (m, 5H), 2.49−2.40 (m, 6H), 2.37−2.30 (m, 1H), 2.00−1.92 (m, 2H), 1.85−1.75 (m, 2H), 1.70−1.64 (m, 4H), 1.55−1.47 (m, 2H), 1.10−1.05 (m, 6H); ¹³C NMR (100 MHz, CDCl₃): δ 177.7, 171.1, 166.6, 143.7, 141.7, 137.9, 134.3, 133.2, 128.7 (2C), 128.0, 127.8 (2C), 127.5, 126.2, 126.1, 122.7, 122.0, 121.3, 115.6, 115.2, 59.5, 57.8, 54.6 (2C), 46.2, 43.6, 31.0, 26.0 (2C), 24.6, 24.4, 23.3, 19.5, 18.5; MS (EI): m/z (relative intensity): 566 ([M]⁺, 12), 368 (10), 207 (17), 138 (40), 98 (100); HRMS (ESI): m/z calcd for C₃₅H₄₃N₄O₃ [M + H]⁺: 567.3330; found: 567.3333.

10-(4′-N-Piperidinobutyl)acridone-2-carboxylic acid benzylamide ( 17 ): A procedure analogous to the preparation of 14 and starting from 12 and benzyl amine was used to obtain compound 17 in 72% yield as a yellow solid: m.p. 77.0 °C−78.0 °C; FTIR, ν_max (cm⁻¹): 1632, 1607, 1480, 1299, 1260, 756; ¹H NMR (300 MHz, CDCl₃): δ 8.92 (s, 1H), 8.47 (d, J = 7.8 Hz, 1H), 8.36 (d, J = 9.0 Hz, 1H), 7.80−7.60 (m, 3H), 7.40−7.20 (m, 5H), 4.66 (d, J = 5.7 Hz, 2H), 4.38 (t, J = 7.2 Hz, 2H), 2.60−2.40 (m, 6H), 2.00−1.60 (m, 8H), 1.55−1.45 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 177.8, 166.2, 143.4, 141.6, 138.2, 133.3, 133.6, 128.6 (2C), 127.8 (2C), 127.4 (2C), 126.7, 125.5, 122.4, 121.9, 121.1, 115.6, 115.1, 57.7, 54.5 (2C), 46.1, 44.1, 25.8 (2C), 24.5, 24.2, 23.2; MS (EI): m/z (relative intensity): 467 ([M]⁺, 20), 376 (3), 341 (3), 207 (9), 138 (39), 98 (100); HRMS (ESI): m/z calcd for C₃₀H₃₄N₃O₂ [M + H]⁺: 468.2645; found: 468.2640.

9(10H)-Acridone-2-methylcarboxylate ( 18 ):²⁸ A procedure analogous to the preparation of 13 and starting from 9 was used to obtain compound 18 in 87% yield as a yellow solid: m.p. >300 °C; FTIR, ν_max (cm⁻¹): 1717, 1633, 1595, 1575, 1293, 764; ¹H NMR (300 MHz, DMSO-d₆): δ 12.16 (s, 1H), 8.82 (d, J = 2.1 Hz, 1H), 8.23 (dd, J = 8.1, 1.5 Hz, 1H), 8.18 (dd, J = 8.7, 2.1 Hz, 1H), 7.80-7.74 (m, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 7.5 Hz, 1H), 7.34-7.28 (m, 1H), 3.89 (s, 3H); ¹³C NMR (75 MHz, DMSO-d₆): δ 176.7, 165.7, 143.6, 140.7, 134.0, 132.9, 128.6, 126.0, 122.0, 121.7, 120.9, 119.6, 117.9, 117.7, 52.1; MS (EI): m/z (relative intensity): 253 ([M]⁺, 100), 222 (84), 195 (20), 167 (29), 149 (76), 97 (37), 57 (50); HRMS (ESI): m/z calcd for C₁₅H₁₂NO₃ [M + H]⁺: 254.0812; found: 254.0807.

N-(9(10H)-Acridone-2-carbonyl)-L-valine benzyl ester ( 19 ): A procedure analogous to the preparation of 14 and starting from 9 was used to obtain 19 in 73% yield as a yellow solid: m.p. 211.0 °C−212.0 °C; FTIR, ν_max (cm⁻¹): 1716, 1642, 1556, 1521, 1479, 1267, 751, 737; ¹H NMR (300 MHz, DMSO-d₆): δ 11.98 (s, 1H), 8.93 (d, J = 7.5 Hz, 1H), 8.87 (s, 1H), 8.27 (d, J = 8.1 Hz, 1H), 8.20 (d, J = 8.7 Hz, 1H), 7.77 (t, J = 7.5 Hz, 1H), 7.58 (d, J = 6.6 Hz, 2H), 7.39−7.28 (m, 5H), 5.10 (d, J = 4.2 Hz, 2H), 4.37 (t, J = 7.5 Hz, 1H), 2.29−2.22 (m, 1H), 1.00 (d, J = 6.0 Hz, 3H), 0.95 (d, J = 6.9 Hz, 3H); ¹³C NMR (75 MHz, DMSO-d₆): δ 176.9, 171.7, 166.5, 142.7, 140.8, 136.0, 133.9, 132.6, 128.4 (2C), 128.1, 128.0 (2C), 126.5, 126.3, 126.1, 121.7, 120.8, 119.5, 117.6, 117.3, 65.8, 59.0, 29.5, 19.3, 19.2; MS (EI): m/z (relative intensity): 428 ([M]⁺, 2), 293 (5), 238 (30), 222 (100), 194 (18), 166 (8), 139 (7); HRMS (APCI): m/z calcd for C₂₆H₂₅N₂O₄ [M + H]⁺: 429.1809; found: 429.1803.

N-(9(10H)-Acridone-2-carbonyl)-L-valine ( 20 ): A procedure analogous to the preparation of 15 and starting from 19 was used to obtain compound 20 in 82% yield as a yellow solid: m.p. 200.0 °C (dec.); FTIR, ν_max (cm⁻¹): 3273, 1628, 1517, 1474, 1311, 1154, 757; ¹H NMR (300 MHz, DMSO-d₆): δ 12.11 (s, 1H), 8.82 (s, 1H), 8.57 (d, J = 7.5 Hz, 1H), 8.25 (d, J = 8.1 Hz, 1H), 8.20 (d, J = 8.7 Hz, 1H), 7.76 (t, J = 7.2 Hz, 1H), 7.60 (d, J = 7.2 Hz, 2H), 7.29 (t, J = 7.2 Hz, 1H), 4.30 (t, J = 6.6 Hz, 1H), 2.30−2.20 (m, 1H), 1.05−0.90 (m, 6H); ¹³C NMR (75 MHz, DMSO-d₆): δ 176.9, 173.4, 166.0, 142.6, 140.8, 133.8, 132.4, 126.7, 126.2, 126.1, 121.6, 120.8, 119.5, 117.6, 117.3, 58.8, 29.7, 19.4, 18.9; MS (EI): m/z (relative intensity): 293 ([M – 45]⁺, 7), 279 (12), 227 (5), 167 (30), 149 (100), 71 (26); HRMS (APCI): m/z calcd for C₁₉H₁₉N₂O₄ [M + H]⁺: 339.1339; found: 339.1345.

9(10H)-Acridone-2-carboxylic acid benzylamide ( 21 ):²⁹ A procedure analogous to the preparation of 14 and starting from 9 and benzyl amine was used to obtain compound 21 in 67% yield as a yellow solid: m.p. > 300 °C; FTIR, ν_max (cm⁻¹): 1622, 1579, 1549, 1523, 1308, 756, 736; ¹H NMR (300 MHz, DMSO-d₆): δ 12.08 (s, 1H), 9.28 (br t, J = 5.2 Hz, 1H), 8.85 (s, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.23 (d, J = 8.8 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 7.5 Hz, 1H), 7.40−7.20 (m, 5H), 4.52 (d, J = 5.4 Hz, 2H); ¹³C NMR (75 MHz, DMSO-d₆): δ 176.9, 165.6, 142.6, 140.8, 139.9, 133.8, 132.2, 128.3 (2C), 127.3 (2C), 126.7 (2C), 126.1, 125.8, 121.7, 120.8, 119.6, 117.6, 117.4, 42.7; MS (EI): m/z (relative intensity): 328 ([M]⁺, 47), 222 (100), 195 (64), 166 (19), 139 (19), 106 (12); HRMS (ESI): m/z calcd for C₂₁H₁₇N₂O₂ [M + H]⁺: 329.1284; found: 329.1287.

In vitro cytotoxicity

The cytotoxic activities of compounds 1, 5, 8, and 11–15 were evaluated against several cancer cell lines, namely, Molt-3, HepG2, HuCCA-1, and A549. Cells in the logarithmic growth phase were seeded in 96-well plates (Costar, 3599, USA) at a density of 10,000–15,000 cells/well. The plates were incubated at 37 °C in a humidified atmosphere with 95% air and 5% CO₂ for 24 h. The culture was then treated with a medium containing either the test compound or the vehicle to the desired final concentration and incubated for another 48 h. The cell monolayer was washed with phosphate-buffered saline (pH 7.2), fixed with 95% ethanol, stained with crystal violet solution, and lysed with 0.1 N HCl in methanol. The absorbance of the lysate was recorded at 540 nm on an automatic microtiter plate reader (Multiskan Ascent, Labsystem). All tests were carried out in triplicate, and the mean value was calculated. Activity is expressed as IC₅₀ (concentration of substance that inhibits 50% of cell growth) by using doxorubicin (Sigma Aldrich) as a standard drug.

Molecular modeling

The chemical structures of the selected compounds were built using the Scigress software suite 2.8.1 and optimized using the MM2 force field. The X-ray crystal structure of DNA was obtained from the Protein Data Bank database, code 2DES. AutoDockTools version 1.5.6 graphic user interface was used to prepare the crystal structure. Hydrogen atoms were added, and crystallographic water and co-crystallized 3′-desamino-3′-(2-methoxy-4-morpholinyl)-doxorubicin ligand were deleted. AutoDock Vina 1.1.2 was used as the docking engine. The grid box was set to 40, 40, and 15 Å for each of the x, y, and z dimensions. The center search space coordinates were set at x, y, z = –0.093, 0.946, and −8.586, respectively. The exhaustiveness that influences the thoroughness of the search pose was set to 15. DNA was kept rigid, and the ligands were free for pose search. The number of docking runs was set to 20 for each ligand, and the docked conformers were ranked according to the ΔG values (kcal mol⁻¹).

MlogP calculation

The Dragon 7.0 software suite was used to calculate the Moriguchi log P (MlogP) values to determine the lipophilicity of the compounds. Cheminformatics software was used to calculate for molecular descriptors.

Supplemental Material

Supplementary_Information – Supplemental material for Synthesis, cytotoxicity evaluation, and molecular modeling studies of 2,N¹⁰-substituted acridones as DNA-intercalating agents

Supplemental material, Supplementary_Information for Synthesis, cytotoxicity evaluation, and molecular modeling studies of 2,N¹⁰-substituted acridones as DNA-intercalating agents by Nisachon Khunnawutmanotham, Watthanachai Jumpathong, Chatchakorn Eurtivong, Nitirat Chimnoi and Supanna Techasakul in Journal of Chemical Research

Footnotes

Acknowledgements

The authors acknowledge the assistance of P Intachote, S Sengsai, and B Saimanee (Biological activity test and screening unit, the Chulabhorn Research Institute) in the cytotoxicity testing.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Supanna Techasakul

Supplemental material

Figure S1 and ¹H NMR and ¹³C NMR spectra are provided in the Supplementary Information.

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Supplementary Material

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Synthesis,cytotoxicity evaluation,and molecular modeling studies of 2,N 10 -substituted acridones as DNA-intercalating agents