Design and synthesis of N-aryl-2-trifluoromethyl-quinazoline-4-amine derivatives as potential Werner-dependent anticancer agents

doi:10.21203/rs.3.rs-3833363/v1

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Design and synthesis of N-aryl-2-trifluoromethyl-quinazoline-4-amine derivatives as potential Werner-dependent anticancer agents

https://doi.org/10.21203/rs.3.rs-3833363/v1

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To discover new Werner (WRN) helicase inhibitors, a series of N-aryl-2-trifluoromethyl-quinazoline-4-amine derivatives were designed and synthesized through structural optimization strategy and the anticancer activities of 25 new target compounds against PC3, K562, and HeLa cell lines were evaluated by MTT assay. Some of it exhibited excellent inhibitory activity against three different cancer cells. In order to further verify whether the anticancer activity of these compounds is dependent on WRN, the PC3 cells with WRN overexpression (PC3-WRN) were constructed to further study their anticancer potence in vitro, the inhibition ratio and IC₂₀ values showed that compounds 6a, 8i, and 13a were more sensitive to PC3-WRN than the control group cells (PC3-NC). The further study demonstrated that 13a was the most sensitivity in PC3-WRN among these tested compounds. In summary, our research provided a series of N-aryl-2-trifluoromethyl-quinazoline-4-amine derivatives as potential WRN-dependent anticancer agents.

N-aryl-2-trifluoromethyl-quinazoline

Werner helicase

Synthetic lethality

Antiproliferative

Genomic instability is one of the key hallmarks of cancer. DNA damage response (DDR) is a protective response to maintain genomic integrity and restore cell homeostasis when cells are subjected to DNA damage and replication stress caused by endogenous factors (such as replication errors, cell metabolism and oxidative stress) or exogenous factors (such as ultraviolet radiation, ionizing radiation and chemical exposure)[1]. Many tumors exhibited abnormal DNA repair or lack of redundancy in DDR which promotes the rapid growth of tumor cells by promoting the accumulation of tumor-driven gene mutations, the generation of tumor heterogeneity and the escape of apoptosis[2]. Major pathways for the repair of different DNA damage include base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR) and so on[3]. Microsatellite Instability (MSI) is the accumulation of insertion or deletion errors at microsatellite repeat sequences in cancerous cells as a result of deficient mismatch repair (dMMR)[4–5]. Features associated with MSI can lead to the formation of a variety of tumors, including CRC (15% with MSI), stomach adenocarcinoma (STAD, 15% with MSI), uterine corpus endometrial carcinoma (UCEC, 20–30% with MSI) and ovarian serous cystadenocarcinoma (OV, 12% with MSI)[6–7].

In view of the importance of mismatch repair (MMR) proteins in suppressing MSI, reports on the interaction between WRN and MMR proteins have highlighted their potential role in MSI cancers[8]. WRN (RecQ3) is one of five human RecQ helicases, the others are RecQ1, BLM (RecQ2), RecQ4 and RecQ5. WRN is engaged with genome caretakers and tumor suppressors to preserve genome homeostasis. Recent developments have elevated WRN helicase to the forefront as a potential target for anticancer therapy[9]. Meanwhile, studies identified WRN helicase as a synthetic lethality (SL) target in cancer cells with MSI that can be used for development of new targeted therapeutic drugs for MSI cancer[10–13]. Up to now, there are few inhibitors of WRN helicase have been reported[14–17]. Therefore, the design and synthesis of novel WRN helicase inhibitors and their application in anticancer research have important application value.

Recently, a number of studies on quinazoline synthesis have been reported and used by several pharmaceutical chemistry groups[18–20]. It is worth pointing out that the quinazoline motifs are frequently found in many anticancer drugs with different mechanisms of action[21–22]. Gefitinib[23], Erlotinib[24], Afatinib[25], and Dacomitinib[26] (Fig. 1), which are quinazoline-based protein kinase inhibitors, have been approved for the treatment of human cancers. Studies have shown that the 2-position without substitution of quinazoline compounds may be oxidized and metabolized by aldehyde oxidase (AOX) in vivo to produce quinazolinone which could cause nephrotoxicity[27]. Therefore, the introduction of metabolically stable groups at the 2-position of the quinazoline ring is a common strategy to avoid the oxidative metabolism. Meanwhile, the adoption of fluorine chemistry in drug discovery is to confer boosted metabolic stability of molecules due to the particular attributes (high electronegativity, low polarizability of C-F bond and small atomic radius) of fluorine[28–30]. As a common fluorine-containing group, the introduction of trifluoromethyl moiety into compounds often enhance pharmacokinetic and pharmacodynamic properties, especially the metabolic stability of the compound[31–33].

Based on the advantages of quinazoline skeleton and trifluoromethyl in drug design, trifluoromethyl was introduced at the 2-position of quinazoline to block its potential metabolic sites. Our group synthesized a series of quinazoline derivatives and constructed a small molecular library. As mentioned above, WRN is a potential anticancer target, but its inhibitors have been less developed. Therefore, in order to obtain a novel small molecule inhibitor of WRN, we screened the available library of small molecules by comparative test of the proliferation inhibitory activity of the compounds against PC3-WRN and PC3-NC, it was found that compound 6a exhibited a WRN-dependent inhibitory activity on cell proliferation, but its anticancer activity is moderate. In this study, we focused on the structure optimization of compound 6a by replacing the piperazine ring group or modifying its 4'-piperazine ring and 25 derivatives were synthesized. Subsequently, the anticancer activities of the target compounds were evaluated using MTT assay in vitro. Then the compounds with better activity were selected for WRN inhibition assay, colony formation assay, and molecular docking to explore the possible mechanism of action.

Chemistry

The target compounds 5a, 5c, 6a-6c, and 7a-7b were prepared by a synthetic route shown in Scheme 1. Firstly, intermediate 3 was synthesized from anthranilamide in the three-steps by trifluoroacetylation reaction, intramolecular cyclization, and chlorination reaction according to the known procedure[34]. Then, the coupling reaction of intermediate 3 with 4-(4-Boc-piperazin-1-yl)aniline, 4-benzyloxyaniline or 4-(4-benzyl-piperazin-1-yl)aniline in the presence of NaH provided 4a-4c, respectively. Methylation of 4a-4c afforded the compounds 5a-5c. The N-Boc of 5a was deprotected by trifluoroacetic acid (TFA) to obtain the target compound 6a. Compounds 5b or 5c was subjected to debenzylation using palladium on charcoal (10% Pd/C) as catalyst to obtain the target compounds 6b or 6c, respectively. Reaction of 6a and 6b with methyl iodide or ethyl iodide generated the corresponding compounds 7a or 7b, respectively.

The synthetic routes of target compounds 8a-8j and 9a-9f are shown in Scheme 2. The target compounds 8a-8j were obtained by various substitutions of the compound 6a. Hydrolysis of the esters (8a) in the presence of lithium hydroxide monohydrate afforded compound 9a. The deprotection of the N-Boc group from compounds 8b or 8c generated 9b or 9c in the presence of TFA. The S_N2 reaction of compound 8d with dimethylamine, phenol, or imidazole to synthesis the compounds 9d-9f, respectively.

The synthetic routes of target compounds 13a-13b and 17a-17c are shown in Scheme 3. Reaction of 3 with N-methyl-4-nitroaniline generated 10. Reduction of nitro group with stannous chloride dihydrate afforded compound 11 which was condensed with corresponding amino acid to yield compounds 12a-12b. The N-Boc of 12a and 12b were deprotected by TFA to obtain compounds 13a-13b, respectively. Reaction of intermediate 3 with 4-aminobenzoic acid generated the intermediate 14. Methylation of intermediate 14 produced the intermediate 15. Subsequently, intermediate 15 was treated with KOH aqueous solution to obtain compound 16 which was condensed with corresponding amine to afford the compounds 17a-17c, respectively.

Biological activity evaluation

To determine whether the anticancer activity of the target compound is WRN-dependent, the WRN expression levels in PC3, HeLa, and K562 cells were assessed by Western blot. The results showed that WRN protein expression was highest in K562 cells, followed by Hela cells, while the expression was lowest in PC3 cells (Fig. 3A). Then the anticancer activity of 25 novel compounds against PC3, HeLa, and K562 cells were evaluated by MTT assay using doxorubicin, paclitaxel, and WRN inhibitor (NSC 617145) as positive control. The results showed that some of the target compounds exhibited better anticancer activity against K562 cell line, the cell inhibition of compounds 6a, 6b, 7b, 8d, 8i, 13a, 13b, 17b, and 17c were around 70% in K562 cells, while most of these compounds were only around 50% in PC3 and HeLa cells which have lower WRN expression than K562 cells (Fig. 2).

Then the half-maximal inhibitory concentration (IC₅₀) values of active title compounds against PC3, K562, and HeLa were further evaluated. As shown in Table 1, compounds 6a, 6b, 7b, 8d, 8i, 13a, 13b, and 17c were more sensitive in K562 than in PC3 cells. Compared with HeLa cells, 6a, 7b, 13a, 13b, and 17c were more sensitive in K562 (Table 1). Compared with 6a, the most of the target compounds showed moderate antiproliferative activity on PC3 cells, K562 cells, and HeLa cells. Compounds 7b and 17b showed better anticancer activity against PC3, with the IC₅₀ values of 0.0048 and 0.0031 µM, than those of paclitaxel (0.0308 µM), doxorubicin (1.2661 µM), and NSC 617145 (0.1846 µM). In addition, compounds 6b, 7b, and 17b exhibited remarkable anticancer activity against K562, their IC₅₀ values were 0.0102, 0.0011, and 0.0034 µM, which were even better than those of paclitaxel (0.0356 µM), doxorubicin (2.689 µM), and NSC 617145 (0.0203 µM). Meanwhile, compounds 6b, 7b, and 17b had stronger anticancer activity against HeLa cells, their IC₅₀ values were 0.0135, 0.0069, and 0.0037 µM, which were even better than those of paclitaxel (0.0750 µM), doxorubicin (1.2400 µM), and NSC 617145 (0.237 µM).

Structure–activity relationships

In summary, 25 target compounds were synthesized by optimizing the structure of compound 6a, including the following two types of derivatives: (1) Piperazine derivatives obtained by introducing different substituents on the NH of the 4'-piperazine ring; (2) Non-piperazine derivatives obtained by replacing the 4'-piperazine ring with hydroxyl, alkoxy, amino, substituted amino, carboxyl, alkoxycarbonyl, alkaminocarbonyl. The anticancer structure-activity relationship analysis is as follows: Piperazine derivatives: (1)The introduction of bulky group to the NH of 4'-piperazine ring in 6a causes a significant decrease in anticancer activity, such as 5a (N-Boc substitution), 5c (N-benzyl), 8j (N-(4-cyanobenzyl)), 9e (N-(2-phenoxyacetyl)). (2) When the NH of 6a was methylated to obtain the target compound 7a, which exhibited reduced anticancer activity against K562 and HeLa cells and no activity against PC3 cells. (3) After acylation modification of compound 6a, 8d (N-(2-chloroacetyl)) and 8i (N-acetyl) were obtained, and demonstrate better activity with IC₅₀ values of 0.355–6.351 µM. (4) The introduction of electrophilic warheads (Michael acceptors) did not give desirable activity, as seen in compounds 8f (N-(4-(dimethylamino)but-2-enoyl) substitution) and 9a (N-(3-carboxyallyl)). Non-piperazine derivatives: (1) When the piperazine ring was replaced by carbamoyl, compound 17b was found to exhibit potent anticancer activity against the three cancer cells reaching IC₅₀ values of 0.003 µM. Nevertheless, compound 17b showed poor selectivity for WRN. (2) Compared with 6a, the replacing of the piperazine ring with hydroxy (6b) or ethoxy (7b) groups leads to a marked enhancement in activity against K562 cells. Compound 7b displayed excellent anticancer activity against K562 cells with an IC₅₀ of 0.001 µM. (3) The replacement of the piperazine ring with the aromatic amino carbonyl (17b), which lead to decrease or loss of anticancer activity. (4) Replacing the piperazine ring with L-leucinamido or L-prolinamido, the anticancer activity of the target compound on K562 cells was maintained and it is worth mentioning that the selectivity of compound 13a (N-Methyl-N-4-(L-leucinamido)phenyl-2-(trifluoromethyl)quinazolin-4-amine) to WRN was significantly improved. To sum up, compounds 7b and 17b demonstrate remarkable anticancer activity, although their selectivity to WRN requires improvement. Meanwhile, compounds 8i and 13a display satisfactory anticancer activity and favorable selectivity against WRN.

Evaluation of WRN-dependent cell survival inhibition by quinazoline compounds

To further explore the sensitivity of N-aryl-2-trifluoromethyl-quinazoline-4-amine derivatives to WRN helicase, the lentiviruses with WRN were transfected in PC3 cells to construct WRN overexpression cell. And the western blot assay confirmed WRN overexpression PC3 cell line (PC3-WRN) was successful, PC3-NC cell as a negative control (Fig. 3B). The WRN sensitivity of the quinazoline derivatives were examined in PC3-NC and PC3-WRN cells. The results are shown in Fig. 3C, 6a, 8i, 8d, 13a, 13b, and 17c were more sensitive to PC3-WRN cells than the control group. The IC₂₀ values of 6a, 8i, and 13a in PC3-WRN were significantly lower than the control group cells PC3-NC (Table 2). These results suggested that the compounds 6a, 8i, and 13a might be potential WRN-targeting anticancer agents.

Table 2

The selective of compounds against WRN overexpression PC3
Compound	IC₂₀(µM)
Compound	PC3 NC ^a	PC3 WRN
6a	3.0823 ± 2.3398	0.0327 ± 0.0358^***
6b	0.0001 ± 0.0001	0.0007 ± 0.0007
7b	0.0004 ± 0.0003	0.0003 ± 0.0001
8d	0.5119 ± 0.1042	0.1155 ± 0.0122^*
8i	0.8437 ± 0.0679	0.0055 ± 0.0010^**
13a	0.1011 ± 0.0668	0.0002 ± 0.0001^***
13b	1.2504 ± 0.0560	0.4536 ± 0.0785^*
17b	0.0004 ± 0.0003	0.0001 ± 0.0001
17c	1.4158 ± 0.5998	0.3256 ± 0.0303^*
* P < 0.05, ** P < 0.01, compared with positive control; ^a Used as a positive control

Evaluation of WRN-dependent clone formation inhibition by compounds 6a, 8i, and 13a

The cell survival inhibition of the active compounds were further evaluated by colony formation assay. As shown in Fig. 4, 6a, 8i, and 13a significantly inhibited the cell proliferation of PC3-WRN at 500 nM and 1000 nM compared with PC3-NC. These results confirmed the N-aryl-2-trifluoromethyl-quinazoline-4-amine derivatives inhibited cancer which with high WRN expression cell survival.

Molecular docking

Compared with PC3-NC, compounds 6a, 8i, and 13a exhibited higher inhibitory activity against PC3-WRN at 500 nM and 1000 nM, thus compounds 6a, 8i, and 13a were selected as ligands for molecular docking calculation to predict their possible binding mode with WRN. The molecular docking study was carried out using Molecular Operating Environment (MOE) 2019. The WRN protein (PDB code: 6YHR) was retrieved from the Protein Data Bank (http://www.rcsb.org/pdb) for the docking calculations. According to the docking results, there is a significant overlap between the binding pocket of 6YHR and compound 6a, 8i, or 13a(Fig. 5). The trifluoromethyl group of three active compounds formed H-bond with the amino acid residues of WRN protein (Compound 6a interacting with Ala823 and Phe824. Compound 8i interacting with Gln605, Gln608, Gln582, and Ser578. Compound 13a interacting with Phe824, Gly825, Leu601, and Asn829). In addition, the compounds 6a, 8i, and 13a can also interact with proteins through various interactions, including as H-donors, H-acceptors and π-cations (Table 3).

Table 3

Three active compounds combine with WRN (PDB: 6YHR) and binding interactions
Compounds	Interaction		Interaction distance	ΔG (kcal/mol)
6a	H-donor	Cys-672	3.73	-8.69
		Ser-847	3.97
		Glu-846	3.38
		Glu-669	3.96
		Thr-703	3.81
		Ala-704	3.40
	H-acceptor	Ala-704	4.42
		Ala-704	4.01
		Gln-850	4.37
		Phe-824	3.32
		Ala-823	3.92
		Phe-824	3.36
8i	H-donor	Gly-825	3.81	-8.96
		Ser-578	3.73
		Met-571	3.51
		Ala-572	3.69
		Met-571	3.68
		Thr-703	3.59
		Ala-704	4.41
	H-acceptor	Gln-605	3.24
		Ser-578	3.39
		Lys-577	3.50
		Ala-704	4.11
		Gln-605	3.61
		Gln-605	3.53
		Gln-605	3.21
		Gln-608	3.69
		Ser-578	3.09
		Gln-582	2.84
		Gln-605	3.36
		Ser-578	3.15
	π-cation	Arg-854	4.41
13a	H-donor	Ala-572	4.22	-9.21
		Glu-846	3.79
		Gln-850	4.44
		Ala-704	4.43
		Glu-846	3.67
		Met-571	3.06
	H-acceptor	Gly-825	3.75
		Asn-829	3.36
		Leu-601	4.21
		Phe-824	3.65
		Phe-824	3.87
		Gly-825	3.03
		Lys-577	3.13
	π-cation	Lys-577	3.39
		Arg-854	3.65
		Arg-857	3.93
		Arg-857	3.56

In summary, we designed and synthesized 25 derivatives bearing trifluoromethyl-quinazoline scaffold through structural modification strategy and their antiproliferative activity against HeLa, PC3, and K562 cancer cell lines were evaluated. Some of these quinazoline derivatives demonstrated a good inhibition on cancer cells. Among them, the IC₅₀ values of compounds 7b and 17b are as low as several nanomolar. The WRN overexpression PC3 cells were further constructed, compounds 6a, 8i, and 13a significantly inhibited the cell proliferation of PC3-WRN compared with PC3-NC. Molecular docking was performed to predict the possible binding mode of compounds 6a, 8i, and 13a with WRN. This research provided a series of N-aryl-2-trifluoromethyl-quinazoline-4-amine derivatives as potential WRN-targeting anticancer agents.

Chemistry

All commercia

l reagents and solvents were used as received without further purification unless otherwise indicated. A dry glassware and syringe technique were used to transfer solutions for all reactions, involving air or moisture sensitive compounds in an argon atmosphere. Column chromatography was performed on silica gel (200–300 mesh), and TLC analysis was carried out on silica gel plates GF254 purchased from Qingdao Ocean Chemical Reagent Co. (Qingdao, China). Nuclear magnetic resonance (¹H NMR, ¹³C NMR and ¹⁹F NMR) spectra were recorded on a Bruker Avance NEO 600 spectrometer with tetramethylsilane (TMS) as an internal standard and CDCl₃, CD₃OD or DMSOd₆ as solvents. High resolution mass spectra (HRMS) were obtained with Q Exactive Focus (Thermofish, USA). Melting points were determined on SGW X-4 (Shanghai, China).

The procedure for synthesis of intermediates 1–3

Compound 3 was synthesized via the procedure according to the literature synthesis method[34].

General procedure for the synthesis of intermediates 4a–4c

A suspension of compound 3 (226 mg, 0.97 mmol), 4-(4-Boc-piperazin-1-yl)aniline (200 mg, 0.72 mmol), and NaH (60% in paraffin oil, 52 mg, 2.16 mmol) in 2.5 mL N,N-Dimethylformamide (DMF) was stirred at room temperature overnight. After disappearance of the starting material, the reaction mixture was poured into water and extracted with ethyl acetate (EtOAc). The organic phases was washed with H₂O and brine, dried over MgSO₄, and concentrated under reduced pressure. The crude product was purified by column chromatography to afford the intermediate 4a (300 mg, yield 87%) as a yellow solid. Compounds 4b-4c were synthesized from the intermediate 3 by a procedure similar to the synthesis of 4a.

N-4-(4-(tert-Butoxycarbonyl)piperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (4a) Yellow solid, (300 mg, yield 87%). ¹H NMR (600 MHz, CDCl₃) δ 8.01 (d, J = 8.3 Hz, 1H), 7.98 (dd, J = 6.5, 1.9 Hz, 2H), 7.82–7.80 (m, 1H), 7.69 (d, J = 8.9 Hz, 2H), 7.60–7.58 (m, 1H), 6.92 (d, J = 9.0 Hz, 1H), 3.58–3.56 (m, 6H), 3.39–3.37 (m, 2H), 1.50 (s, 9H).

N-4-(Benzyloxy)phenyl-2-(trifluoromethyl)quinazolin-4-amine (4b) White solid, (357 mg, yield 56%). ¹H NMR (600 MHz, DMSO-d₆) δ 10.19 (s, 1H), 8.64 (d, J = 8.3 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.2 Hz, 1H), 7.79 (s, 1H), 7.76 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.34 (t, J = 7.3 Hz, 1H), 7.10 (d, J = 8.7 Hz, 2H), 5.14 (s, 2H).

N-4-(4-Benzylpiperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (4c) The crude product was used for the next step without further purification.

General procedure for the synthesis of intermediate 5b, target compounds 5a and 5c

To a solution of 4a (100 mg, 0.21 mmol) in 1.5 mL anhydrous DMF was added NaH (60% in paraffin oil, 29 mg, 0.74 mmol) at 0 ^oC and stirred for 5 min. Then the mixture was added CH₃I (36 mg, 0.25 mmol) and stirred overnight at room temperature. The reaction mixture was poured into water and extracted with ethyl acetate (EtOAc). The product was subjected to column chromatography to afford the target compound 5a (88 mg, yield 86%) as a yellow solid. Compounds 5b and 5c were prepared by a method similar to the synthesis of target compound 5a.

N-Methyl-N-4-(4-(tert-butoxycarbonyl)piperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (5a) Yellow solid, (88 mg, yield 86%). mp: 164.5–165.0 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.91 (dd, J = 8.4, 1.4 Hz, 1H), 7.61 (ddd, J = 8.4, 6.8, 1.5 Hz, 1H), 7.16–7.02 (m, 4H), 6.97–6.92 (m, 2H), 3.67–3.56 (m, 7H), 3.19 (t, J = 5.0 Hz, 4H), 1.49 (s, 9H). ¹³C NMR (150 MHz, CDCl₃) δ 162.00, 154.79, 152.22 (q, J = 35.5 Hz), 151.25, 150.35, 139.37, 132.57, 129.26, 127.22, 126.46, 126.44, 120.21 (q, J = 275.9 Hz), 117.53, 116.49, 80.22, 49.08, 43.09, 28.55. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.95. HRMS (ESI) m/z calcd for C₂₅H₂₈F₃N₅O₂Na [M + Na]⁺ 510.2087, found 510.2075.

N-Methyl-N-4-(benzyloxy)phenyl-2-(trifluoromethyl)quinazolin-4-amine (5b) White solid, (72 mg, yield 69.5%). ¹H NMR (600 MHz, DMSO-d₆) δ 7.88 (d, J = 8.3 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 7.48 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.38–7.33 (m, 3H), 7.25 (t, J = 7.8 Hz, 1H), 7.13 (d, J = 8.4 Hz, 2H), 6.95 (d, J = 8.6 Hz, 1H), 5.17 (s, 2H), 3.57 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 161.82, 157.93, 151.24 (q, J = 34.7 Hz), 150.91, 140.05, 137.22, 133.62, 129.14, 128.94, 128.40, 128.25, 128.08, 127.28, 126.44, 120.47 (q, J = 276.2 Hz), 116.89, 116.24, 70.02, 43.16.

N-Methyl-N-4-(4-benzylpiperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (5c) Yellow solid, (60 mg, yield 58%). mp: 102.3–102.7 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.91 (dd, J = 8.3, 1.3 Hz, 1H), 7.61 (ddd, J = 8.3, 6.8, 1.5 Hz, 1H), 7.38–7.32 (m, 4H), 7.28 (t, J = 6.9 Hz, 1H), 7.12–7.08 (m, 3H), 7.06 (dd, J = 8.7, 1.5 Hz, 1H), 6.93 (d, J = 8.9 Hz, 2H), 3.64 (s, 3H), 3.59 (s, 2H), 3.26 (t, J = 5.0 Hz, 4H), 2.64 (t, J = 5.0 Hz, 4H). ¹³C NMR (150 MHz, CDCl₃) δ 161.96, 152.12 (q, J = 35.3 Hz), 151.24, 150.54, 138.71, 137.97, 132.54, 129.31, 129.29, 129.22, 128.46, 127.37, 127.14, 126.53, 126.44, 120.24 (q, J = 275.9 Hz), 116.94, 116.52, 63.14, 53.04, 48.89, 43.12. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.96. HRMS (ESI) m/z calcd for C₂₇H₂₇F₃N₅ [M + H]⁺ 478.2213, found 478.2199.

General procedure for the synthesis of target compounds 6a-6c

Preparation of N-Methyl-N-4-(piperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (6a)

A solution of target compound 5a (56 mg, 0.11 mmol) in TFA∶CH₂Cl₂ (1∶1, 2 mL) was stirred at room temperature for 30 min (monitored by TLC). The reaction mixture was neutralized with saturated K₂CO₃ aqueous solution and extracted with EtOAc, washed with brine, and dried over MgSO₄. After removal of the solvents under reduced pressure, the crude product was purified by column chromatography to afford the target compound 6a (35 mg, yield 88%) as a yellow solid.

N-Methyl-N-4-(piperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (6a) Yellow solid, (35 mg, yield 88%). mp: 151.9–152.9 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.91 (d, J = 8.4 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.14–7.09 (m, 3H), 7.06 (d, J = 8.6 Hz, 1H), 6.94 (d, J = 8.1 Hz, 2H), 4.85 (s, 1H), 3.63 (s, 3H), 3.28 (t, J = 4.9 Hz, 4H), 3.14 (t, J = 4.8 Hz, 4H). ¹³C NMR (151 MHz, CDCl₃) δ 162.00, 152.56, 152.32, 152.21 (q, J = 35.7 Hz), 152.09, 151.85, 151.22, 150.41, 139.37, 132.59, 129.23, 127.20, 126.50, 126.44, 122.95, 121.12, 120.21 (q, J = 276.3 Hz), 119.29, 117.46, 117.36, 116.49, 49.13, 45.32, 43.09. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.94. HRMS (ESI) m/z calcd for C₂₀H₂₁F₃N₅ [M + H]⁺ 388.1744, found 388.1733.

Preparation of N-Methyl-N-4-hydroxyphenyl-2-(trifluoromethyl)quinazolin-4-amine (6b)

Pd/C (21 mg, 0.83 mmol, 10%, 55% wet) was added to a solution of the intermediate 5b (180 mg, 0.43 mmol) in methanol (4mL) and stirred at room temperature under a hydrogen atmosphere for 20 h. The resulting mixture was filtered through diatomite, and the organic layer was concentrated. The crude product was purified by column chromatography to afford the target compounds 6b (163 mg, yield 96.4%) as a yellow solid. Compound 6c was prepared by a method similar to the synthesis of target compound 6b.

N-Methyl-N-4-hydroxy-phenyl-2-(trifluoromethyl)quinazolin-4-amine (6b) Yellow solid, (163 mg, yield 96.4%). mp: 214.8–215.1 ^oC. ¹H NMR (600 MHz, DMSO-d₆) δ 9.87 (s, 1H), 7.85 (dd, J = 8.3, 1.3 Hz, 1H), 7.74 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H), 7.26 (ddd, J = 8.5, 6.9, 1.4 Hz, 1H), 7.20 (d, J = 8.7 Hz, 2H), 6.96 (dd, J = 8.5, 1.2 Hz, 1H), 6.86 (d, J = 8.7 Hz, 2H), 3.55 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 161.22, 156.90, 150.79 (q, J = 34.7 Hz), 150.41, 137.80, 133.09, 128.61, 127.60, 126.72, 126.06, 120.01 (q, J = 276.0 Hz), 116.83, 115.78, 42.79. ¹⁹F NMR (565 MHz, DMSO-d₆) δ -69.49. HRMS (ESI) m/z calcd for C₁₆H₁₃F₃N₃O [M + H]⁺ 320.1005, found 320.0995.

N-Methyl-N-4-(4-(4-hydroxybutyl)piperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (6c) White solid, (75 mg, yield 78.6%). mp: 51.7–52.2 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.90 (dd, J = 8.4, 1.3 Hz, 1H), 7.61 (ddd, J = 8.3, 6.9, 1.4 Hz, 1H), 7.14–7.07 (m, 3H), 7.03 (dd, J = 8.7, 1.3 Hz, 1H), 6.93 (d, J = 8.9 Hz, 2H), 3.63 (s, 3H), 3.61 (t, J = 4.7 Hz, 2H), 3.30 (t, J = 5.0 Hz, 4H), 2.73 (t, J = 5.0 Hz, 4H), 2.50 (d, J = 5.6 Hz, 2H), 1.75–1.66 (m, 4H). ¹³C NMR (150 MHz, CDCl₃) δ 161.96, 152.19 (q, J = 35.4 Hz), 151.19, 150.20, 139.12, 132.58, 129.17, 127.15, 126.51, 126.45, 120.21 (q, J = 275.6 Hz), 117.21, 116.47, 62.75, 58.56, 52.89, 48.44, 43.09, 32.48, 25.23. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.94. HRMS (ESI) m/z calcd for C₂₄H₂₉F₃N₅O [M + H]⁺ 460.2319, found 460.2303.

General procedure for the synthesis of target compounds 7a-7b

Compounds 7a and 7b were prepared by a method similar to the synthesis of target compound 5a.

N-Methyl-N-4-(4-methylpiperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (7a) Yellow solid, (72 mg, yield 70.0%). mp: 270.7–272.8 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.91 (d, J = 8.4 Hz, 1H), 7.61 (ddd, J = 8.3, 6.6, 1.6 Hz, 1H), 7.13–7.09 (m, 3H), 7.07 (dd, J = 8.8, 1.5 Hz, 1H), 6.94 (d, J = 8.7 Hz, 2H), 3.63 (s, 3H), 3.27 (t, J = 5.0 Hz, 4H), 2.60 (t, J = 4.9 Hz, 4H), 2.37 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 151.65, 143.09 (q, J = 30.7 Hz), 142.22, 141.51, 131.32, 125.81, 122.87, 121.06, 120.51, 120.46, 114.99 (q, J = 242.7 Hz), 112.14, 111.72, 57.76, 52.24, 50.00, 47.24.¹⁹F NMR (565 MHz, CDCl₃) δ -70.94. HRMS (ESI) m/z calcd for C₂₁H₂₃F₃N₅ [M + H]⁺ 402.1900, found 402.1891.

N-Methyl-N-4-ethoxyphenyl-2-(trifluoromethyl)quinazolin-4-amine (7b) Yellow solid, (35 mg, yield 46.0%). mp: 75.1–75.3 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.93 (dd, J = 8.4, 1.3 Hz, 1H), 7.63 (ddd, J = 8.3, 7.0, 1.4 Hz, 1H), 7.15 (d, J = 8.8 Hz, 2H), 7.11 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.01 (dd, J = 8.7, 1.3 Hz, 1H), 6.94 (d, J = 8.8 Hz, 2H), 4.07 (q, J = 7.0 Hz, 2H), 3.65 (s, 3H), 1.46 (t, J = 7.0 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 161.35, 157.72, 150.79 (q, J = 33.9 Hz), 150.44, 139.26, 133.17, 128.67, 127.61, 126.85, 125.99, 120.01 (q, J = 275.5 Hz), 115.93, 115.78, 79.19, 63.44, 42.75 ¹⁹F NMR (565 MHz, DMSO-d₆) δ -69.46. HRMS (ESI) m/z calcd for C₁₈H₁₇F₃N₃O [M + H]⁺ 348.1318, found 348.1305.

General procedure for the synthesis of target compounds 8a, 8d and 8g-8j

To a solution of target compound 6a (50 mg, 0.09 mmol) in 2 mL anhydrous CH₂Cl₂ was added K₂CO₃ (53 mg, 0.38 mmol), then the mixture was added 2-thiophenecarbonyl chloride (40 mg, 0.19 mmol) and stirred for 16 h. The reaction mixture was poured into water and extracted with ethyl acetate (EtOAc). The organic layer was washed with brine, dried over MgSO₄, and concentrated under reduced pressure. The crude product was purified by column chromatography to afford the target compound 8a (53 mg, yield 85.0%) as a yellow solid. Compounds 8d and 8g-8j were prepared by a method similar to the synthesis of compound 8a.

(E)-N-Methyl-N-4-(4-(4-methoxy-4-oxobut-2-en-1-yl)piperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (8a) Yellow solid, (53 mg, yield 85.0%). mp: 99.2–100.7 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.91 (dd, J = 8.4, 1.2 Hz, 1H), 7.61 (ddd, J = 8.3, 6.8, 1.5 Hz, 1H), 7.13–7.08 (m, 3H), 7.06 (dd, J = 8.6, 1.5 Hz, 1H), 7.02–6.96 (m, 1H), 6.93 (d, J = 8.9 Hz, 2H), 6.05 (d, J = 15.8, 1.6 Hz, 1H), 3.75 (s, 3H), 3.63 (s, 3H), 3.26 (t, J = 5.0 Hz, 5H), 3.22 (dd, J = 6.2, 1.7 Hz, 2H), 2.65 (t, J = 5.0 Hz, 4H). ¹³C NMR (151 MHz, CDCl₃) δ 166.69, 161.97, 152.22 (q, J = 35.0 Hz), 151.24, 150.33, 144.96, 138.95, 132.55, 129.22, 127.16, 126.49, 126.45, 123.41, 120.22 (q, J = 275.5 Hz), 117.05, 116.51, 59.26, 53.20, 51.76, 48.84, 43.10. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.96. HRMS (ESI) m/z calcd for C₂₅H₂₇F₃N₅O₂ [M + H]⁺ 486.2111, found 486.2095.

N-Methyl-N-4-(4-(2-chloroacetyl)piperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (8d) Yellow solid, (146 mg, yield 81.0%).mp: 165.1–165.5 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.91 (dd, J = 8.4, 1.3 Hz, 1H), 7.62 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.13 (dd, J = 9.1, 2.4 Hz, 2H), 7.10 (dd, 1H), 7.05 (dd, J = 8.6, 1.4 Hz, 1H), 6.95 (d, J = 8.8 Hz, 2H), 4.12 (s, 2H), 3.82 (t, J = 5.2 Hz, 2H), 3.72 (t, J = 5.2 Hz, 2H), 3.63 (s, 3H), 3.30 (t, J = 5.2 Hz, 2H), 3.25 (t, J = 5.2 Hz, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 165.29, 162.03, 152.20 (q, J = 35.1 Hz), 151.25, 149.82, 139.88, 132.62, 129.30, 127.29, 126.49, 126.37, 120.20 (q, J = 275.6 Hz), 117.73, 116.47, 49.31, 48.93, 46.18, 43.07, 42.06, 40.87. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.91. HRMS (ESI) m/z calcd for C₂₂H₂₁ClF₃N₅ONa [M + Na]⁺ 486.1279, found 486.1265.

N-Methyl-N-4-(4-(thiophene-2-carbonyl)piperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (8g) Yellow solid, (110 mg, yield 86.0%). mp: 146.1–146.6 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.92 (dd, J = 8.4, 1.2 Hz, 1H), 7.62 (ddd, J = 8.3, 6.9, 1.4 Hz, 1H), 7.49 (dd, J = 5.0, 1.1 Hz, 1H), 7.35 (dd, J = 3.7, 1.2 Hz, 1H), 7.14 (d, J = 8.9 Hz, 2H), 7.12–7.10 (m, 1H), 7.08 (dd, J = 5.0, 3.7 Hz, 1H), 7.06 (dd, J = 8.7, 1.3 Hz, 1H), 6.95 (d, J = 8.9 Hz, 2H), 3.97–3.93 (m, 4H), 3.64 (s, 3H), 3.34–3.27 (m, 4H). ¹³C NMR (151 MHz, CDCl₃) δ 163.86, 162.04, 152.24 (q, J = 35.5 Hz), 151.26, 139.72, 136.78, 132.62, 129.30, 129.12, 127.30, 126.97, 126.50, 126.40, 120.21 (q, J = 276.3 Hz), 117.56, 116.49, 49.30,43.10. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.93. HRMS (ESI) m/z calcd for C₂₅H₂₂F₃N₅OSNa [M + Na]⁺ 520.1389, found 520.1379.

N-Methyl-N-4-(4-(furan-2-carbonyl)piperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (8h) Yellow solid, (112 mg, yield 91.0%). mp: 130.8–131.6 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.92 (dd, J = 8.4, 1.3 Hz, 1H), 7.62 (ddd, J = 8.2, 6.8, 1.4 Hz, 1H), 7.51 (dd, J = 1.7, 0.8 Hz, 1H), 7.14 (d, J = 8.9 Hz, 2H), 7.12–7.09 (m, 1H), 7.07 (dd, J = 3.5, 0.8 Hz, 1H), 7.05 (dd, J = 8.8, 1.3 Hz, 1H), 6.96 (d, J = 8.9 Hz, 2H), 6.51 (dd, J = 3.5, 1.8 Hz, 1H), 4.08–3.89 (m, 4H), 3.64 (s, 3H), 3.36–3.29 (m, 4H). ¹³C NMR (151 MHz, CDCl₃) δ 162.02, 159.23, 152.22 (q, J = 35.6 Hz), 151.25, 149.99, 147.95, 144.00, 139.58, 132.60, 129.27, 127.28, 126.48, 126.40, 120.20 (q, J = 275.8 Hz), 117.45, 117.07, 116.49, 111.60, 49.30, 43.08. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.93. HRMS (ESI) m/z calcd for C₂₅H₂₂F₃N₅O₂Na [M + Na]⁺ 504.1618, found 504.1603.

N-Methyl-N-4-(4-acetylpiperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (8i) Yellow solid, (99 mg, yield 90.0%). mp: 156.5–156.8 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.91 (d, J = 8.3 Hz, 1H), 7.61 (ddd, J = 8.3, 6.7, 1.4 Hz, 1H), 7.12 (d, J = 8.8 Hz, 2H), 7.10 (dd, J = 7.0, 1.4 Hz, 1H), 7.04 (dd, J = 8.7, 1.3 Hz, 1H), 6.94 (d, J = 8.9 Hz, 2H), 3.80 (t, J = 5.2 Hz, 2H), 3.65 (t, J = 5.2 Hz, 2H), 3.63 (s, 3H), 3.24 (t, J = 5.2 Hz, 2H), 3.21 (t, J = 5.3 Hz, 2H), 2.15 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 169.15, 162.00, 152.20 (q, J = 35.1 Hz), 151.24, 149.99, 139.63, 132.59, 129.27, 127.25, 126.47, 126.38, 120.19 (q, J = 275.7 Hz), 117.57, 116.47, 49.30, 49.03, 46.16, 43.06, 41.30, 21.45. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.92. HRMS (ESI) m/z calcd for C₂₂H₂₂F₃N₅ONa [M + Na]⁺ 452.1669, found 452.1653.

N-Methyl-N-4-(4-(4-cyanobenzyl)piperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (8j) Yellow solid, (101 mg, yield 79.0%). mp: 126.1–126.5 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.91 (dd, J = 8.4, 1.2 Hz, 1H), 7.63 (d, J = 8.2 Hz, 2H), 7.62–7.59 (m, 1H), 7.50 (d, J = 7.9 Hz, 2H), 7.13–7.07 (m, 3H), 7.06 (dd, J = 8.6, 1.5 Hz, 1H), 6.93 (d, J = 8.9 Hz, 2H), 3.63 (s, 5H), 3.29–3.24 (m, 4H), 2.64 (t, J = 5.0 Hz, 4H). ¹³C NMR (151 MHz, CDCl₃) δ 161.97, 152.22 (q, J = 35.6 Hz), 151.24, 150.36, 143.97, 138.95, 132.54, 132.33, 129.63, 129.23, 127.16, 126.48, 126.43, 120.22 (q, J = 275.6 Hz), 119.02, 117.05, 116.50, 111.21, 62.48, 53.11, 48.89, 43.11. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.93. HRMS (ESI) m/z calcd for C₂₈H₂₆F₃N₆ [M + H]⁺ 503.2166, found 503.2151.

General procedure for the synthesis of intermediates 8b and 8c

To a solution of compound 6a (130 mg, 0.34 mmol), N-Boc-l-proline (86 mg, 402 mmol) and O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 255 mg, 0.67 mmol) in dry CH₂Cl₂ (3 mL) were added N,N-Diisopropylethylamine (DIPEA, 0.01 mmol) at 0 ^oC, the reaction mixture was stirred at room temperature for 0.5 h. The mixture was diluted with EtOAC and washed with saturated K₂CO₃ aqueous solution. The organic layer was washed with brine, dried over MgSO₄, and concentrated under reduced pressure. The crude product was used for the next step without further purification. The following compound 8c was obtained by an approach similar to that for the synthesis of compound 8b.

General procedure for the synthesis of target compounds 8e and 8f

To a solution of target compound 6a (100 mg, 0.26 mmol) and trans-4-dimethylaminocrotonic acid hydrochloride (51 mg, 0.31 mmol) in dry CH₂Cl₂ (2 mL) were added DIPEA (128 µL, 0.77 mmol) at 0 ^oC, and stirred for 3 min. Then the mixture was added isobutyl chloroformate (IBCF, 40 µL, 0.31 mmol) and stirred for 48h. The reaction mixture was decanted to water and extracted with EtOAc (30 mL). The organic layer was washed with brine, dried over MgSO₄, filtered, and concentrated under reduced pressure to get the crude product which was purified by column chromatography to afford the compound 8e as a yellow solid (103 mg, yield 81%). The following compound 8f was obtained by an approach similar to that for the synthesis of compound 8e.

N-Methyl-N-4-(4-(isobutoxycarbonyl)piperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (8e) Yellow solid, (103 mg, yield 81.0%). mp: 109.7–111.1 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.91 (dd, J = 8.4, 1.2 Hz, 1H), 7.62 (ddd, J = 8.3, 6.7, 1.4 Hz, 1H), 7.14–7.09 (m, 3H), 7.05 (dd, J = 8.6, 1.4 Hz, 1H), 6.95 (d, J = 2.3 Hz, 1H), 6.94 (d, J = 2.3 Hz, 1H), 3.91 (d, J = 6.6 Hz, 2H), 3.67 (t, J = 5.2 Hz, 4H), 3.63 (s, 3H), 3.21 (t, J = 5.1 Hz, 4H), 2.00–1.92 (m, 1H), 0.96 (s, 3H), 0.95 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 162.03, 155.65, 152.24 (q, J = 35.0 Hz), 151.27, 150.29, 139.52, 132.58, 129.29, 127.25, 126.46, 126.42, 120.22 (q, J = 276.0 Hz), 117.61, 116.51, 71.89, 49.09, 43.08, 28.15, 19.24. ¹⁹F NMR (565 MHz, DMSO-d₆) δ -75.70. HRMS (ESI) m/z calcd for C₂₅H₂₈F₃N₅O₂Na [M + Na]⁺ 510.2087, found 510.2076.

(E)-N-Methyl-N-4-(4-(4-(dimethylamino)but-2-enoyl)piperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (8f) Yellow solid, (89 mg, yield 35.0%). mp: 113.4–113.7 ^oC. ¹H NMR (600 MHz, DMSO-d₆) δ 7.85 (d, J = 8.4 Hz, 1H), 7.73 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H), 7.24 (dd, J = 8.7, 6.6 Hz, 3H), 7.04 (d, J = 8.9 Hz, 2H), 7.01 (dd, J = 8.7, 1.2 Hz, 1H), 6.64 (s, 2H), 3.70 (d, J = 19.3 Hz, 4H), 3.55 (s, 3H), 3.22 (s, 4H), 3.04 (d, J = 3.3 Hz, 2H), 2.15 (s, 6H). ¹³C NMR (151 MHz, DMSO-d₆) δ 164.18, 161.31, 150.82 (q, J = 34.7 Hz), 150.42, 149.76, 142.19, 137.78, 133.09, 128.60, 126.91, 126.77, 126.05, 122.19, 120.01 (q, J = 276.4 Hz), 116.61, 115.85, 59.93, 45.04, 42.65. ¹⁹F NMR (565 MHz, DMSO-d₆) δ -69.48. HRMS (ESI) m/z calcd for C₂₆H₃₀F₃N₆O [M + H]⁺ 499.2428, found 499.2412.

Procedure for synthesis of target compound 9a

A suspension of target compound 8a (100 mg, 0.20 mmol) and lithium hydroxide monohydrate (17 mg, 0.41 mmol) in THF∶H₂O (1∶1, 2 mL) were stirred at room temperature. EtOAc was added to the reaction mixture, which was washed with brine. The organic phases were dried over MgSO₄, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography to give the target compound 9a (72 mg, yield 74.1%) as a yellow solid .

(E)-N-Methyl-N-4-(4-(3-carboxyallyl)piperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (9a) Yellow solid, (72 mg, yield 74.1%). mp: 113.8–114.2 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.92 (d, J = 8.4 Hz, 1H), 7.61 (ddd, J = 8.2, 6.7, 1.4 Hz, 1H), 7.14–7.09 (m, 3H), 7.06 (d, J = 8.4 Hz, 1H), 7.04–6.98 (m, 1H), 6.94 (d, J = 8.8 Hz, 2H), 6.06 (d, J = 15.6 Hz, 1H), 3.63 (s, 3H), 3.38–3.28 (m, 6H), 2.83–2.72 (m, 4H). ¹³C NMR (151 MHz, CDCl₃) δ 169.69, 162.02, 152.24 (q, J = 35.3 Hz), 151.22, 150.03, 143.30, 139.34, 132.61, 129.23, 127.21, 126.51, 126.46, 120.21 (q, J = 275.8 Hz), 117.31, 116.50, 77.37, 58.93, 52.82, 48.33, 43.13, 29.84. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.93. HRMS (ESI) m/z calcd for C₂₄H₂₅F₃N₅O₂ [M + H]⁺ 472.1955, found 472.1943.

Procedure for synthesis of target compounds 9b and 9c

The following compounds 9b and 9c were obtained by an approach similar to that for the synthesis of compound 6a.

N-4-(4-(L-prolyl)piperazin-1-yl)phenyl-N-methyl-2-(trifluoromethyl)quinazolin-4-amine (9b) Yellow solid, (43 mg, yield 86.0%). mp: 172.1–172.5 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.91 (dd, J = 8.5, 1.2 Hz, 1H), 7.61 (ddd, J = 8.4, 6.7, 1.4 Hz, 1H), 7.13 (d, J = 8.8 Hz, 2H), 7.10 (dd, J = 7.0, 1.4 Hz, 1H), 7.04 (dd, J = 8.6, 1.3 Hz, 1H), 6.94 (d, J = 8.9 Hz, 2H), 4.31 (dd, J = 8.7, 6.2 Hz, 1H), 3.94–3.82 (m, 1H), 3.79–3.70 (m, 2H), 3.68–3.63 (m, 1H), 3.63 (s, 3H), 3.32–3.19 (m, 5H), 3.14–3.05 (m, 1H), 2.34–2.24 (m, 1H), 2.01–1.90 (m, 1H), 1.90–1.71 (m, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 170.87, 162.03, 152.20 (q, J = 35.2 Hz), 151.24, 149.77, 139.93, 132.62, 129.30, 127.28, 126.50, 126.35, 120.18 (q, J = 276.0 Hz), 117.74, 116.46, 58.12, 49.29, 49.05, 47.36, 45.07, 43.06, 42.35, 30.76, 26.04. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.93. HRMS (ESI) m/z calcd for C₂₅H₂₈F₃N₆O [M + H]⁺ 485.2271, found 485.2258.

N-4-(4-(L-valyl)piperazin-1-yl)phenyl-N-methyl-2-(trifluoromethyl)quinazolin-4-amine (9c) Yellow solid, (65 mg, yield 82.5%). mp: 75.4–75.9 ^oC. ¹H NMR (600 MHz, CD₃OD) δ 7.79 (dd, J = 8.3, 1.2 Hz, 1H), 7.63 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.14 (d, J = 2.2 Hz, 1H), 7.13 (d, J = 2.2 Hz, 1H), 7.08 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.04–7.01 (m, 3H), 4.07 (d, J = 5.1 Hz, 1H), 3.87–3.80 (m, 1H), 3.78–3.71 (m, 1H), 3.72–3.64 (m, 2H), 3.57 (s, 3H), 3.31–3.21 (m, 4H), 3.23–3.14 (m, 1H), 2.10–2.01 (m, 1H), 1.03 (d, J = 7.0 Hz, 3H), 0.95 (d, J = 6.9 Hz, 3H). ¹³C NMR (151 MHz, CD₃OD) δ 170.96, 163.28, 153.11 (q, J = 35.3 Hz), 151.98, 151.71, 140.37, 134.09, 129.24, 128.30, 127.74, 127.64, 121.40 (q, J = 275.1 Hz), 118.64, 117.34, 56.44, 50.27, 49.80, 46.72, 43.38, 43.23, 31.94, 19.53, 17.07. ¹⁹F NMR (565 MHz, CD₃OD) δ -72.40. HRMS (ESI) m/z calcd for C₂₅H₃₀F₃N₆O [M + H]⁺ 487.2428, found 487.2415.

Procedure for synthesis of target compounds 9d-9f

To a solution of target compound 8d (60 mg, 0.13 mmol) in 1.5 mL anhydrous CH₃CN was added K₂CO₃ (25 mg, 0.51 mmol), then the mixture was added dimethylamine hydrochloride (15 mg, 0.19 mmol) and stirred for 15h. The reaction mixture was decanted to water and extracted with EtOAc (30 mL). The organic layer was washed with brine, dried over MgSO₄, and concentrated under reduced pressure. The crude product was purified by column chromatography to afford the target compound 9d (54 mg, yield 90%) as a yellow solid. The following compounds 9e and 9f were obtained by an approach similar to that for the synthesis of compound 9d.

N-Methyl-N-4-(4-(dimethylglycyl)piperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (9d) Yellow solid, (54 mg, yield 90.0%). mp: 111.1–111.6 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.90 (dd, J = 8.4, 1.2 Hz, 1H), 7.60 (ddd, J = 8.3, 6.8, 1.4 Hz, 1H), 7.14–7.08 (m, 3H), 7.04 (dd, J = 8.7, 1.4 Hz, 1H), 6.94 (d, J = 8.9 Hz, 2H), 3.82–3.76 (m, 4H), 3.62 (s, 3H), 3.26–3.19 (m, 6H), 2.32 (s, 6H). ¹³C NMR (151 MHz, CDCl₃) δ 168.41, 161.99, 152.17 (q, J = 35.1 Hz), 151.20, 150.06, 139.52, 132.58, 129.23, 127.22, 126.46, 126.38, 120.18 (q, J = 276.3 Hz), 117.50, 116.45, 62.44, 49.50, 49.12, 45.50, 45.32, 43.06, 41.65. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.91. HRMS (ESI) m/z calcd for C₂₄H₂₈F₃N₆O [M + H]⁺ 473.2271, found 473.2257.

N-Methyl-N-4-(4-(2-phenoxyacetyl)piperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (9e) Yellow solid, (54 mg, yield 87.0%). mp: 150.3–150.8 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.92 (dd, J = 8.4, 1.2 Hz, 1H), 7.62 (ddd, J = 8.2, 6.8, 1.4 Hz, 1H), 7.34–7.29 (m, 2H), 7.14–7.09 (m, 3H), 7.05 (dd, J = 8.6, 1.3 Hz, 1H), 7.01 (t, J = 7.3, 1.1 Hz, 1H), 6.97 (d, J = 7.9 Hz, 2H), 6.93 (d, J = 8.9 Hz, 2H), 4.75 (s, 2H), 3.81 (t, J = 4.8 Hz, 4H), 3.63 (s, 3H), 3.23 (t, J = 10.3, 5.1 Hz, 4H). ¹³C NMR (151 MHz, CDCl₃) δ 166.79, 162.03, 157.83, 152.22 (q, J = 35.0 Hz), 151.25, 149.89, 139.72, 132.61, 129.84, 129.29, 127.27, 126.49, 126.38, 121.97, 120.20 (q, J = 275.7 Hz), 117.60, 116.48, 114.68, 68.00, 49.47, 49.00, 45.33, 43.08, 42.05. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.92. HRMS (ESI) m/z calcd for C₂₈H₂₇F₃N₅O₂ [M + H]⁺ 544.1931, found 544.1916.

N-Methyl-N-4-(4-(2-(1H-imidazol-1-yl)acetyl)piperazin-1-yl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (9f) Yellow solid, (81 mg, yield 84.0%). mp: 216.5–216.7 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 7.91 (dd, J = 8.4, 1.2 Hz, 1H), 7.62 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.57 (s, 1H), 7.16–7.08 (m, 4H), 7.04 (dd, J = 8.6, 1.3 Hz, 1H), 6.98 (s, 1H), 6.93 (d, J = 8.9 Hz, 2H), 4.86 (s, 2H), 3.82 (t, J = 5.2 Hz, 2H), 3.65 (t, J = 5.2 Hz, 2H), 3.63 (s, 3H), 3.28–3.21 (m, 4H). ¹³C NMR (151 MHz, CDCl₃) δ 164.76, 162.03, 152.19 (q, J = 35.8 Hz), 151.24, 149.62, 140.03, 138.06, 132.64, 129.46, 129.30, 127.30, 126.50, 126.34, 120.27, 120.19 (q, J = 275.7 Hz), 117.76, 116.46, 49.18, 48.96, 48.21, 45.04, 43.06, 42.17. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.90. HRMS (ESI) m/z calcd for C₂₅H₂₅F₃N₇O [M + H]⁺ 496.2067, found 496.2051.

The procedure for synthesis of intermediate 10

A suspension of intermediate 3 (4.50 g, 19.35 mmol), N-methyl-4-nitroaniline (2.68 g, 17.59 mmol), NaH (1.27 g, 52.77 mmol) and DMF (45 mL) were stirred at room temperature for 3 h. The reaction mixture is slowly added with water and saturated brine. The resulting precipitate was collected by filtration to offer the pure intermediate 10 (5.1 g, yield 82.3%) as a gray solid.

N-Methyl-N-4-nitrophenyl-2-(trifluoromethyl)quinazolin-4-amine (10) Yellow solid, (5.1 g, yield 82.3%). ¹H NMR (600 MHz, DMSO-d₆) δ 8.23 (d, J = 9.0 Hz, 2H), 8.03 (dd, J = 8.4, 1.2 Hz, 1H), 7.90 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H), 7.55 (d, J = 9.0 Hz, 2H), 7.46 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.23 (dd, J = 8.6, 1.3 Hz, 1H), 3.73 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 162.85, 152.75, 150.81, 150.72 (q, J = 34.8 Hz), 144.22, 134.18, 129.07, 128.22, 125.92, 125.36, 124.70, 119.88 (q, J = 276.1 Hz), 116.76, 41.39. HRMS (ESI) m/z calculated for C₁₆H₁₂O₂N₄F₃ [M + H]⁺ 349.0906, found 349.0901.

The procedure for synthesis of intermediate 11

To a solution of intermediate 10 (100 mg, 0.29 mmol), stannous chloride dihydrate (194 mg,0.86 mmol) in acetic acid (3 mL) were added HCl (36%, 74 µL, 0.86 mmol), the reaction mixture was stirred at room temperature for 4 h. After TLC indicated that the reaction was complete. The reaction mixture was poured into water and extracted with EtOAc (30 mL). The filtrate was concentrated in vacuo to give the intermediate 11 (60 mg, yield 75.0%) as a yellow solid.

N-Methyl-N-4-aminophenyl-2-(trifluoromethyl)quinazolin-4-amine (11) Yellow solid, (60 mg, yield 75.0%). ¹H NMR (600 MHz, DMSO-d₆) δ 7.83 (d, J = 8.2 Hz, 1H), 7.72 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.25 (ddd, J = 8.5, 6.9, 1.3 Hz, 1H), 7.06 (d, J = 8.6 Hz, 1H), 7.03 (d, J = 8.5 Hz, 2H), 6.66 (d, J = 8.6 Hz, 2H), 5.65 (s, 2H), 3.52 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 161.07, 150.82 (q, J = 33.8 Hz), 150.38, 147.84, 134.97, 132.95, 128.50, 126.97, 126.51, 126.20, 120.01 (q, J = 276.5 Hz), 115.86, 115.22, 42.84. ¹⁹F NMR (565 MHz, DMSO-d₆) δ -69.53. HRMS (ESI) m/z calculated for C₁₆H₁₃N₄F₃Na [M + Na]⁺ 341.0981, found 341.0984.

Procedure for synthesis of intermediate 12a and 12b

The following compounds 12a and 12b were obtained by an approach similar to that for the synthesis of compound 8b.

N-Methyl-N-4-((tert-butoxycarbonyl)-L-leucinamido)phenyl-2-(trifluoromethyl)quinazolin-4-amine (12a) Yellow solid, (220 mg, yield 66.0%). ¹H NMR (600 MHz, CDCl₃) δ 9.73 (s, 1H), 7.93 (d, J = 8.3 Hz, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.59 (d, J = 8.7 Hz, 2H), 7.18–7.10 (m, 3H), 7.06 (d, J = 8.5 Hz, 1H), 4.59–4.36 (m, 1H), 3.64 (s, 3H), 3.54–3.27 (m, 2H), 2.62–2.45 (m, 1H), 2.07–1.79 (m, 3H), 1.50 (s, 9H).

N-Methyl-N-4-((tert-butoxycarbonyl)-L-prolinamido)phenyl-2-(trifluoromethyl)quinazolin-4-amine (12b) Yellow solid, (202 mg, yield 60.5%). ¹H NMR (600 MHz, CDCl₃) δ 9.73 (s, 1H), 7.93 (d, J = 8.3 Hz, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.59 (d, J = 8.7 Hz, 2H), 7.18–7.10 (m, 3H), 7.06 (d, J = 8.5 Hz, 1H), 4.59–4.36 (m, 1H), 3.64 (s, 3H), 3.54–3.27 (m, 2H), 2.62–2.45 (m, 1H), 2.07–1.79 (m, 3H), 1.50 (s, 9H). ¹³C NMR (151 MHz, CDCl₃) δ 162.13, 152.19 (q, J = 35.5 Hz), 151.26, 132.75, 129.34, 126.83, 126.66, 126.42, 121.25, 120.18 (q, J = 276.1 Hz), 116.45, 81.28, 60.65, 47.47, 43.00, 28.53, 27.05, 24.76.

Procedure for synthesis of target compounds 13a-13b

The following compounds 13a and 13b were obtained by an approach similar to that for the synthesis of compound 6a.

N-Methyl-N-4-(L-leucinamido)phenyl-2-(trifluoromethyl)quinazolin-4-amine (13a) Yellow solid, (44 mg, yield 78.6%). mp: 161.3–162.0 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 9.75 (s, 1H), 7.92 (dd, J = 8.3, 1.2 Hz, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.62 (ddd, J = 8.3, 6.8, 1.4 Hz, 1H), 7.17 (d, J = 8.8 Hz, 2H), 7.12 (ddd, J = 8.4, 6.8, 1.3 Hz, 1H), 7.07 (dd, J = 8.6, 1.3 Hz, 1H), 3.65 (s, 3H), 3.58–3.52 (m, 1H), 1.88–1.76 (m, 4H), 1.49–1.40 (m, 1H), 1.00 (d, J = 6.4 Hz, 3H), 0.97 (d, J = 6.3 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 173.96, 162.15, 152.20 (q, J = 35.5 Hz), 151.26, 143.06, 137.20, 132.74, 129.32, 126.87, 126.65, 126.42, 120.94, 120.19 (q, J = 275.8 Hz), 116.46, 54.00, 43.93, 43.00, 25.15, 23.54, 21.42. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.88. HRMS (ESI) m/z calcd for C₂₂H₂₅F₃N₅O [M + H]⁺432.2006, found 432.1995.

N-Methyl-N-4-(L-prolinamido)phenyl-2-(trifluoromethyl)quinazolin-4-amine (13b) Yellow solid, (35 mg, yield 58.6%). mp: 95.4–95.6 ^oC. ¹H NMR (600 MHz, CDCl₃) δ 9.91 (s, 1H), 7.93 (dd, J = 8.4, 1.2 Hz, 1H), 7.69 (d, J = 8.8 Hz, 2H), 7.63 (ddd, J = 8.3, 6.8, 1.4 Hz, 1H), 7.18 (d, J = 8.8 Hz, 2H), 7.13 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.08 (dd, J = 8.6, 1.5 Hz, 1H), 3.90 (dd, J = 9.3, 5.2 Hz, 1H), 3.66 (s, 3H), 3.15–3.06 (m, 1H), 3.04–2.96 (m, 1H), 2.28–2.15 (m, 1H), 2.11–2.01 (m, 1H), 1.85–1.73 (m, 2H), 1.33–1.16 (m, 1H). ¹³C NMR (150 MHz, CDCl₃) δ 173.75, 162.16, 152.22 (q, J = 35.7 Hz), 151.28, 143.06, 137.14, 132.74, 129.35, 126.87, 126.66, 126.44, 120.90, 120.19 (q, J = 276.1 Hz), 116.47, 61.15, 47.54, 43.02, 30.90, 26.49. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.91. HRMS (ESI) m/z calcd for C₂₁H₂₁F₃N₅O [M + H]⁺ 416.1693, found 416.1676.

The procedure for synthesis of intermediate 14

A suspension of intermediate 3 (1.00 g, 4.30 mmol), 4-aminobenzoic acid (648 mg, 4.73 mmol), and isopropyl alcohol (IPA, 7 mL) was stirred at 80 ^oC for 12 h. The mixture poured into water. The resulting precipitate was filtered and dried over to afford 14 as a white solid (1.37g, yield 95.8%).

4-((2-(Trifluoromethyl)quinazolin-4-yl)amino)benzoic acid (14) White solid, (1.37 g, yield 95.8%). ¹H NMR (600 MHz, DMSO-d₆) δ 10.45 (s, 1H), 8.72 (d, J = 8.3 Hz, 1H), 8.31 (s, 1H), 8.07 (d, J = 8.8 Hz, 2H), 8.04–7.97 (m, 4H), 7.84 (t, J = 7.5 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.89, 158.63, 150.97 (q, J = 34.8 Hz), 148.95, 142.69, 134.48, 130.02, 128.65, 128.53, 126.09, 123.51, 121.43, 119.91 (q, J = 276.2 Hz), 115.32, 79.18.

The procedure for synthesis of intermediate 15

Intermediate 15 was prepared by a method similar to the synthesis of target compound 5a.

N-Methyl-N-4-(methoxycarbonyl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (15) White solid, (953 mg, yield 63.9%). ¹H NMR (600 MHz, CDCl₃) δ 8.06 (d, J = 8.6 Hz, 2H), 7.99 (dd, J = 8.3, 1.2 Hz, 1H), 7.69 (ddd, J = 8.3, 6.9, 1.4 Hz, 1H), 7.23 (d, J = 8.6 Hz, 2H), 7.17 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.07 (dd, J = 8.5, 1.4 Hz, 1H), 3.94 (s, 3H), 3.74 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 166.29, 162.67, 152.25 (q, J = 35.5 Hz), 151.77, 151.42, 133.24, 131.70, 129.66, 128.28, 127.18, 126.19, 125.21, 120.11 (q, J = 276.4 Hz), 116.69, 52.46, 42.34. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.77.

The procedure for synthesis of intermediate 16

A suspension of intermediate 15 (738 mg, 2.04 mmol) and KOH (1.15 g, 20.42 mmol) in H₂O∶EtOH (9∶1, 15 mL), after refluxing for 3 h at 80 ^oC. Then the reaction mixture was poured into water and adjusted to pH 5.0 with HCl (1M). The resulting precipitate was collected by filtration to afford 16 (531 mg, 70.5%) as a yellow solid.

N-Methyl-4-((2-(trifluoromethyl)quinazolin-4-yl)amino)benzoic acid (16) Yellow solid, (531 mg, yield 70.5%). ¹H NMR (600 MHz, CDCl₃) δ 8.10 (d, J = 8.1 Hz, 2H), 7.94 (d, J = 8.4 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.18 (d, J = 8.1 Hz, 2H), 7.10 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 8.6 Hz, 1H), 3.69 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.98, 163.30, 152.37 (q, J = 34.4 Hz), 151.74, 151.04, 134.21, 133.57, 132.23, 129.42, 127.87, 127.17, 126.19, 121.00 (q, J = 269.2 Hz), 117.13, 47.22.

Procedure for synthesis of target compounds 17a-17c

To a stirred solution of intermediate 16 (100 mg, 0.29 mmol), p-anisidine (42.5 mg, 0.35 mmol), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDCI, 82.8 mg, 0.43 mmol), and 4-dimethylaminopyridine (35.2 mg, 0.29 mmol) in DMF (3 mL) were slowly added pyridine (185 µL, 2.3 mmol). The reaction mixture was stirred at room temperature for 14 h. The reaction mixture was poured into water, adjusted to pH 5.0 with HCl (1M), and extracted with EtOAC three times. The combined organic layer was washed with brine, dried over MgSO₄, and evaporated under reduced pressure. The crude product was purified by column chromatography to afford the target compound 17a (85 mg, 68.4%) as a yellow solid. The following compounds 17b and 17c were obtained by an approach similar to that for the synthesis of compound 17a.

N-Methyl-N-4-((4-methoxyphenyl)carbamoyl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (17a) Yellow solid, (85 mg, yield 68.4%). mp: 163.1–163.4 ^oC. ¹H NMR (600 MHz,600 MHz, CDCl₃) δ 8.00 (dd, J = 8.4, 1.2 Hz, 1H), 7.90 (d, J = 8.1 Hz, 2H), 7.78 (s, 1H), 7.70 (ddd, J = 8.3, 6.9, 1.4 Hz, 1H), 7.53 (d, J = 8.5 Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H), 7.19 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.11 (dd, J = 8.6, 1.4 Hz, 1H), 6.91 (d, J = 8.9 Hz, 2H), 3.81 (s, 3H), 3.74 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 164.61, 162.60, 156.95, 152.23 (q, J = 35.5 Hz), 151.38, 150.71, 133.24, 133.10, 130.82, 129.62, 129.18, 127.22, 126.20, 125.63, 122.39, 120.11 (q, J = 275.5 Hz), 116.62, 114.41, 55.63, 42.49. ¹⁹F NMR (565 MHz, CDCl₃) δ -70.73. HRMS (ESI) m/z calcd for C₂₄H₁₉F₃N₄O₂Na [M + Na]⁺475.1352, found 475.1339.

N-Methyl-N-4-carbamoylphenyl-2-(trifluoromethyl)quinazolin-4-amine (17b) White solid, (49.8 mg, yield 50.1%). mp: 229.8–230.2 ^oC. ¹H NMR (600 MHz, DMSO-d₆) δ 8.06 (s, 1H), 7.98–7.88 (m, 3H), 7.80 (d, J = 8.1 Hz, 1H), 7.49–7.42 (m, 3H), 7.32 (t, J = 8.1 Hz, 1H), 7.02 (d, J = 8.8 Hz, 1H), 3.66–3.63 (m, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.93, 162.04, 150.76 (q, J = 34.7 Hz), 150.52, 149.42, 133.54, 132.48, 129.50, 128.84, 127.31, 125.96, 125.36, 119.97 (q, J = 275.7 Hz), 116.05, 42.02. ¹⁹F NMR (565 MHz, DMSO-d₆) δ -69.32. HRMS (ESI) m/z calcd for C₁₇H₁₃F₃N₄ONa [M + Na]⁺ 369.0934, found 369.0921.

N-Methyl-N-4-((1-methyl-1H-indol-6-yl)carbamoyl)phenyl-2-(trifluoromethyl)quinazolin-4-amine (17c) White solid, (94 mg, yield 68.6%). mp: 218.7–219.2 ^oC. ¹H NMR (600 MHz, DMSO-d₆) δ 10.20 (s, 1H), 8.08 (d, J = 8.0 Hz, 2H), 8.02 (s, 1H), 7.96 (d, J = 8.3 Hz, 1H), 7.82 (t, J = 7.7 Hz, 1H), 7.52 (d, J = 8.1 Hz, 2H), 7.48 (d, J = 8.7 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.31 (d, J = 3.1 Hz, 1H), 7.10 (d, J = 8.9 Hz, 1H), 6.41 (d, J = 3.0 Hz, 1H), 3.78 (s, 3H), 3.68 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 164.03, 162.08, 150.78 (q, J = 35.2 Hz), 150.54, 149.43, 133.63, 133.57, 133.44, 131.03, 130.19, 129.59, 128.87, 127.75, 127.40, 126.01, 125.41, 119.99 (q, J = 275.6 Hz), 116.11, 116.05, 112.48, 109.36, 100.35, 42.06, 32.53. ¹⁹F NMR (565 MHz, DMSO-d₆) δ -69.28. HRMS (ESI) m/z calcd for C₂₆H₂₀F₃N₅ONa [M + Na]⁺ 498.1512, found 498.1499.

Biological assays

Cell Culture and lentiviral transfection

PC3, K562, and HeLa cell lines were obtained from the Biology Laboratory of the Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences (Guiyang, China). PC3, HeLa cell lines were maintained in DMEM (BI, Israel) and K562 was maintained in RPMI-1640 media (BI, Israel) with 10% fetal bovine serum (BI, Israel), 1 unit/mL penicillin-streptomycin (Solarbio, Beijing), maintained at 37°C under 5% CO₂. The plasmids were constructed by Hunan Fenghui Biotechnology Co., Ltd. WRN overexpression cell line (PC3-WRN) by lentivirus transfection with pLV-WRN-GFP-Puro. The stable transfer cell lines were screened by Puro. Finally, the WRN expression was detected by Western blot.

Westen blot

The proteins were separated by SDS-PAGE (Solarbio, Beijing) gel electrophoresis, the target proteins were transferred to the PVDF membrane (Millipore, USA). Then washed the PVDF membrane with TBST, blocked in 5% BSA for 2h, and incubated with primary antibody (WRN was purchased in Abcam, β-actin were purchased in Huabio, all antibodies used at a 1:1000 dilution in the experiments) for 4 ^oC overnight. The next day, washed the PVDF membrane with TBST, incubated with secondary antibody (1:30000, CST) for 2h at 22–25 ^oC. Finally, washed the PVDF membrane with TBST, photographed and analyzed them.

MTT assay

The cytotoxicity of target compounds were evaluated by MTT assay. Cells were seeded in 96-well plates at a density of 3 × 10³ -5 × 10³ cells/well for 12 h. Then cells were treated with N-aryl-2-trifluoromethyl-quinazoline-4-amine derivatives (1 × 10^− 4, 1 × 10^− 3, 1 × 10^− 2, 1 × 10^− 1, 1 × 10⁰, 1 × 10¹ µM) for 48 h. The 5 mg/mL MTT solution (Solarbio, Beijing) was added to cells at 37 ^oC for 4 h, supernatant was removed and 150 µL DMSO was added. The absorbance was detected at 490 nm by a microplate reader (Gene, HongKong). DMSO as a negative control. WRN inhibitor (NSC 617145, GLPBIO, USA), doxorubicin and paclitaxel were used as the positive control.

Colony formation Assay

Cells were seeded at a density of 300–800 cells per well in 6-well plates and treated with N-aryl-2-trifluoromethyl-quinazoline-4-amine compounds (100 nM, 500 nM, 1000 nM). After 1–2 weeks, 1 mL 4% paraformaldehyde was added to each well, fixed for 30–60 min, and washed with PBS. Subsequently, 1 mL crystal violet staining solution (Solarbio, Beijing) was added to each well for 10–20 min. Then washed the colony with PBS. The colony cells were air-dried and photographed with a camera.

Statistical analysis

The data were analyzed by Graghpad prism 7.0 software. Data were expressed as mean ± SD. For all measurements, one-way ANOVA were used for comparison between multiple groups. P < 0.05 was considered statistically significant.

Molecular docking

The molecular docking of compounds 6a, 8i and 13a at the WRN protein (PDB: 6YHR) was performed by Molecular Operating Environment (MOE) 2019. Chemical structures of the compounds were obtained by ChemDraw (version 18.0). Protein PDB files were downloaded from the website (http://www.rcsb.org/pdb/).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

We have shared the link to my data at the Attach File step named Supporting Information

Acknowledgments

This work was supported by the National Natural Science Foundations of China (82260675), the Department of Science and Technology of Guizhou Province (No. QKHPTRC[2016]5678, No. QKHZYD[2022]4015), Natural Science Foundation of Guizhou province (No. QKHJC-ZK[2021]YB07, No. QKHJC-ZK[2022]ZD031). The Science and Technology Planning Project of Guizhou Province (No. QKHZC [2023]YB241, No. QKHZC[2020]4Y161), and Guizhou province “Hundred” level innovative talent training project (No. QKHPTRC-GCC[2022]034-1), Natural Science Foundation of Guizhou Medical University (No. 21NSFCP42, No. 22NSFCP24).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org.

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Table 1 is available in the Supplementary Files section.

Schemes are available in the Supplementary Files section.

No competing interests reported.

Supplementaryinformation.docx
Appendix A. Supplementary data
GraphicalAbstrct.png
Scheme1.png
Scheme 1. General procedures of preparing compounds 5a, 5c, 6a-6c, and 7a-7b. Reagents and conditions: (i) Trifluoroacetic anhydride, K₂CO₃, CH₂Cl₂, RT, 2 h; (ii) KOH, CH₃CN, 80 ^oC, 4 h; (iii) SOCl₂, DMF, CHCl₃, 80 ^oC, reflux, 6 h; (iv) Aniline derivatives, NaH, DMF, RT, 1 h; (v) CH₃I, NaH, DMF, RT, 3 h; (vi) TFA, CH₂Cl₂, RT, 5 h; (vii) Pd/C, THF, RT, 15 h; (viii) CH₃I or CH₃CH₂I, NaH, DMF, RT, 5 h.
Scheme2.png
Scheme 2. General procedures of preparing compounds 8a-8j, and 9a-9f. Reagents and conditions: (i) K₂CO₃, CH₂Cl₂, RT, 3 h; (ii) IBCF, DIPEA, CH₂Cl₂, RT, 10 h; (iii) HATU, DIPEA, CH₂Cl₂, RT, 4 h; (iv) K₂CO₃, CH₃CN, RT, 15 h; (v) LiOH·H₂O, THF/H₂O, RT, 10 h; (vi) TFA, CH₂Cl₂, RT, 6 h.
Scheme3.png
Scheme 3. General procedures of preparing compounds 13a-13b and 17a-17c. Reagents and conditions: (i) N-methyl-4-nitroaniline, NaH, DMF, RT, 3 h; (ii) SnCl₂·2H₂O, HCl, CH₃COOH, 30 ^oC, 15 h; (iii) HATU, DIPEA, CH₂Cl₂, RT, 7 h; (iv) TFA, CH₂Cl₂, RT, 5 h; (v) 4-aminobenzoic acid, IPA, 80 ^oC, 10 h; (vi) CH₃I, NaH, DMF, RT, 9 h; (vii) KOH, EtOH/H₂O, 80 ^oC, 3 h; (viii) substituted aniline or ammonium chloride, EDCI, DMAP, Py, DMF, 80 ^oC, 10 h.
Table1.docx

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Design and synthesis of N-aryl-2-trifluoromethyl-quinazoline-4-amine derivatives as potential Werner-dependent anticancer agents

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Abstract

Figures

Introduction

Results and discussion

Chemistry

Biological activity evaluation

Structure–activity relationships

Evaluation of WRN-dependent cell survival inhibition by quinazoline compounds

Molecular docking

Conclusion

Experimental

Chemistry

The procedure for synthesis of intermediates 1–3

General procedure for the synthesis of intermediates 4a–4c

General procedure for the synthesis of intermediate 5b, target compounds 5a and 5c

General procedure for the synthesis of target compounds 6a-6c

General procedure for the synthesis of target compounds 7a-7b

General procedure for the synthesis of target compounds 8a, 8d and 8g-8j

General procedure for the synthesis of intermediates 8b and 8c

General procedure for the synthesis of target compounds 8e and 8f

Procedure for synthesis of target compound 9a

Procedure for synthesis of target compounds 9b and 9c

Procedure for synthesis of target compounds 9d-9f

The procedure for synthesis of intermediate 10

The procedure for synthesis of intermediate 11

Procedure for synthesis of intermediate 12a and 12b

Procedure for synthesis of target compounds 13a-13b

The procedure for synthesis of intermediate 14

The procedure for synthesis of intermediate 15

The procedure for synthesis of intermediate 16

Procedure for synthesis of target compounds 17a-17c

Biological assays

Cell Culture and lentiviral transfection

Westen blot

MTT assay

Colony formation Assay

Statistical analysis

Molecular docking

Declarations

References

Table 1

Schemes

Additional Declarations

Supplementary Files

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