New 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazoline and 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinoline Derivatives: Synthesis and Biological Evaluation as Novel Anticancer Agents by Targeting G-Quadruplex

Abstract

The syntheses of novel 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazolines 12 and 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinolines 13 are reported here in six steps starting from various halogeno-quinazoline-2,4-(1H,3H)-diones or substituted anilines. The antiproliferative activities of the products were determined in vitro against a panel of breast (MCF-7 and MDA-MB-231), human adherent cervical (HeLa and SiHa), and ovarian (A2780) cell lines. Disubstituted 6- and 7-phenyl-bis(3-dimethylaminopropyl)aminomethylphenyl-quinazolines 12b, 12f, and 12i displayed the most interesting antiproliferative activities against six human cancer cell lines. In the series of quinoline derivatives, 6-phenyl-bis(3-dimethylaminopropyl)aminomethylphenylquinoline 13a proved to be the most active. G-quadruplexes (G4) stacked non-canonical nucleic acid structures found in specific G-rich DNA, or RNA sequences in the human genome are considered as potential targets for the development of anticancer agents. Then, as small aza-organic heterocyclic derivatives are well known to target and stabilize G4 structures, their ability to bind G4 structures have been determined through FRET melting, circular dichroism, and native mass spectrometry assays. Finally, telomerase inhibition ability has been also assessed using the MCF-7 cell line.

1. Introduction

Cancer is one of the most devastating diseases and the second leading cause of death around the world. In 2020, over 18.1 million cancer cases were estimated around the world; 9.3 million in men and 8.8 million in women [1]. Among them, the most common and frequently encountered types of cancers were breast, lung, colorectal, prostate, stomach, liver, cervix uteri, esophagus, and thyroid, and the most common causes of cancer-related mortality included lung, colon and rectum, liver, stomach, and breast cancers [1,2]. Different cancer treatments including chemotherapy, radiation therapy, hormone therapy, immunotherapy, photodynamic therapy, hyperthermia, stem cell therapy, targeted therapy, and surgery have been used to treat them [3]. In the case of chemotherapy, cancer cells increasingly display multidrug resistance, and patients develop severe side effects to these drugs, hindering their efficacy. Moreover, despite the recent and promising development of targeted therapies, all patients are not responsive to current drugs, fostering the need for new cytotoxic molecules.

Telomerase has emerged as an interesting primary target in cancer therapy, as 85–90% of cancer cells display telomerase activity, enhancing their indefinite proliferation and immortality. Indeed, this ribonucleoprotein reverse transcriptase enzyme, which adds telomere repeats to telomere DNA to maintain chromosome stability and integrity, acts as a key regulator of long-term proliferation. A working hypothesis in the targeting of telomeric DNA with drugs is that, if the telomere overhang DNA forms higher order structures, such as G-quadruplexes, the telomere capping machinery, including the telomerase, will be perturbed and proliferation arrested [4].

G-quadruplexes (G4) are single-stranded guanine-rich nucleic acid sequences which may fold into non-canonical four-stranded secondary structures. These structures are formed through π–π stacking of G-quartets which involve four guanines organized in a plane via eight Hoogsteen type H-bonding. G4-structures are stabilized in the presence of monovalent cations such as K⁺ or Na⁺. In addition, G4-forming motifs have been identified in oncogene promoter regions, highlighting the therapeutic potential for targeted gene regulation at the transcriptional level [5,6]. The most studied oncogene promoters of G4s include c-MYC, BCL-2, h-RAS, K-RAS, and c-KIT. Thus, both the inhibition of telomerase activity through G4 structure stabilization and the induction of the DNA damage response through telomere uncapping can prompt a proliferation arrest. Hence, small molecules which target and stabilize G4-structures in human genome could act as potential and interesting cancer therapeutic agents [7,8,9].

The large planar aromatic surface of a terminal G-quartet provided a rationale for the design and development of planar G4 ligands such as polyaromatic fused molecules, for instance, acridines, phenanthroline, quinolone, and quinone [10,11,12,13,14,15]. Based on these chemical pharmacophores, a number of G4 binding molecules have been designed and developed over the last two decades, such as SYUIQ-5, SYUIQ-FM05, Quarfloxin, GTC365, Tz 1, or LZ-11c (Figure 1) [11,12,13,14,15,16,17]. Many of these heterocyclic ligands are selective for G4 structures over duplex DNA, but the design of a ligand specific for a given G4 structure remains challenging. In addition, quinoline and quinazoline have emerged as valuable scaffolds in medicinal chemistry possessing a diversity of biological activities, such as anticancer activities [18,19,20,21].

In the course of our research devoted to the design and the discovery of novel heterocycles for cancer chemotherapies [22,23,24,25,26,27,28,29], we previously designed and prepared two series of new substituted 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthrolines A and diquinolinyl-pyridines B (Figure 1) designed to bind to DNA G-quadruplexes [25,28], and endowed with interesting and promising activity towards human leukemia cells. In this context, and through considering the pharmacological activities of these previous series A and B on human leukemia cells [25,28], we undertook the design and the synthesis of a new series of 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazoline and 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinoline derivatives 12,13 (series C), novel structural analogues of these latter series A and B. The antiproliferative activities of these new derivatives were herein determined in vitro against a panel of breast (MCF-7 and MDA-MB-231), human adherent cervical (HeLa and SiHa), and ovarian (A2780) cell lines. In addition, the antiproliferative activity of the obtained derivatives 12,13 was then evaluated in vitro against the hematologic malignant K562 cell line.

We also evaluated ligand-induced stabilization, specificity, and selectivity of the newly synthesized 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazoline and 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinoline derivatives 12,13 for various oncogene promoter G4 topologies, including c-MYC, BCL-2, and K-RAS through FRET-melting experiments.

2. Results & Discussion

2.1. Chemistry

These new 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazoline and 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinoline derivatives 12,13 were each prepared in six steps starting from various halogeno-quinazoline-2,4-(1H,3H)-diones 1 or substituted anilines 2 (Scheme 1).

Suzuki coupling reaction with compounds 1a–d with phenyl boronic acid provided the different phenyl-quinazoline-2,4-(1H,3H)-diones 3a–d, which then reacted with phosphorous oxychloride in the presence of diisopropylethylamine to generate dichloro derivatives 4a–d. The addition of dimethyl malonate to phenylanilines 2a,b provided amido-esters 5a,b. Quinoline-2,4-diones 6a,b were then prepared by intramolecular cyclization of amido-esters 5a,b in chlorobenzene using AlCl₃. The lactams 6a,b were subsequently chlorodehydroxylated with phosphorous oxychloride, leading to 2,4-dichloroquinolines 7a,b. The intermediates bis-(formylphenyl)-quinazolines and -quinolines 8–9 were synthesized by a double-Suzuki-Miyaura cross-coupling reaction of the dichloro derivatives 4,7 with 4-formylphenylboronic acids in the presence of Pd(PPh₃)₄ as a catalyst and in the presence of sodium carbonate. The 3D structural determination of two new substituted derivatives 8b and 9a was established by X-ray crystallography (Figure 2 and Figure 3) [30] and confirmed the structure in the solid state as anticipated on the basis of NMR data.

During the synthesis of compound 8a, we also isolated the 2-(4-formylphenyl)-5-phenyl-3H-quinazolin-4-one 14 as a side product (Scheme 2).

The structure of the latter derivative 14 was confirmed by a complete NMR analysis and was consistent with previously described 5-substituted-3H-quinazolin-4-ones [31]. Condensation of various primary amines with these dialdehydes 8,9 provided the di-imines 10,11, which were immediately reduced into the 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazolines 12a–n and 2,4-bis[(substituted-aminomethyl)phenyl]quinolines 13a–e using sodium borohydride as a reductive agent in refluxing methanol as previously described by our team (Scheme 1).

Our quinazolines and quinolines 12,13 were then converted into ammonium oxalate salts via treatment with oxalic acid in refluxing isopropanol. These oxalate salts were less hygroscopic than the hydrochloride ones and were also soluble in water. Table S1 (in the Supplementary Materials) summarizes the physical properties of the new synthesized 12,13 oxalates.

The different attempts to synthesize halogeno-quinoline-2,4-diones 16a and 16b from the diamide 15, previously prepared from 3-chloroaniline with malonyl chloride, and using a mixture of phosphorus pentoxide and methanesulfonic acid, led to a mixture of compounds 16a,b for which the separation has always failed (Scheme 3) [32].

2.2. Biology

The in vitro antiproliferative capacity of 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazolines 12a–n and 2,4-bis[(substituted-aminomethyl)phenyl]quinolines 13a–e was then evaluated on a panel of human adherent cancer cell lines. These novel derivatives were tested on cervical (HeLa and SiHa), ovarian (A2780), and breast (MCF-7 and MDA-MB-231) cell lines. The antiproliferative IC₅₀ values of 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazolines 12a–n and 2,4-bis[(substituted-aminomethyl)phenyl]quinolines 13a–e ranged from 0.33 to 7.10 μM. Additionally, the antiproliferative activity of the new derivatives 12,13 was also evaluated in vitro using the hematologic malignant K562 cell line. MTT assays were conducted and revealed that all compounds exhibited considerable activities (Figure 4,

Table 1. Antiproliferative activities of compounds 12,13.

Compound	IC50 Values (μM) ± SEM a
	HeLa	SiHa	MCF-7	MDA-MB-231	A2780	K562	HEK293
12a	1.36 ± 0.04	0.62 ± 0.12	1.12 ± 0.13	1.45 ± 0.14	2.60 ± 0.03	14.0 ± 0.2	n.d. b
12b	0.82 ± 0.31	1.08 ± 0.10	0.58 ± 0.02	0.70 ± 0.16	1.20 ± 0.27	14.0 ± 0.3	>30
12c	2.21 ± 0.86	0.76 ± 0.01	1.44 ± 0.03	1.07 ± 0.42	2.04 ± 0.54	7.0 ± 0.2	n.d. b
12d	1.32 ± 0.02	1.28 ± 0.27	1.01 ± 0.30	1.44 ± 0.17	1.74 ± 0.11	17.0 ± 0.4	n.d. b
12e	1.27 ± 0.02	0.63 ± 0.05	1.27 ± 0.01	0.89 ± 0.46	2.19 ± 0.41	13.0 ± 0.2	n.d. b
12f	1.51 ± 0.09	0.75 ± 0.10	0.58 ± 0.08	0.55 ± 0.01	0.79 ± 0.19	14.0 ± 0.3	>30
12g	0.95 ± 0.51	0.52 ± 0.22	0.63 ± 0.35	0.67 ± 0.35	1.74 ± 0.89	12.0 ± 0.3	3.98 ± 0.12
12h	2.31 ± 0.76	0.75 ± 0.01	1.19 ± 0.27	1.10 ± 0.38	1.43 ± 0.07	13.0 ± 0.2	n.d. b
12i	1.15 ± 0.36	0.92 ± 0.24	0.67 ± 0.31	0.52 ± 0.02	1.14 ± 0.16	23.0 ± 0.5	>30
12j	1.48 ± 0.80	0.33 ± 0.08	0.58 ± 0.25	1.10 ± 0.15	1.44 ± 0.10	8.0 ± 0.2	4.90 ± 0.15
12k	1.19 ± 0.24	1.01 ± 0.27	1.24 ± 0.14	1.36 ± 0.06	1.34 ± 0.14	7.0 ± 0.1	n.d. b
12l	1.39 ± 0.16	1.14 ± 0.51	1.45 ± 0.02	1.55 ± 0.12	2.08 ± 0.48	7.0 ± 0.2	n.d. b
12m	1.17 ± 0.23	0.88 ± 0.14	1.22 ± 0.19	2.00 ± 0.45	2.10 ± 0.48	7.0 ± 0.2	n.d. b
12n	3.51 ± 0.17	1.89 ± 0.74	4.17 ± 0.50	3.94 ± 0.54	7.10 ± 1.15	14.0 ± 0.3	n.d. b
13a	0.50 ± 0.01	0.99 ± 0.15	0.54 ± 0.01	0.58 ± 0.02	0.90 ± 0.04	10.0 ± 0.4	>30
13b	3.58 ± 0.18	1.76 ± 0.61	3.03 ± 0.64	3.68 ± 0.80	6.49 ± 1.76	13.0 ± 0.4	n.d. b
13c	1.30 ± 0.06	1.45 ± 0.13	0.86 ± 0.38	1.33 ± 0.13	1.58 ± 0.10	34.0 ± 0.7	n.d. b
13d	2.27 ± 0.89	1.11 ± 0.37	1.62 ± 0.09	1.64 ± 0.11	2.10 ± 0.09	14.0 ± 0.3	n.d. b
13e	1.28 ± 0.21	1.07 ± 0.45	1.12 ± 0.10	1.21 ± 0.03	1.50 ± 0.34	7.0 ± 0.1	n.d. b

^a Standard error from two independent determinations duplicate. ^b n.d.: not done.

). Furthermore, adherent cell lines of gynecological origin were substantially more sensitive than leukemia cells. Based on these obtained biological activities, some conclusions could be formulated concerning the possible structure–activity relationships.

Against the cervical HeLa cell line, the new derivatives 12,13 showed some significant antiproliferative activities, with an IC₅₀ between 0.50 and 3.58 μM. Among these compounds 12,13, the 2,4-bis{4-[(3-dimethylaminopropyl)aminomethyl]phenyl}-6-phenylquinoline 13a exhibited the best antiproliferative activity (IC₅₀ = 0.50 μM). In terms of structure–activity relationships, when we compared quinazolines 12a, 12b, 12f, and 12k bearing two (3-dimethylaminopropyl)aminomethylphenyl side chains and particularly the phenyl ring in positions 5, 6, 7, and 8 of the quinazoline heterocyclic moiety, respectively, derivative 12b was more active than its other homologous (IC₅₀ = 0.82 μM versus 1.19–1.51 μM for compounds 12a, 12f, and 12k). The same experiments in the quinoline series showed that derivative 13a, substituted by the phenyl group in position 6, was more active against the HeLa cell line than its analogue 13e, substituted at position 8; i.e., IC₅₀ = 0.50 μM for 13a and 1.28 μM for 13e. A comparison of compounds 12b and 13a, both substituted by (3-dimethylaminopropyl)aminomethylphenyl side chains and also by a phenyl in position 6, but different by the basic heterocyclic system, quinazoline versus quinoline, revealed that the quinoline skeleton presented a more promising antiproliferative activity (IC₅₀ = 0.50 μM for 13a and 0.82 μM for 12b). In addition, the disubstituted 7-phenylquinazoline 12g, which was disubstituted with two C4 dimethylaminoalkylaminobenzyl chains, exhibited a better biological activity (IC₅₀ = 0.95 μM) than its C3 or C5 homologs, compounds 12f and 12h, with IC₅₀ values of 1.51 μM and 2.31 μM, respectively. Similar comparisons between the disubstituted 6-phenylquinazolines 12b and 12c showed that elongation of the alkyl side chain (propyl versus butyl) led to a drop in the antiproliferative activity against HeLa cells (IC₅₀ = 0.82 μM for 12b and 2.21 μM for 12c). This observation was consistent with 8-phenyl-derivatives 12m and 12n, substituted by 3-(4-methylpiperazin-1-yl)propyl)aminobenzyl or 4-(4-methylpiperazin-1-yl)butyl)aminobenzyl side chains, as in quinoline compounds 13c and 13d with a phenyl ring in position 6 and displayed an IC₅₀ =1.30 and 2.27 μM, respectively.

In the case of the cervical SiHa cancer cell line, quinazoline 12j was the most active compound with an IC₅₀ of 0.33 μM. By comparing compounds 12a, 12b, 12f and 12k all bearing two (3-dimethylaminopropyl)aminomethylphenyl side chains and a phenyl ring linked to the quinazoline skeleton in positions 5, 6, 7 and 8, respectively, 12a was slightly more active than its analogues (IC₅₀ = 0.62 μM versus 0.75–1.08 μM). When we compared 6-phenylquinazolines 12d versus 12e, then 7-phenylquinazolines 12i versus 12j, we noticed that the heterocyclic moieties substituted by 4-(4-methylpiperazin-1-yl)butyl)aminobenzyl side chains exhibited more interesting cytotoxicity than those bearing 3-(4-methylpiperazin-1-yl)propyl)aminobenzyl side chains; i.e., IC₅₀ = 0.63 μM for 12e and 1.28 μM for 12d, IC₅₀ = 0.33 μM for 12j and 0.92 μM for 12i. However, the displacement of the phenyl nucleus in position 8 of these disubstituted 3-(4-methylpiperazin-1-yl)propyl)aminobenzyl and 4-(4-methylpiperazin-1-yl)butyl)aminobenzylquinazolines (compounds 12m and 12n) did not lead to the same conclusion as SAR, derivative 12m being more active than 12n (IC₅₀ = 0.88 μM for 12m and 1.89 μM for 12n). Concerning the quinoline compounds, 13a was slightly more active than its other substituted homologs with an IC₅₀ = 0.99 μM versus 1.07–1.76 μM.

For the MCF-7 breast adenocarcinoma, the introduction of the phenyl ring in position 6 or 7 of bis(3-dimethylaminopropyl)aminomethylphenylquinazolines (compounds 12b and 12f) was twice as active on this MCF-7 line than substitution of this aromatic nucleus in position 5 or 8 (compounds 12a and 12k) with IC₅₀ of 0.58 μM for 12b and 12f, then ranging from 1.12 to 1.24 μM for 12a and 12k. The best antiproliferative activity was observed for quinoline 13a with an IC₅₀ of 0.54 μM. Moreover, the chain elongation with one additional carbon atom (compound 13b) resulted in a decrease in the biological activity (IC₅₀ = 3.03 μM). Similar observations were also made between compounds 12m and 12n (IC₅₀ = 1.22 μM for 12m versus 4.17 μM for 12n).

Among the nineteen compounds tested for antiproliferative activities on the breast cancer MDA-MB-231 cell line, 7-phenyl substituted quinazoline 12i bearing 3-(4-methylpiperazin-1-yl)propyl)aminobenzyl side chains was the most active compound with an IC₅₀ of 0.52 µM. From a general point of view, the 8-phenyl substituted quinazolines 12k–n were less active against this breast cancer cell line (IC₅₀ ranging from 1.36 to 3.94 μM) than their 5-phenyl, 6-phenyl, and 7-phenyl analogues, compounds 12a (IC₅₀ = 1.451 μM), 12b–e (IC₅₀ ranging from 0.70 to 1.44 μM) and 12f–j (IC₅₀ ranging from 0.52 to 1.10 μM), respectively. The bio-isosteric replacement of the quinazoline moiety by a quinoline displayed no benefit in terms of antiproliferative activity against the MDA-MB-231 cell line, except for derivative 13a that was relatively active, with an IC₅₀ of 0.58 μM.

Against the ovarian A2780 cancer cell line, the bisaminoalkylaminobenzyl substituted quinazolines 12f–j, bearing the phenyl nucleus in position 7 of the heterocyclic skeleton, presented better antiproliferative activities than their 5-, 6-, or 8-phenyl analogues; i.e., IC₅₀ = 0.79–1.74 μM for 12f–j versus 1.20–7.10 μM for 12a–e and 12k–n. In addition, disubtituted quinoline 13a with an IC₅₀ of 0.90 μM was found 7.2 more active than its quinoline homolog 13b bearing only one additional carbon atom in the diaminoalkyl side chains (IC₅₀ = 6.49 μM).

The antiproliferative properties of these new derivatives 12,13 were also examined using the human myeloid leukemia cell line K562. The disubstituted quinazolines and quinolines showed significant antiproliferative activity, with IC₅₀ values ranging from 7 to 34 μM. From a general point of view, compounds 12k–n, substituted in position 8 of the quinazoline moiety by a phenyl, had the best pharmacological activity against the K562 cell line with an IC₅₀ ranging from 7 to 14 μM.

The most bioactive compounds were then tested on the human epithelial cell line HEK293 to evaluate their cytotoxicity on normal cells. Compounds 12b, 12f, 12i, and 13a displayed a low cytotoxicity against this HEK293 cell line (IC₅₀ > 30 μM), while derivatives 12g and 12j were moderately cytotoxic at 3.98 and 4.90 μM, respectively. The index of selectivity (IS) was defined as the ratio of the IC₅₀ value of the normal cell line HEK293 to the IC₅₀ value of the different cancer cell lines (

Table 2. Selectivity indexes of selected compounds 12,13.

Compound	Selectivity Index a
	HEK293/HeLa	HEK293/SiHa	HEK293/MCF-7	HEK293/MDA-MB-231	HEK293/A2780	HEK293/K562
12b	>36.6	>27.8	>51.7	>42.8	>25.0	>2.14
12f	>19.9	>40.0	>51.7	>54.5	>38.0	>2.14
12g	4.19	7.65	6.32	5.94	2.29	0.33
12i	>26.1	>32.6	>44.8	>57.7	>26.3	>1.30
12j	3.31	14.85	8.45	4.45	3.40	0.61
13a	>60.0	>30.3	>55.5	>51.7	>33.3	>3.0

^a SI was defined as the ratio between the IC₅₀ value on the HEK293 normal cells and the IC₅₀ value against the leukemia HeLa, SiHa, MCF-7, MDA-MB-231, A2780 or K562 cells.

). Derivative 13a displayed a promising index of selectivity towards the HeLa cell line (IS > 60) and breast adenocarcinoma MCF-7 cells (IS > 55.5). Quinazoline 12f demonstrated an interesting IS towards the cervical SiHa cancer cell line (IS > 40), as well as the ovarian A2780 cancer cell line (IS > 38). Moreover, derivative 12i showed interesting selectivity towards the breast cancer MDA-MB-231 cell line (SI > 57.7).

Consequently, comparing the two heterocyclic skeletons, we found no crucial differences between the corresponding analogs (12b–13a, 12d–13c, 12k–13e) and phenylquinazolines seemed more effective than their phenylquinoline analogs (12c–13b, 12e–13d). Substituents containing dimethylamino functions were generally favored over methylpiperazin analogs (e.g., 12b–d, 12l–n, 13a–c) except for 13d, which was more active than 13b. At the level of the position of the phenyl group, position 7 was more advantageous than position 6 (12c–g, 12d–i, 12e–j), while phenyl at position 5 or 8 resulted in less active molecules (e.g., 12a, 12m, 12n, 13e). The linker length between the two lateral chains (the n in Scheme 1) had limited action on the calculated IC₅₀ values. However, a shorter linker (n = 1) tended to result in analogs with more pronounced cell growth-inhibiting action (12b, 12c, 12m, 12n, 13a–d). Collectively, the most promising compounds, i.e., 13a, 12f, 12b, 12i, 12g, and 12j, exhibited nanomolar or low micromolar IC₅₀ values on the used adherent cell panel. Therefore, among these molecules, compounds 13a, 12f, and 12i (Figure 5) may be regarded as drug candidates and their further development, including mechanistic cell-based studies, is highly advocated.

2.3. FRET-Melting Experiments

G4-forming motifs have previously been identified in oncogene promoter regions, bringing to light the therapeutic potential for targeted gene regulation at the transcriptional level. Among them, the most studied oncogene G4 promoters include c-MYC, K-RAS, BCL-2, h-Ras, and c-KIT. We thus decided to evaluate ligand-induced stabilization, specificity, and selectivity of these newly synthesized quinazolines 12a–n and quinolines 13a–e for various oncogene promoter G4 topologies including c-MYC, BCL-2, and K-RAS through FRET-melting experiments (

Table 3. FRET-melting values for compounds 12,13 and PhenDC3.

Compound	ΔT1/2 (°C) a
	Fc-MYCT	FK-RAST	FBCL-2T	F21T	FdxT
PhenDC3	11.5 ± 0.3	16.9 ± 0.4	13.1 ± 0.1	21.2 ± 0.5	−0.6 ± 0.1
12a	9.3 ± 0.3	10.6 ± 0.6	7.4 ± 0.9	6.8 ± 0.8	0.9 ± 0.2
12b	5.8 ± 0.9	17.4 ± 0.6	8.9 ± 0.7	23.7 ± 1.7	0.4 ± 0.3
12c	17.7 ± 0.8	24.5 ± 0.7	16.4 ± 0.4	28.0 ± 0.4	3.8 ± 0.8
12d	8.4 ± 0.9	15.5 ± 0.5	9.7 ± 0.06	15.8 ± 1.2	0.5 ± 0.3
12e	19.5 ± 1.0	33.1 ± 2.1	22.7 ± 0.4	33.9 ± 0.5	5.4 ± 1.6
12f	9.1 ± 1.4	20.1 ± 0.3	12.6 ± 0.6	16.3 ± 1.5	2.4 ± 0.1
12g	14.7 ± 0.8	24.3 ± 1.1	17.5 ± 0.6	19.2 ± 1.0	3.2 ± 0.3
12h	18.7 ± 0.2	28.6 ± 2.1	18.2 ± 1.7	28.1 ± 2.3	2.7 ± 1.0
12i	12.5 ± 0.7	20.2 ± 0.5	14.8 ± 0.7	18.6 ± 0.2	1.9 ± 0.1
12j	14.1 ± 0.3	18.7 ± 1.6	9.8 ± 1.0	14.5 ± 1.2	1.5 ± 0.4
12k	9.9 ± 1.4	13.0 ± 0.4	9.5 ± 0.2	17.7 ± 0.3	0.8 ± 0.1
12l	14.2 ± 1.1	18.7 ± 0.8	17.2 ± 1.2	24.9 ± 0.3	2.9 ± 0.2
12m	14.6 ± 0.9	18.2 ± 0.6	15.5 ± 0.3	23.3 ± 0.7	2.2 ± 0.2
12n	17.0 ± 2.0	18.0 ± 2.5	12.0 ± 2.0	20.7 ± 3.1	2.4 ± 0.7
13a	7.3 ± 1.0	15.0 ± 0.3	7.7 ± 0.6	21.5 ± 0.4	0.4 ± 0.1
13b	13.8 ± 3.0	19.6 ± 3.5	11.1 ± 2.6	13.6 ± 2.9	1.5 ± 0.6
13c	6.8 ± 1.4	14.5 ± 0.8	7.5 ± 0.5	11.3 ± 0.9	0.1 ± 0.2
13d	10.6 ± 0.7	12.7 ± 2.2	5.0 ± 1.1	8.0 ± 1.5	0.8 ± 0.2
13e	5.5 ± 0.9	11.6 ± 1.4	8.3 ± 0.1	15.9 ± 0.4	0.2 ± 0.1

^a All experiments were conducted in K⁺ conditions at 2 μM. ΔT_m of Fc-MYCT, FK-RAST, FBCL-2T, F21T, and FdxT (0.2 μM) were recorded in 10 mM lithium cacodylate (pH 7.2), 10 mM KCl, 90 mM LiCl. Compound PhenDC3 was tested at 0.5 μM. Error margins correspond to SD of three replicates.

, Figure S1).

The stabilization of our new derivatives was also investigated on the fluorescently labeled human telomeric sequence F21T. To probe the G4 selectivity of our novel heterocyclic compounds 12,13 over duplex DNA, a FRET-melting assay was performed using a duplex control sequence, FdxT. For comparison, the G4 ligand PhenDC3 was used as a reference compound. To compare levels of selectivity, the difference of melting temperature (ΔT_m) between the T_m of the G-quadruplex formed by c-MYC, BCL-2, K-RAS, F21T, or FdxT in the presence or absence of each selected compound was calculated. For these novel disubstituted quinazoline and quinoline derivatives 12,13, the ΔT_m values for the c-MYC, BCL-2, K-RAS, F21T sequences ranged from 5.0 to 33.9 °C at a 2 µM ligand concentration.

The best nitrogen heterocyclic ligands which stabilized the three pro-oncogene (c-MYC, BCL-2, K-RAS) and also the human F21T G-quadruplexes sequences (

Table 4. Labelled DNA sequences used in the present study.

Name	Sequences a	Topology
FK-RAST	FAM-A GGG C GG TGT GGG AAGA GGG A-TAMRA	Parallel
FBCL-2T	FAM-GGG CGC GGG A GG AAG GGG GC GGG-TAMRA	Parallel
F21T	FAM-GGG TTA GGG TTA GGG TTA GGG-TAMRA	Hybrid
Fc-MYCT	FAM-GGG T GGG TA GGG T GGG TAA-TAMRA	Parallel
FdxT	FAM-TAT AGC TAT A-hexaethylene glycol-T ATA GCT ATA-TAMRA	Hairpin duplex

^a All sequences are provided in the 5′ => 3′direction.

) were derivatives 12c, 12e, and 12h, bearing substituted alkylaminobutylaminobenzyl or alkylaminopentylaminobenzyl side chains at positions 2 and 4 of the heterocyclic moiety, and mainly compound 12e that showed the highest ΔT_m values (ΔT_m = 19.5, 33.1, 22.7 and 33.9 °C, respectively on the different c-MYC, K-RAS, BCL-2, and F21T sequences). Thus, the stabilization effects on the various quadruplex sequences seem to be dependent on the length of the alkylaminoalkylaminobenzyl side chains of the quinazolines 12.

We also observed that quinazoline compounds 12a, 12b, 12f, and 12k (all bearing two (3-dimethylaminopropyl)aminomethylphenyl side chains and a phenyl ring linked to the heterocyclic skeleton in the various positions 5, 6, 7 and 8, respectively) displayed lower stabilization than their other substituted quinazolinyl analogs 1: ΔT_m = 5.8–9.9 °C on c-MYC, 10.6–20.1 °C on K-RAS, 7.4–12.6 °C on BCL-2 and 6.8–23.7 °C on F21T for 12a, 12b, 12f and 12k versus 8.4–19.5 °C on c-MYC, 15.5–33.1 °C on K-RAS, 9.5–22.7 °C on BCL-2 and 14.5–33.9 °C on F21T for the other quinazoline derivatives 12.

For each G-quadruplex sequence, the disubstituted quinoline ligands 13a–e exhibited lower stabilization profiles in comparison with their quinazoline disubstituted homologs 12a–n; these latter being better stabilizing ligands.

Concerning the position of the substitution of the phenyl group on the nitrogen heterocycle, we cannot draw conclusions on their structure–activity relationships. In addition, quinazolines 12 and quinolines 13 were no more specific for the hybrid G4 topology of F21T sequence than for the parallel G4 structures of c-MYC, BCL-2, or K-RAS.

Hence, FRET assays showed that there was no stabilization of our heterocyclic structures on duplex DNA sequences.

2.4. Native Electrospray Mass Spectrometry

The binding affinity and stoichiometry of quinazoline (12b, 12e) and quinoline (13a, 13d) derivatives for G-quadruplexes were determined by native electrospray mass spectrometry [33]. We selected three G-quadruplex-forming oligonucleotides: the oncogene promoters c-MYC (5′-TGAG₃TG₃TAG₃TG₃TA₂) and BCL-2 (5′-G₃CGCG₃AG₂A₂T₂G₃CG₃) and the human telomeric sequence 24TTG (5′-T₂(G₃T₂A)₃G₃A). Circular dichroism (CD) experiments revealed that in the 100 mM ammonium acetate buffer used for native MS experiments, c-MYC forms a parallel G-quadruplex (positive band at 260 nm, negative band at 240 nm; Figure 6 and Figure S2), whereas BCL-2 and 24TTG had hybrid or mixed signatures (positive bands at 290 and shoulder at 270 nm). Native MS confirms the formation of 3-tetrad G-quadruplexes specifically binding two ammonium cations (Figure 6). ds26, an oligonucleotide forming a hairpin duplex (CA₂TCG₂ATCGA₂T₂CGATC₂GAT₂G) was used as a control.

The four ligands form complexes with all three G-quadruplexes with both 1:1 and 2:1 ligand:DNA stoichiometries (Figures S3–S5). The dissociation constants of the 1:1 complexes (Kd₁) formed by 12b, 12e, and 13a with BCL-2 and 24TTG were in the micromolar range, whereas 13d was a weaker binder by an order of magnitude (

Table 5. Binding affinities of 12b, 12e, 13a, and 13d for BCL-2, 24TTG, c-MYC, and ds26 determined by native mass spectrometry, expressed as percentage of bound DNA and dissociation constants (Kd₁ and Kd₂ correspond to two successive binding events). The second binding event is cooperative if Kd₁/Kd₂ > 4 [34].

	12b	12e	13a	13d
BCL-2
Bound DNA (%)	79	79	92	33
Kd1	5.6	6.2	1.6	53
Kd2	2.8	2.2	0.75	20
Kd1/Kd2	2.0	2.8	2.1	2.6
24TTG
Bound DNA (%)	77	72	81	39
Kd1	9.5	14	3.7	29
Kd2	1.5	1.9	7.1	83
Kd1/Kd2	6.3	7.2	0.5	0.4
c-MYC
Bound DNA (%)	70	75	78	44
Kd1	5.8	4.3	3.8	20
Kd2	100	74	36	440
Kd1/Kd2	0.1	0.1	0.1	0.1
ds26
Bound DNA (%)	48	36	53	18
Kd1	17	30	14	85
Kd2	130	280	95	460
Kd1/Kd2	0.1	0.1	0.2	0.2

). The Kd₂ of the second binding reaction of 12b, 12e, and 13a was also in the micromolar range and in most cases lower. Remarkably, the second binding event was cooperative for 12b and 12e binding 24TTG (Kd₁/Kd₂ > 4) [34]. This suggests that the conformation of 24TTG and BCL-2 may be altered upon binding of these ligands to accommodate the second ligand more tightly. This is supported by (i) the change in the CD signature towards a more antiparallel signature (decrease in the shoulder at 270 nm; Figure 6B) and (ii) the displacement of an ammonium cation (particularly for 2:1 complexes). A decrease in cation stoichiometry may be the result of a switch towards two-tetrad conformers [35] or the intercalation of the ligand [36].

Binding of 12b, 12e, and 13a to the parallel c-MYC G-quadruplex was similar in magnitude to that of 24TTG and BCL-2, but mostly restricted to 1:1 complexes (Kd₁~4–6 µM vs. Kd₂~36–100 µM). Consistently, there was no significant change in ammonium stoichiometry or CD signature. Again, 13d was a weaker binder than its counterparts.

Finally, all four ligands bound moderately to the duplex control ds26 (Figure S6). The lack of stabilization of FdxT in the FRET-melting experiments could be explained by (i) the use of much lower oligonucleotide concentrations (0.2 µM vs. 10 µM here), leading to low binding resulting in large K_d values, and (ii) the difference in GC content between FdxT (20% GC) and ds26 (46% GC), the latter favoring intercalation of small molecules compared to the former.

2.5. Detection of Telomerase Activity in Cell Lysates

Additionally, the more bioactive ligands 12b, 12f, and 12i were also tested for telomerase activity in MCF-7 protein extracts. For MCF-7 cell lysate, we observed that 5 µM of 12b, 12f, or 12i reduced drastically the telomerase activity compared to control. In our experimental conditions, MCF-7 cell lysates were more sensitive to 12b than 12i. Indeed, while compound 12b decreased MCF-7 telomerase activity from 0.5 µM, for compound 12i it was necessary to use 5 µM to drastically reduce MCF-7 telomerase activity (Figure 7).

3. Materials and Methods

3.1. Chemistry

Commercially available reagents were used without additional purification. Melting points were determined with an SM-LUX-POL Leitz hot-stage microscope (Leitz GMBH, Midland, ON, USA) and are uncorrected.

IR spectra were recorded on a NICOLET 380FT-IR spectrophotometer (Bruker BioSpin, Wissembourg, France). NMR spectra were recorded with tetramethylsilane as an internal standard using a BRUKER AVANCE 300 spectrometer (Bruker BioSpin, Wissembourg, France). Splitting patterns have been reported as follows: s = singlet; bs = broad singlet; d = doublet; t = triplet; q = quartet; dd = double doublet; ddd = double double doublet; qt = quintuplet; and m = multiplet.

Analytical TLC were carried out on 0.25 precoated silica gel plates (POLYGRAM SIL G/UV254) (Merck KGaA, Darmstadt, Germany) and compounds were visualized after UV light irradiation. A silica gel 60 (70–230 mesh) was used for column chromatography. Mass spectra were recorded on an ESI LTQ Orbitrap Velos mass spectrometer (ThermoFisher, Bremen, Germany). Ionization was performed using an Electrospray ion source operating in positive ion mode with a capillary voltage of 3.80 kV and capillary temperature of 250 °C. The scan type analyzed was full scan, all MS recordings were in the m/z range between 150 to 2000 m/z. No fragmentation was carried out and the resolution used for the analysis was 60,000.

• General procedure for generating 2,4-bis(4-formylphenyl)-phenylquinazolines 8a–d and 2,4-bis(4-formylphenyl)-phenylquinolines 9a,b

To a solution of 4.37 mmol of the appropriate 2,4-dichloroquinazoline 6a–d or 2,4-dichloroquinoline 7a,b 1.44 g of 3- or 4-formylphenyl boronic acid (9.63 mmol, 2.2 eq.) and 506 mg (0.437 mmol, 0.1 eq.) of tetrakis(triphenylphosphine)palladium(0) in 45 mL of 1,2-dimethoxyethane, 5 mL of 2 M K₂CO₃ aqueous solution, previously degassed for 10 min with nitrogen, were added at room temperature. The mixture was then warmed to reflux and stirred for 24 h under nitrogen-positive pressure. The reaction mix was cooled down to room temperature and the solvent was evaporated under vacuum. The organic layer was extracted with CH₂Cl₂, and the organic phase was filtered on filter paper, then washed with water (20 mL × 3 times), dried over anhydrous sodium sulfate and activated charcoal, filtered and evaporated under vacuum. The residue was cooled and triturated with a minimum of EtOH and EtO₂ and filtered on sintered glassware to obtain the crude product. The residue was purified using silica gel column chromatography (CH₂Cl₂/CH₃OH 95:5), then cooled and triturated in EtOH, filtered on sintered glassware, washed with a minimum of EtOH, EtO₂, and petroleum ether, and dried under pressure to generate the solid product 8,9.

• 2,4-bis(4-formylphenyl)-5-phenylquinazoline (8a)

Pale yellow crystals (39%); m.p. = 214–216 °C; ¹H NMR (CDCl₃) δ ppm: 10.16 (s, 1H, CHO), 9.94 (s, 1H, CHO), 8.90 (d, 2H, J = 8.40 Hz, H-3″, and H-5″), 8.28 (dd, 1H, J = 8.40, and 1.20 Hz, H-8), 8.08 (d, 2H, J = 8.40 Hz, H-3′, and H-5′), 8.04 (dd, 1H, J = 8.40, and 7.20 Hz, H-7), 7.68 (dd, 1H, J = 7.20, and 1.50 Hz, H-6), 7.61 (d, 2H, J = 8.40 Hz, H-2″, and H-6″), 7.53 (d, 2H, J = 8.40 Hz, H-2′, and H-6′), 7.12–6.95 (m, 5H, H-phe).

• 2,4-bis(4-formylphenyl)-6-phenylquinazoline (8b)

Yellow crystals (41%); m.p. = 221–223 °C; ¹H NMR (CDCl₃) δ ppm: 10.22 (s, 1H, CHO), 10.16 (s, 1H, CHO), 8.90 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.32 (d, 1H, J = 7.80 Hz, H-8), 8.26 (dd, 1H, J = 7.80, and 2.10 Hz, H-7), 8.24 (d, 1H, J = 2.10 Hz, H-5), 8.18 (d, 2H, J = 8.10 Hz, H-3′, and H-5′), 8.12 (d, 2H, J = 8.10 Hz, H-2″, and H-6″), 8.08 (d, 2H, J = 8.10 Hz, H-2′, and H-6′), 7.66 (d, 2H, J = 7.00 Hz, H-2, and H-6_phe), 7.52 (t, 2H, J = 7.00 Hz, H-3, and H-5_phe), 7.46 (t, 1H, J = 7.00 Hz, H-4_phe).

• 2,4-bis(4-formylphenyl)-7-phenylquinazoline (8c)

Pale-yellow crystals (51%); m.p. = 202–204 °C; ¹H NMR (CDCl₃) δ ppm: 10.23 (s, 1H, CHO), 10.17 (s, 1H, CHO), 8.90 (d, 2H, J = 8.20 Hz, H-3″, and H-5″), 8.46 (d, 1H, J = 1.60 Hz, H-8), 8.20 (d, 2H, J = 8.40, H-3′, and H-5′), 8.19 (d, 1H, J = 8.70 Hz, H-5), 8.11 (d, 2H, J = 8.20 Hz, H-2″, and H-6″), 8.08 (d, 2H, J = 8.20 Hz, H-2′, and H-6′), 7.93 (dd, 1H, J = 8.70 Hz, and J = 1.60 Hz, H-6), 7.86–7.82 (d, 2H, J = 7.40 Hz, H-2, and H-6_phe), 7.58 (t, 2H, J = 7.40 Hz, H-3, and H-5_phe), 7.50 (t, 1H, J = 7.40 Hz, H-4_phe). ¹³C NMR (CDCl₃) δ ppm: 192.3 (CHO), 191.8 (CHO), 166.9 (C-2), 159.4 (C-4), 152.4 (C-8a), 149.6 (C-1″), 143.4 (C-7), 143.0 (C-1_phe), 139.1 (C-1′), 137.6 (C-4′), 137.2 (C-4″), 130.8 (C-3″ and C-5″), 130.0 (C-3′ and C-5′), 129.9 (C-2′ and C-6′), 129.3 (C-3, C-5_phe and C-4_phe), 129.2 (C-2″ and C-6″), 128.9 (C-4_phe), 127.9 (C-8), 127.6 (C-2 and C-6_phe), 126.9 (C-5), 126.8 (C-6), 120.7 (C-4a).

• 2,4-bis(4-formylphenyl)-8-phenylquinazoline (8d)

Pale-yellow crystals (51%); m.p. = 212–214 °C; ¹H NMR (CDCl₃) δ ppm: 10.22 (s, 1H, CHO), 10.11 (s, 1H, CHO), 8.76 (d, 2H, J = 8.40 Hz, H-3″, and H-5″), 8.16 (d, 2H, J = 8.40 Hz, H-3′, and H-5′), 8.09 (d, 2H, J = 8.40 Hz, H-2″, and H-6″), 8.07 (dd, 1H, J =7.80, and 1.20 Hz, H-7), 8.05 (dd, 1H, J = 7.35, and 1.20 Hz, H-5), 8.01(d, 2H, J = 8.40 Hz, H-2′, and H-6′), 7.88 (d, 2H, J = 8.10 Hz, H-2_phe, and H-6_phe), 7.71 (dd, 1H, J =7.80 H,z, and J = 7.35 Hz, H-6), 7.62 (t, 2H, J = 8.10 Hz, H-3, and H-5_phe),7.56 (t, 1H, J = 8.10 Hz, H-4_phe).

• 2,4-bis(4-formylphenyl)-6-phenylquinoline (9a)

Pale-orange crystals (66%); m.p. = 207–209 °C; ¹H NMR (CDCl₃) δ ppm: 10.19 (s, 1H, CHO), 10.15 (s, 1H, CHO), 8.43 (d, 2H, J = 8.25 Hz, H-3″, and H-5″), 8.37 (d, 1H, J = 8.70 Hz, H-8), 8.13 (d, 2H, J = 8.25 Hz, H-3′, and H-5′), 8.08 (dd, 1H, J = 8.70, and 1.80 Hz, H-7), 8.07 (d, 2H, J = 8.25 Hz, H-2″, and H6″), 8.02 (d, 1H, J = 1.80 Hz, H-5), 7.92 (s, 1H, H-3), 7.82 (d, 2H, J = 8.25 Hz, H-2′, and H-6′), 7.64 (d, 2H, J = 7.20 Hz, H-2, and H-6_phe), 7.48 (t, 2H, J = 7.20 Hz, H-3, and H-5_phe),7.41 (t, 1H, J = 7.20 Hz, H-4_phe).

• 2,4-bis(4-formylphenyl)-8-phenylquinoline (9b)

Pale-yellow crystals (27%); m.p. = 127–129 °C; ¹H NMR (CDCl₃) δ ppm: 10.20 (s, 1H, CHO), 10.01 (s, 1H, CHO), 8.34 (d, 2H, J = 8.25 Hz, H-3″, and H-5″), 8.12 (d, 2H, J = 8.25 Hz, H-3′, and H-5′), 8.01 (d, 2H, J = 8.25 Hz, H-2″, and H6″), 7.98 (dd, 1H, J =7.80, and 1.25 Hz, H-7), 7.96 (dd, 1H, J = 7.70, and 1.25 Hz, H-5), 7.93 (s, 1H, H-3), 7.88–7.76 (m, 5H, H-2′, H-6′, H-6, H-2_phe, and H-6_phe), 7.65–7.50 (m, 3H, H-3, H-4, and H-5_phe).

• General procedure for 2,4-bis[(substituted-iminomethyl)phenyl]-phenylquinazolines 10a–n and 2,4-bis[(substituted-iminomethyl)phenyl]-phenylquinolines 11a–e

To a solution of diamine (0.126 mmol, 2.1 eq.) in ethanol (7 mL), we added 2,4-bis(4-formylphenyl)quinazoline 8 or 1,3-bis(4-formylphenyl)quinoline 9 (0.6 mmol). The reaction mix was then heated under reflux for 5 h and evaporated to dryness under reduced pressure. After cooling, the residue was extracted with dichloromethane (40 mL). The organic layer was dried over sodium sulfate and activated charcoal and evaporated to dryness. Products were then used without further purification.

• 2,4-bis{4-[(3-dimethylaminopropyl)iminomethyl]phenyl}-5-phenylquinazoline (10a)

Yellow oil (98%); ¹H NMR (CDCl₃) δ ppm: 8.72 (d, 2H, J = 8.40 Hz, H-3″, and H-5″), 8.34 (s, 1H, CH=N), 8.13 (dd, 1H, J = 8.40, and 1.20 Hz, H-8), 8.09 (s, 1H, CH=N), 7.86 (dd, 1H, J = 8.40, and 7.20 Hz, H-7), 7.84 (d, 2H, J = 8.40 Hz, H-3′, and H-5′), 7.52 (dd, 1H, J = 7.20, and 1.50 Hz, H-6), 7.35–7.33 (m, 4H, H-2″, H-6″, H-2′, and H-6′), 7.03–6.93 (m, 5H, H-phe), 3.65 (t, 2H, J = 7.20 Hz, NCH₂), 3.60 (t, 2H, J = 7.20 Hz, NCH₂), 2.34 (t, 2H, J = 7.20 Hz, NCH₂), 2.30 (t, 2H, J = 7.20 Hz, NCH₂), 2.21 (s, 6H, N(CH₃)₂), 2.20 (s, 6H, N(CH₃)₂), 1.91–1.80 (m, 4H, 2CH₂).

• 2,4-bis{4-[(3-dimethylaminopropyl)iminomethyl]phenyl}-6-phenylquinazoline (10b)

Yellow crystals (97%); m.p. = 116–118 °C; ¹H NMR (CDCl₃) δ ppm: 8.77 (d, 2H, J = 8.40 Hz, H-3″, and H-5″), 8.46 (s, 1H, CH=N), 8.40 (s, 1H, CH=N), 8.29 (d, 1H, J = 1.95 Hz, H-5), 8.24 (d, 1H, J = 8.40 Hz, H-8), 8.18 (dd, 1H, J = 8.40, and 1.95 Hz, H-7), 8.03–7.98 (m, 4H, H-3′,H-5′, H-2″, and H-6″), 7.90 (d, 2H, J = 8.40 Hz, H-2′, and H-6′), 7.66 (d, 2H, J = 8.40 Hz, H-2, and H-6_phe), 7.51 (t, 2H, J = 8.40 Hz, H-3, and H-5_phe), 7.43 (t, 1H, J = 8.40 Hz, H-4_phe), 3.74 (t, 2H, J = 7.05 Hz, NCH₂), 3.72 (t, 2H, J = 7.05 Hz, NCH₂), 2.43 (t, 2H, J = 7.05 Hz, NCH₂), 2.40 (t, 2H, J = 7.05 Hz, NCH₂), 2.28 (s, 6H, N(CH₃)₂), 2.27 (s, 6H, N(CH₃)₂), 1.99–1.87 (m, 4H, 2CH₂).

• 2,4-bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-6-phenylquinazoline (10c)

Orange oil (98%); ¹H NMR (CDCl₃) δ ppm: 8.72 (d, 2H, J = 8.70 Hz, H-3″ and H-5″), 8.39 (s, 1H, CH=N), 8.34 (s, 1H, CH=N), 8.23 (d, 1H, J = 1.80 Hz, H-5), 8.18 (d, 1H, J = 8.70 Hz, H-8), 8.12 (dd, 1H, J = 8.70, and 1.80 Hz, H-7), 7.96–7.93 (m, 4H, H-3′,H-5′, H-2″, and H-6″), 7.85 (d, 2H, J = 8.70 Hz, H-2′, and H-6′), 7.59 (d, 2H, J = 7.50 Hz, H-2, and H-6_phe),7.44 (t, 2H, J = 7.50 Hz, H-3, and H-5_phe), 7.36 (t, 1H, J = 7.50 Hz, H-4_phe), 3.68 (t, 2H, J = 6.90 Hz, NCH₂), 3.64 (t, 2H, J = 6.90 Hz, NCH₂), 2.32 (t, 2H, J = 6.90 Hz, NCH₂), 2.28 (t, 2H, J = 6.90 Hz, NCH₂), 2.22 (s, 6H, N(CH₃)₂), 2.21 (s, 6H, N(CH₃)₂), 1.80–1.69 (m, 4H, 2CH₂), 1.60–1.52 (m, 4H, 2CH₂).

• 2,4-bis{4-[(3-(4-methylpiperazin-1-yl)propyl)iminomethyl]phenyl}-6-phenylquinazoline (10d)

Yellow crystals (98%); m.p. = 129–131 °C; ¹H NMR (CDCl₃) δ ppm: 8.66 (d, 2H, J = 8.10 Hz, H-3″ and H-5″), 8.34 (s, 1H, CH=N), 8.27 (s, 1H, CH=N), 8.16 (d, 1H, J = 1.80 Hz, H-5), 8.11 (d, 1H, J = 8.20 Hz, H-8), 8.04 (dd, 1H, J = 8.70, and 1.80 Hz, H-7), 7.90–7.86 (m, 4H, H-3′,H-5′, H-2″, and H-6″), 7.79 (d, 2H, J = 8.10 Hz, H-2′, and H-6′), 7.52 (d, 2H, J = 8.10 Hz, H-2, and H-6_phe), 7.37 (t, 2H, J = 8.10 Hz, H-3, and H-5_phe) 7.30 (t, 1H, J = 8.10 Hz, H-4_phe), 3.65 (t, 2H, J = 6.90 Hz, NCH₂), 3.61 (t, 2H, J = 6.90 Hz, NCH₂), 2.52–2.30 (m, 20H, 2NCH_2, and 8NCH_2pip), 2.22 (s, 6H, 2NCH₃), 1.90–1.86 (m, 4H, 2CH₂).

• 2,4-bis{4-[(4-(4-methylpiperazin-1-yl)butyl)iminomethyl]phenyl}-6-phenylquinazoline (10e)

Yellow-orange oil (57%); ¹H NMR (CDCl₃) δ ppm: 8.72 (d, 2H, J = 8.25 Hz, H-3″, and H-5″), 8.38 (s, 1H, CH=N), 8.32 (s, 1H, CH=N), 8.22 (d, 1H, J = 1.80 Hz, H-5), 8.18 (d, 1H, J = 8.75 Hz, H-8), 8.12 (dd, 1H, J = 8.75, and 1.80 Hz, H-7), 7.97–7.90 (m, 4H, H-3′, H-5′, H-2″, and H-6″), 7.84 (d, 2H, J = 8.25 Hz, H-2′, and H-6′), 7.58 (d, 2H, J = 8.40 Hz, H-2, and H-6_phe), 7.43 (t, 2H, J = 8.40 Hz, H-3, and H-5_phe), 7.34 (t, 1H, J = 8.40 Hz, H-4_phe), 3.70–3.61 (m, 4H, 2NCH₂), 3.65–2.32 (m, 20H, 2 NCH_2, and 8 NCH_2pip), 2.24 (s, 6H, 2NCH₃), 1.78–1.70 (m, 4H, 2CH₂), 1.61–1.55 (m, 4H, 2CH₂).

• 2,4-bis{4-[(3-dimethylaminopropyl)iminomethyl]phenyl}-7-phenylquinazoline (10f)

Yellow oil (98%); ¹H NMR (CDCl₃) δ ppm: 8.71 (d, 2H, J = 8.40 Hz, H-3″, and H-5″), 8.38 (s, 1H, CH=N), 8.32 (s, 1H, CH=N), 8.28 (d, 1H, J = 1.50 Hz, H-8), 8.05 (d, 1H, J = 8.70 Hz, H-5), 7. 92 (d, 2H, J = 8.40 Hz, H-3′, and H-5′), 7.89 (d, 2H, J = 8.40 Hz, H-2″ and H-6″), 7.83 (d, 2H, J = 8.40 Hz, H-2′, and H-6′), 7.77 (dd, 1H, J = 8.70, and 1.50 Hz, H-6), 7.72 (d, 2H, J = 7.50 Hz, H-2, and H-6_phe), 7.46 (t, 2H, J = 7.50 Hz, H-3, and H-5_phe), 7.38 (t, 1H, J = 7.50 Hz, H-4_phe), 3.69 (t, 2H, J = 6.90 Hz, NCH₂), 3.66 (t, 2H, J = 6.90 Hz, NCH₂), 2.37 (t, 2H, J = 6.90 Hz, NCH₂), 2.35 (t, 2H, J = 6.90 Hz, NCH₂), 2.24 (s, 6H, N(CH₃)₂), 2.23 (s, 6H, N(CH₃)₂), 1.97–1.83 (m, 4H, 2CH₂).

• 2,4-bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-7-phenylquinazoline (10g)

Yellow oil (98%); ¹H NMR (CDCl₃) δ ppm: 8.73 (d, 2H, J = 8.40 Hz, H-3″, and H-5″), 8.40 (s, 1H, CH=N), 8.35 (s, 1H, CH=N), 8.33 (d, 1H, J = 1.65 Hz, H-8), 8.11 (d, 1H, J = 8.70 Hz, H-5), 7.95 (d, 2H, J = 8.40, H-3′, H-5′), 7.93 (d, 2H, J = 8.40, H-2″, and H-6″), 7.86 (d, 2H, J = 8.40 Hz, H-2′, and H-6′), 7.78 (dd, 1H, J = 8.70, and 1.65 Hz, H-6), 7.75 (d, 2H, J = 7.80 Hz, H-2, and H-6_phe), 7.51 (t, 2H, J = 7.80 Hz, H-3, and H-5_phe), 7.43 (t, 1H, J = 7.80 Hz, H-4_phe), 3.70 (t, 2H, J = 6.90 Hz, NCH₂), 3.67 (t, 2H, J = 6.90 Hz, NCH₂), 2.36 (t, 2H, J = 6.90 Hz, NCH₂), 2.32 (t, 2H, J = 6.90 Hz, NCH₂), 2.25 (s, 6H, N(CH₃)₂), 2.23 (s, 6H, N(CH₃)₂), 1.82–1.70 (m, 4H, 2CH₂), 1.65–1.54 (m, 4H, 2CH₂).

• 2,4-bis{4-[(5-dimethylaminopentyl)iminomethyl]phenyl}-7-phenylquinazoline (10h)

Orange oil (98%); ¹H NMR (CDCl₃) δ ppm: 8.72 (d, 2H, J = 8.40 Hz, H-3″, and H-5″), 8.37 (s, 1H, CH=N), 8.31 (s, 1H, CH=N), 8.30 (d, 1H, J = 1.80 Hz, H-8), 8.09 (d, 1H, J = 8.70 Hz, H-5), 7.93 (d, 2H, J = 8.40, H-3′, H-5′), 7.91 (d, 2H, J = 8.40, H-2″, and H-6″), 7.85 (d, 2H, J = 8.40 Hz, H-2′, and H-6′), 7.76 (dd, 1H, J = 8.70, and 1.80 Hz, H-6), 7.73 (d, 2H, J = 7.20 Hz, H-2, and H-6_phe), 7.48 (t, 2H, J = 7.20 Hz, H-3, and H-5_phe), 7.41 (t, 1H, J = 7.20 Hz, H-4_phe), 3.67 (t, 2H, J = 6.90 Hz, NCH₂), 3.63 (t, 2H, J = 6.90 Hz, NCH₂), 2.31 (t, 2H, J = 6.90 Hz, NCH₂), 2.28 (t, 2H, J = 6.90 Hz, NCH₂), 2.23 (s, 6H, N(CH₃)₂), 2.22 (s, 6H, N(CH₃)₂), 1.80–1.72 (m, 4H, 2CH₂), 1.56–1.45 (m, 4H, 2CH₂), 1.43–1.35 (m, 4H, 2CH₂).

• 2,4-bis{4-[(3-(4-methylpiperazin-1-yl)propyl)iminomethyl]phenyl}-7-phenylquinazoline (10i)

Yellow oil (98%); ¹H NMR (CDCl₃) δ ppm: 8.69 (d, 2H, J = 8.40 Hz, H-3″, and H-5″), 8.36 (s, 1H, CH=N), 8.31 (s, 1H, CH=N), 8.29 (d, 1H, J = 1.50 Hz, H-8), 8.07 (d, 1H, J = 8.70 Hz, H-5), 7.91–7.85 (m, 4H, H-3′, H-5′, H-2″, and H-6″), 7.82 (d, 2H, J = 8.40 Hz, H-2′, and H-6′), 7.73 (dd, 1H, J = 8.70 and 1.50 Hz, H-6), 7.71 (d, 2H, J = 8.00 Hz, H-2, and H-6_phe), 7.46 (t, 2H, J = 8.00 Hz, H-3, and H-5_phe), 7.38 (t, 1H, J = 8.00 Hz, H-4_phe), 3.67 (t, 2H, J = 6.90 Hz, NCH₂), 3.63 (t, 2H, J = 6.90 Hz, NCH₂), 2.56–2.31 (m, 20H, 2 NCH_2, and 8 NCH_2pip), 2.24 (s, 3H, NCH₃), 2.23 (s, 3H, NCH₃), 1.95–1.83 (m, 4H, 2CH₂).

• 2,4-bis{4-[(4-(4-methylpiperazin-1-yl)butyl)iminomethyl]phenyl}-7-phenylquinazoline (10j)

Orange-yellow oil (98%); ¹H NMR (CDCl₃) δ ppm: 8.74 (d, 2H, J = 8.40 Hz, H-3″, and H-5″), 8.39 (s, 1H, CH=N), 8.35 (s, 1H, CH=N), 8.34 (d, 1H, J = 1.80 Hz, H-8), 8.13 (d, 1H, J = 8.70 Hz, H-5), 7.95–7.92 (m, 4H, H-3′,H-5′, H-2″, and H-6″), 7.86 (d, 2H, J = 8.40 Hz, H-2′, and H-6′), 7.80 (dd, 1H, J = 8.70, and 1.80 Hz, H-6), 7.77 (d, 2H, J = 7.50 Hz, H-2, and H-6_phe), 7.51 (t, 2H, J = 7.50 Hz, H-3, and H-5_phe), 7.43 (t, 1H, J = 7.50 Hz, H-4_phe), 3.69 (t, 2H, J = 6.60 Hz, NCH₂), 3.65 (t, 2H, J = 6.60 Hz, NCH₂), 2.53–2.30 (m, 20H, 2 NCH_2, and 8 NCH_2pip), 2.28 (s, 3H, NCH₃), 2.27 (s, 3H, NCH₃), 1.80–1.70 (m, 4H, 2CH₂), 1.65–1.53 (m, 4H, 2CH₂).

• 2,4-bis{4-[(3-dimethylaminopropyl)iminomethyl]phenyl}-8-phenylquinazoline (10k)

Pale-yellow crystals (95%); m.p. = 117–119 °C; ¹H NMR (CDCl₃) δ ppm: 8.67 (d, 2H, J = 8.40 Hz, H-3″, and H-5″), 8.46 (s, 1H, CH=N), 8.37 (s, 1H, CH=N), 8.11 (dd, 1H, J = 8.40, and 1.50 Hz, H-7), 7.99 (dd, 1H, J = 8.40, and 1.50 Hz, H-5), 7.98 (d, 2H, J = 8.40 Hz, H-3′, and H-5′), 7.97 (d, 2H, J = 8.40 Hz, H-2″, and H-6″), 7.89 (d, 2H, J = 7.20 Hz, H-2, and H-6_phe), 7.84 (d, 2H, J = 8.40 Hz, H-2′, and H-6′), 7.67–7.51 (m, 4H, H-6, H-3, H-4, and H-5_phe), 3.76 (t, 2H, J = 6.90 Hz, NCH₂), 3.70 (t, 2H, J = 6.90 Hz, NCH₂), 2.42 (t, 2H, J = 6.90 Hz, NCH₂), 2.39 (t, 2H, J = 6.90 Hz, NCH₂), 2.29 (s, 6H, N(CH₃)₂), 2.27 (s, 6H, N(CH₃)₂), 2.01–1.87 (m, 4H, 2CH₂).

• 2,4-bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-8-phenylquinazoline (10l)

Pale-yellow crystals (98%); m.p. = 90–92 °C; ¹H NMR (CDCl₃) δ ppm: 8.61 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.40 (s, 1H, CH=N), 8.31 (s, 1H, CH=N), 8.06 (dd, 1H, J = 8.20, and 1.50 Hz, H-7), 7.96–7.90 (m, 5H, H-5, H-3′, H-5′, H-2″, and H-6″), 7.85 (d, 2H, J = 7.20, H-2, H-6_phe), 7.78 (d, 2H, J = 8.10, H-2′, and H-6′), 7.59 (t, 1H, J = 8.10 Hz, H-6), 7.54 (t, 2H, J = 7.20 Hz, H-3, and H-5_phe), 7.46 (t, 1H, J = 7.20 Hz, H-4_phe), 3.69 (t, 2H, J = 5.40 Hz, NCH₂), 3.63 (t, 2H, J = 5.40 Hz, NCH₂), 2.32 (t, 2H, J = 5.40 Hz, NCH₂), 2.29 (t, 2H, J = 5.40 Hz, NCH₂), 2.22 (s, 6H, N(CH₃)₂), 2.20 (s, 6H, N(CH₃)₂), 1.80–1.70 (m, 4H, 2CH₂), 1.61–1.49 (m, 4H, 2CH₂).

• 2,4-bis{4-[(3-(4-methylpiperazin-1-yl)propyl)iminomethyl]phenyl}-8-phenylquinazoline (10m)

Pale yellow oil (98%); ¹H NMR (CDCl₃) δ ppm: 8.61 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.41 (s, 1H, CH=N), 8.32 (s, 1H, CH=N), 8.07 (dd, 1H, J = 8.40, and 1.50 Hz, H-7), 7.95 (dd, 1H, J = 8.40, and 1.50 Hz, H-5), 7.94–7.92 (m, 4H, H-3′,H-5′, H-2″, and H-6″), 7.84 (d, 2H, J = 7.20 Hz, H-2, and H-6_phe), 7.78 (d, 2H, J = 8.10 Hz, H-2′, and H-6′), 7.62–7.57 (m, 1H, H-6), 7.54 (t, 2H, J = 7.20 Hz, H-3, and H-5_phe), 7.47 (t, 1H, J = 7.20 Hz, H-4_phe), 3.71 (t, 2H, J = 5.10 Hz, NCH₂), 3.65 (t, 2H, J = 5.10 Hz, NCH₂), 2.61–2.38 (m, 20H, 2 NCH_{2, and} 8 NCH_2pip), 2.28 (s, 3H, NCH₃), 2.26 (s, 3H, NCH₃), 1.98–1.86 (m, 4H, 2CH₂).

• 2,4-bis{4-[(4-(4-methylpiperazin-1-yl)butyl)iminomethyl]phenyl}-8-phenylquinazoline (10n)

Yellow oil (98%); ¹H NMR (CDCl₃) δ ppm: 8.61 (d, 2H, J = 8.40 Hz, H-3″, and H-5″), 8.38 (s, 1H, CH=N), 8.29 (s, 1H, CH=N), 8.05 (dd, 1H, J = 8.40, and 1.20 Hz, H-7), 7.94–7.89 (m, 5H, H-5, H-3′, H-5′, H-2″, and H-6″), 7.84 (d, 2H, J = 7.20 Hz, H-2, and H-6_phe), 7.78 (d, 2H, J = 8.40 Hz, H-2′, and H-6′), 7.60–7.44 (m, 4H, H-3, H-6, H-4, and H-5_phe), 3.68 (t, 2H, J = 7.20 Hz, NCH₂), 3.63 (t, 2H, J = 7.20 Hz, NCH₂), 2.65–2.29 (m, 20H, 2 NCH_{2, and} 8 NCH_2pip), 2.27 (s, 3H, NCH₃), 2.25 (s, 3H, NCH₃), 1.79–1.68 (m, 4H, 2CH₂), 1.61–1.50 (m, 4H, 2CH₂).

• 2,4-bis{4-[(3-dimethylaminopropyl)iminomethyl]phenyl}-6-phenylquinoline (11a)

Yellow-orange oil (98%); ¹H NMR (CDCl₃) δ ppm: 8.42 (s, 1H, CH=N), 8.38 (s, 1H, CH=N), 8.31 (d, 1H, J = 8.60 Hz, H-8), 8.28 (d, 2H, J = 8.40 Hz, H-3″, and H-5″), 8.08 (d, 1H, J = 2.00 Hz, H-5), 8.01 (dd, 1H, J = 8.60, and 2.00 Hz, H-7), 7.93 (d, 2H, J = 8.40 Hz, H-3′,H-5′), 7.89 (d, 2H, J = 8.40 Hz, H-2″, and H-6″), 7.87 (s, 1H, H-3), 7.66 (d, 2H, J = 8.40 Hz, H-2′, and H-6′), 7.62 (d, 2H, J = 7.50 Hz, H-2, and H-6_phe), 7.44 (d, 2H, J = 7.50 Hz, H-3, and H-5_phe), 7.38 (t, 1H, J = 7.50 Hz, H-4_phe), 3.73 (t, 2H, J = 6.90 Hz, NCH₂), 3.70 (t, 2H, J = 6.90 Hz, NCH₂), 2.41 (t, 2H, J = 6.90 Hz, NCH₂), 2.38 (t, 2H, J = 6.90 Hz, NCH₂), 2.28 (s, 6H, N(CH₃)₂), 2.26 (s, 6H, N(CH₃)₂), 1.97–1.85 (m, 4H, 2CH₂).

• 2,4-bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-6-phenylquinoline (11b)

Pale-orange oil (98%); ¹H NMR (CDCl₃) δ ppm: 8.40 (s, 1H, CH=N), 8.35 (s, 1H, CH=N), 8.30 (d, 1H, J = 8.70 Hz, H-8), 8.27 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.07 (d, 1H, J = 1.80, H-5), 8.00 (dd, 1H, J = 8.70, and 1.80 Hz, H-7), 7.92 (d, 2H, J = 8.10 Hz, H-3′and H-5′), 7.88 (d, 2H, J = 8.10 Hz, H-2″, and H-6″), 7.86 (s, 1H, H-3), 7.64 (d, 2H, J = 8.10 Hz, H-2′, and H-6′), 7.61 (d, 2H, J = 7.80 Hz, H-2, and H-6_phe), 7.44 (t, 2H, J = 7.80 Hz, H-3, and H-5_phe), 7.32 (t, 1H, J = 7.80 Hz, H-4_phe), 3.70 (t, 2H, J = 6.90 Hz, NCH₂), 3.68 (t, 2H, J = 6.90 Hz, NCH₂), 2.34 (t, 2H, J = 6.90 Hz, NCH₂), 2.31 (t, 2H, J = 6.90 Hz, NCH₂), 2.23 (s, 6H, N(CH₃)₂), 2.21 (s, 6H, N(CH₃)₂), 1.81–1.71 (m, 4H, 2CH₂), 1.63–1.53 (m, 4H, 2CH₂).

• 2,4-bis{4-[(3-(4-methylpiperazin-1-yl)propyl)iminomethyl]phenyl}-6-phenylquinoline (11c)

Yellow oil (98%); ¹H NMR (CDCl₃) δ ppm: 8.41 (s, 1H, CH=N), 8.37 (s, 1H, CH=N), 8.31 (d, 1H, J = 8.70 Hz, H-8), 8.28 (d, 2H, J = 8.40 Hz, H-3″, and H-5″), 8.07 (d, 1H, J = 1.80 Hz, H-5), 8.01 (dd, 1H, J = 8.70, and 1.80 Hz, H-7), 7.92 (d, 2H, J = 8.40 Hz, H-3′, and H-5′), 7.88 (d, 2H, J = 8.40 Hz, H-2″, and H-6″), 7.87 (s, 1H, H-3), 7.66 (d, 2H, J = 8.40 Hz, H-2′, and H-6′), 7.61 (d, 2H, J = 7.20 Hz, H-2, and H-6_phe), 7.45 (t, 2H, J = 7.20 Hz, H-3, and H-5_phe), 7.36 (t, 1H, J = 7.20 Hz, H-4_phe), 3.72 (t, 2H, J = 6.90 Hz, NCH₂), 3.68 (t, 2H, J = 6.90 Hz, NCH₂), 2.71–2.31 (m, 20H, 2 NCH₂ and 8 NCH₂), 2.30 (s, 6H, 2NCH₃), 2.00–1.89 (m, 4H, 2CH₂).

• 2,4-bis{4-[(4-(4-methylpiperazin-1-yl)butyl)iminomethyl]phenyl}-6-phenylquinoline (11d)

Yellow oil (98%); ¹H NMR (CDCl₃) δ ppm: 8.35 (s, 1H, CH=N), 8.31 (s, 1H, CH=N), 8.26 (d, 1H, J = 8.70 Hz, H-8), 8.23 (d, 2H, J = 8.40 Hz, H-3″, and H-5″), 8.02 (d, 1H, J = 1.80 Hz, H-5), 7.97 (dd, 1H, J = 8.70, and 1.80 Hz, H-7), 7.87 (d, 2H, J = 8.40 Hz, H-3′and H-5′), 7.83 (d, 2H, J = 8.40 Hz, H-2″, and H-6″), 7.82 (s, 1H, H-3), 7.62 (d, 2H, J = 8.40 Hz, H-2′, and H-6′), 7.57 (d, 2H, J = 7.30 Hz, H-2, and H-6_phe), 7.40 (t, 2H, J = 7.30 Hz, H-3, and H-5_phe), 7.31 (t, 1H, J = 7.30 Hz, H-4_phe), 3.65 (t, 2H, J = 6.90 Hz, NCH₂), 3.61 (t, 2H, J = 6.90 Hz, NCH₂), 2.68–2.30 (m, 20H, 2 NCH_2, and 8 NCH₂), 2.32 (s, 6H, 2NCH₃), 1.80–1.71 (m, 4H, 2CH₂), 1.67–1.60 (m, 4H, 2CH₂).

• 2,4-bis{4-[(3-dimethylaminopropyl)iminomethyl]phenyl}-8-phenylquinoline (11e)

Yellow oil (88%); ¹H NMR (CDCl₃) δ ppm: 8.44 (s, 1H, CH=N), 8.34 (s, 1H, CH=N), 8.24 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 7.94 (d, 2H, J = 8.10 Hz, H-3′, and H-5′), 7.92 (s, 1H, H-3), 7.87 (dd, 1H, J = 8.40, and 1.50 Hz, H-7), 7.85 (dd, 1H, J = 8.40, and 1.50 Hz, H-5), 7.84–7.81 (m, 4H, H-2″, H-6″, H-2′, and H-6′), 7.65 (d, 2H, J = 8.10 Hz, H-2, and H-6_phe), 7.58–7.52 (m, 3H, H-3_phe, H-5_phe, and H-6), 7.49 (t, 1H, J = 8.10 Hz, H-4_phe), 3.74 (t, 2H, J = 6.90 Hz, NCH₂), 3.68 (t, 2H, J = 6.90 Hz, NCH₂), 2.42 (t, 2H, J = 6.90 Hz, NCH₂), 2.38 (t, 2H, J = 6.90 Hz, NCH₂), 2.29 (s, 6H, N(CH₃)₂), 2.23 (s, 6H, N(CH₃)₂), 1.98–1.85 (m, 4H, 2CH₂).

• General procedure for 2,4-bis[(substituted-aminomethyl)phenyl]-phenylquinazolines 12a–n and 2,4-bis[(substituted-aminomethyl)phenyl]-phenylquinolines 13a–e

To a solution of compounds 10,11 (0.4 mmol) in methanol (10 mL), we added portion-wise at 0 °C sodium borohydride (3.2 mmol, 8 eq.). The reaction mix was then stirred at room temperature for 1 h and subsequently heated under reflux for 1 h. It was then evaporated to dryness under reduced pressure. After cooling, the residue was triturated in water and extracted with dichloromethane (40 mL). The organic layer was separated, dried over sodium sulfate and activated charcoal, and evaporated to dryness. Oils were used without further purification to generate compounds 12,13.

• 2,4-bis{4-[(3-dimethylaminopropyl)aminomethyl]phenyl}-5-phenylquinazoline (12a)

Yellow oil (75%); ¹H NMR (CDCl₃) δ ppm: 8.72 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.16 (dd, 1H, J = 8.40, and 1.20 Hz, H-8), 7.91 (dd, 1H, J = 8.40, and 7.20 Hz, H-7), 7.57 (dd, 1H, J = 7.20, and 1.50 Hz, H-6), 7.48 (d, 2H, J = 8.10 Hz, H-3′, and H-5′), 7.31 (d, 2H, J = 8.10 Hz, H-2″, and H-6″), 7.09–6.95 (m, 7H, H-2′, H-6′, and H-phe), 3.89 (s, 2H, NCH₂Ar), 3.66 (s, 2H, NCH₂Ar), 2.68 (t, 2H, J = 7.20 Hz, NCH₂), 2.58 (t, 2H, J = 7.20 Hz, NCH₂), 2.37–2.33 (m, 4H, 2NCH₂), 2.22 (s, 6H, N(CH₃)₂), 2.20 (s, 6H, N(CH₃)₂), 1.76–1.63 (m, 4H, 2CH₂); ¹³C NMR (CDCl₃) δ ppm: 169.3 (C-2), 160.3 (C-4), 155.0 (C-8a), 144.5 (C-1_phe), 142.4 (C-5, C-4′, and C-4″), 140.0 (C-1″), 137.9 (C-1′), 134.1 (C-7), 131.8 (C-4_phe), 131.6 (C-3 and C-5_phe), 131.1 (C-3″ and C-5″), 130.1 (C-3′ and C-5′), 130.0 (C-8), 129.6 (C-2 and C-6_phe), 129.0 (C-2″ and C-6″), 128.5 (C-2′ and C-6′), 127.9 (C-6), 121.3 (C-4a), 59.5 (NCH₂), 55.2 (NCH₂), 54.9 (NCH₂), 49.3 (NCH₂), 48.8 (NCH₂), 47.0 (N(CH₃)₂), 29.5 (CH₂); MALDI-TOF MS m/z [M + H]⁺ Calcd for C₃₈H₄₇N₆: 587.3862, Found: 587.3845.

• 2,4-bis{4-[(3-dimethylaminopropyl)aminomethyl]phenyl}-6-phenylquinazoline (12b)

Yellow oil (96%); ¹H NMR (CDCl₃) δ ppm: 8.66 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.29 (d, 1H, J = 1.80 Hz, H-5), 8.19 (d, 1H, J = 8.85 Hz, H-8), 8.11 (dd, 1H, J = 8.85, and 1.80 Hz, H-7), 7.90 (d, 2H, J = 8.10 Hz, H-3′, and H-5′), 7.62 (d, 2H, J = 8.10 Hz, H-2″, and H-6″), 7.54 (d, 2H, J = 8.10 Hz, H-2′, and H-6′), 7.49–7.35 (m, 5H, H_phe), 3.93 (s, 2H, NCH₂Ar), 3.88 (s, 2H, NCH₂Ar), 2.75 (t, 2H, J = 7.20 Hz, NCH₂), 2.70 (t, 2H, J = 7.20 Hz, NCH₂), 2.36 (t, 2H, J = 7.20 Hz, NCH₂), 2.33 (t, 2H, J = 7.20 Hz, NCH₂), 2.24 (s, 6H, N(CH₃)₂), 2.22 (s, 6H, N(CH₃)₂), 1.79–1.64 (m, 4H, 2CH₂); ¹³C NMR (CDCl₃) δ ppm: 169.5 (C-2), 161.4 (C-4), 152.8 (C-8a), 144.1 (C-1″), 144.0 (C-6), 141.3 (C-1_phe), 141.0 (C-1′), 138.4 (C-4′), 137.7 (C-4″), 134.5 (C-4_phe), 131.7 (C-3″ and C-5″), 130.9 (C-8), 130.4 (C-3′ and C-5′), 130.1 (C-2′ and C-6′), 129.7 (C-2″, C-6″, C-3, C-5_phe, and C-4_phe), 129.3 (C-7), 128.7 (C-2 and C-6_phe), 125.9 (C-5), 123.2 (C-4a), 59.5 (NCH₂), 55.1 (NCH₂), 49.5 (NCH₂), 49.2 (NCH₂), 46.9 (N(CH₃)₂), 29.3 (CH₂); MALDI-TOF MS m/z [M+3H]⁺ Calcd for C₃₈H₄₉N₆: 589.4018, Found: 589.551.

• 2,4-bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-6-phenylquinazoline (12c)

Yellow oil (97%); ¹H NMR (CDCl₃) δ ppm: 8.66 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.30 (d, 1H, J = 1.20 Hz, H-5), 8.20 (d, 1H, J = 8.70 Hz, H-8), 8.13 (dd, 1H, J = 8.70, and 1.20 Hz, H-7), 7.91 (d, 2H, J = 8.10 Hz, H-3′, H-5′), 7.65 (d, 2H, J = 8.10 Hz, H-2″, and H-6″), 7.57 (d, 2H, J = 8.10 Hz, H-2′, and H-6′), 7.50–7.44 (m, 4H, H-2, H-3, H-5, and H-6_phe), 7.40–7.36 (m, 1H, H-4_phe), 3.94 (s, 2H, NCH₂Ar), 3.89 (s, 2H, NCH₂Ar), 2.78–2.66 (m, 4H, 2NCH₂), 2.30–2.25 (m, 4H, 2NCH₂), 2.21 (s, 6H, N(CH₃)₂), 2.21 (s, 6H, N(CH₃)₂), 1.83–1.74 (m, 4H, 2CH₂), 1.59–1.47 (m, 4H, 2CH₂); ¹³C NMR (CDCl₃) δ ppm: 169.5 (C-8), 161.4 (C-4), 152.8 (C-8a), 144.2 (C-1″), 143.9 (C-6), 141.3 (C-1_phe), 141.1 (C-1′), 138.4 (C-4′), 137.7 (C-4″), 134.5 (C-4_phe), 131.7 (C-3″ and C-5″), 130.9 (C-8), 130.4 (C-3′ and C-5′), 130.1 (C-2′ and C-6′), 129.7 (C-2″, C-6″, C-3, C-5_phe, and C-4_phe), 129.3 (C-7), 128.7 (C-2 and C-6_phe), 125.9 (C-5), 123.2 (C-4a), 65.9 (NCH₂), 61.0 (NCH₂), 55.1 (NCH₂), 50.9 (NCH₂), 50.6 (NCH₂), 46.8 (N(CH₃)₂), 29.3 (CH₂), 29.1 (CH₂), 26.9 (CH₂); MALDI-TOF MS m/z [M + H]⁺ Calcd for C₄₀H₅₁N₆: 615.4175, Found: 615.4152.

• 2,4-bis{4-[(4-(3-methylpiperazin-1-yl)propyl)aminomethyl]phenyl}-6-phenylquinazoline (12d)

Yellow oil (97%); ¹H NMR (CDCl₃) δ ppm: 8.59 (d, 2H, J = 8.25 Hz, H-3″ and H-5″), 8.21 (d, 1H, J = 1.90 Hz, H-5), 8.09 (d, 1H, J = 8.85 Hz, H-8), 8.01 (dd, 1H, J = 8.85, and 1.90 Hz, H-7), 7.82 (d, 2H, J = 8.25 Hz, H-3′and H-5′), 7.52 (d, 2H, J = 8.25 Hz, H-2″, and H-6″), 7.48 (d, 2H, J = 8.25 Hz, H-2′, and H-6′), 7.39 (d, 2H, J = 8.10 Hz, H-2, and H-6_phe), 7.31 (t, 2H, J = 8.10 Hz H-3, and H-5_phe), 7.28 (t, 1H, J = 8.10 Hz H-4_phe), 3.83 (s, 2H, NCH₂Ar), 3.79 (s, 2H, NCH₂Ar), 2.67 (t, 2H, J = 6.90 Hz, NCH₂), 2.62 (t, 2H, J = 6.90 Hz, NCH₂), 2.50–2.25 (m, 20H, 2NCH_{2, and} 8NCH_2pip), 2.19 (s, 3H, J = 7.20 Hz, NCH₃), 2.17 (s, 3H, NCH₃), 1.73–1.61 (m, 4H, 2CH₂); ¹³C NMR (CDCl₃) δ ppm: 169.4 (C-2), 161.3 (C-4), 152.7 (C-8a), 144.1 (C-1″), 144.0 (C-6), 141.2 (C-1_phe), 140.9 (C-1′), 138.2 (C-4′), 137.6 (C-4″), 134.4 (C-4 phe), 131.7 (C-3″ and C-5″), 130.7 (C-8), 130.3 (C-3′ and C-5′), 130.0 (C-2′ and C-6′), 129.5 (C-2″, C-6″, C-3, C-5_phe, and C-4_phe), 129.3 (C-7), 128.6 (C-2 and C-6_phe), 123.1 (C-4a), 58.4 (NCH₂), 56.5 (NCH_2pip), 55.10 (NCH₂), 54.6 (NCH_2pip), 49.7 (NCH₂), 49.5 (NCH₂), 47.4 (NCH₃), 28.3 (CH₂); MALDI-TOF MS m/z [M+2H]⁺ Calcd for C₄₄H₅₈N₈: 698.4784, Found: 698.167.

• 2,4-bis{4-[(4-(4-methylpiperazin-1-yl)butyl)aminomethyl]phenyl}-6-phenylquinazoline (12e)

Yellow oil (97%); ¹H NMR (CDCl₃) δ ppm: 8.64 (d, 2H, J = 8.25 Hz, H-3″, and H-5″), 8.31 (d, 1H, J = 1.80 Hz, H-5), 8.20 (d, 1H, J = 8.70 Hz, H-8), 8.13 (dd, 1H, J = 8.70, and 1.80 Hz, H-7), 7.90 (d, 2H, J = 8.25 Hz, H-3′and H-5′), 7.64 (d, 2H, J = 8.25 Hz, H-2″, and H-6″), 7.56 (d, 2H, J = 8.25 Hz, H-2′, and H-6′), 7.49–7.44 (m,4H-2, H-3, H-5, and H-6 _phe), 7.41–7.36 (m, 1H, H-4 _phe), 3.92 (s, 2H, NCH₂Ar), 3.88 (s, 2H, NCH₂Ar), 2.72–2.58 (m, 4H, 2NCH₂), 2.50–2.29 (m, 20H, 2NCH_{2, and} 8NCH_2pip), 2.27 (s, 6H, 2NCH₃), 2.58–1.53 (m, 8H, 4CH₂); ¹³C NMR (CDCl₃) δ ppm: 169.5 (C-2), 161.4 (C-4), 152.8 (C-8a), 144.1 (C-1″), 144.0 (C-6), 141.3 (C-1_phe), 141.1 (C-1′), 138.3 (C-4′), 137.7 (C-4″), 134.5 (C-4 phe), 131.7 (C-3″ and C-5″), 130.8 (C-8), 130.4 (C-3′ and C-5′), 130.1 (C-2′ and C-6′), 129.7 (C-2″ and C-6″), 129.6 (C-3, C-5_phe, and C-4_phe), 129.2 (C-7), 128.7 (C-2 and C-6_phe), 123.1 (C-4a), 59.9 (NCH₂), 56.5 (NCH_2pip), 55.20 (NCH₂), 54.5 (NCH_2pip), 50.9 (NCH₂), 50.6 (NCH₂), 47.4 (NCH₃), 29.5 (CH₂), 26.1 (CH₂); MALDI-TOF MS m/z [M + H]⁺ Calcd for C₄₆H₆₁N₈: 725.5019, Found: 725.4992.

• 2,4-bis{4-[(3-dimethylaminopropyl)aminomethyl]phenyl}-7-phenylquinazoline (12f)

Pale yellow oil (98%); ¹H NMR (CDCl₃) δ ppm: 8.63 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.29 (d, 1H, J = 1.50 Hz, H-8), 8.12 (d, 1H, J = 8.70 Hz, H-5), 7.83 (d, 2H, J = 8.10 Hz, H-3′, and H-5′), 7.73 (d, 2H, J = 8.10 Hz, H-2″, and H-6″), 7.71 (dd, 1H, J = 8.70, and 1.50 Hz, H-6), 7.52 (d, 2H, J = 8.10 Hz, H-2′, and H-6′), 7.49–7.37 (m, 5H, H_phe), 3.89 (s, 2H, NCH₂Ar), 3.84 (s, 2H, NCH₂Ar), 2.71 (t, 2H, J = 7.05 Hz, NCH₂), 2.66 (t, 2H, J = 7.05 Hz, NCH₂), 2.31 (t, 2H, J = 7.05 Hz, NCH₂), 2.29 (t, 2H, J = 7.05 Hz, NCH₂), 2.21 (s, 6H, N(CH₃)₂), 2.18 (s, 6H, N(CH₃)₂), 1.75–1.61 (m, 4H, 2CH₂); ¹³C NMR (CDCl₃) δ ppm: 169.1 (C-2), 161.9 (C-4), 153.8 (C-8a), 147.3 (C-1″), 144.5 (C-7), 144.1 (C-1_phe), 140.8 (C-1′), 138.3 (C-4′), 137.6 (C-4″), 131.6 (C-3″ and C-5″), 130.4 (C-3′ and C-5′), 129.9 (C-2′and C-6′), 129.5 (C-2″, C-6″,C-3, C-5, C-4_phe), 128.8 (C-2, C-6_phe, and C-8), 127.8 (C-6 and C-5), 122.0 (C-4a), 59.5 (NCH₂), 55.2 (NCH₂), 49.4 (NCH₂), 49.3 (NCH₂), 46.9 (N(CH₃)₂), 29.4 (CH₂); MALDI-TOF MS m/z [M+2H]⁺ Calcd for C₃₈H₄₈N₆: 588.394, Found: 588.988.

• 2,4-bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-7-phenylquinazoline (12g)

Pale yellow oil (96%); ¹H NMR (CDCl₃) δ ppm: 8.65 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.33 (d, 1H, J = 1.50 Hz, H-8), 8.17 (d, 1H, J = 8.70 Hz, H-5), 7.88 (d, 2H, J = 8.10 Hz, H-3′and H-5′), 7.80–7.76 (m, 3H, H-2″, H-6″, and H-6), 7.56 (d, 2H, J = 8.10 Hz, H-2′, and H-6′), 7.52–7.40 (m, 5H, H_phe), 3.93 (s, 2H, NCH₂), 3.88 (s, 2H, NCH₂), 2.70 (t, 2H, J=6.90 Hz, NCH₂), 2.67 (t, 2H, J=6.90 Hz, NCH₂), 2.31 (t, 2H, J = 6.90 Hz, NCH₂), 2.29 (t, 2H, J=6.90 Hz, NCH₂), 2.22 (s, 6H, N(CH₃)₂), 2.20 (s, 6H, N(CH₃)₂), 1.58–1.52 (m, 8H, 4CH₂); ¹³C NMR (CDCl₃) δ ppm: 169.2 (C-2), 161.9 (C-4), 153.8 (C-8a), 147.4 (C-1″), 144.1 (C-7), 143.9 (C-1_phe), 140.9 (C-1′), 138.5 (C-4′), 137.7 (C-4″), 131.7 (C-3″ and C-5″), 130.4 (C-3′ and C-5′), 130.1 (C-2′ and C-6′), 129.9 (C-4_phe), 129.7 (C-2″, C-6″, and C-8), 127.8 (C-6, C-5), 122.0 (C-4a), 61.0 (NCH₂), 55.0 (NCH₂), 50.6 (NCH₂), 50.2 (NCH₂), 46.7 (N(CH₃)₂), 29.3 (CH₂), 26.9 (CH₂); MALDI-TOF MS m/z [M + H]⁺ Calcd for C₄₀H₅₁N₆: 615.4175, Found: 615.4171.

• 2,4-bis{4-[(5-dimethylaminopentyl)aminomethyl]phenyl}-7-phenylquinazoline (12h)

Yellow oil (93%); ¹H NMR (CDCl₃) δ ppm: 8.65 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.32 (d, 1H, J = 1.50 Hz, H-8), 8.16 (d, 1H, J = 8.70 Hz, H-5), 7.86 (d, 2H, J = 8.10 Hz, H-3′, and H-5′), 7.78–7.52 (m, 3H, H-2″, H-6″, and H-6), 7.54 (d, 2H, J = 8.10 Hz, H-2′, and H-6′), 7.50–7.41 (m, 5H, H_phe), 3.91 (s, 2H, NCH₂), 3.86 (s, 2H, NCH₂), 2.69 (t, 2H, J = 6.90 Hz, NCH₂), 2.66 (t, 2H, J = 6.90 Hz, NCH₂), 2.26 (t, 2H, J = 6.90 Hz, NCH₂), 2.23 (t, 2H, J = 6.90 Hz, NCH₂), 2.20 (s, 6H, N(CH₃)₂), 2.19 (s, 6H, N(CH₃)₂), 1.60–1.44 (m, 8H, 4CH₂), 1.39–1.30 (m, 4H, 2CH₂); ¹³C NMR (CDCl₃) δ ppm: 169.2 (C-2), 161.9 (C-4), 153.8 (C-8a), 147.4 (C-1″), 144.4 (C-7), 144.0 (C-1_phe), 140.9 (C-1′), 138.3 (C-4′), 137.7 (C-4″), 131.6 (C-3″ and C-5″), 130.4 (C-3′, C-5′and C-4_phe), 130.1 (C-2′ and C-6′), 129.9 (C-8), 129.6 (C-2″, C-6″, C-3, and C-5_phe), 128.9 (C-2 and C-6_phe), 127.8 (C-6 and C-5), 122.0 (C-4a), 61.1 (NCH₂), 55.2 (NCH₂), 50.8 (NCH₂), 50.7 (NCH₂), 46.8 (N(CH₃)₂), 31.4 (CH₂), 28.9 (CH₂), 26.6 (CH₂); MALDI-TOF MS m/z [M + H]⁺ Calcd for C₄₂H₅₅N₆: 643.4488, Found: 643.4495.

• 2,4-bis{4-[(4-(3-methylpiperazin-1-yl)propyl)aminomethyl]phenyl}-7-phenylquinazoline (12i)

Yellow oil (97%); ¹H NMR (CDCl₃) δ ppm: 8.66 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.35 (d, 1H, J = 1.50 Hz, H-8), 8.18 (d, 1H, J = 8.70 Hz, H-5), 7.89 (d, 2H, J = 8.10, H-3′, and H-5′), 7.79 (d, 2H, J = 8.10 Hz, H-2″and H-6″), 7.78 (dd, 1H, J = 8.70, and 1.50 Hz, H-6); 7.56 (d, 2H, J = 8.10 Hz, H-2′, and H-6′), 7.52–7.42 (m, 5, H_phe), 3.93 (s, 2H, NCH₂Ar), 3.88 (s, 2H, NCH₂Ar), 2.76 (t, 2H, J = 6.90 Hz, NCH₂), 2.70 (t, 2H, J = 6.90 Hz, NCH₂), 2.58–2.35 (m, 20H, 2NCH_{2, and} 8NCH_2pip), 2.28 (s, 3H, NCH₃), 2.27 (s, 3H, NCH₃),1.82–1.70 (m, 4H, 2CH₂); ¹³C NMR (CDCl₃) δ ppm: 169.2 (C-2), 162.0 (C-4), 153.8 (C-8a), 147.4 (C-1″), 144.0 (C-7), 144.1 (C-1_phe), 140.9 (C-1′), 138.4 (C-4′), 137.7 (C-4″), 131.7 (C-3″ and C-5″), 130.5 (C-3′ and C-5′), 130.1 (C-2′ and C-6′), 129.9 (C-8), 129.6 (C-2″, C-6″, C-3, C-5_phe, and C-4_phe), 128.9 (C-2 and C-6_phe), 127.9 (C-5 and C-6), 122.0 (C-4a), 58.4 (NCH₂), 56.6 (NCH_2pip), 55.2 (NCH₂), 54.7 (NCH_2pip), 49.6 (NCH₂), 49.5 (NCH₂), 47.4 (NCH₃), 28.4 (CH₂); MALDI-TOF MS m/z [M + H]⁺ Calcd for C₄₄H₅₇N₈: 697.4706, Found: 697.516.

• 2,4-bis{4-[(4-(4-methylpiperazin-1-yl)butyl)aminomethyl]phenyl}-7-phenylquinazoline (12j)

Yellow oil (96%); ¹H NMR (CDCl₃) δ ppm: 8.67 (d, 2H, J = 8.20 Hz, H-3″, and H-5″), 8.37 (d, 1H, J = 1.80 Hz, H-8), 8.20 (d, 1H, J = 8.70 Hz, H-5), 7.90 (d, 2H, J = 8.20 Hz, H-3′, and H-5′), 7.81 (dd, 1H, J = 8.70, and 1.80 Hz, H-6), 7.80 (d, 2H, J = 8.20 Hz, H-2″, and H-6″), 7.59 (d, 2H, J = 8.20 Hz, H-2′, and H-6′), 7.53 (d, 2H, J = 7.80 Hz, H-2, and H-6_phe), 7.51–7.45 (m, 3H-2, H-3, H-4, and H-5 _phe), 3.96 (s, 2H, NCH₂), 3.91 (s, 2H, NCH₂), 2.76–2.67 (m, 4H, 2NCH₂), 2.53–2.32 (m, 20H, 2NCH_{2, and} 8NCH_2pip), 2.29 (s, 3H, NCH₃), 2.28 (s, 3H, NCH₃), 1.62–1.55 (m, 8H, 4CH₂); ¹³C NMR (CDCl₃) δ ppm: 169.1 (C-2), 161.9 (C-4), 153.8 (C-8a), 147.4 (C-1″), 143.7 (C-7), 143.5 (C-1_phe), 140.9 (C-1′), 138.3 (C-4′), 137.7 (C-4″), 131.7 (C-3″ and C-5″), 130.5 (C-3′,C-5′, and C-4_phe), 130.2 (C-2′ and C-6′), 129.8 (C-2″, C-6″,C-3, and C-5_phe), 128.9 (C-2, C-6_phe, and C-8), 127.9 (C-5 and C-6), 122.0 (C-4a), 59.8 (NCH₂), 56.4 (NCH_2pip), 54.80 (NCH₂), 54.5 (NCH_2pip), 50.6 (NCH₂), 50.5 (NCH₂), 47.4 (NCH₃), 29.5 (CH₂), 26.1 (CH₂); MALDI-TOF MS m/z [M + H]⁺ Calcd for C₄₆H₆₁N₈: 725.5019, Found: 725.5022.

• 2,4-bis{4-[(3-dimethylaminopropyl)aminomethyl]phenyl}-8-phenylquinazoline (12k)

Pale yellow oil (86%); ¹H NMR (CDCl₃) δ ppm: 8.53 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.08 (dd, 1H, J = 8.40, and 1.50 Hz, H-7), 7.91 (dd, 1H, J = 8.40, and 1.50 Hz, H-5), 7.86–7.83 (m, 4H, H-3′,H-5′, H-2″, and H-6″), 7.57–7.51 (m, 5H, H-2′,H-6′, H-6, H-2_phe, and H-6_phe), 7.47–7.42 (m, 1H, H-4_phe), 7.39–7.35 (m, 2H, H-3, and H-5_phe), 3.91 (s, 2H, NCH₂), 3.82 (s, 2H, NCH₂), 2.74 (t, 2H, J = 6.90 Hz, NCH₂), 2.65 (t, 2H, J = 7.20 Hz, NCH₂), 2.37 (t, 2H, J = 6.90 Hz, NCH₂), 2.32 (t, 2H, J = 6.90 Hz, NCH₂), 2.25 (s, 6H, N(CH₃)₂), 2.21 (s, 6H, N(CH₃)₂), 1.80–1.64 (m, 4H, 2CH₂); ¹³C NMR (CDCl₃) δ ppm: 167.5 (C-2), 158.3 (C-4), 148.5 (C-8a), 141.7 (C-1″), 141.4 (C-8), 139.3 (C-1_phe), 137.5 (C-1′), 136.2 (C-4′), 135.6 (C-4″), 132.8 (C-4_phe), 130.0 (C-3″ and C-5″, C-3′ and C-5′), 129.4 (C-3 and C-5_phe), 127.8 (C-2″ and C-6″), 127.3 (C-2′and C-6′), 126.9 (C-2, C-8, and C-6_phe), 126.5 (C-7), 125.5 (C-5), 125.4 (C-6), 121.1 (C-4a) 57.1 (NCH₂), 52.8 (NCH₂), 52.4 (NCH₂), 47.0 (NCH₂), 46.8 (NCH₂), 44.6 (N(CH₃)₂), 44.5 (N(CH₃)₂), 27.0 (CH₂), 26.9 (CH₂); MALDI-TOF MS m/z [M + H]⁺ Calcd for C₃₈H₄₇N₆: 587.3862, Found: 587.3851.

• 2,4-bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-8-phenylquinazoline (12l)

Pale yellow oil (91%); ¹H NMR (CDCl₃) δ ppm: 8.52 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.06 (dd, 1H, J = 8.10, and 1.50 Hz, H-7), 7.88 (dd, 1H, J = 8.10, and 1.50 Hz, H-5), 7.84–7.81 (m, 4H, H-3′, H-5′, H-2″, and H-6″), 7.54–7.50 (m, 5H, H-2′,H-6′, H-2, H-6, and H-6_phe), 7.45–7.42 (m, 1H, H-4_phe), 7.39–7.36 (m, 2H, H-3, and H-5_phe), 3.90 (s, 2H, NCH₂), 3.81 (s, 2H, NCH₂), 2.69 (t, 2H, J = 5.70 Hz, NCH₂), 2.62 (t, 2H, J = 5.70 Hz, NCH₂), 2.26 (t, 2H, J = 5.70 Hz, NCH₂), 2.24 (t, 2H, J = 5.70 Hz, NCH₂), 2.19 (s, 6H, N(CH₃)₂), 2.15 (s, 6H, N(CH₃)₂), 1.54–1.47 (m, 8H, 4CH₂); ¹³C NMR (CDCl₃) δ ppm: 167.5 (C-2), 158.3 (C-4), 148.4 (C-8a), 141.5 (C-1″), 141.3 (C-8), 139.2 (C-1_phe), 137.4 (C-1′), 136.2 (C-4′), 135.6 (C-4″), 132.8 (C-4_phe), 130.0 (C-3″ and C-5″), 129.3 (C-3′ and C-5′), 127.8 (C-3 and C-5_phe), 127.2 (C-2″, C-6″, C-2′, and C-6′), 126.8 (C-2 and C-6_phe), 126.5 (C-7), 125.5 (C-5), 125.4 (C-6), 121.1 (C-4a), 58.6 (NCH₂), 58.5 (NCH₂), 52.7 (NCH₂), 52.6 (NCH₂), 48.3 (NCH₂), 48.1 (NCH₂), 44.4 (N(CH₃)₂), 44.3 (N(CH₃)₂), 26.9 (CH₂), 26.8 (CH₂), 24.5 (CH₂), 24.4 (CH₂); MALDI-TOF MS m/z [M + H]⁺ Calcd for C₄₀H₅₁N₆: 615.4175, Found: 615.4169.

• 2,4-bis{4-[(4-(3-methylpiperazin-1-yl)propyl)aminomethyl]phenyl}-8-phenylquinazoline (12m)

Pale yellow oil (71%); ¹H NMR (CDCl₃) δ ppm: 8.51 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.06 (dd, 1H, J = 8.30, and 1.50 Hz, H-7), 7.88 (dd, 1H, J = 8.30, and 1.50 Hz, H-5), 7.84–7.81 (m, 4H, H-3′, H-5′, H-2″, and H-6″), 7.55–7.48 (m, 5H, H-2′, H-6′, H-2_phe, H-6_phe, and H-6), 7.44–7.42 (m, 1H, H-4 phe), 7.37–7.34 (m, 2H, H-3, and H-5_phe), 3.88 (s, 2H, NCH₂), 3.79 (s, 2H, NCH₂), 2.70 (t, 2H, J = 5.10 Hz, NCH₂), 2.63 (t, 2H, J = 5.10 Hz, NCH₂), 2.53–2.29 (m, 20H, 2NCH_{2, and} 8NCH_2pip), 1.72 (qt, 2H, J = 5.10 Hz, CH₂), 1.66 (qt, 2H, J = 5.10 Hz, 2CH₂); ¹³C NMR (CDCl₃) δ ppm: 167.5 (C-2), 158.3 (C-4), 148.4 (C-8a), 141.7 (C-1″), 141.4 (C-8), 139.3 (C-1_phe), 137.4 (C-1′), 136.1 (C-4′), 135.6 (C-4″), 132.8 (C-4_phe), 130.0 (C-3″, C-5″,C-3′, and C-5′), 129.3 (C-3 and C-5_phe), 127.8 (C-2″ and C-6″), 127.1 (C-2′ and C-6′), 126.8 (C-2 and C-6_phe), 126.5 (C-7), 125.5 (C-5), 125.4 (C-6), 121.1 (C-4a), 56.0 (NCH₂), 55.9 (NCH₂), 54.2 (NCH_2pip), 54.1 (NCH_2pip), 52.7 (NCH₂), 52.3 (NCH_2pip), 52.2 (NCH_2pip), 47.2 (NCH₂), 47.0 (NCH₂), 45.1 (NCH₃), 45.0 (NCH₃), 26.0 (CH₂), 25.8 (CH₂); MALDI-TOF MS m/z [M + H]⁺ Calcd for C₄₄H₅₇N₈: 697.4706, Found: 697.4691.

• 2,4-bis{4-[(4-(4-methylpiperazin-1-yl)butyl)aminomethyl]phenyl}-8-phenylquinazoline (12n)

Pale yellow oil (93%); ¹H NMR (CDCl₃) δ ppm: 8.53 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.09 (dd, 1H, J = 8.40, and 1.20 Hz, H-7), 7.91 (dd, 1H, J = 8.40, and 1.20 Hz, H-5), 7.88–7.83 (m, 4H, H-3′, H-5′, H-2″, and H-6″), 7.58–7.50 (m, 5H, H-2′, H-6′, H-2_phe, H-6_phe, and H-6), 7.48–7.44 (m, 1H, H-4_phe), 7.42–7.40 (m, 2H, H-3, and H-5_phe), 3.92 (s, 2H, NCH₂), 3.83 (s, 2H, NCH₂), 2.71 (t, 2H, J = 5.70 Hz, NCH₂), 2.63 (t, 2H, J = 5.70 Hz, NCH₂), 2.54–2.30 (m, 20H, 2NCH_{2, and} 8NCH_2pip), 1.59–1.50 (m, 8H, 4CH₂); ¹³C NMR (CDCl₃) δ ppm: 168.5 (C-2), 159.3 (C-4), 149.4 (C-8a), 143.0 (C-1″), 142.5 (C-8), 140.2 (C-1_phe), 138.4 (C-1′), 137.1 (C-4′), 136.6 (C-4″), 133.8 (C-4_phe) 131.0 (C-3″ and C-5″), 130.3 (C-3′ and C-5′), 128.8 (C-3 and C-5_phe), 128.2 (C-2″ and C-6″), 128.1 (C-2′ and C-6′), 127.8 (C-2, C-6_phe), 127.5 (C-7), 126.5 (C-5), 126.4 (C-6), 122.1 (C-4a), 58.6 (NCH₂), 58.5 (NCH₂), 55.1 (NCH_2pip), 53.8 (NCH₂), 53.3 (NCH_2pip), 53.2 (NCH_2pip), 49.4 (NCH₂), 49.2 (NCH₂), 46.1 (NCH₂), 28.2 (CH₂), 28.1 (CH₂), 24.8 (CH₂), 24.7 (CH₂); MALDI-TOF MS m/z [M + H]⁺ Calcd for C₄₆H₆₁N₈: 725.5019, Found: 725.5015.

• 2,4-bis{4-[(3-dimethylaminopropyl)aminomethyl]phenyl}-6-phenylquinoline (13a)

Yellow oil (97%); ¹H NMR (CDCl₃) δ ppm: 8.31 (d, 1H, J = 8.70 Hz, H-8), 8.18 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.11 (d, 1H, J = 1.65 Hz, H-5), 7.99 (dd, 1H, J = 8.70, and 1.65 Hz, H-7), 7.83 (s, 1H, H-3), 7.62 (d, 2H, J = 8.10 Hz, H-3′and H-5′), 7.57 (d, 2H, J = 8.10 Hz, H-2″, and H-6″), 7.52 (d, 2H, J = 8.10 Hz, H-2′, and H-6′), 7.48 (d, 2H, J = 7.20 Hz, H-2, and H-6_phe), 7.45 (t, 2H, J = 7.20 Hz, H-3, and H-5_phe), 7.36 (t, 1H, J = 7.20 Hz, H-4 _phe), 3.92 (s, 2H, NCH₂), 3.89 (s, 2H, NCH₂), 2.77 (t, 2H, J = 7.20 Hz, NCH₂), 2.71 (t, 2H, J = 7.20 Hz, NCH₂), 2.38 (t, 2H, J = 7.20 Hz, NCH₂), 2.34 (t, 2H, J = 7.20 Hz, NCH₂), 2.25 (s, 6H, N(CH₃)₂), 2.24 (s, 6H, N(CH₃)₂), 1.81–1.66 (m, 4H, 2CH₂); ¹³C NMR (CDCl₃) δ ppm: 158.0 (C-2), 150.5 (C-4), 149.6 (C-8a), 143.2 (C-1″), 142.3 (C-6), 142.0 (C-1_phe), 140.3 (C-1′), 139.6 (C-4′), 138.3 (C-4″), 131.9 (C-4_phe), 131.0 (C-3″ and C-5″), 130.6 (C-8), 130.2 (C-3′ and C-5′), 129.9 (C-3 and C-5_phe), 129.8 (C-2′ and C-6′), 128.9 (C-2″, C-6″, and C-7), 128.8 (C-2 and C-6_phe), 127.3 (C-4a), 124.8 (C-5), 121.1 (C-3), 59.5 (NCH₂), 55.2 (NCH₂), 49.2 (NCH₂), 47.0 (N(CH₃)₂), 29.4 (CH₂); MALDI-TOF MS m/z [M + H]⁺ Calcd for C₃₉H₄₈N₅: 586.3910, Found: 586.4230.

• 2,4-bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-6-phenylquinoline (13b)

Yellow oil (yield, 97%); ¹H NMR (CDCl₃) δ ppm: 8.29 (d, 1H, J = 8.70 Hz, H-8), 8.17 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.11 (d, 1H, J = 1.80 Hz, H-5), 7.99 (dd, 1H, J = 8.70, and 1.80 Hz, H-7), 7.83 (s,1H,H-3), 7.62 (d, 2H, J = 8.10 Hz, H-3′, and H-5′), 7.58 (d, 2H, J = 8.10 Hz, H-2″, and H-6″), 7.52 (d, 2H, J = 8.10 Hz, H-2′, and H-6′), 7.47 (d, 2H, J = 7.20 Hz, H-2, and H-6_phe), 7.45 (t, 2H, J = 7.20 Hz, H-3, and H-5_phe), 7.35 (t, 1H, J = 7.20 Hz, H-4_phe), 3.92 (s, 2H, NCH₂), 3.88 (s, 2H, NCH₂), 2.74 (t, 2H, J = 6.90 Hz, NCH₂), 2.68 (t, 2H, J = 6.90 Hz, NCH₂), 2.30 (t, 2H, J = 6.90 Hz, NCH₂), 2.28 (t, 2H, J = 6.90 Hz, NCH₂), 2.23 (s, 6H, N(CH₃)₂), 2.22 (s, 6H, N(CH₃)₂), 1.60–1.52 (m, 4H, 2CH₂); ¹³C NMR (CDCl₃) δ ppm: 158.0 (C-2), 150.5 (C-4), 149.6 (C-8a), 143.1 (C-1″), 142.2 (C-6), 142.0 (C-1_phe), 140.3 (C-1′), 139.7 (C-4′), 138.3 (C-4″), 131.8 (C-4_phe), 131.0 (C-3″ and C-5″), 130.6 (C-8), 130.2 (C-3′ and C-5′), 130.0 (C-3 and C-5_phe), 129.8 (C-2′ and C-6′), 129.0 (C-2″, C-6″, and C-7) 128.8 (C-2 and C-6_phe), 127.3 (C-4a), 124.8 (C-5), 121.1 (C-3), 61.0 (NCH₂), 55.1 (NCH₂), 50.9 (NCH₂), 50.6 (NCH₂), 46.8 (N(CH₃)₂), 29.3 (CH₂), 26.8 (CH₂); MALDI-TOF MS m/z [M + H]⁺ Calcd for C₄₁H₅₂N₅: 614.4223, Found: 614.4225.

• 2,4-bis{4-[(4-(3-methylpiperazin-1-yl)propyl)aminomethyl]phenyl}-6-phenylquinoline (13c)

Yellow oil (65%); ¹H NMR (CDCl₃) δ ppm: 8.29 (d, 1H, J = 9.00 Hz, H-8), 8.18 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.11 (d, 1H, J = 1.80, H-5), 7.99 (dd, 1H, J = 9.00, and 1.80 Hz, H-7), 7.83 (s,1H, H-3), 7.62 (d, 2H, J = 8.10 Hz, H-3′and H-5′), 7.57 (d, 2H, J = 8.10 Hz, H-2″, and H-6″), 7.51 (d, 2H, J = 8.10 Hz, H-2′, and H-6′), 7.47 (d, 2H, J = 7.50 Hz, H-2, and H-6_phe), 7.44 (t, 2H, J = 7.50 Hz, H-3, and H-5_phe), 7.36 (t, 1H, J = 7.50 Hz, H-4_phe), 3.92 (s, 2H, NCH₂), 3.88 (s, 2H, NCH₂), 2.78 (t, 2H, J = 6.90 Hz, NCH₂), 2.73 (t, 2H, J = 6.90 Hz, NCH₂), 2.65–2.50 (m, 20H, 2NCH_{2, and} 8NCH_2pip), 2.28 (s, 3H, NCH₃), 2.26 (s, 3H, NCH₃), 1.83–1.69 (m, 4H, 2CH₂); ¹³C NMR (CDCl₃) δ ppm: 158.0 (C-2), 150.5 (C-4), 149.6 (C-8a), 143.2 (C-1″), 142.3 (C-6), 142.0 (C-1_phe), 140.3 (C-1′), 139.7 (C-4′), 138.3 (C-4″), 131.9 (C-4_phe) 131.0 (C-3″ and C-5″), 130.6 (C-8), 130.3 (C-3′ and C-5′), 129.9 (C-3 and C-5_phe), 129.7 (C-2′ and C-6′), 128.9 (C-7, C-2″ and C-6″), 128.8 (C-2 and C-6_phe), 127.3 (C-4a), 124.8 (C-5), 121.0 (C-3), 58.4 (NCH₂), 56.6 (NCH_2pip), 55.1 (NCH₂), 54.7 (NCH_2pip), 47.4 (NCH₃), 28.4 (CH₂); MALDI-TOF MS m/z [M + H]⁺ Calcd for C₄₇H₆₂N₇: 696.4753, Found: 696.4740.

• 2,4-bis{4-[(4-(4-methylpiperazin-1-yl)butyl)aminomethyl]phenyl}-6-phenylquinoline (13d)

Yellow oil (76%); ¹H NMR (CDCl₃) δ ppm: 8.28 (d, 1H, J = 8.70 Hz, H-8), 8.17 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 8.10 (d, 1H, J = 1.80, H-5), 7.99 (dd, 1H, J = 8.70, and 1.80 Hz, H-7),7.82 (s,1H, H-3), 7.62 (d, 2H, J = 8.10 Hz, H-3′and H-5′), 7.57 (d, 2H, J = 8.10 Hz, H-2″, and H-6″), 7.50 (d, 2H, J = 8.10 Hz, H-2′, and H-6′), 7.45 (d, 2H, J = 7.50 Hz, H-2, and H-6_phe), 7.41 (t, 2H, J = 7.50 Hz, H-3, and H-5_phe), 7.33 (t, 1H, J = 7.50 Hz, H-4_phe), 3.92 (s, 2H, NCH₂), 3.88 (s, 2H, NCH₂), 2.74 (t, 2H, J = 6.60 Hz, NCH₂), 2.67 (t, 2H, J = 6.60 Hz, NCH₂), 2.50–2.45 (m, 20H, 2NCH_{2, and} 8NCH_2pip), 2.28 (s, 6H, 2NCH₃), 1.59–1.53 (m, 8H, 4CH₂); ¹³C NMR (CDCl₃) δ ppm: 158.0 (C-2), 150.5 (C-4), 149.6 (C-8a), 143.2 (C-1″), 142.3 (C-6), 142.0 (C-1_phe), 140.3 (C-1′), 139.7 (C-4′), 138.4 (C-4″), 131.9 (C-4_phe) 131.0 (C-3″ and C-5″), 130.6 (C-8), 130.0 (C-3′ and C-5′), 129.8 (C-2′ and C-6′), 128.9 (C-2″ and C-6″), 128.8 (C-7, C-2_phe, and C-6_phe), 127.3 (C-4a), 124.8 (C-5), 121.0 (C-3), 59.9 (NCH₂), 56.5 (NCH_2pip), 55.1 (NCH₂), 54.6 (NCH_2pip), 47.4 (NCH₃), 29.5 (CH₂), 26.2 (CH₂); MALDI-TOF MS m/z [M + H]⁺ Calcd for C₄₇H₆₂N₇: 724.5067, Found: 724.5074.

• 2,4-bis{4-[(3-dimethylaminopropyl)aminomethyl]phenyl}-8-phenylquinoline (13e)

Yellow oil (83%); ¹H NMR (CDCl₃) δ ppm: 8.16 (d, 2H, J = 8.10 Hz, H-3″, and H-5″), 7.92 (dd, 1H, J = 8.40, and 1.50 Hz, H-7), 7.90 (d, 2H, J = 8.10 Hz, H-3′, and H-5′), 7.88 (s, 1H, H-3), 7.79 (dd, 1H, J = 8.40, and 1.50 Hz, H-5), 7.58–7.35 (m, 10H, H-2″, H-6″, H-2′, H-6′, H-6, and H_phe), 3.94 (s, 2H, NCH₂), 3.85 (s, 2H, NCH₂), 2.79 (t, 2H, J = 6.90 Hz, NCH₂), 2.69 (t, 2H, J = 6,90 Hz, NCH₂), 2.37 (t, 2H, J = 6.90 Hz, NCH₂), 2.33 (t, 2H, J = 6.90 Hz, NCH₂), 2.27 (s, 6H, N(CH₃)₂), 2.26 (s, 6H, N(CH₃)₂), 1.80–1.68 (m, 4H, 2CH₂); ¹³C NMR (CDCl₃) δ ppm: 156.6 (C-2), 150.6 (C-4), 147.5 (C-8a), 143.1 (C-1″), 142.4 (C-8), 142.1 (C-1_phe), 141.3 (C-1′), 139.5 (C-4′), 138.8 (C-4″), 132.6 (C-3″ and C-5″), 131.7 (C-4_phe), 131.1 (C-3′ and C-5′), 129.9 (C-3 and C-5_phe), 129.7 (C-2″ and C-6″), 129.0 (C-2′and C-6′), 128.9 (C-2 and C-6_phe), 128.4 (C-7), 127.7 (C-4a), 127.3 (C-5), 126.7 (C-6), 119.8 (C-3), 59.4 (NCH₂), 55.1 (NCH₂), 49.4 (NCH₂), 49.2 (NCH₂), 46.9 (N(CH₃)₂), 29.4 (CH₂); MALDI-TOF MS m/z [M + H]⁺ Calcd for C₃₉H₄₈N₅: 586.3910, Found: 586.5060.

• 2-(4-Formylphenyl)-5-phenyl-3H-quinazolin-4-one (14)

White crystals (10%); m.p. = 252 °C. ¹H NMR (CDCl₃) δ ppm: 11.99 (s, 1H, NH), 10.13 (s, 1H, CHO), 8.16 (d, 2H, J = 8.40 Hz, H-3′, and H-5′), 7.85 (dd, 1H, J = 7.50, and 1.40 Hz, H-6), 7.82 (d, 2H, J = 8.40 Hz, H-2′and H-6′), 7.79 (dd, 1H, J = 8.40, and 7.50 Hz, H-7), 7.47–7.29 (m, 6H, H-8, and H-phe); ¹³C NMR (CDCl₃) δ ppm: 191.7 (CHO), 163.6 (CON), 150.6 (C-2), 143.5 (C-8a), 141.6 (C-4′), 137.7 (C-1_phe), 137.4 (C-1′), 133.8 (C-7), 131.0 (C-5), 130.1 (C-3′ and C-5′), 129.0 (C-3_phe and C-5_phe), 128.1 (C-4_phe), 127.9 (C-2_phe and C-6_phe), 127.5 (C-2′ and C-6′), 127.2 (C-6), 117.6 (C-8); MS-ES⁺ m/z [M + H]⁺ Calcd for C₂₁H₁₅N₂O₂: 327.1134, Found: 327.1130.

3.2. FRET-Melting Experiments

FRET-melting experiments were conducted on a Stratagene MX3005P (Thermo Fisher Scientific, Illkirch-Graffenstaden, France) real-time PCR equipment in 96-well plates on the DNA sequences reported in Table 4. Experiments were performed in 10 mM lithium cacodylate buffer (pH 7.2) and either 10 mM KCl and 90 mM LiCl (FK-RAST, FBCL-2T, F21T, and FdxT) or 1 mM KCl and 99 mM LiCl (Fc-MYCT) concentrations, depending on the T_m of the G4s alone. The DNA concentration was 0.2 µM. The stabilization induced by the compounds was calculated as the difference (ΔTm) between the average transition temperature of the nucleic acid alone and that measured with the appropriate ligand concentration (2 µM). Data are presented as an average of three independent measurements, each conducted in duplicate conditions (λ_exc = 492 nm, λ_em = 516 nm, T interval = 25–95 °C, ramp: 25 °C for 5 min, then 1 °C/min, measurements every 1 °C, 8× magnification of the fluorescence signal).

3.3. Native MS

The affinity and stoichiometry of binding to oligonucleotides were determined by native electrospray ionization mass spectrometry in the negative ion mode on a Thermo Orbitrap Exactive mass spectrometer calibrated daily, and operated in negative mode, on a 400–3000 m/z scan range, using the 50,000 resolution setting, with the following tuning parameters: spray voltage: 3.2 kV, capillary temperature: 170 °C, sheath gas: 60, aux gas: 0, heater temperature: 35 °C, tube lens voltage: −175 V, capillary voltage: −17 V, skimmer voltage: −22 V. The syringe injection flow rate was 3 µL/min. These parameters ensured a good signal intensity without disrupting noncovalent complexes, which was verified using d[G₄T₄G₄]₂ as a control [37]. Oligonucleotides were purchased lyophilized, and RP-cartridge purified from Eurogentec (Kanaka Eurogentec, Seraing, Belgium), then buffer exchanged with ammonium acetate (5 M; Sigma-Aldrich, Saint-Quentin-Fallavier, France) using Amicon Ultra (3 K cut-off; Merck Millipore, Cork, Ireland). Sample solutions were prepared by diluting the proper volume of oligonucleotide stock solutions to reach 10 µM of DNA and 20 µM of ligand, in 100 mM ammonium acetate. The solutions were incubated for 16 h at 4 °C before analysis. Kd were calculated as already described [29].

3.4. Circular Dichroism

CD experiments were performed with a JASCO J-1500 spectropolarimeter (JASCO, Lisses, France) using quartz cells of 2 mm path length. The scans were recorded at 22 °C, from 220 to 450 nm with the following parameters: 1.0 nm data pitch, 2 nm bandwidth, 0.5 s response, 50 nm/min scanning speed; they are the result of three accumulations. Solutions were prepared as in Section 3.3. CD data were blank subtracted then normalized against molar dichroic absorption (Δε, in cm⁻¹·M⁻¹) using the equation below, where θ is the ellipticity in millidegrees, C is the oligonucleotide concentration in mol/l, and l is the path length in centimeters:

$$∆\epsilon =\frac{\theta }{\mathrm{32,980}\times C\times l}$$

3.5. Determination of Antiproliferative Effects (MTT Assay)

The growth-inhibitory effects of the compounds were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on selected cell lines, including HeLa and SiHa (cervical cancers), MDA-MB-231 and MCF-7 (breast cancers), and A2780 ovarian cancer cells [38]. All cell lines were purchased from the European Collection of Cell Cultures (Salisbury, UK) except SiHa, which was obtained from the American Tissue Culture Collection (Manassas, VA, USA). The cells were kept in minimum essential medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin-amphotericin-B at 37 °C in a humidified atmosphere containing 5% CO₂. Media and supplements were obtained from Lonza Group Ltd., (Basel, Switzerland). Cancer cells were seeded onto 96-well plates (5000 cells/well), after an overnight incubation the test compounds were added in six concentrations (0.1, 0.3, 1.0, 3.0, 10.0, 30.0 µM) and incubated for 72 h under cell-culture condition. After the incubation, 20 μL of 5 mg/mL MTT solution was added to each well and incubated for a further 4 h. The medium was removed, and the precipitated formazan crystals were dissolved in DMSO for 30 min under shaking at 37 °C. The absorbance was measured at 545 nm with a microplate reader. Untreated cells were included as controls. Calculations were performed by means of the GraphPad Prism 5.01 software (GraphPad Software Inc., San Diego, CA, USA). At least two independent determinations were performed with 5 parallel wells for each condition.

The human leukemic cell line K562 was grown in RPMI 1640 medium (Life Technology-Invitrogen by Thermo Fisher Scientific, Saint-Aubin, France) supplemented with 10% fetal calf serum (FCS), antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin), and L-glutamine, (Eurobio, Les Ulis, France) at 37 °C, 5% CO₂ in air. The MTS cell proliferation assay (Promega, Charbonnières-les-Bains, France) is a colorimetric assay system, which measures the transformation of a tetrazolium component (MTS) into formazan produced by the mitochondria of viable cells. Cells were washed twice in PBS (Phosphate Buffer Saline) and plated in quadruplicate into microtiter-plate wells in 100 μL culture media with or without our various compounds at increasing concentrations (0, 1, 5, 10, 20, and 50 μM) for 1, 2, and 3 days. After 3 h of incubation at 37 °C with 20 μL MTS/well, the plates were read using an ELISA microplate reader (Thermo, Electrocorporation, Waltham, MA, USA) at 490 nm. The amount of color produced was directly proportional to the number of viable cells. The results are expressed as the concentrations inhibiting cell growth by 50% after a 3-day incubation period. The 50% cytotoxic concentrations (CC₅₀) were determined by linear regression analysis, expressed in μM ± SD (Microsoft Excel).

The MTT proliferation test on the epithelial cell line HEK293 was performed as pre-viously described [39]. The 50% inhibiting concentrations (IC₅₀) were determined by linear regression analysis.

3.6. Telomerase Assay

Telomerase activity was assessed using the TRAP assay kit (TRAPeze^® RT telomerase detection kit, Chemicon, Millipore Sigma S7700, Merck KGaA, Darmstadt, Germany) according to manufacturer’s instructions with some modifications. Briefly, 106 were resuspended in CHAPS lysis buffer and proteins were extracted. Protein extracts and various concentrations of compounds (0 to 5 µM) were used to extend a synthetic telomeric DNA using an enzymatic reaction (30 °C during 20 min), followed by a quantification of telomeric DNA using a telomeric PCR amplification: 95 °C for 3 min, 2 cycles of 95 °C for 20 s, and 49 °C for 20 s, followed by 30 cycles 95 °C for 20 s and 60 °C for 20 s with signal acquisition) in a Stratagene Mx3005P system (Agilent, Les Ulis, France) using specific telomeric PCR quantitative primers [40]. Each sample was measured in technical duplicate with control DNA. For each drug and each dilution, we used biological triplicate of cell lysates.

4. Conclusions

In this study, our team designed and synthesized a series of new 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazoline and 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinoline derivatives 12,13 and their cytotoxic activities against five human cancer cell lines (HeLa, SiHa, MCF-7, MDA-MB-231, A2780) were investigated. Their cytotoxicity on the human leukemic K562 cell line was also evaluated. Their ability to stabilize various oncogene promoter G4-structures (c-MYC, K-RAS, and BCL-2) was also determined through a FRET-melting assay. Native mass spectrometry experiments showed that 12b, 12e, or 13a can bind to the human telomeric sequence and the oncogene promoters BCL-2 in the micromolar range. Significant conformational changes allowed the binding of a second ligand; again, in the micromolar range. Conversely, binding to the parallel G-quadruplex formed by the c-MYC promoters was mostly 1:1 and with no conformational change. By using these biological and biophysical data, preliminary SAR studies on these quinazoline and quinoline compounds were discussed. The pharmacological evaluations of these new nitrogen compounds 12,13 showed antiproliferative activities against the different human and gynecologic cancer cell lines, and also the K562 leukemic cell line. The disubstituted 6- and 7-phenyl-bis(3-dimethylaminopropyl)aminomethylphenyl-quinazolines 12b, 12f, and 12i displayed the most interesting antiproliferative activities against these human cancer cell lines, as well as 6-phenyl-bis(3-dimethylaminopropyl)aminomethylphenylquinoline 13a. Based on our presented results, these novel substituted phenylquinazolines and phenylquinolines can be regarded as attractive cores for the design and synthesis of innovative molecules with potent anticancer actions. The most effective members of the current library are suitable for further mechanistic studies.