Synthesis and biological activity of new 2,4,6-trisubstituted triazines as potential phosphoinositide 3-kinase inhibitors

Abstract

Twenty-five novel 2,4,6-trisubstituted triazines were synthesized and biologically evaluated. Most of the compounds synthesized showed good antiproliferative activity against HCT-116 and MCF-7. Compounds B18 and B19 showed the best antiproliferative activity. Further study showed B18 and B19 inhibited four phosphoinositide 3-kinase isoforms and mammalian target of rapamycin with good potency. These results demonstrate that 2,4,6-trisubstituted triazines are potentially useful phosphoinositide 3-kinase inhibitors for the development of new anticancer drugs.

Keywords

anticancer antiproliferative effects PI3K inhibitor synthesis triazine derivatives

The phosphoinositide 3-kinase (PI3K) is a very important node within the PI3K/AKT/mTOR pathway that regulates a variety of cellular processes including cell proliferation, differentiation, survival, and migration.¹ Aberrant activation of PI3K signaling has been implicated in multiple cancer types including colorectal, breast, leukemia, and so on.² Therefore, PI3K is considered as a promising target for solid and hematologic tumor therapy.³

During the past 20 years, a great number of small molecule PI3K inhibitors have been investigated. Some early agents such as wortmannin and LY294002 did not enter clinical development due to their narrow therapeutic window and severe toxicity.⁴ Fortunately, the selective second generation PI3K or PI3K/mTOR inhibitors, such as idelalisib (CAL-101, 1),⁵ copanlisib (BAY80-6946, 2),⁶ apitolisib (GDC-0980, 3),⁷ omipalisib (GSK2126458, 4),⁸ dactolisib (NVP-BEZ235, 5),⁹ buparlisib (NVP-BKM120, 6),¹⁰ and VS-5584 (7)¹¹ were advanced into clinical development, and some agents showed promising clinical efficacy (Figure 1). Particularly, idelalisib (selective PI3Kδ inhibitor) and copanlisib (pan-PI3K inhibitor) were recently approved by the Food and Drug Administration (FDA) for treatment of hematologic malignancy.^5,6 These encouraging facts refueled the discovery and development of new PI3K inhibitors (Figure 1).

Recently, we reported our drug design and synthesis effort on the discovery of new potent PI3K inhibitors. The first strategy was to develop novel selective PI3Kδ inhibitors with distinct chemotypes which may elevate the therapeutic index and provide clinical benefit.^12,13 For example, several 6-aryl substituted quinazoline derivatives were synthesized and some showed promising potency against PI3Kδ in vitro.^14,15 The other strategy was to develop pan-PI3K or PI3K/mTOR inhibitors, exemplified by the preparation of several series of derivatives such as quinazolin-4(3H)-ones,^16,17 [1,2,4]triazolo[1,5-a]pyridines,¹⁸ benzo[d]thiazoles,^19,20 4-morpholinoquinazolines,²¹ and 4-(morpholin-4-yl)-1,3,5-triazines.²² In particular, in the series of 4-(morpholin-4-yl)-1,3,5-triazines, representative derivatives displayed potent antitumor effects in vitro and in vivo. Thus, in an attempt to develop new anticancer agents and based on our previous reports and the chemical structure of VS-5584, we intend to open the purine ring of VS-5584 and replace it with a triazine core so as to design a series of 1,3,5-triazines as new PI3K inhibitors (indicted in Figure 2). Certainly, the 4-(morpholin-4-yl)-1,3,5-triazine is facile to be constructed and its derivatives were reported to show good antitumor effects. Furthermore, the 4-(morpholin-4-yl)-1,3,5-triazine scaffold was a good mimic of the main pharmacophore of VS-5584 which generated several key H-bond interactions with PI3Kα. Therefore, in this paper we design and synthesize a series of 4-(morpholin-4-yl)-1,3,5-triazines by introducing a variety of aliphatic amino groups at the 6-position as potential PI3K inhibitors, and investigate the primary structure–activity relationship (Figure 2).

Results and discussion

The title compounds A were prepared by the route shown in Scheme 1. The key intermediates a1–a6 were constructed by amination of the starting material 2,4-dichloro-6-morpholinyl-1,3,5-triazine with substituted aliphatic amines. The target compounds A1–A6 were then synthesized in one-pot by PdCl₂(dppf)-catalyzed Suzuki coupling of a1–a6 and the boronic ester intermediate 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine which was newly prepared from 2-amino-5-bromopyrimidine and bis(pinacolato)diboron using Miyaura borylation in the presence of PdCl₂(dppf) and potassium acetate (Scheme 1).

As outlined in Scheme 2, the compounds B1–B2 were obtained similarly to Scheme 1. The compounds B3–B4 were prepared by deprotection of the Boc group in B1 or B2 using trifluoroacetic acid (TFA) in dichloromethane. The reductive amination of B3 with cyclopropanecarbaldehyde gave compound B5. The piperidinyl base is stronger than the amino group linked to the pyrimidinyl group, and so the nucleophilic substitution of B3 with benzyl chloride or 4-nitrobenzyl bromide reacted at the piperidinyl site to yield compound B6 or B7. Similarly, the reaction of compound B3 or B4 with cyclopropanesulfonyl chloride, tosyl chloride, or acetic anhydride, respectively, produced the title compounds B8–B11 and in the presence of a condensing agent, the reaction of B3 or B4 with a carboxylic acid provided the title compounds B12–B19 (Scheme 2).

Compounds A1–A6 and B1–B19 were first evaluated for their antiproliferative activity by MTT assays.¹⁶ Two cancer cell lines including HCT-116 (PI3CA mutant: H1047R) and MCF-7 (PI3CA mutant: E545K) were used in this evaluation, and the results are summarized in Table 1. Albeit sharing the same pharmacophore, 2-(2-aminopyrimidin-5-yl)-4-(morpholin-4-yl)triazine, the compounds A1–A6 and B1–B19 exhibited significantly different antiproliferation, which was mainly related to the 6-substituted groups linked to the triazine scaffold. Compounds A1–A3 with a simple substituted amino group at the 6-position of the triazine showed weak antiproliferative effect and compounds A4–A6 with a piperidinyl substituent displayed loss of cell-based activity. Subsequently, the piperid-1-yl substituent was moved to the piperidine-4-yl amino group leading to synthesis of compounds B1–B7. Compounds B1 and B2 with the Boc group attached to the 1-position of piperidine-4-yl ring, showed more potent antiproliferative activity. However, removal of the Boc group resulted in compounds B3 and B4 that showed weaker activity. These results suggested that the presence of the NH group of the piperidine-4-yl substituent was unfavorable. Thereafter, alkyl, sulfonyl, or acyl groups were evaluated (B5–B19). Compounds (B5–B7) with an alkyl group at the 1-position of the piperidinyl ring displayed weak or loss antiproliferative activity. A cyclopropyl sulfonyl group in B8 and B10 was tolerated; however, the larger 4-methylbenzenesulfonyl group (B9) was not. A survey of acyl substituents at the 1-position of the piperidinyl ring was investigated. As the data showed in Table 1, the antiproliferative activity of the acyl substituted compounds B11–B19 was remarkably improved. In particular, the N-cyclopropyl-N-(1-acylpiperidin-4-yl)amino substituted compounds B17–B19 showed more potent antiproliferative effect against both HCT-116 and MCF-7 cell lines, and more importantly, both compound B18 and B19 were comparable to the positive drug VS-5584, despite being slightly weaker than dactolisib (BEZ235). Building on their structure–activity relationship, it was found that the N-cyclopropyl-N-(1-acylpiperidin-4-yl)amino moiety is a suitable substituent at the 6-position of the triazine scaffold. Compounds B18 and B19 were the best antiproliferative agents, and were further examined for their enzymic activity (Table 1).

Table 1.

Antiproliferative effects of compounds A and B ( $\bar{x} \pm s$ , n = 3).

Compound	R¹	R²	IC₅₀ (µM)
Compound	R¹	R²	HCT-116	MCF-7
A1	−Et	−Pr-i	2.95 ± 0.19	4.45 ± 0.40
A2	−Pr-i	−Pr-i	9.54 ± 1.13	>10
A3			9.89 ± 0.60	>10
A4			>10	>10
A5			>10	>10
A6			>10	>10
B1	−CH₃		2.29 ± 0.44	3.60 ± 0.40
B2			1.89 ± 0.14	1.95 ± 0.08
B3	−CH₃		8.09 ± 0.36	7.23 ± 1.10
B4			5.34 ± 0.11	3.49 ± 0.50
B5	−CH₃		>10	>10
B6	−CH₃		>10	7.62 ± 1.50
B7	−CH₃		>10	6.98 ± 0.50
B8	−CH₃		2.84 ± 0.32	3.37 ± 0.52
B9	−CH₃		>10	>10
B10			1.39 ± 0.30	1.92 ± 0.05
B11	−CH₃		1.56 ± 0.23	2.93 ± 0.10
B12	−CH₃		2.33 ± 0.08	3.15 ± 0.15
B13	−CH₃		2.07 ± 0.06	3.06 ± 0.14
B14	−CH₃		1.42 ± 0.15	4.66 ± 0.10
B15	−CH₃		1.18 ± 0.11	2.34 ± 0.22
B16	−CH₃		1.31 ± 0.16	2.76 ± 0.44
B17			1.08 ± 0.12	2.18 ± 0.10
B18			0.68 ± 0.01	1.58 ± 0.12
B19			0.79 ± 0.03	1.55 ± 0.15
Dactolisib	−	−	0.37 ± 0.21	0.35 ± 0.31
VS-5584	−	−	0.60 ± 0.12	1.06 ± 0.10

B18 and B19 were evaluated for their inhibitory activity against PI3Ks and mTOR by performing an ATP depletion assay. VS-5584 and Dactolisib were used as the positive drugs. As the data showed in Table 2, B18 displayed good inhibitory activity against four PI3K isoforms and mTOR with IC₅₀ values of 62, 286, 870, 627, and 772 nM, respectively, and B19 showed respective IC₅₀ values of 78, 423, 521, 422 and 263 nM. Although the IC₅₀ values of both B18 and B19 were higher than that of VS-5584, it was noted that both B18 and B19 afforded comparable PI3Kα inhibition to Dactolisib. However, B18 and B19 showed potent antiproliferative activity in cell-based effects but weaker PI3K inhibition which may be attributed to their diverse efficacy (Table 2).

Table 2.

Enzymatic activity of B18 and B19 (IC₅₀, nM, n = 2).

Compound	PI3Kα	PI3Kβ	PI3Kγ	PI3Kδ	mTOR
B18	62	286	870	627	772
B19	78	423	521	422	263
Dactolisib	49	478	72	138	44
VS-5584	7	23	9	9	52

To explain further the good PI3Kα inhibitory activities of compound B18, docking studies with human PI3Kα (PDB code, 5UK8) were performed. As depicted in Figure 3, the oxygen atom of the morpholine moiety formed a hydrogen bond with Val851, and the two hydrogen atoms of the amino group on the pyrimidine ring formed hydrogen bonds with Asp810 and Asp802. In addition, the nitrogen atom of the pyrimidine ring formed hydrogen bonds with Asp933. The presence of these hydrogen bonds suggested that B18 can undoubtedly interact with the catalytic domain of PI3Kα (Figure 3).

In summary, a series of novel 2,4,6-trisubstituted triazines were synthesized and characterized. Most compounds exhibited significant antiproliferative effects against HCT-116 and MCF-7 cell lines. The discussion of the SAR reveals that the antiproliferative effect is closely related to the substituent at the 6-position of the triazine, and the N-cyclopropyl-N-(1 acylpiperidin-4-yl)amino moiety is a suitable substituent at the 6-position contributing to the potent antiproliferation. The cell-based potencies of compounds B18 and B19 are comparable to that of VS-5584. The compounds B18 and B19 displayed good inhibitory activity against PI3Ks and mTOR. These results suggest that our compounds can serve as antiproliferative agents and potential PI3K inhibitors.

Experimental

All starting materials, reagents, and solvents were commercially available, unless specified otherwise. Reactions were monitored by thin-layer chromatography on silica gel plates (GF-254) and visualized with UV light. The melting points were determined on a Shanghai micro melting-point apparatus (model: SGW^® X-4B) and are uncorrected. NMR spectra were recorded on a 400 Bruker NMR spectrometer with tetramethylsilane (TMS) as an internal reference. Chemical shifts are reported in parts per million (ppm). Mass measurements were performed using electrospray ionization (positive mode) on a quadrupole time-of-flight (QTOF) mass spectrometer (Skyray Instrument).

General procedure for the synthesis of intermediates a1–a6

To a solution of 4-(4,6-dichloro-1,3,5-triazin-2-yl)morpholine (0.10 g, 0.43 mmol) in dry dichloromethane (10 mL) was added dropwise a mixture of the substituted aliphatic amine (0.43 mmol) and triethylamine (178 μL) at 0 °C. The reaction mixture was stirred at room temperature until all the starting materials have been consumed. The solvent was removed under reduced pressure. Water (30 mL) was added to the residue and the mixture was stirred at room temperature for 10 min. The mixture was extracted with ethyl acetate (15 mL × 3). The organic extracts were combined and washed with water. After drying with anhydrous Na₂SO₄, the organic extracts were evaporated to provide compounds a1–a6.

4-Chloro-N-ethyl-N-isopropyl-6-morpholino-1,3,5-triazin-2-amine (a1):²³ colorless oil, yield 88%. ESI-MS m/z: 286.2 [M+ H]⁺.

4-Chloro-N,N-diisopropyl-6-morpholino-1,3,5-triazin-2-amine (a2):²³ Colorless oil, yield 83%. ESI-MS m/z: 300.2 [M + H]⁺.

4-(4-Chloro-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)morpholine (a3):²³ colorless oil, yield 80%. ESI-MS m/z: 299.2 [M + H]⁺.

(1-(4-Chloro-6-morpholino-1,3,5-triazin-2-yl)piperidin-4-yl)(pyrrolidin-1-yl)methanone (a4): colorless oil, yield 71%. ¹H NMR (CDCl₃) δ 4.75–4.70 (m, 2H), 3.73–3.70 (m, 8H), 3.58–3.40 (m, 4H), 2.95–2.90 (m, 2H), 2.61 (d, J = 5.4 Hz, 1H), 1.98 (d, J = 6.1 Hz, 2H), 1.88–1.78 (m, 6H). ESI-MS m/z: 381.2 [M + H]⁺.

(1-(4-Chloro-6-morpholino-1,3,5-triazin-2-yl)piperidin-4-yl)(piperidin-1-yl)methanone (a5): colorless oil, yield 60%. ¹H NMR (CDCl₃) δ 4.72–4.68 (m, 2H), 3.62–3.56 (m, 8H), 3.50–3.41 (m, 4H), 2.99–2.81 (m, 2H), 2.69 (d, J = 5.9 Hz, 1H), 1.68–1.55 (m, 6H), 1.50–1.45 (m, 4H). ESI-MS m/z: 395.2 [M + H]⁺.

(1-(4-Chloro-6-morpholino-1,3,5-triazin-2-yl)piperidin-4-yl)(morpholino) methanone (a6): colorless oil, yield 65%. ¹H NMR (CDCl₃) δ 4.62–4.52 (m, 2H), 3.62–3.56 (m, 8H), 3.57–3.55 (m, 3H), 3.52–3.38 (m, 4H), 2.93–2.76 (m, 2H), 2.70–2.63 (m, 2H), 1.66–1.60 (m, 4H). ESI-MS m/z: 397.2 [M + H]⁺.

General procedure for the synthesis of compound A1–A6

The mixture of 5-bromopyrimidin-2-amine (65 mg, 0.37 mmol), bis(pinacolato)diboron (113 mg, 0.44 mmol), potassium acetate (109 mg, 1.11 mmol), PdCl₂(dppf) (21 mg, 0.02 mmol), and 1,4-dioxane (10 mL) was heated under reflux under Ar atmosphere for 2.5 h, then concentrated under reduced pressure. To the resulting residue was added b1 (98 mg, 0.25 mmol), K₂CO₃ (154 mg, 1.11 mmol), PdCl₂(dppf) (21 mg, 0.02 mmol), 1,4-dioxane (10 mL), and water (4 mL). The mixture obtained was heated under reflux in Ar atmosphere for 4 h. The volatile material was removed under reduced pressure and the residue was purified by a column chromatography on silica gel with CH₂Cl₂ / MeOH (v: v = 50:1) as eluent to produce A1 as an off-white solid.

4-(2-Aminopyrimidin-5-yl)-N-ethyl-N-isopropyl-6-morpholino-1,3,5-triazin-2-amine (A1): off-white solid, yield 86%. mp 216–218 °C. ¹H NMR (CDCl₃) δ 9.19 (s, 2H), 5.41 (s, 2H), 5.22–4.86 (m, 1H), 3.90–3.85 (m, 4H), 3.80–3.68 (m, 4H), 3.60–3.39 (m, 2H), 1.22 (d, J = 5.2 Hz, 9H). ¹³C NMR (DMSO-d₆): δ 165.2, 164.7 (2C), 164.1, 158.9 (2C), 119.4, 66.5 (2C), 46.0, 45.9, 43.7 (2C), 20.7, 20.5, 15.0. ESI-MS m/z: 345.2 [M + H]⁺.

4-(2-Aminopyrimidin-5-yl)-N,N-diisopropyl-6-morpholino-1,3,5-triazin-2-amine (A2): off-white solid, yield 76%. mp 224–226 °C. ¹H NMR (CDCl₃) δ 9.19 (s, 2H), 5.40 (s, 2H), 4.45–4.40 (m, 2H), 3.90–3.85 (m, 4H), 3.79–3.72 (m, 4H), 1.37–1.30 (m, 12H). ¹³C NMR (DMSO-d₆): δ 166.9, 165.2, 164.4, 164.1, 158.8 (2C), 119.5, 66.5 (2C), 45.4 (2C), 43.80 (2C), 20.9 (2C), 20.6 (2C). ESI-MS m/z: 359.8 [M + H]⁺.

5-(4-(4-Methylpiperazin-1-yl)-6-morpholino-1,3,5-triazin-2-yl)pyrimidin-2-amine (A3): off-white solid, yield 86%. mp >250 °C. ¹H NMR (CDCl₃) δ 9.18 (s, 2H), 5.36 (s, 2H), 3.87 (s, 8H), 3.77–3.73 (m, 4H), 2.54–2.42 (m, 4H), 2.36 (s, 3H). ¹³C NMR (DMSO-d₆) δ 167.7, 165.3, 164.7, 164.3, 159.0 (2C), 119.1, 66.5 (2C), 54.9 (2C), 46.2 (2C), 43.7 (2C), 43.1. ESI-MS m/z: 357.2 [M + H]⁺.

(1-(4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)piperidin-4-yl)(pyrrolidin-1-yl)methanone (A4): off-white solid, yield 87%. mp >250 °C. ¹H NMR (CDCl₃) δ 9.22 (s, 2H), 5.53 (s, 2H), 4.90–4.75 (m, 2H), 3.87–3.75 (m, 8H), 3.58–3.45 (m, 4H), 2.95–2.90 (m, 2H), 2.67–2.57 (m, 1H), 2.03–1.92 (m, 2H), 1.88–1.72 (m, 6H). ¹³C NMR (DMSO-d₆) δ 172.6, 167.7, 165.3, 164.7, 164.4, 159.0 (2C), 119.1, 66.5 (2C), 63.3, 54.0 (2C), 46.2 (2C), 45.9 (2C), 26.1, 24.3, 18.5, 17.2. ESI-MS m/z: 440.2 [M + H]⁺.

(1-(4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)piperidin-4-yl)(piperidin-1-yl)methanone (A5): white solid, yield 66%. mp >250 °C. ¹H NMR (DMSO-d₆) δ 9.03 (s, 2H), 7.25 (s, 2H), 4.90–4.74 (m, 2H), 3.72–3.68 (m, 8H,), 3.48–3.38 (m, 5H), 2.94–2.82 (m, 3H), 1.67–1.58 (m, 4H), 1.48–1.42 (m, 6H). ¹³C NMR (DMSO-d₆) δ 172.4, 167.7, 165.2, 164.8, 164.4, 159.0 (2C), 119.4, 66.5 (2C), 55.4 (2C), 46.2 (2C), 42.5 (2C), 37.64, 28.7, 27.0, 25.8, 24.6 (2C). ESI-MS m/z: 454.2 [M + H]⁺.

(1-(4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)piperidin-4-yl)(morpholino)methanone (A6): white solid, yield 76%. mp >250 °C. ¹H NMR (DMSO-d₆) δ 9.04 (s, 2H), 7.25 (s, 2H), 4.90–4.74 (m, 2H), 3.90–3.62 (m, 8H), 3.57–3.50 (m, 6H), 3.49–3.39 (m, 2H), 3.01–2.91 (m, 3H), 1.75–1.62 (m, 2H), 1.50–1.42 (m, 2H). ¹³C NMR (DMSO-d₆) δ 172.9, 167.7, 165.2, 164.5, 164.4, 159.0 (2C), 119.1, 66.8, 66.7, 66.5 (2C), 56.5 (2C), 45.9 (2C), 42.0 (2C), 37.5, 28.5, 19.1. ESI-MS m/z: 456.2 [M + H]⁺.

Tert-butyl 4-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)(methyl)amino) piperidine-1-carboxylate (b1): To a solution of 4-(4,6-dichloro-1,3,5-triazin-2-yl)morpholine (4.00 g,17.0 mmol) in dry dichloromethane (20 mL) was added dropwise the mixture of tert-butyl 4-(methylamino)piperidine-1-carboxylate (3.65 g, 17.0 mmol) and triethylamine (4.71 mL) at 0 °C. The reaction mixture was stirred at room temperature until all starting materials consumed. The solvent was removed under reduced pressure. To the residue was added ethanol (4 mL) and water (30 mL), and the mixture was stirred at room temperature for 10 min. The solid was filtered, washed with water (5 mL× 3), and dried to afford compound b1 (4.32 g) with a yield of 62%. mp 117–119 °C. ¹H NMR (CDCl₃) δ 4.11–3.80 (m, 8H), 3.76 (s, CH₃), 3.10–2.82 (m, 5H), 1.75–1.64 (m, 4H), 1.48 (s, 9H). ESI-MS m/z: 413.2 [M + H]⁺.

Tert-Butyl 4-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)(cyclopropyl) amino) piperidine-1-carboxylate (b2): To a solution of 4-(4,6-dichloro-1,3,5-triazin-2-yl)morpholine (1.76 g, 7.52 mmol) in dry tetrahydrofuran (20 mL) were added the mixture of tert-butyl 4-(cyclopropylamino)piperidine-1-carboxylate (2.16 g, 9.02 mmol), 4-methy lbenzenesulfonic acid (0.20 g, 1.16 mmol), and N,N-diisopropylethylamine (3.95 mL). The reaction mixture was heated at reflux for 4 h. The solvent was removed under reduced pressure. The residue was suspended in water (50 mL) and extracted with dichloromethane (20 mL × 3). The organic layer was washed by saturated sodium chloride, dried, filtered, and concentrated to afford compound b2 (1.30 g). Yield: 71%. ¹H NMR (CDCl₃) δ 4.34–4.00 (m, 2H), 3.85–3.59 (m, 8H), 2.72–2.37 (m, 4H), 2.04–1.72 (s, 2H), 1.43 (s, 9H), 0.86–0.80 (m, 2H), 0.75–0.63 (m, 2H). ESI-MS m/z: 439.2 [M + H]⁺.

General procedure for the synthesis of compound B1 and B2

The mixture of 5-bromopyrimidin-2-amine (242 mg, 1.38 mmol), bis(pinacolato)diboron (386 mg, 1.52 mmol), KOAc (541 mg, 4.14 mmol), PdCl₂(dppf) (76 mg, 0.10 mmol), and 1,4-dioxane (20 mL) was heated at reflux under Ar atmosphere for 2.5 h, then concentrated in vacuum. To the resulted residue was added b1 (400 mg, 0.97 mmol), K₂CO₃ (116 mg, 0.84 mmol), PdCl₂(dppf) (76 mg, 0.10 mmol), 1,4-dioxane (20 mL), and water (4 mL). The obtained mixture was heated at reflux under Ar atmosphere for 4 h. The volatile was removed under vacuum and the residue was purified through a column chromatography on silica gel with CH₂Cl₂ / MeOH (v:v = 50:1) as eluent to produce B1 or B2.

Tert-Butyl-4-((4-(2-aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl) (methyl)amino) piperidine-1-carboxylate (B1): white solid, yield 74%. mp 227–228 °C. ¹H NMR (CDCl₃) δ 9.18 (s, 2H), 5.45 (s, 2H), 4.11–3.80 (m, 8H), 3.77 (s, CH₃), 3.10–2.82 (m, 5H), 1.75–1.64 (m, 4H), 1.48 (s, 9H). ¹³C NMR (CDCl₃) δ 164.8, 164.1, 159.1 (3C), 154.7, 121.3, 79.7, 66.9 (2C), 52.3 (2C), 43.6 (2C), 28.3 (7C). ESI-MS m/z: 472.3 [M + H]⁺.

Tert-Butyl-4-((4-(2-aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl) (cyclopropyl)amino)piperidine-1-carboxylate (B2): off-white solid, yield 45%. mp 222–225 °C. ¹H NMR (CDCl₃) δ 9.19 (s, 2H), 5.48 (s, 2H), 4.34–4.00 (m, 2H), 3.85–3.59 (m, 8H), 2.72–2.37 (m, 4H), 2.04–1.72 (s, 2H), 1.43 (s, 9H), 0.86–0.80 (m, 2H), 0.75–0.63 (m, 2H). ¹³C NMR (CDCl₃) δ 167.1, 167.0, 164.6, 164.2, 159.1 (2C), 154.8, 121.4, 79.6, 66.8 (2C), 57.2, 43.6, 30.4, 28.5 (7C), 26.8, 9.2 (2C). ESI-MS m/z: 498.3 [M + H]⁺.

General procedure for the synthesis of compound B3 and B4

To a solution of B1 or B2 (300 mg, 0.63 mmol) in dichloromethane (10 mL), TFA (2 mL) was added. The resulting solution was stirred at room temperature for 2 h, concentrated in vacuum. The residue was suspended in saturated sodium carbonate solution (5.0 mL) and extracted with dichloromethane (20 mL × 3). The organic layer was washed by saturated sodium chloride, dried, filtered, and concentrated to afford compound B3 or B4 as a white solid.

4-(2-Aminopyrimidin-5-yl)-N-methyl-6-morpholino-N-(piperidin-4-yl)-1,3,5-triazin-2-amine (B3): white solid, yield 65%. mp >250 °C. ¹H NMR (CDCl₃) δ 9.19 (s, 2H), 5.38 (s, 2H), 4.00–3.75 (m, 8H), 4.87–4.65 (m, 1H), 3.71 (s, CH₃), 3.25–3.03 (m, 5H), 2.80–2.60 (m, 2H), 2.00–1.71 (m, 4H). ¹³C NMR (CDCl₃) δ 164.8 (2C), 164.0, 159.2 (3C), 121.5, 66.9 (2C), 52.4, 46.4 (3C), 43.6, 30.4, 30.2, 28.3. ESI-MS m/z: 372.2 [M + H]⁺.

4-(2-Aminopyrimidin-5-yl)-N-cyclopropyl-6-morpholino-N-(piperidin-4-yl)-1,3,5-triazin-2-amine (B4): white solid. yield: 94%. mp: >250 °C. ¹H NMR (DMSO-d₆) δ 9.09 (s, 2H), 7.26 (s, 2H), 4.40–4.35 (m, 1H), 3.81–3.66 (m, 8H), 3.40–3.25 (m, 2H), 3.10–2.95 (m, 2H), 2.63–2.50 (m, 1H), 2.40–2.25 (m, 2H), 2.00–1.90 (m, 2H), 0.92–0.86 (m, 2H), 0.75–0.63 (m, 2H). ¹³C NMR (DMSO-d₆): δ 167.3, 166.9, 165.2, 164.4, 159.2 (2C), 66.52 (2C), 54.7, 43.7 (4C), 27.9, 27.4 (2C), 9.4 (2C). ESI-MS m/z: 398.3 [M + H]⁺.

4-(2-Aminopyrimidin-5-yl)-N-(1-(cyclopropylmethyl)piperidin-4-yl)-N-methyl-6-morpholino-1,3,5-triazin-2-amine (B5): To a solution of B3 (50 mg, 0.14 mmol), cyclopropylformaldehyde (10 mg, 11 μL), NaBH(OAc)₃ (45 mg, 0.22 mmol), and acetic acid (10 μL) were added. The resulted suspension was stirred overnight, quenched with aqueous sodium hydrogen carbonate and extracted with dichloromethane (10 mL×2). The combined organic phase was washed with brine, and dried over Na₂SO₄, filtered, concentrated in vacuum. The residue was purified by column chromatography on silica gel to produce B5 (10 mg). Yield 17%. mp >250 °C. ¹H NMR (CDCl₃) δ 9.17 (s, 2H), 5.50 (s, 2H), 5.09–5.03 (m, 1H), 3.80–3.60 (m, 8H), 3.20–3.04 (m, 3H), 2.90–2.75 (m, 6H), 1.90–1.84 (m, 6H), 1.33–1.20 (m, 1H), 0.82–0.75 (m, 2H), 0.50–0.40 (m, 2H). ESI-MS m/z: 426.3 [M + H]⁺.

General procedure for the synthesis of compounds B6 and B7

The mixture containing compound B3 (50 mg, 0.14 mmol), benzyl bromide (24 mg, 0.14 mmol), potassium carbonate (58 mg), and dimethylformamide (DMF) (10 mL) was stirred at room temperature overnight, concentrated in vacuum. The residue was purified by column chromatography on silica gel to produce B6 or B7.

4-(2-Aminopyrimidin-5-yl)-N-(1-benzylpiperidin-4-yl)-N-methyl-6-morpholino-1,3,5-triazin-2-amine (B6): white solid, yield 89%. mp 232–234 °C. ¹H NMR (CDCl₃) δ 9.19 (s, 2H), 7.35–7.26 (m, 5H), 5.33 (s, 2H), 4.68–4.49 (m, 1H), 3.98–3.71 (m, 8H), 3.56 (s, 2H), 3.06–3.01 (m, 5H), 2.13–2.00 (m, 2H), 1.95–1.75 (m, 2H), 1.70–1.65 (m, 2H). ¹³C NMR (CDCl₃) δ 164.8, 164.0, 159.2 (2C), 129.2 (2C), 128.3 (4C), 127.1 (2C), 121.5, 66.9 (2C), 63.2 (2C), 53.32 (2C), 52.4, 43.6, 29.1, 28.9, 28.3. ESI-MS m/z: 462.3 [M + H]⁺.

4-(2-Aminopyrimidin-5-yl)-N-methyl-6-morpholino-N-(1-(4-nitrobenzyl) piperidin-4-yl)-1,3,5-triazin-2-amine (B7): white solid, yield 87%. mp >250 °C. ¹H NMR (CDCl₃) δ 9.18 (s, 2H), 8.20 (d, J = 8.3 Hz, 2H), 7.53 (d, J = 7.6 Hz, 2H), 5.38 (s, 2H), 4.80–4.69 (m, 1H), 3.97–3.73 (m, 8H), 3.62 (s, 2H), 3.09–2.95 (m, 5H), 2.19–2.04 (m, 2H), 1.87–1.72 (m, 2H), 1.69–1.60 (m, 2H). ¹³C NMR (CDCl₃): δ 164.9, 164.0, 159.2 (2C), 147.1, 146.7, 129.5, 123.6 (4C), 121.9, 121.5, 66.9 (2C), 62.2 (2C), 53.4 (2C), 43.6 (2C), 29.2, 28.8, 28.3. ESI-MS m/z: 507.3 [M + H]⁺.

General procedure for the synthesis of compound B8 and B10

The mixture containing B3 or B4 (50 mg, 0.14 mmol), cyclopropylsulfonyl chloride (16 mg, 0.14 mmol), 4-dimethylaminopyridine (DMAP) (16 mg, 0.07 mmol), triethylamine (60 μL), and dichloromethane (15 mL) was stirred at room temperature for 2 h, concentrated in vacuum. The residue was purified by column chromatography on silica gel to produce B8 or B10.

4-(2-Aminopyrimidin-5-yl)-N-(1-(cyclopropylsulfonyl)piperidin-4-yl)-N-methyl-6-morpholino-1,3,5-triazin-2-amine (B8): white solid, yield 65%. mp >250 °C. ¹H NMR (CDCl₃) δ 9.20 (s, 2H), 5.47 (s, 2H), 4.89–4.79 (m, 1H), 3.92–3.69 (m, 8H), 3.09–3.05 (m, 3H), 2.97–2.95 (m, 2H), 2.37–2.26 (m, 2H), 1.99–1.86 (m, 3H), 1.81–1.69 (m, 2H), 1.25–1.16 (m, 2H), 1.10–1.00 (m, 2H). ¹³C NMR (DMSO-d₆) δ 167.5, 165.2, 164.8, 164.6, 159.0 (2C), 119.1, 66.51 (2C), 46.0 (4C), 43.6, 28.7, 28.5, 26.1, 26.0, 4.53 (2C). ESI-MS m/z: 498.2 [M + Na]⁺.

4-(2-Aminopyrimidin-5-yl)-N-cyclopropyl-N-(1-(cyclopropylsulfonyl) piperidin-4-yl)-6-morpholino-1,3,5-triazin-2-amine (B10): white solid, yield 62%. mp 240–243 °C. ¹H NMR (CDCl₃) δ 9.19 (s, 2H), 5.52 (s, 2H), 4.41–4.38 (m, 1H), 3.95–3.75 (m, 9H), 2.90–2.85 (m, 2H), 2.50 (s, 1H), 2.28–2.19 (m, 4H), 1.95–1.90 (m, 2H), 1.22–1.16 (m, 2H), 1.07–0.99 (m, 2H), 0.95–0.90 (m, 2H), 0.81–0.70 (m, 2H). ¹³C NMR (CDCl₃) δ 167.1 (2C), 164.6, 164.0, 159.1 (2C), 121.2, 66.8 (2C), 58.4, 56.4, 46.6 (2C), 43.6, 30.2 (2C), 26.0, 18.4, 9.2 (2C), 4.4 (2C). ESI-MS m/z: 502.3 [M + H]⁺.

4-(2-Aminopyrimidin-5-yl)-N-methyl-6-morpholino-N-(1-tosylpiperidin-4-yl)-1,3,5-triazin-2-amine (B9): To a solution of 4-methylbenzenesulfonyl chloride (26 mg, 0.14 mmol) in dry dichloromethane (10 mL) was added dropwise the mixture of B3 (50 mg, 0.14 mmol), and triethylamine (4.71 mL) at 0 °C. The reaction mixture was stirred at room temperature until all starting materials consumed. The solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel with CH₂Cl₂ / MeOH (v: v = 50: 1) as eluent to produce B9 as a white solid. Yield 74%. mp >250 °C. ¹H NMR (CDCl₃) δ 9.18 (s, 2H), 7.34 (d, J = 7.6 Hz, 2H), 7.22 (d, J = 7.9 Hz, 2H), 5.43 (s, 2H), 4.93–4.80 (m, 1H), 3.99–3.74 (m, 10H), 3.07–3.00 (m, 5H), 2.88–2.80 (m, 2H), 2.38 (s, 3H), 1.83–1.75 (m, 4H). ¹³C NMR (DMS-d₆): δ 164.8, 164.6, 159.0, 143.9, 133.8, 130.4 (3C), 127.9 (4C), 119.1, 66.48 (2C), 56.50 (3C), 46.03 (2C), 43.6, 21.5, 19.0 (2C). ESI-MS m/z: 548.3 [M + Na]⁺.

General procedure for the synthesis of compound B11– B19

A mixture of B3 or B4 (50 mg, 0.14 mmol), substituted carboxylic acid (0.14 mmol), HATU (54 mg, 0.14 mmol), N, N-diisopropylethylamine (0.42 mmol) and dichloromethane (15 mL) was stirred at room temperature for 2 h, then concentrated in vacuum. The residue was purified by column chromatography on silica with CH₂Cl₂ / MeOH (v: v = 50:1) as eluent to produce B11–B19 as a white solid.

1-(4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)(methyl)amino)piperidin-1-yl)ethan-1-one (B11): white solid, yield 56%. mp >250 °C. ¹H NMR (CDCl₃) δ 9.18 (s, 2H), 5.51 (s, 2H), 5.10–4.70 (m, 2H), 4.01–3.67 (m, 9H), 3.20–3.04 (m, 3H), 2.63–2.58 (m, 2H), 2.14 (s, 3H), 1.84–1.62 (m, 4H). ¹³C NMR (DMSO-d₆) δ 168.5, 165.3 (2C), 164.7, 163.8, 159.0 (2C), 119.2, 66.5 (2C), 52.4, 49.1 (4C), 45.8, 43.7, 21.71 (2C). ESI-MS m/z: 414.3 [M + H]⁺.

(4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)(methyl)amino)piperidin-1-yl)(cyclopropyl)methanone (B12): white solid, yield 79%. mp:>250 °C. ¹H NMR (CDCl₃) δ 9.19 (s, 2H), 5.52 (s, 2H), 5.20–5.05 (m, 1H), 4.90–4.40 (m, 2H), 4.00–3.75 (m, 8H), 3.22–3.03 (m, 4H), 2.67–2.58 (m, 1H), 1.84–1.63 (m, 4H), 1.24 (t, J = 7.0 Hz, 1H), 1.00–0.90 (m, 2H), 0.80–0.75 (m, 2H). ¹³C NMR (DMSO-d₆): δ 171.3 (2C), 165.2 (2C), 164.6, 159.1, 159.0, 119.1, 66.5 (2C), 56.5, 55.4, 52.6 (2C), 44.9, 43.7, 28.7, 19.0, 10.8, 7.3 (2C). ESI-MS m/z: 462.3 [M + Na]⁺.

(4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)(methyl)amino)piperidin-1-yl)(cyclobutyl)methanone (B13): white solid, yield 51%. mp >250 °C. ¹H NMR (CDCl₃) δ 9.18 (s, 2H), 5.52 (s, 2H), 5.15–4.66 (m, 2H), 3.99–3.68 (m, 9H), 3.29–3.20 (m, 1H), 3.03–2.95 (m, 4H), 2.70–2.50 (m, 1H), 2.40–2.25 (m, 2H), 2.25–2.17 (m, 2H), 2.09–1.81 (m, 2H), 1.80–1.52 (m, 4H). ¹³C NMR (DMSO-d₆): δ 172 (2C), 165.2 (2C), 164.6, 159.1, 159.0, 119.1, 66.5 (2C), 52.5, 52.4, 44.4, 44.3, 43.7, 41.2, 36.8, 28.6, 25.1 (2C), 17.8 (2C). ESI-MS m/z: 476.3 [M +Na]⁺.

(4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)(methyl)amino)piperidin-1-yl)(cyclopentyl)methanone (B14): white solid, yield 51%. mp 233–235 °C. ¹H NMR (CDCl₃) δ 9.19 (s, 2H), 5.45 (s, 2H), 5.20–4.75 (m, 2H), 4.12–4.05 (m, 1H), 4.02–3.67 (m, 8H), 3.23–2.92 (m, 5H), 2.63–2.50 (m, 1H), 1.93–1.64 (m, 12H). ¹³C NMR (DMSO-d₆): δ 173.6 (2C), 165.2 (2C), 164.6, 159.1, 158.9, 119.1, 66.51 (2C), 52.6, 52.5, 44.8, 43.7, 41.3, 30.2 (2C), 30.1 (2C), 26.2 (2C), 26.1 (2C). ESI-MS m/z: 468.3 [M + H]⁺.

(4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)(methyl)amino)piperidin-1-yl)(cyclohexyl)methanone (B15): white solid, yield 53%. mp 181–182 °C. ¹H NMR (CDCl₃) δ 9.19 (s, 2H), 5.55 (s, 2H), 5.05–4.75 (m, 2H), 4.07–4.00 (m, 1H), 3.82–3.70 (m, 8H), 3.29–2.99 (m, 4H), 2.62–2.51 (m, 2H), 1.80–1.75 (m, 8H), 1.51–1.46 (m, 4H), 1.43–1.25 (m, 2H). ¹³C NMR (DMSO-d₆): δ 173.7 (2C), 165.2 (2C), 164.6, 159.1, 159.0, 119.1, 66.51 (2C), 54.0, 52.5, 44.7, 43.7, 42.3, 41.1, 38.7, 29.7, 26.1, 25.7 (2C), 18.5, 17.2, 13.0. ESI-MS m/z: 482.3 [M + H]⁺.

(4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)(methyl)amino)piperidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone (B16): white solid, yield 67%. mp 237–239 °C. ¹H NMR (CDCl₃) δ 9.18 (s, 2H), 5.44 (s, 2H), 5.16–4.70 (m, 2H), 4.04–4.00 (m, 3H), 3.97–3.63 (m, 8H), 3.48–3.44 (m, 2H), 3.18–3.01 (m, 4H), 2.80–2.64 (s, 2H), 2.06–1.79 (m, 4H), 1.70–1.45 (m, 4H, CH₂). ¹³C NMR (DMSO-d₆): δ 172.6, 165.2 (2C), 164.6, 159.1, 159.0, 119.1, 66.8 (4C), 66.5 (2C), 52.6 (2C), 44.7, 43.7, 36.8, 29.5, 29.5. ESI-MS m/z: 506.3 [M +Na]⁺.

(4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)(cyclopropyl)amino)piperidin-1-yl)(cyclopropyl)methanone (B17): white solid, yield 46%. mp: >250 °C. ¹H NMR (CDCl₃) δ 9.19 (s, 2H), 5.53 (s, 2H), 4.77–4.60 (m, 1H), 4.59–4.25 (m, 2H), 4.00–3.78 (m, 8H), 3.19–3.15 (m, 1H), 2.65–2.53 (m, 2H), 2.19–2.02 (m, 2H), 2.02–1.79 (m, 3H), 1.09–0.85 (m, 4H), 0.80–0.73 (m, 4H). ¹³C NMR (CDCl₃) δ 171.8, 167.1, 167.0, 164.6, 164.0, 159.1 (2C), 121.31, 66.8 (2C), 57.4 (2C), 45.7, 43.6 (2C), 42.4, 11.0 (2C), 9.3, 7.3 (2C). ESI-MS m/z: 466.3 [M + H]⁺.

(4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)(cyclopropyl)amino)piperidin-1-yl)(cyclopentyl)methanone (B18): white solid, yield 63%. mp 233–236 °C. Anal. calcd for C₂₅H₃₅N₉O₂: C, 60.83; H, 7.15; N, 25.54; found: C, 60.86; H, 7.13; N, 25.57%; ¹H NMR (CDCl₃) δ 9.18 (s, 2H), 5.52 (s, 2H), 4.81 (d, J = 8.7 Hz, 1H), 4.43 (s, 1H), 4.09 (d, J = 8.7 Hz, 1H), 3.80–3.73 (m, 8H), 3.11–2.85 (m, 2H), 2.60–2.50 (m, 2H), 2.18–1.97 (m, 2H), 1.95–1.74 (m, 8H), 1.66–1.51 (m, 2H), 0.92 (m, 2H), 0.79–0.66 (m, 2H). ¹³C NMR (CDCl₃): δ 174.4, 167.1, 167.0, 164.6, 164.1, 159.1 (2C), 121.1, 66.8 (2C), 58.3, 57.5, 45.6, 43.6, 42.2, 41.1 (2C), 31.2, 30.3, 30.1, 26.1, 26.0, 18.4, 9.3, 9.2. ESI-MS m/z: 494.4 [M + H]⁺.

4-((4-(2-Aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)(cyclopropyl)amino)piperidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone (B19): white solid, yield 58%. mp 149–151 °C. Anal. calcd for C₂₅H₃₅N₉O₃: C, 58.92; H, 6.92; N, 24.74; found: C, 58.96; H, 6.89; N, 24.78%; ¹H NMR (CDCl₃) δ 9.17 (s, 2H), 5.52 (s, 2H), 4.81 (d, J = 8.7 Hz, 1H), 4.43–4.35 (m, 1H), 4.03–4.00 (m, 2H), 3.80–3.75 (m, 8H), 3.48–3.44 (m, 2H), 3.15–3.11 (m, 1H), 2.80–2.74 (m, 1H), 2.63–2.49 (m, 2H), 2.21–1.83 (m, 7H), 1.63–1.50 (m, 2H), 0.95–0.84 (m, 2H), 0.79–0.66 (m, 2H). ¹³C NMR (CDCl₃): δ 172.7, 167.1, 167.0, 164.6, 164.2, 159.1 (2C), 121.1, 67.3 (2C), 66.8, 58.2, 57.4, 45.5, 43.6, 42.1, 37.7 (2C), 29.3, 29.1, 27.3, 18.4 (2C), 9.3 (2C). ESI-MS m/z: 510.3 (M + H)⁺.

Antiproliferative assays by MTT method

VS-5584 was purchased from Shanghai Biochempartner Company (Purity: 99%, high-performance liquid chromatography), and 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Sigma (St. Louis, MO, USA). The cellular chemosensitivity was determined by using a modified MTT method assay in human cell lines HCT-116 and MCF-7, which was similar to the procedures described.¹⁶ In brief, HCT-116, MCF-7 cells in 200 μL culture medium were seeded into 96-well microplates at 3000–5000 cells/well, respectively, and cultured in Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum (FBS), or RPMI-1640 with 10% calf serum, incubated at 37 °C for 24 h prior to drug exposure. Cell numbers were titrated to keep control cells growing in the exponential phase throughout the 72 h incubation period. Cells were treated with final concentrations of 10.0, 5.0, 1.0, and 0.5 μM of tested compounds simultaneously and incubated for 72 h and then 20 μL of MTT solution (5 mg/mL in PBS) was added to each well at lucifugal condition and incubated for 4 h at 37 °C. The formed purple formazan crystals were pelleted at the bottom of the well, separated from the supernatant, and dissolved in 200 μL of DMSO. The optical density at 570 nm was determined by Varioskan Flash Multimode Reader (Thermo scientific). Three separate experiments with triplicate data were performed to obtain mean cell viability. The IC₅₀ value was calculated according to the inhibition ratios.

PI3K enzymatic activity assay

PI3K and mTOR enzymatic assay was carried out as described in Wang et al.¹⁸

Molecular modeling

The PI3Kα (PDB code: 5UK8) was used in the molecular modeling, and the docking procedure was performed as described in Hei et al.¹⁷ The protein-ligand complex crystal structure was chosen as the template, to elucidate the binding mode of B18. Protein structure was downloaded from Protein Data Bank (PDB 5UK8). The molecular docking procedure was performed within SYBYLX 2.0 software. The EGFR enzyme was defined as a receptor and the site sphere was selected based on the ligand binding location. Ligand was removed and compound B18 was placed, other parameters were set as default. After accomplishment of the molecular docking procedure, 20 docking poses were scored and selected based on calculated energy. The best docking pose was prepared using PyMOL as showed in Figure 3.

Footnotes

Declaration of conflicting interests

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

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Natural Science Foundation of Shaanxi Province, and China (2019JM-023 and 81673354).

ORCID iD

Minhang Xin

References

Engelman

Luo

Cantley

LC.

Nat Rev Genet 2006; 7: 606.

Liu

Cheng

Roberts

, et al. Nat Rev Drug Discov 2009; 8: 627.

Fruman

Rommel

Nat Rev Drug Discov 2014; 13: 140.

Janku

Yap

Meric-Bernstam

Nat Rev Clin Oncol 2018; 15: 273.

Markham

Drugs 2014; 74: 1701.

Markham

Drugs 2017; 77: 2057.

Sutherlin

Bao

Berry

, et al. J Med Chem 2011; 54: 7579.

Knight

Adams

Burgess

, et al. ACS Med Chem Lett 2010; 1: 39.

Maira

Stauffer

Brueggen

, et al. Mol Cancer Ther 2008; 7: 1851.

10.

Burger

Pecchi

Wagman

, et al. ACS Med Chem Lett 2011; 2: 774.

11.

Kolev

Wright

Vidal

, et al. Cancer Res 2015; 75: 446.

12.

Xin

Hei

Zhang

, et al. Lett Drug Des Disc 2017; 14: 167.

13.

Xin

Hei

Zhang

, et al. Bioorg Med Chem Lett 2017; 27: 1972.

14.

Xin

Duan

Feng

, et al. Bioorg Med Chem 2018; 26: 2028.

15.

Xin

Duan

Feng

, et al. J Enzyme Inhit Med Chem 2018; 33: 651.

16.

Zhang

Xin

Xie

, et al. Bioorg Med Chem 2015; 23: 7765.

17.

Hei

Xin

Zhang

, et al. Bioorg Med Chem Lett 2016; 26: 4408.

18.

Wang

Mao

Cao

, et al. Bioorg Med Chem 2015; 23: 5662.

19.

Wang

, et al. Bioorg Med Chem 2014; 22: 3739.

20.

Xie

Wang

, et al. Bioorg Med Chem 2015; 23: 6477.

21.

Wang

Xin

, et al. Eur J Med Chem 2015; 96: 382.

22.

Wang

Xin

, et al. Bioorg Med Chem Lett 2015; 25: 1730.

23.

Nikolaeva

Kolbin

Sapozhnikov

, et al. Khim Geterotsikl Soedin 1990; 10: 1370.