Design, Synthesis, and Biological Evaluation of 4-Methyl Quinazoline

Article Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/jmc

Design, Synthesis, and Biological Evaluation of 4‑Methyl Quinazoline Derivatives as Anticancer Agents Simultaneously Targeting Phosphoinositide 3‑Kinases and Histone Deacetylases Kehui Zhang,†,‡,∥ Fangfang Lai,†,∥ Songwen Lin,†,‡ Ming Ji,† Jingbo Zhang,†,‡ Yan Zhang,†,‡ Jing Jin,† Rong Fu,† Deyu Wu,†,‡ Hua Tian,†,‡ Nina Xue,† Li Sheng,§ Xiaowen Zou,§ Yan Li,§ Xiaoguang Chen,*,† and Heng Xu*,†,‡ Downloaded via BUFFALO STATE on July 18, 2019 at 01:32:50 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, ‡Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, and §Beijing Key Laboratory of Non-Clinical Drug Metabolism and PK/PD Study, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China S Supporting Information *

ABSTRACT: Polypharmacology is a promising paradigm in modern drug discovery. Herein, we have discovered a series of novel PI3K and HDAC dual inhibitors in which the hydroxamic acid moiety as the zinc binding functional group was introduced to a quinazoline-based PI3K pharmacophore through an appropriate linker. Systematic structure−activity relationship studies resulted in lead compounds 23 and 36 that simultaneously inhibited PI3K and HDAC with nanomolar potencies and demonstrated favorable antiproliferative activities. Compounds 23 and 36 efficiently modulated the expression of p-AKT and Ac-H3, arrested the cell cycle, and induced apoptosis in HCT116 cancer cells. Following pharmacokinetic studies, 23 was further evaluated in HCT116 and HGC-27 xenograft models to show significant in vivo anticancer efficacies with tumor growth inhibitions of 45.8% (po, 150 mg/kg) and 62.6% (ip, 30 mg/kg), respectively. Overall, this work shows promise in discovering new anticancer therapeutics by the approach of simultaneously targeting PI3K and HDAC pathways with a single molecule.

■

INTRODUCTION Combination therapy, a therapy modality that combines two or more therapeutic treatments, is becoming routine practice in cancer treatment.1−3 Because of the nature of this malignant disease that usually involves complex signaling networks interacting through cross talk and feedback loops, combination therapy may potentiate efficacy in a synergistic or additive manner and is also a possible way to tackle drug-resistant cancers.4−6 However, co-administration of two or more drugs at the same time could cause undesirable pharmacokinetic (PK) changes of either agent, through drug−drug interactions, and therefore possible increased toxicities, posing limitations for their use in combination therapy.7,8 An alternative to combination therapy, polypharmacology drugs that simultaneously target multiple mechanisms with a single molecule, are emerging as a new paradigm in drug discovery.9−15 Particularly relevant to cancer, which often results from multiple genetic alterations, inhibition of a single oncogenic target or pathway essential for tumor cell proliferation may not be sufficient to produce sustained efficacy.16 Polypharmacology drugs that modulate a spectrum of relevant disease pathways have thus © XXXX American Chemical Society

proven to be a successful therapeutic strategy in cancer treatment.17−24 For instance, sorafenib, the first multikinase inhibitor blocking Raf isoforms, VEGFR1-3 and several other tyrosine kinases such as PDG-FRβ, c-Kit, Flt-3, and RET, has been a landmark for targeted cancer therapy.25 Class I phosphoinositide 3-kinases (PI3Ks), a family of lipid kinases which play key regulatory roles in many cellular processes including cell survival, proliferation and differentiation, have been one of the most intensively pursued targets for therapeutic intervention in cancer.26−28 Class I PI3Ks comprised four isoforms (α, β, γ, and δ), of which α and β isoforms are ubiquitously expressed in all cells, while γ and δ isoforms are largely restricted to leucocytes.26 Activated by cell surface receptors such as tyrosine kinase receptors (RTKs) or G protein-coupled receptors (GPCRs), class I PI3Ks convert phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3), which in turn activates the serine/threonine kinase AKT and other downstream effector Received: March 3, 2019 Published: May 22, 2019 A

DOI: 10.1021/acs.jmedchem.9b00390 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Selected chemical structures of PI3K inhibitors (1−4), HDAC inhibitors (5−9), and PI3K/HDAC dual inhibitors (10−12).

pathways.29 Recent human cancer genomic studies have revealed that PI3K pathway aberrations are among the most common in cancer.30 To date, over 30 structurally distinct class I PI3K inhibitors31,32 have entered into clinical trials and can be subdivided into pan-PI3K inhibitors, dual PI3K/mTOR inhibitors, and isoform-selective PI3K inhibitors according to their isoform selectivity profiles. Among them, the PI3Kδ selective inhibitor idelalisib,33 the pan-PI3K inhibitor copanlisib,34 and the PI3Kγ/δ selective inhibitor duvelisib35 have been approved by the FDA for the treatment of blood cancers (Figure 1). While validated for hematological malignancies, PI3K inhibitors yield limited response to patients with solid tumors in the clinical trials, potentially because of negative feedback resulting in activation of compensatory signaling pathways.36,37 Histone deacetylases (HDACs) are a class of epigenetic enzymes that catalyze the removal of an acetyl group from lysine residues of histones and other proteins, thereby regulating chromatin structure and transcriptional activity.38−40 HDACs are overexpressed in many types of cancer, and aberrant activity of HDACs is linked to key oncogenic events.38 Inhibition of HDACs has been shown to exert a range of anticancer effects such as induction of cell cycle arrest, apoptosis, and inhibition of angiogenesis and metastasis.41−43 To date, five HDAC inhibitors (vorinostat, romidepsin, panobinostat, belinostat, and chidamide) have been approved

by the regulatory agencies for the treatment of hematologic cancers (Figure 1).44,45 Interestingly, recent studies revealed that the combination of HDAC and PI3K inhibitors showed synergistic anticancer activities. For example, the PI3K/mTOR inhibitor BEZ235 markedly potentiates HDAC inhibitor activity in NHL cells,46 while the combination of HDAC and PI3K inhibitors synergistically induces apoptosis in human endometrial carcinoma cells.47 In particular, CUDC-907, a hybrid derivative of pictilisib and quisinostat developed by Curis, potently inhibited both PI3K and HDAC enzymes and showed greater efficacies than single-target PI3K or HDAC inhibitors alone or in combination in cell proliferation assays and in vivo xenograft models, paving the way to be the first PI3K/HDAC dual inhibitor to be investigated in clinical trials.48−50 Notably, CUDC-907 has recently been granted fast-track designation by the FDA for the treatment of relapsed or refractory diffuse large B-cell lymphoma (DLBCL) after positive results from phase II studies (Figure 1).51 A couple of other structurally related PI3K/HDAC dual inhibitors have also demonstrated promising profiles that may establish a new class of anticancer agents. For example, purine-based hydroxamic acid derivatives (e.g., 11) simultaneously targeting PI3K and HDAC were reported to have marked in vitro and in vivo antitumor activities,52 and fused pyrimidine-based hydroxamate anaB



Article

Figure 2. Design strategy of 4-methyl quinazoline-based PI3K/HDAC dual inhibitors.

acid) essential for HDAC inhibition to this 8-position of the quinazoline PI3K scaffold. First, hydroxamic acids were attached to the quinazoline PI3K scaffold using different alkyl linkers (Table 1). Compound 15 with a one-carbon (methylene) linker displayed potent inhibitory activities against PI3Kα and HDAC1 with IC50 values of 68 and 166 nM, respectively. Herein, PI3Kα and HDAC1 were chosen to drive our primary SAR study, mainly because PI3Kα harbors the frequent somatic mutations in various solid tumors,27 while HDAC1 is well documented with its crucial role in cancer cell proliferation.39 As the length of the linker was increased to three carbons in compound 16, a similar PI3Kα potency was observed, and the HDAC1 activity was enhanced about 2-fold (91 vs 166 nM). As the linker length increased further (e.g., 17, 18, and 19), HDAC1 potency was boosted by over 60-fold (IC50s: 1.3 vs 84 nM for 19 vs 17). Of particular note, compound 19 with a six-carbon linker exhibited a notable HDAC1 potency with an IC50 value of 1.3 nM, comparable to that of the marketed HDAC inhibitors (e.g., SAHA). However, compounds with different linker lengths retained a similar potency against PI3Kα (e.g., IC50s = 51−102 nM for 15−19). It was thought that the alkyl linker was situated in the solvent-exposed region and did not affect the key binding interactions with the target protein. It is noteworthy that linkers longer than six-carbon resulted in lower potencies for both PI3Kα and HDAC1. For example, relative to 19, compound 20 with a seven-carbon linker showed an approximate 1.5-fold and 3-fold decrease in PI3Kα and HDAC1 potencies, respectively (PI3Kα IC50: 102 vs 143 nM; HDAC1 IC50: 1.3 vs 3.7 nM), while compound 21 with an eight-carbon linker displayed a further decrease in PI3K potency (PI3Kα IC50 = 162 nM). Clearly, our SAR studies revealed that the quinazoline PI3K scaffold could be a favorable “capping region” (e.g., surface recognition domain) and the six-carbon linker is optimal for HDAC1 inhibition.52,55 Furthermore, we evaluated their antiproliferative effects on

logues (e.g., 12) showed significant efficacies in various liver cancer xenograft models (Figure 1).53 Designing PI3K/HDAC dual inhibitors typically requires a strategy of merging PI3K and HDAC pharmacophores. While HDAC activity can be obtained from incorporation of a zincbinding group through a linker, PI3K activity requires an appropriate chemical scaffold that can accommodate the HDAC functionality without compromising its PI3K potency. It should be noted that simply bridging PI3K and HDAC pharmacophores through a linker does not often work for the dual design and a thorough understanding of their respective structure−activity relationships (SARs) is essential. To date, a large majority of reported PI3K/HDAC dual inhibitors (e.g., CUDC-907, 11, and 12) feature a morpholinopyrimidine core; not many other PI3K scaffolds have been explored for this class of dual-action agents aiming to achieve the optimal balance of PI3K and HDAC inhibitory activities. Furthermore, the new scaffold is likely to display different PI3K and/or HDAC isoform selectivity profiles to existing PI3K/HDAC dual inhibitors, which may offer a potential for balancing efficacy and safety to improve clinical outcomes.

■

RESULTS AND DISCUSSIONS To find an efficient PI3K scaffold, we focused on our recently disclosed quinazoline PI3K inhibitors (e.g., 13, Figure 2) which establish hydrogen bonding interactions with Val851, a conserved water molecule bridge with Tyr836 and Asp810 and one charge interaction between the deprotonated sulfonamide and Lys802 in the ATP binding pocket of PI3Kα.54 In addition to the above critical interactions, this quinazoline scaffold allows synthetic manipulation to its 8-position through an ether linkage for further derivatization. These ether functionalities are located in the solvent-exposed region to have the minimum effect on the PI3K activity. In our design of novel PI3K/HDAC dual inhibitors, we therefore planned to introduce the zinc-binding functional group (e.g., hydroxamic C



Article

Table 1. SARs of Alkyl Linkers and Substituted Methoxyl Pyridines

IC50 (nM)b

IC50 (μM)c

cmpd

n

R1

clog Pa

PI3Kα

HDAC1

HCT116

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 SAHA BKM120 CUDC-907

1 3 4 5 6 7 8 5 6 7 8 5 6 7 8 3 4 5 6 7 8

H H H H H H H F F F F MeSO2NH− MeSO2NH− MeSO2NH− MeSO2NH− 2,4-diF-PhSO2NH− 2,4-diF-PhSO2NH− 2,4-diF-PhSO2NH− 2,4-diF-PhSO2NH− 2,4-diF-PhSO2NH− 2,4-diF-PhSO2NH−

1.4 2.0 2.0 2.5 3.0 3.5 4.1 2.5 3.0 3.5 4.1 1.9 2.5 3.0 3.5 3.5 3.4 4.0 4.5 5.0 5.5 1.0 1.6 2.1

68 73 51 58 102 143 162 30 42 54 116 5.1 2.3 3.9 4.0 1.3 0.60 0.66 0.69 0.63 0.90

166 91 84 30 1.3 3.7 3.0 37 1.4 3.3 2.2 42 1.3 2.1 4.9 37 303 39 6.3 1.8 8.9 32

>10 >10 1.4 0.54 0.14 0.80 0.76 1.2 0.15 1.4 0.58 >10 37 3.4 11 >10 0.35 0.54 7.2 1.5 4.0 1.9 1.3 0.005

20 69

0.36

a

Calculated from ChemBioDraw Ultra 14.0. bData represent the mean value from at least two independent experiments. cData represent the mean value from at least three independent experiments.

rise to even higher PI3Kα potencies (0.60−1.3 nM), largely because of the resonance effect that rendered a more acidic sulfonamide NH proton and resulted in a stronger charge interaction. To our delight, the above modifications on the 3position of the methoxyl pyridine did not affect HDAC1 activity. It was noted that the significantly increased PI3Kα potency induced by the sulfonamide functionality did not cause an improvement in antiproliferative activity on HCT116 cells, possibly attributed to the unfavorable permeability caused by the polar sulfonamide group. In addition to alkyl linkers, we also explored pyrimidyl linkers that have featured in several HDAC clinical candidates (Table 2).57,58 Whereas compound 36 with an amino-2pyrimidinyl linker displayed a slight decrease in PI3Kα potency relative to 19 that has a six-carbon alkyl linker (IC50: 226 vs 102 nM), these two compounds showed an equal potency against HDAC1 (IC50 = 1.3 nM). Impressively, 36 potently inhibited the proliferation of HCT116 cells with an IC50 value of 0.007 μM, a 20-fold enhancement over 19. By increasing the length of the pyrimidyl linker (from two carbons to three carbons between the oxygen and the amino-2-pyrimidine), 37 maintained its PI3Kα potency, but its HDAC1 activity decreased about 2-fold. Similar to the compounds with alkyl linkers, introducing fluorine or phenyl sulfonamide to the 3-

human colon carcinoma HCT116 cells harboring both activating K-Ras and PIK3CA mutations. Generally, their antiproliferative IC50s fell into the micromolar range and mostly correlated with their HDAC1 potencies. Among them, compound 19 was the most potent against HCT116 cells with an IC50 value of 0.14 μM, which is greater than 10-fold more potent than that of SAHA (IC50 = 1.9 μM). Notably, compounds 15 and 16 with one or three-carbon linkers were inactive in cellular assays. This is likely attributed to their high polarity as indicated by their low clog P values (clog P ≤ 2). Next, we focused on reaching the desired level of PI3K potency by introducing various functionalities that could form an interaction with Lys802 to the 3-position of the pyridine moiety adjacent to the methoxyl group (Table 1). When the fluoro atom was attached, PI3Kα potency was increased by 1.5−3 fold (22 vs 18, 23 vs 19, 24 vs 20, and 25 vs 21). This potency enhancement can be attributed to the hydrogen bonding interaction between the F atom and the Lys802 residue.56 As expected, sulfonamide functionalities that can form a charge interaction with Lys802 further boosted the PI3Kα potency. Methyl sulfonamides 26−29 displayed IC50 values between 2.3 and 5.1 nM, an 11−44-fold increase in PI3Kα potencies over their corresponding methoxyl pyridines 18−21. Notably, 2,4-diflurophenyl sulfonamides 30−35 gave D



Article

Table 2. SARs of Amino-2-pyrimidinyl Linkers and Substituted Methoxyl Pyridines

IC50 (nM)b cmpd

n

R2

R3

clog P

36 37 38 39 40 41 42 43 44 45 46 47 48 49 SAHA BKM120 CUDC-907

2 3 2 3 4 2 3 2 2 2 2 2 3 4

H H F F F 2,4-diF-PhSO2NH− 2,4-diF-PhSO2NH− H F H F F F F

Me Me Me Me Me Me Me Et Et iPr iPr H H H

2.6 3.0 2.5 3.0 3.2 4.0 4.5 3.1 3.1 3.4 3.4 2.4 2.9 3.1 1.0 1.6 2.1

a

a

IC50 (μM)c

PI3Kα

HDAC1

HCT116

226 213 73 94 48 1.8 1.1 195 72 82 43 43 43 41

1.3 2.5 1.3 2.4 3.3 1.7 11 2.1 2.6 1.6 2.0 5.3 4.0 11 32

0.007 0.10 0.030 0.082 0.25 0.53 2.9 0.022 0.039 0.039 0.096 4.6 2.1 9.3 1.9 1.3 0.005

20 69

0.36

b

Calculated from ChemBioDraw Ultra 14.0. Data represent the mean value from at least two independent experiments. cData represent the mean value from at least three independent experiments.

Table 3. PI3K and HDAC Isoform Profiling of Compounds 23 and 36a PI3K isoforms IC50 (nM)

IC50 (nM)

HDAC isoforms IC50 (nM)

cmpd

PI3Kα

PI3Kβ

PI3Kγ

PI3Kδ

mTOR

HDAC1

HDAC2

HDAC4

HDAC6

HDAC8

HDAC11

23 36 CUDC-907

42 226 69

101 279 16

67 467 374

8.1 29 359

2861 6262 431

1.4 1.1 0.36

3.0 3.4 1.6

>1000 972 445

6.6 17 8.0

18 12 1.4

>1000 >1000 132

a

Data represent the mean value from at least two independent experiments.

23 and 36 also potently inhibited HDAC2 (class I), HDAC6 (class IIb), and HDAC8 (class I) with IC50 values in the range of 3.0−18 nM. Similar to SAHA, these two compounds were ineffective against HDAC11 (class IV). Notably, 36 showed some inhibitory effect on HDAC4 (class IIa) with an IC50 value of 972 nM. Compound 23 was also screened at a 1 μM concentration to assess its kinase selectivity using DiscoverX’s kinomescan technology (Figure 3). Among 97 kinases tested, 23 exhibited good affinity for class I PI3Ks as expected. It also displayed moderate affinity for class II PI3K (e.g., PIK3C2B) and DYRK1B. Overall, 23 demonstrated an excellent selectivity profile against kinases. 23 and 36 were further evaluated in a broad panel of hematologic and solid tumor cell lines including lymphoma/leukemia, breast, brain, colon, gastric, lung, pancreas, liver, and prostate cancer. As shown in Table 4, 23 and 36 were generally more potent than the reference HDAC inhibitor SAHA and PI3K inhibitor BKM120 in most of the cancer cell lines tested. IC90 values of compounds 23 and 36 against the HCT116 cell line were also determined to be 48 and 2.8 μM, respectively. In particular, 36, with a pyrimidyl linker, exhibited remarkable antiproliferative activities with IC50 values in a single-digit or double-digit nanomolar range

position of the methoxyl pyridine also significantly enhanced the PI3Kα potency (e.g., 38−42). Notably, the difluorophenyl sulfonamide substituent negatively affected the cellular proliferation activity (IC50: 0.53 vs 0.007 μM for 41 vs 36; 2.9 vs 0.10 μM for 42 vs 37). We also varied the alkyl groups on the amino-2-pyrimidyl linker and found that relative to methyl group (36 and 38), ethyl (43 and 44) and isopropyl (45 and 46) groups afforded similar PI3Kα and HDAC1 potencies. However, removal of the alkyl group caused a significant decrease in antiproliferative activity against HCT116 cells (e.g., IC50s > 2 μM for 47−49), likely because of their poor permeability. On the basis of their favorable PI3K and HDAC inhibitory potencies, as well as their potent antiproliferative activities, compounds 23 and 36 featuring an alkyl and a pyrimidyl linker, respectively, were selected for further profiling. As shown in Table 3, these two compounds potently inhibited all four class I PI3K isoforms, with >5-fold selectivity for PI3Kδ over the other isoforms. Notably, they showed a degree of selectivity for class I PI3Ks over mTOR (>68-fold for 23; >27fold for 36). Compounds 23 and 36 were also tested against different classes of HDAC enzymes. Besides HDAC1 (class I), E



Article

Figure 3. Kinase selectivity screening of compound 23 at a concentration of 1 μM (DiscoveRx). (A) TREEspot interaction maps for compound 23. (B) Among 97 kinases tested, only four were hit with 100 5.1 3.0 3.8 1.8 0.35 2.0 4.3 7.1

colon brain NSCLC lung pancreas prostate stomach liver

BKM120 Hematologic 12 7.9 Solid 5.7 0.37 1.7 1.3 4.8 3.2 3.6 42 1.3 45 0.99 3.2 2.0 13

CUDC-907

23

36

NDb 0.28

5.4 6.8

0.91 1.2

0.041 0.009 0.005 0.005 0.007 0.15 0.025 0.007 0.18 0.56 0.041 0.015 0.56 0.013

2.0 0.30 0.32 0.15 3.2 3.0 1.0 0.43 0.90 0.54 0.11 1.1 2.5 5.2

1.1 0.004 0.059 0.007 0.34 2.7 0.18 0.27 0.23 0.066 0.014 0.076 0.24 1.6

a

Data represent the mean value from at least three independent experiments. bND: not determined. F



Article

Figure 5. Compounds 23 and 36 dose-dependently induced cell cycle arrest and apoptosis in HCT116 cancer cells. (A) Flow cytometry analysis of the cell cycle distribution following 23, 36, SAHA, and BKM120 treatment to HCT116 cells at the indicated concentrations for 24 h. (B) Percentage of HCT116 cells in different phases of the cell cycle. (C) Flow cytometry analysis of annexin-V/propidium iodide (PI) double staining of apoptotic cells following 23, 36, SAHA, and BKM120 treatment to HCT116 cells at the indicated concentrations for 48 h. (D) Quantitative analysis of apoptotic cells.

Figure 6. Predicted binding modes of compound 23 (yellow) and 36 (pink) with PI3Kα [PDB ID: 4JPS, (A,B)] and HDAC1 [PDB ID: 1C3S, (C,D)]. Hydrogen bonds are shown as yellow dashed lines. Key residues as well as water molecules and the zinc ion interacting with the compounds are highlighted. Images were generated with Pymol.

predominantly induced G2/M phase cell cycle arrest. The percentage of apoptotic cells increased with the treatment of 23 and 36 in a dose-dependent manner. Compared to the

Figure 5A,B, cell cycle analysis revealed that 23 and 36 dosedependently arrested the cell cycle at the G1 phase in HCT116 cells, while the reference compounds SAHA and BKM120 G



Article

Table 5. Mouse PK Parameters of Compounds 23 and 36 cmpd

route

23

a,c

36 a

iv pob,d iva,c pob,d

dose (mg/kg) T1/2β (h) Tmax (h) Cmax (ng/mL) 3 30 3 30

0.33 2.07 e e

0.03 0.30 0.03 1.80

AUC(0−t) (h·ng/mL)

AUC(0−∞) (h·ng/mL)

551 230 133 23.1

558 274 143 51.4

3051 143 417 9.39

Vz (L/kg) CL (mL min−1 kg−1) 2.57

F%

90.0 4.2

50.0

352 1.7

b

Three mice per study for iv. Five mice per study for po. civ formulation: 10% DMSO/saline. dpo formulation: 0.5% cmc/0.3% Tween 80/water. e Not determined because of the lack of sufficient (≥3) data points in the elimination phase.

Figure 7. In vivo antitumor efficacy of compound 23 in the HCT116 tumor xenograft model. (A) Tumor volume (TV) changes following the treatment of 23 at oral doses of 100 and 150 mg/kg and SAHA at an intraperitoneal dose of 100 mg/kg. (B) Average body weights for 23, SAHA, and vehicle-treated mice groups. (C) Concentrations of 23 in tumor tissues for the 23-treated mice group. (D) Immunohistochemical staining of Ki67, Ac-H3, and p-AKT (S473) in tumor tissues from 23-treated mice and vehicle groups (200×). (E) Integrated optical density (IOD) analysis of IHC staining. Results are expressed as the mean ± SEM (n = 5−7 for each group), one-way ANOVA analysis, *p < 0.05 vs vehicle.

Phe198, and Leu265. Specifically, the pyrimidine linker in 36 interacted with the phenyl groups of Phe141 and Phe198 through π−π stacking. Compounds 23 and 36 were further evaluated in in vivo PK studies (Table 5). Similar to other hydroxamate-based HDAC inhibitors, 23 and 36 showed high clearance and poor oral bioavailability in mice. At an oral dose of 30 mg/kg, the area under the curve (AUC(0−∞)) was determined to be 274 and 51.4 h·ng/mL, and oral bioavailability (% F) was calculated to be 4.2 and 1.7% for 23 and 36, respectively. It is well documented that hydroxamic acid is rapidly metabolized through glucuronidation and hydrolysis, contributing the main reason for poor PK profiles of clinical hydroxamate HDAC inhibitors.59−63 The glucuronide and hydrolyzed metabolites of 23 were consistently found in the rat bile (Supporting Information). Furthermore, in vitro ADME assays revealed that compound 23 was quickly cleared in mouse plasma (t1/2 = 1.20 h) and mouse liver microsomes (t1/2 = 0.41 h), rationalizing its in vivo PK properties. Relative to 36, 23 with an alkyl linker showed a better PK profile (slower clearance and enhanced oral exposure).

HDAC inhibitor SAHA and PI3K inhibitor BKM120, 23 and 36 were much more potent in inducing apoptosis at a concentration of 0.5 μM (Figure 5C,D). Molecular docking studies were performed to investigate the binding modes of compounds 23 and 36 with PI3Kα and HDAC1. As shown in Figure 4, the quinazoline scaffold fits well into the PI3K ATP binding pocket and forms a hydrogen bond interaction with the backbone NH group of Val851 in the hinge region as well as a water bridge with Typ836 and Asp810 (Figure 6A,B). These interactions were identified as the key driving force for the PI3K binding. It was also revealed that the hydroxamic acid moiety and its linkers were located in the solvent-exposed region and had minimum effect on PI3K binding. Docking compounds 23 and 36 into HDAC1 showed that the hydroxamic acid moieties, as expected, effectively coordinated the catalytic Zn2+ in a bidentate manner and formed multiple hydrogen bonds with Asp258, Asp168, and His170, while the large quinazoline cap was positioned on the highly solvent-exposed surface (Figure 6C,D). The alkyl linker in 23 and the pyrimidine linker in 36 occupied the hydrophobic channel, formed by three residues Phe141, H



Article

Figure 8. In vivo antitumor efficacy of compound 23 in the HGC-27 tumor xenograft model. (A) TV changes following the treatment of 23 at doses of 100 mg/kg (po), 200 mg/kg (po), and 30 mg/kg (ip) and SAHA at an oral dose of 100 mg/kg. (B) Average body weights for 23, SAHA, and vehicle-treated mice groups. (C) Concentrations of 23 in blood and tumor tissues for 23-treated mice groups. (D) Immunohistochemical staining of Ki67, Ac-H3, and p-AKT(S473) in tumor tissues from 23-treated mice and vehicle groups (200×). (E) IOD analysis of IHC staining. Results are expressed as the mean ± SEM (n = 5−7 for each group), one-way ANOVA analysis, *p < 0.05 vs vehicle.

Table 6. Preliminary Safety Assessment of Compounds 23 and 36 Single dose toxicityd (MTD, mg/kg)

SI cmpd

hERG (IC50, μM)

AMESa

PBMC IC50b/HCT116 IC50

LO2 IC50c/HCT116 IC50

ip

po

23 36

>30 >30

negative negative

23 8240

147 1430

>100 >100

>2000 >2000

Tested at concentrations from 5 to 500 μg/plate with or without metabolic activation using S. typhimurium strains TA97a, TA98, TA100, TA102, and TA1535. bPBMC: human primary peripheral blood mononuclear cell. cLO2: human normal liver cell. dICR mice.

a

Figure 8, an oral dose of 200 mg/kg resulted in a TGI of 45.9%. Notably, an ip dose of 30 mg/kg achieved a more pronounced efficacy with a TGI of 62.6%, but caused a significant body weight loss (20%). At the end of treatment (0.5 h after the final dosing), a PK study was performed to analyze both plasma and tumor tissue concentrations of 23. The drug concentrations in plasma were determined to be 179, 232, and 577 ng/mL for the 100 mg/kg (po), 200 mg/kg (po), and 30 mg/kg (ip) dosing groups, while the corresponding concentrations in tumor tissue were 36.6, 105, and 370 ng/g, respectively. The in vivo antitumor efficacy was shown to correlate well with the drug concentrations in both plasma and tumor tissues. Further IHC staining revealed that the expression levels of p-AKT and Ki-67 were downregulated and the expression levels of Ac-H3 were upregulated in the tumor tissues of the 23 treatment groups, validating its dual antitumor mechanisms. Compounds 23 and 36 were further subjected to a preliminary safety assessment (Table 6). In a hERG assay, 23 and 36 did not show any inhibitory effect with an IC50 value of >30 μM, indicating their low probability for drug-induced cardiovascular effects. In a bacterial reverse mutation assay (AMES test), 23 and 36 were negative in all Salmonella

On the basis of its favorable in vitro potencies as well as acceptable PK profiles, 23 was progressed into in vivo antitumor activity studies. In a HCT116 human colon cancer xenograft model, oral dosing of mice with 100 and 150 mg/kg of 23 achieved a dose-dependent tumor growth inhibition (TGI) of 18.3% for 100 mg/kg and 45.8% for 150 mg/kg (Figure 7). The antitumor efficacy of 23 at the highest oral dose tested (150 mg/kg) is comparable to that of SAHA at an intraperitoneal (ip) dose of 100 mg/kg. It was also found that 23 induced body weight loss at the doses tested in this model. At 1 h after the final dosing, concentrations of 23 in HCT116 tumor tissues were determined to 72.7 and 74.8 ng/g for the 100 and 150 mg/kg dosing groups, respectively. Immunohistochemistry (IHC) staining showed that the expression levels of p-AKT were dose-dependently suppressed, and the expression levels of Ac-H3 were dose-dependently increased in tumor tissues of the 23 treatment groups, indicating the efficient inhibition of both PI3K and HDAC pathways in the in vivo setting. IHC staining also revealed that the expression level of Ki-67 (widely accepted as a cell proliferation marker) was also dose-dependently decreased in the 23 treatment groups. In a HGC-27 gastric cancer xenograft model, mice were dosed orally or intraperitoneally with 23. As shown in I



Article

Scheme 1. Synthesis of Compounds 15−35a

a

Reagents and conditions: (a) HNO3, rt, 72 h, 38%; (b) Fe, NH4Cl, THF/EtOH/water, reflux, 2 h, 98%; (c) NBS, DCM, rt, 3 h, 75%; (d) HCONH2, HCONH4, 150 °C, Ar, 10 h, 53%; (e) AlCl3, DCE, 80 °C, 2 h, 55%; (f) ω-bromo substituted alkyl carboxylate, K2CO3, MeCN, reflux, 16 h, 59−71%; (g) pyridinyl boronic acid or ester, K2CO3, PdCl2(dppf), toluene/water, 80 °C, 8 h, 21−56%; (h) NH2OH (aq), KOH, MeOH, rt, 2 h, 37−55%.

Scheme 2. Synthesis of Compounds 36−49a

a Reagents and conditions: (a) Boc-amino alcohol, Ph3P, DEAD, THF, Ar, rt, 16 h, 65−77%; (b) TFA, DCM, rt, 1.5 h; (c) ethyl 2chloropyrimidine-5-carboxylate, DIPEA, MeCN, rt, 16 h, 63−70%. (d) Pyridinyl boronic acid or ester, K2CO3, PdCl2(dppf), toluene/water, 80 °C, 8 h, 53−71%; (e) NH2OH (aq), KOH, MeOH, rt, 2 h, 33−69%.

typhimurium strains investigated in both the presence and absence of S9 mix, displaying low carcinogenic potentials. Additionally, compounds 23 and 36 exhibited high selectivity indexes (SIs) between normal human primary cells and HCT116 cancer cells. In the single dose toxicity studies, the maximum tolerated doses (MTD) of compounds 23 and 36 were determined to be >100 mg (ip) and >2 g (po), respectively. Overall, compounds 23 and 36 show favorable safety profiles that merit further preclinical assessment.

reduction with iron powder. Bromination of 52 with NBS afforded 53, which was reacted with ammonium formate in formamide to give 54. The subsequent demethylation of 54 afforded 55, which was converted to 56a−g by a nucleophilic substitution reaction with ω-bromo-substituted alkyl carboxylate. In the other route shown in Scheme 2, 55 was converted to 58a−h via a Mitsunobu reaction. The following deprotection of the Boc group afforded 59a−h, which were reacted with ethyl 2-chloropyrimidine-5-carboxylate to give 60a−h. Compounds 56a−g and 60a−h were converted to 57a−u and 61a−n via Suzuki reaction, respectively. Hydroxamic acids 15−49 were prepared from the corresponding carboxylates 57a−u and 61a−n. X-ray diffraction technology was used to determine the crystal structure of compound 36 (Figure 9).

■

CHEMISTRY The synthetic routes of compounds 15−49 with alkyl and pyrimidine linkers are illustrated in Schemes 1 and 2, respectively. As shown in Scheme 1, the starting material 50 was nitrated to afford 51, which was converted to 52 by J



Article

mm, 25 °C, 5−95% mobile phase B in 3.0 min, 95% in 2.0 min, 95− 5% in 0.1 min, 5% in 1.0 min, 0.6 mL/min, UV detector at 254 nm, Agilent G1946D with APCI mode as the MS detector. All final compounds passed the PAINS filter64 with False Positive Remover.65 General Procedure for the Synthesis of Compounds 15−35 and 36−49. To a solution of compounds 57a−u (1.0 equiv) and potassium hydroxide (10 equiv) in methanol was added hydroxylamine solution (50 wt % in H2O) (30 equiv). The reaction mixture was stirred at room temperature for 2 h, cooled in an ice bath, and acidified with 3 M HCl solution until the pH value was 5−6. The crude product was obtained by filtration as a light yellow solid and then purified by column chromatography (silica gel, DCM/MeOH = 10:1) to afford the pure product as a white or light yellow solid. 4-Methyl-6-(2-methoxy-5-pyridinyl)-8-(N-hydroxyacetamide-2oxy)quinazoline (15). Compound 15 was prepared from compound 57a (light yellow solid, 37% yield). 1H NMR (400 MHz, MeOD-d4): δ 9.14 (s, 1H), 8.63 (d, J = 2.6 Hz, 1H), 8.19 (dd, J = 8.7, 2.6 Hz, 1H), 8.09 (d, J = 1.6 Hz, 1H), 7.76 (d, J = 1.6 Hz, 1H), 6.98 (d, J = 8.6 Hz, 1H), 4.96 (s, 2H), 4.01 (s, 3H), 3.07 (s, 3H). 13C NMR (101 MHz, MeOD-d4): δ 172.7, 170.8, 165.9, 156.0, 153.7, 146.9, 141.8, 139.7, 139.6, 130.8, 127.0, 115.1, 113.4, 112.1, 71.5, 64.6, 54.9. HRMS (ESI) m/z: [M + H]+ calcd for C17H16O4N4, 341.1244; found, 341.1234, Δ −3.08 ppm. 4-Methyl-6-(2-methoxy-5-pyridinyl)-8-(N-hydroxybutanamide4-oxy)quinazoline (16). Compound 16 was prepared from compound 57b (light yellow solid, 39% yield). 1H NMR (400 MHz, MeOD-d4): δ 9.05 (s, 1H), 8.58 (d, J = 1.6 Hz, 1H), 8.20−8.04 (dd, J = 8.5 1.6 Hz, 1H), 7.95 (s, 1H), 7.64 (s, 1H), 6.95 (d, J = 8.5 Hz, 1H), 4.37 (t, J = 5.6 Hz, 2H), 3.99 (s, 3H), 3.01 (s, 3H), 2.44 (m, 2H), 2.36−2.23 (m, 2H). 13C NMR (101 MHz, MeOD-d4): δ 170.9, 165.9, 155.9, 153.8, 146.9, 141.9, 139.6, 131.0, 130.7, 127.0, 115.2, 113.8, 112.1, 111.6, 69.5, 64.5, 54.4, 26.5, 22.3. HRMS (ESI) m/z: [M + H]+ calcd for C19H20O4N4, 369.1557; found, 369.1552, Δ −1.49 ppm. 4-Methyl-6-(2-methoxy-5-pyridinyl)-8-(N-hydroxypentanamide5-oxy)quinazoline (17). Compound 17 was prepared from compound 57d (light yellow solid, 43% yield). 1H NMR (400 MHz, MeOD-d4): δ 9.04 (s, 1H), 8.56 (d, J = 1.9 Hz, 1H), 8.13 (dd, J = 8.5, 1.8 Hz, 1H), 7.90 (d, J = 1.5 Hz, 1H), 7.58 (d, J = 1.5 Hz, 1H), 6.94 (d, J = 8.6 Hz, 1H), 4.34 (m, 2H), 3.98 (s, 3H), 3.00 (s, 3H), 2.33 (t, J = 6.7 Hz, 2H), 1.98 (m, 4H). 13C NMR (101 MHz, MeODd4): δ 172.9, 170.8, 165.9, 156.0, 153.7, 146.8, 141.8, 139.7, 139.6, 130.7, 126.9, 114.9, 113.3, 112.1, 71.1, 54.4, 33.6, 28.6, 24.8, 22.3. HRMS (ESI) m/z: [M + H]+ calcd for C20H22O4N4, 383.1714; found, 383.1706, Δ −2.14 ppm. 4-Methyl-6-(2-methoxy-5-pyridinyl)-8-(N-hydroxyhexanamide6-oxy)quinazoline (18). Compound 18 was prepared from compound 57f (white solid, 50% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.39 (s, 1H), 9.05 (s, 1H), 8.72 (d, J = 2.3 Hz, 1H), 8.69 (s, 1H), 8.26 (dd, J = 8.6, 2.4 Hz, 1H), 7.93 (d, J = 1.3 Hz, 1H), 7.64 (d, J = 1.2 Hz, 1H), 6.97 (d, J = 8.6 Hz, 1H), 4.26 (t, J = 6.4 Hz, 2H), 3.93 (s, 3H), 2.93 (s, 3H), 2.01 (t, J = 7.2 Hz, 2H), 1.92−1.82 (m, 2H), 1.69−1.57 (m, 2H), 1.55−1.44 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 169.1, 168.0, 163.5, 154.6, 153.0, 145.7, 140.5, 138.3, 136.5, 128.7, 124.9, 113.4, 111.8, 110.6, 68.6, 53.4, 32.3, 28.5, 25.3, 25.0, 22.0. HRMS (ESI) m/z: [M + H]+ calcd for C21H24O4N4, 397.1870; found, 397.1853, Δ −4.36 ppm. 4-Methyl-6-(2-methoxy-5-pyridinyl)-8-(N-hydroxyheptanamide7-oxy)quinazoline (19). Compound 19 was prepared from compound 57j (light yellow solid, 53% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.34 (s, 1H), 9.06 (s, 1H), 8.74 (d, J = 2.5 Hz, 1H), 8.65 (s, 1H), 8.28 (dd, J = 8.6, 2.6 Hz, 1H), 7.96 (d, J = 1.3 Hz, 1H), 7.66 (d, J = 1.3 Hz, 1H), 6.98 (d, J = 8.6 Hz, 1H), 4.28 (t, J = 6.5 Hz, 2H), 3.93 (s, 3H), 2.95 (s, 3H), 1.97 (t, J = 7.3 Hz, 2H), 1.91−1.81 (m, 2H), 1.52 (dt, J = 14.8, 7.4 Hz, 4H), 1.37 (d, J = 6.8 Hz, 2H). 13C NMR (101 MHz, DMSO-d6): δ 169.1, 168.0, 163.5, 154.7, 153.0, 145.7, 140.5, 138.3, 136.5, 128.7, 124.9, 113.4, 111.8, 110.6, 68.6, 53.4, 32.2, 28.6, 28.4, 25.4, 25.1, 22.0. HRMS (ESI) m/z: [M + H]+ calcd for C22H26O4N4, 411.2027; found, 411.2019, Δ −1.90 ppm.

Figure 9. X-ray crystal structure of compound 36. Two molecules of 36 were bound with one methanol through intermolecular hydrogen bonding.

■

CONCLUSIONS In summary, we discovered a series of novel PI3K/HDAC dual inhibitors through rational design. These PI3K/HDAC dual inhibitors feature the efficient hybridization of a quinazolinebased PI3K pharmacophore and a hydroxamate-based HDAC functionality. SAR studies revealed that substitutions at the 3position of the methoxyl pyridine on the PI3K scaffold allow control over PI3K potency. Variation of the linkers for the hydroxamic acid serves as a useful tool to manipulate HDAC potency, thereby providing an effective strategy to achieve the optimal combination for PI3K and HDAC activities. Compounds 23 and 36 with an alkyl linker and a pyrimidine linker, respectively, were selected as representatives for further profiling, and both exhibited nanomolar enzymatic potencies against PI3K and HDAC. Relative to BKM120 (PI3K inhibitor) or SAHA (HDAC inhibitor), 23 and 36 showed more favorable antiproliferative activities in a broad panel of cancer cell lines, illustrating that simultaneous inhibition of both PI3K and HDAC is beneficial over single target inhibition. Compounds 23 and 36 also showed target modulation in cancer cells by western blotting analysis (e.g., decreased p-AKT and increased Ac-H3). Compound 23 displayed a high selectivity profile against 97 kinase targets. Compounds 23 and 36 also induced cell-cycle arrest in the G1 phase and increased cell apoptosis. Similar to other hydroxamate-based HDAC inhibitors, 23 and 36 exhibited fast clearance and poor oral bioavailability in PK studies. Moreover, 23 resulted in in vivo antitumor efficacies in xenograft models, in which both PI3K and HDAC pathways were sufficiently inhibited as indicated by reduction of p-AKT and increase of Ac-H3 in IHC-stained tumor tissues. Overall, these structurally differentiated dual inhibitors shows promise in discovery of new anti-cancer therapeutics by simultaneously targeting PI3K and HDAC pathways with a single molecule.

■

EXPERIMENTAL PROCEDURES

Chemistry. All starting materials, solvents and reagents were purchased from commercial suppliers and used without further purification. 1H NMR spectra were recorded on a Varian 400 MHz NMR spectrometer or a JOEL 400 MHz NMR spectrometer with tetramethylsilane as internal standard. 13C NMR spectra were recorded on a Bruker 400 MHz NMR spectrometer. Chemical shifts were given as δ units in ppm (in NMR description, s = singlet, d = doublet, t = triplet, q = quartet and m = multiple). HRMS spectra were acquired on a Thermo LCQ Deca XP Maz mass spectrometer with electrospray ionization (ESI) in positive ion mode. Purity of all final compounds for biological tests was determined to be >95% with LCMS analysis, which was also used to record MS spectra of intermediates. The LCMS method was as follows: HPLC-Agilent 1100, water with 0.1% formic acid as mobile phase A, acetonitrile as mobile phase B, Agela Bonshell C18 plus column, 2.7 μm, 4.6 × 50 K



Article

DMSO-d6): δ 169.1, 168.2, 154.7, 153.2, 152.2 (d, JC−F = 11.6 Hz), 146.8 (d, JC−F = 257 Hz), 140.7, 140.1 (d, JC−F = 5.6 Hz), 135.2, 129.7, 124.9, 122.8 (d, JC−F = 16.2 Hz), 114.0, 111.8, 68.7, 53.8, 32.2, 28.8, 28.7, 28.6, 26.3, 25.6, 25.1, 22.1. HRMS (ESI) m/z: [M + H]+ calcd for C24H29O4N4F, 457.2246; found, 457.2228, Δ −3.83 ppm. 4-Methyl-6-(2-methoxy-3-methanesulfonamido-5-pyridinyl)-8(N-hydroxyhexanamide-6-oxy)quinazoline (26). Compound 26 was prepared from compound 57h (light yellow solid, 42% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.36 (s, 1H), 9.08 (s, 1H), 8.67 (s, 1H), 8.55 (d, J = 2.3 Hz, 1H), 8.08 (d, J = 2.3 Hz, 1H), 7.93 (d, J = 1.6 Hz, 1H), 7.63 (d, J = 1.5 Hz, 1H), 4.28 (t, J = 6.4 Hz, 2H), 4.00 (s, 3H), 3.10 (s, 3H), 2.95 (s, 3H), 2.01 (t, J = 7.6 Hz, 2H), 1.92− 1.79 (m, 2H), 1.66−1.56 (m, 2H), 1.56−1.44 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 169.0, 168.1, 157.1, 154.7, 153.1, 142.0, 140.6, 136.2, 132.6, 129.4, 124.9, 121.2, 113.9, 112.1, 68.6, 53.9, 41.0, 32.3, 28.4, 25.3, 24.9, 22.0. HRMS (ESI) m/z: [M + H]+ calcd for C22H27O6N5S, 490.1755; found, 490.1739, Δ −3.12 ppm. 4-Methyl-6-(2-methoxy-3-methanesulfonamido-5-pyridinyl)-8(N-hydroxyheptanamide-7-oxy)quinazoline (27). Compound 27 was prepared from compound 57l (light yellow solid, 47% yield). 1 H NMR (400 MHz, MeOD-d4): δ 9.02 (s, 1H), 8.38 (d, J = 1.8 Hz, 1H), 8.10 (d, J = 1.8 Hz, 1H), 7.85 (s, 1H), 7.54 (s, 1H), 6.84 (s, 1H), 4.30 (t, J = 6.6 Hz, 2H), 4.08 (s, 3H), 3.07 (s, 3H), 3.01 (s, 3H), 2.12 (t, J = 7.5 Hz, 2H), 2.00−1.97 (m, 2H), 1.71−1.56 (m, 4H), 1.48−1.45 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 169.1, 168.0, 157.1, 154.7, 153.1, 142.0, 140.6, 136.2, 132.6, 129.4, 124.9, 121.1, 113.9, 112.0, 68.7, 53.9, 41.0, 32.3, 28.6, 28.4, 25.4, 25.1, 22.0. HRMS (ESI) m/z: [M + H]+ calcd for C23H29O6N5S, 504.1911; found, 504.1897, Δ −2.90 ppm. 4-Methyl-6-(2-methoxy-3-methanesulfonamido-5-pyridinyl)-8(N-hydroxyoctanamide-8-oxy)quinazoline (28). Compound 28 was prepared from compound 57p (light yellow solid, 43% yield). 1H NMR (400 MHz, MeOD-d4): δ 9.01 (s, 1H), 8.37 (d, J = 2.2 Hz, 1H), 8.10 (d, J = 2.2 Hz, 1H), 7.86 (s, 1H), 7.53 (s, 1H), 4.30 (t, J = 6.5 Hz, 2H), 4.08 (s, 3H), 3.07 (s, 3H), 2.99 (s, 3H), 2.10 (t, J = 7.4 Hz, 2H), 2.05−1.93 (m, 2H), 1.70−1.57 (m, 4H), 1.51−1.36 (m, 4H). 13C NMR (101 MHz, DMSO-d6): δ 169.1, 168.0, 157.1, 154.7, 153.1, 142.0, 140.6, 136.2, 132.6, 129.4, 124.9, 121.1, 113.9, 112.0, 68.7, 53.9, 41.0, 32.3, 28.7, 28.6, 28.5, 25.5, 25.1, 22.0. HRMS (ESI) m/z: [M + H]+ calcd for C24H31O6N5S, 518.2068; found, 518.2053, Δ −2.93 ppm. 4-Methyl-6-(2-methoxy-3-methanesulfonamido-5-pyridinyl)-8(N-hydroxynonanamide-9-oxy)quinazoline (29). Compound 29 was prepared from compound 57t (light yellow solid, 44% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.32 (s, 1H), 9.07 (s, 1H), 8.64 (s, 1H), 8.55 (d, J = 1.8 Hz, 1H), 8.08 (d, J = 2.2 Hz, 1H), 7.92 (s, 1H), 7.62 (s, 1H), 4.28 (t, J = 6.5 Hz, 2H), 4.00 (s, 3H), 3.10 (s, 3H), 2.95 (s, 3H), 1.94 (t, J = 7.3 Hz, 2H), 1.91−1.82 (m, 2H), 1.57−1.44 (m, 4H), 1.38−1.23 (m, 6H). 13C NMR (101 MHz, DMSO-d6): δ 169.1, 168.0, 157.1, 154.7, 153.1, 142.0, 140.6, 136.2, 132.5, 129.4, 124.9, 121.2, 113.9, 112.0, 68.7, 53.8, 40.9, 32.2, 28.8, 28.7, 28.6, 26.3, 25.6, 25.1, 22.0. HRMS (ESI) m/z: [M + H]+ calcd for C25H33O6N5S, 532.2224; found, 532.2210, Δ −2.78 ppm. 4-Methyl-6-(2-methoxy-3-(2,4-difluorobenzenesulfonamido)-5pyridinyl)-8-(N-hydroxybutanamide-4-oxy)quinazoline (30). Compound 30 was prepared from compound 57c (light yellow solid, 37% yield). 1H NMR (400 MHz, DMSO-d6): δ10.47 (s, 1H), 10.29 (s, 1H), 9.03 (s, 1H), 8.71 (d, J = 2.1 Hz, 1H), 8.56 (d, J = 2.1 Hz, 1H), 8.13−8.10 (m, 1H), 7.92 (d, J = 1.1 Hz, 1H), 7.77−7.72 (m, 1H), 7.62−7.56 (m, 2H), 7.24−7.20 (m, 1H), 4.30 (t, J = 6.0 Hz, 2H), 3.66 (s, 3H), 2.96 (s, 3H), 2.25 (t, J = 7.2 Hz, 2H), 2.11−2.09 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 168.7, 168.2, 165.1 (dd, JC−F = 254, 11.8 Hz), 159.5 (dd, JC−F = 254, 11.8 Hz), 158.1, 154.6, 153.2, 143.4, 140.6, 135.8, 135.3, 131.8 (d, JC−F = 10.7 Hz), 129.1, 125.3 (dd, JC−F = 14.4, 3.3 Hz), 124.9, 119.6, 114.0, 112.0, 111.8 (dd, JC−F = 22.0, 2.7 Hz), 105.8 (t, JC−F = 26.3 Hz), 68.2, 53.4, 28.8, 24.8, 22.1. HRMS (ESI) m/z: [M + H]+ calcd for C25H23O6N5F2S, 560.1410; found, 560.1407, Δ −0.53 ppm. 4-Methyl-6-(2-methoxy-3-(2,4-difluorobenzenesulfonamido)-5pyridinyl)-8-(N-hydroxypentanamide-5-oxy)quinazoline (31).

4-Methyl-6-(2-methoxy-5-pyridinyl)-8-(N-hydroxyoctanamide-8oxy)quinazoline (20). Compound 20 was prepared from compound 57n (white solid, 50% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.33 (s, 1H), 9.06 (s, 1H), 8.74 (d, J = 2.4 Hz, 1H), 8.65 (s, 1H), 8.27 (dd, J = 8.6, 2.5 Hz, 1H), 7.95 (d, J = 1.2 Hz, 1H), 7.66 (d, J = 1.1 Hz, 1H), 6.98 (d, J = 8.6 Hz, 1H), 4.28 (t, J = 6.5 Hz, 2H), 3.93 (s, 3H), 2.95 (s, 3H), 1.95 (t, J = 7.3 Hz, 2H), 1.91−1.81 (m, 2H), 1.56−1.45 (m, 4H), 1.40−1.32 (m, 2H), 1.31−1.21 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 169.1, 168.0, 163.5, 154.7, 153.0, 145.7, 140.5, 138.3, 136.5, 128.7, 124.9, 113.4, 111.8, 110.6, 68.7, 53.4, 32.3, 28.7, 28.6, 28.5, 25.5, 25.1, 22.0. HRMS (ESI) m/z: [M + H]+ calcd for C23H28O4N4, 425.2183; found, 425.2166, Δ −3.98 ppm. 4-Methyl-6-(2-methoxy-5-pyridinyl)-8-(N-hydroxynonanamide9-oxy)quinazoline (21). Compound 21 was prepared from compound 57r (white solid, 55% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.32 (s, 1H), 9.06 (s, 1H), 8.74 (d, J = 2.5 Hz, 1H), 8.64 (d, J = 1.6 Hz, 1H), 8.27 (dd, J = 8.6, 2.6 Hz, 1H), 7.96 (d, J = 1.3 Hz, 1H), 7.66 (d, J = 1.2 Hz, 1H), 6.98 (d, J = 8.6 Hz, 1H), 4.28 (t, J = 6.5 Hz, 2H), 3.93 (s, 3H), 2.95 (s, 3H), 2.00−1.81 (m, 4H), 1.58−1.43 (m, 4H), 1.43−1.20 (m, 6H). 13C NMR (101 MHz, DMSO-d6): δ 169.1, 168.0, 163.5, 154.7, 153.0, 145.7, 140.5, 138.3, 136.5, 128.7, 124.9, 113.4, 111.9, 110.6, 68.7, 53.4, 32.2, 28.9, 28.8, 28.7, 28.6, 25.6, 25.1, 22.0. HRMS (ESI) m/z: [M + H]+ calcd for C24H30O4N4, 439.2340; found, 439.2323, Δ −3.83 ppm. 4-Methyl-6-(2-methoxy-3-fluoro-5-pyridinyl)-8-(N-hydroxyhexanamide-6-oxy)quinazoline (22). Compound 22 was prepared from compound 57g (white solid, 52% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.37 (s, 1H), 9.07 (s, 1H), 8.67 (s, 1H), 8.60 (d, J = 2.1 Hz, 1H), 8.36 (dd, J = 12.0, 2.1 Hz, 1H), 8.03 (d, J = 1.6 Hz, 1H), 7.70 (d, J = 1.5 Hz, 1H), 4.28 (t, J = 6.5 Hz, 2H), 4.03 (s, 3H), 2.96 (s, 3H), 2.01 (t, J = 7.4 Hz, 2H), 1.92−1.81 (m, 2H), 1.68−1.57 (m, 2H), 1.54−1.43 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 169.0, 168.2, 154.7, 153.2, 152.3 (d, J = 11.3 Hz), 146.8 (d, JC−F = 257 Hz), 140.7, 140.1, 135.2, 129.7 (d, JC−F = 11.0 Hz), 124.9, 122.8 (d, JC−F = 16.2 Hz), 114.0, 111.9, 68.7, 53.8, 32.3, 28.4, 25.3, 24.9, 22.1. HRMS (ESI) m/z: [M + H]+ calcd for C21H23O4N4F, 415.1776; found, 415.1761, Δ −3.59 ppm. 4-Methyl-6-(2-methoxy-3-fluoro-5-pyridinyl)-8-(N-hydroxyheptanamide-7-oxy)quinazoline (23). Compound 23 was prepared from compound 57k (light yellow solid, 50% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.34 (s, 1H), 9.07 (s, 1H), 8.59 (s, 1H), 8.36 (d, J = 12.0 Hz, 1H), 8.01 (s, 1H), 7.69 (s, 1H), 4.28 (t, J = 6.5 Hz, 2H), 4.03 (s, 3H), 2.95 (s, 3H), 1.97 (t, J = 7.3 Hz, 2H), 1.92−1.81 (m, 2H), 1.61−1.44 (m, 4H), 1.43−1.31 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 169.1, 168.2, 154.7, 153.2, 152.2 (d, JC−F = 11.3 Hz), 146.8 (d, JC−F = 256.8 Hz), 140.7, 140.1 (d, JC−F = 5.7 Hz), 135.2, 129.7, 124.9, 122.8 (d, JC−F = 16.2 Hz), 114.0, 111.8, 68.7, 53.8, 32.2, 28.6, 28.4, 25.4, 25.1, 22.0. HRMS (ESI) m/z: [M + H]+ calcd for C22H25O4N4F, 429.1933; found, 429.1917, Δ −3.54 ppm. 4-Methyl-6-(2-methoxy-3-fluoro-5-pyridinyl)-8-(N-hydroxyoctanamide-8-oxy)quinazoline (24). Compound 24 was prepared from compound 57o (light yellow solid, 48% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.35 (s, 1H), 9.05 (s, 1H), 8.66 (s, 1H), 8.57 (d, J = 2.0 Hz, 1H), 8.32 (dd, J = 12.0, 1.9 Hz, 1H), 7.98 (d, J = 1.3 Hz, 1H), 7.65 (d, J = 1.2 Hz, 1H), 4.26 (t, J = 6.5 Hz, 2H), 4.02 (s, 3H), 2.93 (s, 3H), 1.96 (t, J = 7.3 Hz, 2H), 1.91−1.79 (m, 2H), 1.57−1.43 (m, 4H), 1.38−1.25 (m, 4H). 13C NMR (101 MHz, DMSO-d6): δ 169.1, 168.1, 154.7, 153.2, 152.2 (d, JC−F = 11.4 Hz), 146.8 (d, JC−F = 257 Hz), 140.7, 140.1 (d, JC−F = 5.5 Hz), 135.2, 129.7, 124.8, 122.8 (d, JC−F = 16.2 Hz), 113.9, 111.7, 68.7, 53.7, 32.3, 28.7, 28.6, 28.5, 25.5, 25.1, 22.0. HRMS (ESI) m/z: [M + H]+ calcd for C23H27O4N4F, 443.2089; found, 443.2070, Δ −4.31 ppm. 4-Methyl-6-(2-methoxy-3-fluoro-5-pyridinyl)-8-(N-hydroxynonanamide-9-oxy)quinazoline (25). Compound 25 was prepared from compound 57s (white solid, 51% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.32 (s, 1H), 9.07 (s, 1H), 8.64 (d, J = 1.7 Hz, 1H), 8.60 (d, J = 2.0 Hz, 1H), 8.36 (dd, J = 12.0, 2.1 Hz, 1H), 8.02 (d, J = 1.5 Hz, 1H), 7.69 (d, J = 1.5 Hz, 1H), 4.28 (t, J = 6.5 Hz, 2H), 4.03 (s, 3H), 2.95 (s, 3H), 1.94 (t, J = 7.3 Hz, 2H), 1.91−1.82 (m, 2H), 1.55−1.45 (m, 4H), 1.37−1.21 (m, 6H). 13C NMR (101 MHz, L



Article

DMSO-d6): δ 169.1, 168.1, 165.1 (dd, JC−F = 254, 11.8 Hz), 159.5 (dd, JC−F = 254, 11.8 Hz), 158.1, 154.8, 153.1, 143.5, 140.6, 135.8, 135.3, 131.8 (d, JC−F = 10.7 Hz), 129.2, 125.3 (dd, JC−F = 14.5, 3.5 Hz), 124.9, 119.5, 113.8, 111.8 (dd, JC−F = 22.0, 3.2 Hz), 111.7, 105.8 (t, JC−F = 26.2 Hz), 68.7, 53.4, 32.3, 28.8, 28.7, 28.6, 25.6, 25.1, 22.0. HRMS (ESI) m/z: [M + H]+ calcd for C30H33O6N5F2S, 630.2192; found, 630.2189, Δ −0.58 ppm. N-Hydroxy-2-((2-((6-(6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)ethyl)(methyl)amino)pyrimidine-5-carboxamide (36). Compound 36 was prepared from compound 61a (light yellow solid, 51% yield). 1H NMR (400 MHz, DMSO-d6): δ 11.07 (s, 1H), 9.06 (s, 1H), 9.00 (s, 1H), 8.73 (d, J = 2.2 Hz, 1H), 8.68 (s, 2H), 8.25 (dd, J = 8.6, 1.7 Hz, 1H), 7.98 (s, 1H), 7.76 (s, 1H), 6.96 (d, J = 8.6 Hz, 1H), 4.52 (t, J = 5.6 Hz, 2H), 4.16 (t, J = 5.6 Hz, 2H), 3.93 (s, 3H), 3.37 (s, 3H), 2.95 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 168.0, 163.5, 161.7, 157.0, 154.2, 153.1, 145.7, 140.4, 138.2, 136.5, 128.5, 125.0, 114.4, 113.8, 112.1, 110.5, 66.3, 53.4, 47.9, 36.6, 22.0. HRMS (ESI) m/z: [M + H]+ calcd for C23H23O4N7, 462.1884; found, 462.1876, Δ −1.75 ppm. N-Hydroxy-2-((3-((6-(6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)propyl) (methyl)amino)pyrimidine-5-carboxamide (37). Compound 37 was prepared from compound 61d (light yellow solid, 57% yield). 1H NMR (400 MHz, DMSO-d6): δ 11.02 (s, 1H), 9.08 (s, 1H), 8.73 (d, J = 2.5 Hz, 1H), 8.62 (s, 2H), 8.26 (dd, J = 8.7, 2.6 Hz, 1H), 7.98 (d, J = 1.2 Hz, 1H), 7.68 (d, J = 1.3 Hz, 1H), 6.98 (d, J = 8.7 Hz, 1H), 4.37 (t, J = 6.0 Hz, 2H), 3.94 (s, 3H), 3.90 (t, J = 7.0 Hz, 2H), 3.19 (s, 3H), 2.96 (s, 3H), 2.25−2.15 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 168.0, 163.5, 161.7, 156.9, 156.8, 154.5, 153.1, 145.7, 140.6, 138.2, 136.5, 128.6, 125.0, 114.0, 113.7, 112.3, 110.6, 66.7, 53.4, 46.3, 35.7, 26.6, 22.1. HRMS (ESI) m/z: [M + H]+ calcd for C24H25O4N7, 476.2041; found, 467.2021, Δ −4.24 ppm. 2-((2-((6-(5-Fluoro-6-methoxypyridin-3-yl)-4-methylquinazolin8-yl)oxy)ethyl) (methyl)amino)-N-hydroxypyrimidine-5-carboxamide (38). Compound 38 was prepared from compound 61b (light yellow solid, 50% yield). 1H NMR (400 MHz, DMSO-d6): δ 11.06 (s, 1H), 9.07 (s, 1H), 8.99 (s, 1H), 8.68 (s, 2H), 8.60 (d, J = 2.0 Hz, 1H), 8.36 (dd, J = 12.0, 2.0 Hz, 1H), 8.05 (d, J = 1.5 Hz, 1H), 7.79 (d, J = 1.4 Hz, 1H), 4.53 (t, J = 5.8 Hz, 2H), 4.17 (t, J = 5.8 Hz, 2H), 4.03 (s, 3H), 3.37 (s, 3H), 2.96 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 168.2, 161.7, 157.0, 154.3, 153.3, 152.3 (d, JC−F = 11.3 Hz), 146.8 (d, JC−F = 257 Hz), 140.7, 140.2 (d, JC−F = 5.5 Hz), 135.2, 129.6 (d, JC−F = 4.0 Hz), 124.9, 122.9, 122.7, 114.4, 112.0, 109.5, 66.4, 53.8, 47.9, 36.6, 22.1. HRMS (ESI) m/z: [M + H]+ calcd for C23H22O4N7F, 480.1790; found, 480.1767, Δ −4.74 ppm. 2-((3-((6-(5-Fluoro-6-methoxypyridin-3-yl)-4-methylquinazolin8-yl)oxy)propyl) (methyl)amino)-N-hydroxypyrimidine-5-carboxamide (39). Compound 39 was prepared from compound 61e (light yellow solid, 55% yield). 1H NMR (400 MHz, DMSO-d6): δ 11.02 (s, 1H), 9.08 (s, 1H), 8.97 (s, 1H), 8.62 (s, 2H), 8.57 (s, 1H), 8.33 (d, J = 12.0 Hz, 1H), 8.02 (s, 1H), 7.70 (s, 1H), 4.36 (t, J = 5.9 Hz, 2H), 4.02 (s, 3H), 3.89 (t, J = 6.9 Hz, 2H), 3.19 (s, 3H), 2.95 (s, 3H), 2.24−2.12 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 168.2, 162.1, 161.7, 156.9, 154.5, 153.3, 152.2 (d, JC−F = 11.3 Hz), 146.8 (d, JC−F = 257 Hz), 140.8, 140.1 (d, JC−F = 5.5 Hz), 135.1, 129.6, 124.9, 122.8 (d, JC−F = 16.2 Hz), 114.2, 114.0, 112.2, 66.7, 53.8, 46.3, 35.7, 26.5, 22.1. HRMS (ESI) m/z: [M + H]+ calcd for C24H24O4N7F, 494.1947; found, 494.1926, Δ −4.22 ppm. 2-((4-((6-(5-Fluoro-6-methoxypyridin-3-yl)-4-methylquinazolin8-yl)oxy)butyl) (methyl)amino)-N-hydroxypyrimidine-5-carboxamide (40). Compound 40 was prepared from compound 61g (light yellow solid, 59% yield). 1H NMR (400 MHz, DMSO-d6): δ 9.05 (s, 1H), 8.66 (s, 2H), 8.57 (s, 1H), 8.33 (d, J = 12.0 Hz, 1H), 7.99 (s, 1H), 7.67 (s, 1H), 4.32 (t, J = 5.2 Hz, 2H), 4.02 (s, 3H), 3.77 (t, J = 6.6 Hz, 2H), 3.16 (s, 5H), 2.94 (s, 3H), 1.87−1.73 (m, 4H). 13C NMR (101 MHz, DMSO-d6): δ 168.2, 162.1, 161.8, 157.0, 154.6, 153.2, 152.3 (d, JC−F = 11.2 Hz), 146.8 (d, JC−F = 257 Hz), 140.7, 140.1 (d, JC−F = 5.5 Hz), 135.2, 129.7, 124.9, 122.8 (d, JC−F = 16.1 Hz), 114.1, 113.9, 111.9, 68.7, 53.8, 48.6, 35.2, 26.0, 23.6, 22.1. HRMS (ESI) m/z: [M + H]+ calcd for C25H26O4N7F, 508.2103; found, 508.2087, Δ −3.14 ppm.

Compound 31 was prepared from compound 57e (light yellow solid, 40% yield). 1H NMR (400 MHz, MeOD-d4): δ 9.03 (s, 1H), 8.32 (d, J = 2.2 Hz, 1H), 8.06 (d, J = 2.2 Hz, 1H), 7.95−7.85 (m, 1H), 7.78 (d, J = 1.2 Hz, 1H), 7.45 (d, J = 1.2 Hz, 1H), 7.27−7.15 (m, 1H), 7.06 (m, 1H), 4.30 (d, J = 4.9 Hz, 2H), 3.82 (s, 3H), 2.97 (s, 3H), 2.34 (t, J = 6.4 Hz, 2H), 2.07−1.90 (m, 4H). 13C NMR (101 MHz, MeOD-d4): δ 173.8, 168.4, 165.5 (dd, JC−F = 260, 12.1 Hz), 159.4 (dd, JC−F = 260, 12.3 Hz), 158.1, 156.2, 153.9, 143.7, 141.9, 139.1, 133.6 (d, JC−F = 11.1 Hz), 130.1, 128.5, 126.9, 122.0 (dd, JC−F = 13.9, 3.7 Hz), 120.3, 115.2, 113.3 (dd, JC−F = 21.6, 3.8 Hz), 113.2, 109.6 (t, JC−F = 23.4 Hz), 71.1, 54.5, 33.6, 28.6, 22.2, 14.6. HRMS (ESI) m/z: [M + H]+ calcd for C26H25O6N5F2S, 574.1566; found, 574.1564, Δ −0.47 ppm. 4-Methyl-6-(2-methoxy-3-(2,4-difluorobenzenesulfonamido)-5pyridinyl)-8-(N-hydroxyhexanamide-6-oxy)quinazoline (32). Compound 32 was prepared from compound 57i (light yellow solid, 40% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.37 (s, 1H), 10.34 (s, 1H), 9.07 (s, 1H), 8.67 (d, J = 1.4 Hz, 1H), 8.56 (d, J = 2.3 Hz, 1H), 8.10 (d, J = 2.3 Hz, 1H), 7.91 (d, J = 1.3 Hz, 1H), 7.76 (td, J = 8.5, 6.5 Hz, 1H), 7.65−7.55 (m, 2H), 7.22 (td, J = 8.5, 2.3 Hz, 1H), 4.27 (t, J = 6.4 Hz, 2H), 3.66 (s, 3H), 2.95 (s, 3H), 2.01 (t, J = 7.2 Hz, 2H), 1.93−1.81 (m, 2H), 1.69−1.58 (m, 2H), 1.57−1.45 (m, 2H). 13 C NMR (101 MHz, DMSO-d6): δ 169.0, 168.1, 165.1 (dd, JC−F = 254, 12.1 Hz), 159.5 (dd, JC−F = 254, 12.1 Hz), 158.1, 154.7, 153.2, 143.5, 140.6, 135.8, 135.4, 131.8 (d, JC−F = 10.8 Hz), 129.2, 125.3 (dd, JC−F = 14.8, 3.4 Hz), 124.9, 119.5, 113.8, 111.8, 111.7 (dd, J = 22.0, 3.2 Hz), 105.8 (t, J = 26.2 Hz), 68.6, 53.4, 32.3, 28.4, 25.3, 25.0, 22.0. HRMS (ESI) m/z: [M + H]+ calcd for C27H27O6N5F2S, 588.1723; found, 588.1702, Δ −3.50 ppm. 4-Methyl-6-(2-methoxy-3-(2,4-difluorobenzenesulfonamido)-5pyridinyl)-8-(N-hydroxyheptanamide-7-oxy)quinazoline (33). Compound 33 was prepared from compound 57m (light yellow solid, 41% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.35 (s, 2H), 9.07 (s, 1H), 8.65 (s, 1H), 8.55 (d, J = 2.3 Hz, 1H), 8.09 (d, J = 2.3 Hz, 1H), 7.90 (d, J = 1.4 Hz, 1H), 7.76 (td, J = 8.5, 6.4 Hz, 1H), 7.64−7.54 (m, 2H), 7.22 (td, J = 8.5, 2.4 Hz, 1H), 4.27 (t, J = 6.5 Hz, 2H), 3.66 (s, 3H), 2.95 (s, 3H), 1.97 (t, J = 7.3 Hz, 2H), 1.92−1.79 (m, 2H), 1.60−1.47 (m, 4H), 1.42−1.30 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 169.1, 168.1, 165.0 (dd, JC−F = 254, 12.1 Hz), 159.4 (dd, JC−F = 253, 9.2 Hz), 158.1, 154.7, 153.2, 143.3, 140.6, 135.8, 135.2, 131.8 (d, JC−F = 10.6 Hz), 129.2, 125.4 (dd, JC−F = 14.7, 3.4 Hz), 124.9, 119.7, 113.8, 111.8, 111.7 (dd, JC−F = 22.0, 3.0 Hz), 105.8 (t, JC−F = 26.2 Hz), 68.7, 53.4, 32.3, 28.6, 28.4, 25.4, 25.1, 22.0. HRMS (ESI) m/z: [M + H]+ calcd for C28H29O6N5F2S, 602.1879; found, 602.1857, Δ −3.76 ppm. 4-Methyl-6-(2-methoxy-3-(2,4-difluorobenzenesulfonamido)-5pyridinyl)-8-(N-hydroxyoctanamide-8-oxy)quinazoline (34). Compound 34 was prepared from compound 57q (light yellow solid, 40% yield). 1H NMR (400 MHz, MeOD-d4): δ 8.99 (s, 1H), 8.30 (s, 1H), 8.04 (s, 1H), 7.85 (dd, J = 14.5, 8.2 Hz, 1H), 7.75 (s, 1H), 7.44 (s, 1H), 7.21 (t, J = 9.3 Hz, 1H), 7.06 (t, J = 8.0 Hz, 1H), 4.26 (t, J = 5.1 Hz, 2H), 3.81 (s, 3H), 2.95 (s, 3H), 2.10 (t, J = 7.3 Hz, 2H), 2.03− 1.92 (m, 2H), 1.72−1.53 (m, 4H), 1.51−1.36 (m, 4H). 13C NMR (101 MHz, MeOD-d4): δ 173.2, 170.7, 168.9, 168.8, 166.4, 166.3, 167.7 (dd, JC−F = 257, 11.1 Hz), 161.7 (dd, JC−F = 260, 13.1 Hz), 158.6, 156.3, 153.8, 143.6, 142.0, 139.0, 133.7 (d, JC−F = 10.1 Hz), 133.5, 131.2, 126.9, 126.2 (dd, JC−F = 14.1, 4.0 Hz), 122.0, 115.0, 113.4, 112.8 (dd, JC−F = 22.2, 3.0 Hz), 106.7 (t, JC−F = 26.2 Hz), 70.7, 54.5, 33.9, 30.3, 30.2, 30.1, 27.2, 26.9, 22.3. HRMS (ESI) m/z: [M + H]+ calcd for C29H31O6N5F2S, 616.2036; found, 616.2030, Δ −0.95 ppm. 4-Methyl-6-(2-methoxy-3-(2,4-difluorobenzenesulfonamido)-5pyridinyl)-8-(N-hydroxynonanamide-9-oxy)quinazoline (35). Compound 35 was prepared from compound 57u (light yellow solid, 43% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.33 (s, 2H), 9.07 (s, 1H), 8.65 (s, 1H), 8.56 (d, J = 2.2 Hz, 1H), 8.10 (d, J = 2.2 Hz, 1H), 7.90 (d, J = 0.9 Hz, 1H), 7.76 (dd, J = 14.9, 8.4 Hz, 1H), 7.65−7.54 (m, 2H), 7.22 (t, J = 7.7 Hz, 1H), 4.27 (t, J = 6.4 Hz, 2H), 3.66 (s, 3H), 2.95 (s, 3H), 1.94 (t, J = 7.3 Hz, 2H), 1.91−1.82 (m, 2H), 1.57−1.44 (m, 4H), 1.43−1.20 (m, 6H). 13C NMR (101 MHz, M



Article

2-((2-((6-(5-Fluoro-6-methoxypyridin-3-yl)-4-methylquinazolin8-yl)oxy)ethyl) (isopropyl)amino)-N-hydroxypyrimidine-5-carboxamide (46). Compound 46 was prepared from compound 61k (light yellow solid, 66% yield). 1H NMR (400 MHz, DMSO-d6): δ 11.06 (s, 1H), 9.08 (s, 1H), 9.00 (s, 1H), 8.71 (s, 2H), 8.60 (d, J = 1.5 Hz, 1H), 8.36 (dd, J = 11.8, 1.6 Hz, 1H), 8.05 (s, 1H), 7.84 (d, J = 1.5 Hz, 1H), 5.11−4.96 (m, 1H), 4.47 (t, J = 6.8 Hz, 2H), 4.15−3.96 (m, 5H), 2.97 (s, 3H), 1.28 (d, J = 6.7 Hz, 6H). 13C NMR (101 MHz, DMSO-d6): δ 168.2, 162.0, 161.4, 157.1, 154.2, 153.2, 152.3 (d, JC−F = 11.3 Hz), 146.8 (d, JC−F = 257 Hz), 140.6, 140.2 (d, JC−F = 5.8 Hz), 135.3, 129.7, 124.9, 122.8 (d, JC−F = 16.5 Hz), 114.8, 114.3, 112.0, 66.0, 53.8, 46.5, 40.9, 22.1, 19.9. HRMS (ESI) m/z: [M + H]+ calcd for C25H26O4N7F, 508.2103; found, 508.2095, Δ −1.53 ppm. 2-((2-((6-(5-Fluoro-6-methoxypyridin-3-yl)-4-methylquinazolin8-yl)oxy)ethyl)amino)-N-hydroxypyrimidine-5-carboxamide (47). Compound 47 was prepared from compound 61l (light yellow solid, 54% yield). 1H NMR (400 MHz, DMSO-d6): δ 11.05 (s, 1H), 9.06 (s, 1H), 8.99 (s, 1H), 8.61 (d, J = 1.8 Hz, 2H), 8.36 (dd, J = 12.0, 1.6 Hz, 1H), 8.07−8.00 (m, 2H), 7.82 (s, 1H), 4.46 (t, J = 6.0 Hz, 2H), 4.03 (s, 3H), 3.90−3.80 (m, 2H), 2.96 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 168.3, 162.8, 162.0, 154.3, 153.2, 152.3 (JC−F = 11.1 Hz), 146.8 (JC−F = 259 Hz), 140.7, 140.1, 135.2, 129.6, 124.9, 122.8 (JC−F = 16.2 Hz), 115.2, 114.4, 112.4, 66.9, 53.8, 22.1, 18.6. HRMS (ESI) m/z: [M + H]+ calcd for C22H20O4N7F, 466.1634; found, 466.1617, Δ −3.53 ppm. 2-((3-((6-(5-Fluoro-6-methoxypyridin-3-yl)-4-methylquinazolin8-yl)oxy)propyl)amino)-N-hydroxypyrimidine-5-carboxamide (48). Compound 48 was prepared from compound 61m (light yellow solid, 61% yield). 1H NMR (400 MHz, DMSO-d6): δ 11.00 (s, 1H), 9.08 (s, 1H), 8.96 (s, 1H), 8.66−8.58 (m, 2H), 8.35 (d, J = 11.9 Hz, 1H), 8.03 (s, 1H), 7.93 (t, J = 5.5 Hz, 1H), 7.72 (s, 1H), 4.40 (t, J = 5.8 Hz, 2H), 4.03 (s, 3H), 3.58 (dd, J = 12.4, 6.4 Hz, 2H), 2.96 (s, 3H), 2.21−2.10 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 168.3, 162.8, 162.1, 157.3, 154.6, 153.2, 152.3 (JC−F = 11.1 Hz), 146.8 (JC−F = 258 Hz), 140.7, 140.1 (JC−F = 6.1 Hz), 135.2, 129.7, 124.9, 122.8 (JC−F = 16.2 Hz), 114.8, 114.2, 112.1, 66.9, 53.8, 38.1, 28.4, 22.1. HRMS (ESI) m/z: [M + H]+ calcd for C23H22O4N7F, 480.1790; found, 480.1777, Δ −2.72 ppm. 2-((4-((6-(5-Fluoro-6-methoxypyridin-3-yl)-4-methylquinazolin8-yl)oxy)butyl)amino)-N-hydroxypyrimidine-5-carboxamide (49). Compound 49 was prepared from compound 61n (light yellow solid, 51% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 9.06 (s, 1H), 8.95 (s, 1H), 8.67−8.54 (m, 2H), 8.35 (dd, J = 12.0, 2.0 Hz, 1H), 8.02 (d, J = 1.5 Hz, 1H), 7.89 (t, J = 5.8 Hz, 1H), 7.70 (d, J = 1.4 Hz, 1H), 4.33 (t, J = 6.3 Hz, 2H), 4.03 (s, 3H), 3.43 (q, J = 6.6 Hz, 2H), 2.95 (s, 3H), 1.99−1.88 (m, 2H), 1.84−1.73 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 168.2, 162.9, 162.2, 157.3, 154.6, 153.2, 152.3 (d, JC−F = 11.5 Hz), 146.8 (d, JC−F = 257 Hz), 140.7, 140.1 (d, JC−F = 5.6 Hz), 135.2, 129.7, 124.9, 122.8 (d, JC−F = 16.2 Hz), 114.6, 114.1, 111.9, 68.6, 53.8, 40.4, 26.2, 25.6, 22.1. HRMS (ESI) m/z: [M + H]+ calcd for C24H24O4N7F, 494.1947; found, 494.1924, Δ −4.61 ppm. 2-Nitro-3-methoxyacetophenone (51). To 65% nitric acid (1.0 L) was added 3-methoxyacetophenone 50 (180 g, 1.20 mol) under vigorous stirring. The reaction mixture was stirred at room temperature for 72 h. The mixture was then poured into ice water (4 L) under stirring. The resulting orange yellow was collected by filtration, washed with water (1 L) followed by absolute ethanol (1 L), and dried to afford the product 51 as a light yellow solid (89.2 g, 38% yield). 1H NMR (400 MHz, CDCl3): δ 7.53 (dd, J = 8.4, 7.9 Hz, 1H), 7.37 (dd, J = 7.9, 1.1 Hz, 1H), 7.24 (dd, J = 8.4, 1.0 Hz, 1H), 3.93 (s, 3H), 2.58 (s, 3H). MS (ESI+) m/z: 195.9 [M + H]+. 2-Amino-3-methoxyacetophenone (52). To a solution of 2-nitro3-methoxyacetophenone 51 (46.1 g, 0.24 mol) in THF/EtOH/water (400 mL/400 mL/200 mL) were added ammonium chloride (25.3 g, 0.47 mol) and reduced iron powder (52.9 g, 0.94 mol). The reaction mixture was refluxed for 2 h and filtered with diatomite to remove an insoluble solid. The volatiles were then removed under reduced pressure. The residue was resolved in DCM, washed sequentially with water (300 mL) and brine (200 mL), dried over anhydrous Na2SO4,

2-((2-((6-(5-((2,4-Difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)ethyl) (methyl)amino)-N-hydroxypyrimidine-5-carboxamide (41). Compound 41 was prepared from compound 61c (light yellow solid, 33% yield). 1H NMR (400 MHz, DMSO-d6): δ 11.09 (s, 1H), 10.33 (s, 1H), 9.07 (s, 1H), 8.99 (s, 1H), 8.69 (s, 2H), 8.53 (s, 1H), 8.09 (d, J = 1.8 Hz, 1H), 7.91 (d, J = 1.0 Hz, 1H), 7.76 (td, J = 8.5, 6.5 Hz, 1H), 7.69 (s, 1H), 7.60−7.52 (m, 1H), 7.20 (td, J = 8.4, 2.1 Hz, 1H), 4.52 (t, J = 5.7 Hz, 2H), 4.17 (t, J = 5.6 Hz, 2H), 3.66 (s, 3H), 3.38 (s, 3H), 2.95 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 168.1, 165.0 (dd, JC−F = 254, 11.7 Hz), 161.8, 161.4 (dd, JC−F = 254, 11.7 Hz), 158.1 (d, JC−F = 13.3 Hz), 158.0, 157.0, 154.3, 153.2, 143.2, 140.6, 135.8, 135.1, 131.9, 131.8, 129.7, 129.0, 125.0, 114.4, 114.1, 112.0, 111.7 (dd, JC−F = 21.1, 2.4 Hz), 105.8 (t, JC−F = 26.0 Hz), 66.5, 53.4, 48.0, 36.6, 22.0. HRMS (ESI) m/z: [M + H]+ calcd for C29H26O6N8F2S, 653.1737; found, 653.1708, Δ −4.35 ppm. 2-((3-((6-(5-((2,4-Difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)propyl) (methyl)amino)-Nhydroxypyrimidine-5-carboxamide (42). Compound 42 was prepared from compound 61f (light yellow solid, 41% yield). 1H NMR (400 MHz, DMSO-d6): δ 11.02 (s, 1H), 10.30 (s, 1H), 9.09 (s, 1H), 8.97 (s, 1H), 8.63 (s, 2H), 8.56 (s, 1H), 8.12 (d, J = 1.6 Hz, 1H), 7.93 (d, J = 1.2 Hz, 1H), 7.74 (td, J = 8.4, 6.3 Hz, 1H), 7.64 (s, 1H), 7.61− 7.52 (m, 1H), 7.20 (td, J = 8.4, 2.0 Hz, 1H), 4.36 (t, J = 5.6 Hz, 2H), 3.90 (t, J = 6.8 Hz, 2H), 3.67 (s, 3H), 3.19 (s, 3H), 2.96 (s, 3H), 2.22−2.17 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 168.2, 165.1 (dd, JC−F = 254, 11.8 Hz), 162.1, 161.7, 159.5 (dd, JC−F = 246, 11.4 Hz), 158.1, 156.9, 154.6, 153.2, 143.5, 140.7, 135.7, 135.5, 131.8 (d, JC−F = 10.5 Hz), 130.4, 129.1, 125.2 (dd, JC−F = 14.5, 4.1 Hz), 124.9, 119.5, 114.1, 112.2, 111.8 (d, JC−F = 21.6 Hz), 105.8 (t, JC−F = 26.3 Hz), 66.7, 53.4, 46.4, 35.7, 26.6, 22.0. HRMS (ESI) m/z: [M + H]+ calcd for C30H28O6N8F2S, 667.1893; found, 667.1862, Δ −4.76 ppm. 2-(Ethyl(2-((6-(6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)ethyl)amino)-N-hydroxypyrimidine-5-carboxamide (43). Compound 43 was prepared from compound 61h (light yellow solid, 62% yield). 1H NMR (400 MHz, DMSO-d6): δ 11.06 (s, 1H), 9.06 (s, 1H), 9.00 (s, 1H), 8.74 (d, J = 2.3 Hz, 1H), 8.69 (s, 2H), 8.26 (dd, J = 8.7, 2.5 Hz, 1H), 7.99 (d, J = 1.0 Hz, 1H), 7.78 (s, 1H), 6.97 (d, J = 8.6 Hz, 1H), 4.51 (t, J = 6.0 Hz, 2H), 4.12 (t, J = 5.9 Hz, 2H), 3.93 (s, 3H), 3.84 (q, J = 6.9 Hz, 2H), 2.95 (s, 3H), 1.20 (t, J = 6.9 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6): δ 168.0, 163.5, 161.3, 157.1, 154.3, 153.1, 145.7, 140.4, 138.2, 136.5, 128.6, 125.0, 114.5, 113.8, 112.0, 110.5, 66.3, 53.4, 46.2, 43.3, 22.0, 12.6. HRMS (ESI) m/z: [M + H]+ calcd for C24H25O4N7, 476.2041; found, 476.2022, Δ −3.86 ppm. 2-(Ethyl(2-((6-(5-fluoro-6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)ethyl)amino)-N-hydroxypyrimidine-5-carboxamide (44). Compound 44 was prepared from compound 61i (light yellow solid, 67% yield). 1H NMR (400 MHz, DMSO-d6): δ 11.09 (s, 1H), 9.07 (s, 1H), 8.99 (s, 1H), 8.69 (s, 2H), 8.59 (d, J = 1.7 Hz, 1H), 8.34 (dd, J = 11.9, 1.6 Hz, 1H), 8.03 (s, 1H), 7.79 (s, 1H), 4.51 (t, J = 5.9 Hz, 2H), 4.12 (t, J = 5.8 Hz, 2H), 4.02 (s, 3H), 3.84 (q, J = 7.0 Hz, 2H), 2.95 (s, 3H), 1.20 (t, J = 7.0 Hz, 5H). 13C NMR (101 MHz, DMSO-d6): δ 168.2, 162.0, 161.2, 157.1, 154.3, 153.3, 152.3 (d, JC−F = 11.3 Hz), 146.8 (d, JC−F = 257 Hz), 140.6, 140.2 (d, JC−F = 5.6 Hz), 135.2, 129.6, 124.9, 122.8 (d, JC−F = 16.2 Hz), 114.5, 114.3, 111.9, 66.4, 53.8, 46.2, 43.3, 22.1, 12.6. HRMS (ESI) m/z: [M + H]+ calcd for C24H24O4N7F, 494.1947; found, 494.1926, Δ −4.16 ppm. N-Hydroxy-2-(isopropyl(2-((6-(6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)ethyl)amino)pyrimidine-5-carboxamide (45). Compound 45 was prepared from compound 61j (light yellow solid, 69% yield). 1H NMR (400 MHz, DMSO-d6): δ 11.07 (s, 1H), 9.07 (s, 1H), 9.00 (s, 1H), 8.74 (d, J = 2.1 Hz, 1H), 8.71 (s, 2H), 8.27 (dd, J = 8.7, 1.9 Hz, 1H), 7.99 (s, 1H), 7.83 (s, 1H), 6.97 (d, J = 8.6 Hz, 1H), 5.09−4.97 (m, 1H), 4.47 (t, J = 6.7 Hz, 2H), 4.00 (t, J = 6.7 Hz, 2H), 3.93 (s, 3H), 2.96 (s, 3H), 1.28 (d, J = 6.7 Hz, 6H). 13C NMR (101 MHz, DMSO-d6): δ 168.1, 163.5, 162.0, 161.4, 157.0, 154.2, 153.0, 145.8, 140.4, 138.3, 136.6, 128.6, 125.0, 114.8, 113.7, 112.0, 110.5, 65.9, 53.4, 46.5, 40.8, 22.1, 19.9. HRMS (ESI) m/z: [M + H]+ calcd for C25H27O4N7, 490.2197; found, 490.2174, Δ −4.73 ppm. N



Article

filtered, and concentrated. The obtained yellow oil 52 (39.0 g, 98% yield) was used directly in the next step without further purification. 1 H NMR (400 MHz, CDCl3): δ 7.33 (dd, J = 8.4, 0.8 Hz, 1H), 6.84 (dd, J = 7.6, 0.8 Hz, 1H), 6.57 (dd, J = 8.4, 7.6 Hz, 1H), 3.87 (s, 3H), 2.57 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 200.9, 147.4, 141.7, 123.5, 117.8, 114.2, 113.0, 55.9, 28.3. MS (ESI+) m/z: 166.1 [M + H]+. 2-Amino-3-methoxy-5-bromoacetophenone (53). To a solution of 2-amino-3-methoxyacetophenone 52 (39.0 g, 0.236 mol) in DCM (500 mL) was added NBS (43.4 g, 0.244 mol). The reaction mixture was stirred at room temperature for 3 h, poured into water (500 mL), and extracted with DCM (200 mL × 3). The combined organic layers were washed with water (200 mL) and brine (200 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography (silica gel, PE/EA = 3:1, v/v) to afford the product 53 as a light yellow solid (43.0 g, 75% yield). 1H NMR (400 MHz, CDCl3): δ 7.44 (d, J = 2.0 Hz, 1H), 6.90 (d, J = 2.0 Hz, 1H), 3.87 (s, 3H), 2.55 (s, 3H). MS (ESI+) m/z: 243.9, 246.0 [M + H]+. 4-Methyl-6-bromo-8-methoxyquinazoline (54). To a solution of 2-amino-3-methoxy-5-bromoacetophenone 53 (7.59 g, 31 mmol) in formamide (90 mL) was added ammonium formate (9.11 g, 144 mmol). The resulting reaction mixture was stirred at 150 °C under argon for 10 h. The mixture was then quenched with water (500 mL) and extracted with EA (80 mL × 10). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (silica gel, DCM/EA = 2:1) to afford the product 54 as a light yellow solid (4.2 g, 53% yield). 1H NMR (400 MHz, DMSO-d6): δ 9.08 (s, 1H), 7.98 (d, J = 1.9 Hz, 1H), 7.53 (d, J = 1.9 Hz, 1H), 4.00 (s, 3H), 2.86 (s, 3H). MS (ESI+) m/z: 253.0, 255.0 [M + H]+. 4-Methyl-6-bromo-8-hydroxyquinazoline (55). To a solution of 4-methyl-6-bromo-8-methoxyquinazoline 54 (4.2 g, 16.6 mmol) in DCE (250 mL) was added aluminum chloride (6.75 g, 50.6 mmol). The reaction mixture was stirred at 80 °C for 2 h, quenched with ice water (250 mL), and extracted with DCM (60 mL × 5). The combined organic layers were washed with water (100 mL) and brine (100 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by column chromatography (silica gel, DCM/ MeOH = 15:1) to afford the product 55 as a brown solid (2.2 g, 55% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.82 (s, 1H), 9.09 (s, 1H), 7.85 (d, J = 1.9 Hz, 1H), 7.40 (d, J = 1.9 Hz, 1H), 2.85 (s, 3H). 13 C NMR (101 MHz, DMSO-d6): δ 167.5, 154.7, 152.7, 139.2, 125.5, 120.9, 118.8, 117.4, 22.0. MS (ESI+) m/z: 239.0, 240.9 [M + H]+. General Procedure for the Synthesis of 56a−g. To a solution of 4-methyl-6-bromo-8-hydroxyquinazoline 55 (1.0 equiv) and potassium carbonate (2.0 equiv) in anhydrous acetonitrile was added ethyl ω-bromo substituted alkyl carboxylate (1.2 equiv). The resulting mixture was refluxed for 16 h and filtered to remove an insoluble solid. The volatiles were removed under reduced pressure. The residue was purified by column chromatography (silica gel, PE/ EA = 1:2) to afford the desired products 56a−g as a yellow or light yellow solid. 4-Methyl-6-bromo-8-(ethyl acetate-2-oxy)quinazoline (56a). Compound 56a was prepared from 4-methyl-6-bromo-8-hydroxyquinazoline (55) and ethyl bromoacetate (yellow solid, 63% yield). 1H NMR (400 MHz, CDCl3): δ 9.10 (s, 1H), 7.77 (d, J = 1.8 Hz, 1H), 7.14 (d, J = 1.8 Hz, 1H), 4.87 (s, 2H), 4.22 (q, J = 7.1 Hz, 2H), 2.82 (s, 3H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 167.9, 167.3, 154.4, 154.0, 141.1, 126.4, 121.1, 120.4, 117.7, 66.6, 61.9, 22.4, 14.4. MS (ESI+) m/z: 325.0, 327.0 [M + H]+. 4-Methyl-6-bromo-8-(ethyl butyrate-4-oxy)quinazoline (56b). Compound 56b was prepared from compound 55 and ethyl 4bromobutyrate (yellow solid, 56% yield). 1H NMR (400 MHz, CDCl3): δ 9.20 (s, 1H), 7.79 (d, J = 1.5 Hz, 1H), 7.31 (s, 1H), 4.27 (t, J = 6.4 Hz, 2H), 4.15 (q, J = 7.1 Hz, 2H), 2.90 (s, 3H), 2.60 (t, J = 7.1 Hz, 2H), 2.31 (p, J = 6.8 Hz, 2H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 173.1, 167.1, 155.3, 153.9, 141.1, 126.2, 121.5, 119.0, 116.6, 68.6, 60.7, 30.7, 24.2, 22.4, 14.4. MS (ESI+) m/z: 353.0, 355.0 [M + H]+.

4-Methyl-6-bromo-8-(ethyl pentanoate-5-oxy)quinazoline (56c). Compound 56c was prepared from compound 55 and ethyl 5bromopentanoate (light yellow solid, 63% yield). 1H NMR (400 MHz, CDCl3): δ 9.18 (s, 1H), 7.77 (d, J = 1.6 Hz, 1H), 7.26 (d, J = 1.6 Hz, 1H), 4.19 (t, J = 6.6 Hz, 2H), 4.11 (q, J = 7.1 Hz, 2H), 2.89 (d, J = 0.7 Hz, 3H), 2.40 (t, J = 7.4 Hz, 2H), 2.08−2.00 (m, 2H), 1.91−1.83 (m, 2H), 1.25−1.21 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 367.1, 369.1 [M + H]+. 4-Methyl-6-bromo-8-(ethyl hexanoate-6-oxy)quinazoline (56d). Compound 56d was prepared from compound 55 and ethyl 6bromohexanoate (light yellow solid, 60% yield). 1H NMR (400 MHz, CDCl3): δ 9.22 (s, 1H), 7.82 (d, J = 1.9 Hz, 1H), 7.33 (d, J = 1.7 Hz, 1H), 4.22 (t, J = 6.7 Hz, 2H), 4.13 (q, J = 7.1 Hz, 2H), 2.98 (s, 3H), 2.35 (t, J = 7.4 Hz, 2H), 2.13−1.93 (m, 2H), 1.76 (m, 2H), 1.67− 1.49 (m, 2H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 173.8, 167.0, 155.5, 151.1, 141.0, 125.9, 123.1, 119.0, 118.0, 69.9, 60.5, 34.3, 28.7, 25.7, 24.8, 21.4, 14.5. MS (ESI+) m/z: 381.1, 383.0 [M + H]+. 4-Methyl-6-bromo-8-(ethyl heptanoate-7-oxy)quinazoline (56e). Compound 56e was prepared from compound 55 and ethyl 7-bromoheptanoate (light yellow solid, 68% yield). 1H NMR (400 MHz, CDCl3): δ 9.22 (s, 1H), 7.81 (d, J = 1.9 Hz, 1H), 7.32 (d, J = 1.8 Hz, 1H), 4.21 (t, J = 6.8 Hz, 2H), 4.13 (q, J = 7.1 Hz, 2H), 2.96 (s, 3H), 2.32 (t, J = 7.5 Hz, 2H), 2.09−1.97 (m, 2H), 1.68 (dt, J = 15.1, 7.4 Hz, 2H), 1.62−1.52 (m, 2H), 1.50−1.40 (m, 2H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 173.9, 167.1, 155.6, 154.0, 141.2, 126.2, 121.6, 118.7, 116.4, 77.6, 77.2, 76.9, 69.7, 60.4, 34.4, 29.0, 28.8, 25.9, 25.0, 22.4, 14.4. MS (ESI+) m/z: 395.2, 397.0 [M + H]+. 4-Methyl-6-bromo-8-(ethyl octanoate-8-oxy)quinazoline (56f). Compound 56f was prepared from compound 55 and ethyl 8bromooctanoate (light yellow solid, 71% yield). 1H NMR (400 MHz, CDCl3): δ 9.19 (s, 1H), 7.77 (d, J = 1.8 Hz, 1H), 7.26 (d, J = 1.8 Hz, 1H), 4.17 (t, J = 6.9 Hz, 2H), 4.10 (q, J = 7.1 Hz, 2H), 2.89 (s, 3H), 2.28 (t, J = 7.5 Hz, 2H), 2.05−1.94 (m, 2H), 1.68−1.58 (m, 2H), 1.56−1.47 (m, 2H), 1.46−1.31 (m, 4H), 1.23 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 173.9, 167.0, 155.6, 153.9, 141.2, 126.2, 121.6, 118.6, 116.3, 69.8, 60.4, 34.5, 29.2, 28.9, 26.0, 25.0, 22.4, 14.4. MS (ESI+) m/z: 409.0, 411.0 [M + H]+. 4-Methyl-6-bromo-8-(ethyl nonanoate-9-oxy)quinazoline (56g). Compound 56g was prepared from compound 55 and ethyl 9bromononanoate (light yellow solid, 62% yield). 1H NMR (400 MHz, CDCl3): δ 9.21 (s, 1H), 7.79 (d, J = 1.9 Hz, 1H), 7.29 (d, J = 2.0 Hz, 1H), 4.19 (t, J = 6.9 Hz, 2H), 4.12 (q, J = 7.1 Hz, 2H), 2.92 (s, 3H), 2.28 (t, J = 7.5 Hz, 2H), 2.06−1.96 (m, 2H), 1.67−1.57 (m, 2H), 1.57−1.47 (m, 2H), 1.43−1.29 (m, 6H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 174.0, 167.0, 155.6, 153.8, 141.2, 126.2, 121.7, 118.7, 116.4, 69.9, 60.4, 34.5, 29.3, 29.2, 28.9, 26.1, 25.1, 22.3, 14.4. MS (ESI+) m/z: 423.1, 425.1 [M + H]+. General Procedure for the Synthesis of 57a−u and 61a−n. To a mixture of compound 56a−g or 60a−h (1.0 equiv), pyridinyl boronic acid or ester (1.5 equiv) and PdCl2(dppf) (0.1 equiv) in toluene/water (2:1, v/v) was added 2 M aqueous K2CO3 solution (3.0 equiv). The reaction mixture was stirred under argon at 80 °C for 8 h. After being cooled to room temperature, the mixture was diluted with water and extracted with EA three times. The combined organic layers were washed with water and brine, dried over anhydrous Na2SO4, and concentrated. The resulting residue was purified by column chromatography (silica gel, PE/EA = 1:6) to afford the desired products 57a−u as a light yellow solid. 4-Methyl-6-(2-methoxy-5-pyridinyl)-8-(ethyl acetate-2-oxy)quinazoline (57a). Compound 57a was prepared from 4-methyl-6bromo-8-(ethyl acetate-2-oxy)quinazoline (56a) and (6-methoxypyridin-3-yl)boronic acid (light yellow solid, 23% yield). 1H NMR (400 MHz, CDCl3): δ 9.17 (s, 1H), 8.42 (d, J = 2.3 Hz, 1H), 7.82 (dd, J = 8.6, 2.6 Hz, 1H), 7.75 (d, J = 1.6 Hz, 1H), 7.29 (d, J = 1.6 Hz, 1H), 6.85 (d, J = 8.7 Hz, 1H), 5.00 (s, 2H), 4.25 (q, J = 7.1 Hz, 2H), 3.98 (s, 3H), 2.95 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 168.5, 168.2, 164.5, 154.2, 153.4, 145.7, 141.4, 137.8, O



Article

(s, 1H), 8.23 (d, J = 1.8 Hz, 1H), 7.67 (s, 1H), 7.63 (dd, J = 10.8, 1.9 Hz, 1H), 7.29 (d, J = 0.6 Hz, 1H), 4.25 (t, J = 6.4 Hz, 2H), 4.15−4.06 (m, 5H), 2.95 (s, 3H), 2.33 (t, J = 7.3 Hz, 2H), 2.11−1.98 (m, 2H), 1.79−1.66 (m, 2H), 1.64−1.51 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 173.7, 168.1, 155.4, 154.0, 153.4 (d, JC−F = 11.3 Hz), 147.7 (d, JC−F = 260 Hz), 141.7, 139.8 (d, JC−F = 5.7 Hz), 136.4, 130.5, 125.7, 122.3 (d, JC−F = 16.0 Hz), 114.1, 111.6, 69.4, 60.5, 54.3, 34.3, 28.8, 25.8, 24.8, 22.5, 14.4. MS (ESI+) m/z: 428.1 [M + H]+. 4-Methyl-6-(2-methoxy-3-methanesulfonamido-5-pyridinyl)-8(ethyl hexanoate-6-oxy)quinazoline (57h). Compound 57h was prepared from compound 56d and N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)-methanesulfonamide (light yellow solid, 41% yield). 1H NMR (400 MHz, CDCl3): δ 9.20 (s, 1H), 8.24 (d, J = 2.2 Hz, 1H), 8.07 (d, J = 2.2 Hz, 1H), 7.69 (d, J = 1.4 Hz, 1H), 7.31 (d, J = 1.2 Hz, 1H), 6.81 (s, 1H), 4.27 (q, J = 7.0 Hz, 2H), 4.13−4.09 (m, 2H), 4.08 (s, 3H), 3.04 (s, 3H), 2.96 (s, 3H), 2.33 (t, J = 7.4 Hz, 2H), 2.11−2.01 (m, 2H), 1.76−1.70 (m, 2H), 1.66−1.45 (m, 2H), 1.30−1.17 (t, J = 7.0 Hz, 3H). MS (ESI+) m/z: 503.0 [M + H]+. 4-Methyl-6-(2-methoxy-3-(2,4-difluorobenzenesulfonamido)-5pyridinyl)-8-(ethyl hexanoate-6-oxy)quinazoline (57i). Compound 57i was prepared from compound 56d and N-(2-methoxy-5-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)-2,4-difluorobenzenesulfonamide (light yellow solid, 41% yield). 1H NMR (400 MHz, CDCl3): δ 9.20 (s, 1H), 8.16 (s, 1H), 8.02 (s, 1H), 7.86 (d, J = 5.3 Hz, 1H), 7.63 (s, 1H), 7.35−7.31 (m, 1H), 7.27−7.24 (m, 1H), 7.00−6.91 (m, 2H), 4.32−4.23 (m, 2H), 4.10 (q, J = 7.1 Hz, 2H), 3.94 (d, J = 1.2 Hz, 3H), 2.96 (s, 3H), 2.34 (t, J = 7.3 Hz, 2H), 2.10− 2.00 (m, 2H), 1.74−1.67 (m, 2H), 1.59−1.50 (m, 2H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 173.8, 168.1, 166.4 (dd, JC−F = 259, 11.6 Hz), 160.1 (dd, JC−F = 259, 12.8 Hz), 155.3, 154.8, 153.8, 141.6, 141.1, 137.0, 132.6 (d, JC−F = 10.4 Hz), 130.5, 127.4, 125.7, 123.6 (dd, JC−F = 13.6, 3.4 Hz), 120.7, 114.1, 112.1 (dd, JC−F = 21.9, 2.9 Hz), 111.8, 106.0 (t, JC−F = 25.4 Hz), 69.4, 60.5, 54.4, 34.3, 28.8, 25.8, 24.9, 22.4, 14.4. MS (ESI+) m/z: 600.9 [M + H]+. 4-Methyl-6-(2-methoxy-5-pyridinyl)-8-(ethyl heptanoate-7-oxy)quinazoline (57j). Compound 57j was prepared from compound 56e and (6-methoxypyridin-3-yl)boronic acid (light yellow solid, 51% yield). 1H NMR (400 MHz, CDCl3): δ 9.20 (s, 1H), 8.48 (d, J = 2.3 Hz, 1H), 7.89 (dd, J = 8.6, 2.5 Hz, 1H), 7.70 (d, J = 1.3 Hz, 1H), 7.35 (d, J = 1.1 Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H), 4.27 (t, J = 6.8 Hz, 2H), 4.11 (q, J = 7.1 Hz, 2H), 4.00 (d, J = 1.8 Hz, 3H), 2.98 (s, 3H), 2.30 (t, J = 7.5 Hz, 2H), 2.09−1.99 (m, 2H), 1.66 (dd, J = 15.2, 7.6 Hz, 2H), 1.56 (dd, J = 15.3, 7.7 Hz, 2H), 1.49−1.40 (m, 2H), 1.26−1.21 (t, 3H). 13C NMR (101 MHz, CDCl3): δ 173.9, 168.0, 164.4, 155.3, 153.5, 145.7, 141.5, 137.9, 137.8, 129.6, 125.7, 113.7, 112.0, 111.4, 69.5, 60.4, 53.9, 34.4, 29.0, 26.0, 25.0, 22.4, 14.4. MS (ESI+) m/z: 424.2 [M + H]+. 4-Methyl-6-(2-methoxy-3-fluoro-5-pyridinyl)-8-(ethyl heptanoate-7-oxy)quinazoline (57k). Compound 57k was prepared from compound 56e and (5-fluoro-6-methoxypyridin-3-yl)boronic acid (light yellow solid, 47% yield). 1H NMR (400 MHz, CDCl3): δ 9.23 (s, 1H), 8.26 (d, J = 1.8 Hz, 1H), 7.71 (s, 1H), 7.66 (dd, J = 10.8, 1.9 Hz, 1H), 7.35 (d, J = 0.8 Hz, 1H), 4.28 (t, J = 6.6 Hz, 2H), 4.17−4.06 (m, 5H), 3.02 (s, 3H), 2.32 (t, J = 7.4 Hz, 2H), 2.12−1.97 (m, 2H), 1.75−1.63 (m, 2H), 1.62−1.52 (m, 2H), 1.54−1.40 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 173.8, 168.0, 155.4, 153.7, 153.3 (d, JC−F = 11.2 Hz), 147.6 (d, JC−F = 261 Hz), 141.6, 139.7 (d, JC−F = 5.1 Hz), 136.5, 130.4 (d, JC−F = 1.0 Hz), 125.6, 122.2 (d, JC−F = 16.1 Hz), 114.0, 111.7, 69.5, 60.3, 54.2, 34.3, 28.9, 28.8, 25.8, 24.9, 22.4, 14.3. MS (ESI+) m/z: 442.2 [M + H]+. 4-Methyl-6-(2-methoxy-3-methanesulfonamido-5-pyridinyl)-8(ethyl heptanoate-7-oxy)quinazoline (57l). Compound 57l was prepared from compound 56e and N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)-methanesulfonamide (light yellow solid, 43% yield). 1H NMR (400 MHz, CDCl3): δ 9.22 (s, 1H), 8.27 (d, J = 1.9 Hz, 1H), 8.09 (d, J = 1.9 Hz, 1H), 7.71 (s, 1H), 7.35 (s, 1H), 6.84 (s, 1H), 4.28 (t, J = 6.8 Hz, 2H), 4.11 (q, J = 7.1 Hz, 2H), 4.10 (s, 3H), 3.06 (s, 3H), 3.01 (s, 3H), 2.31 (t, J = 7.5

137.7, 129.2, 125.9, 115.4, 113.7, 111.5, 66.8, 61.9, 54.0, 22.3, 14.4. MS (ESI+) m/z: 354.0 [M + H]+. 4-Methyl-6-(2-methoxy-5-pyridinyl)-8-(ethyl butyrate-4-oxy)quinazoline (57b). Compound 57b was prepared from compound 56b and (6-methoxypyridin-3-yl)boronic acid (light yellow solid, 31% yield). 1H NMR (400 MHz, CDCl3): δ 9.12 (s, 1H), 8.42 (d, J = 2.3 Hz, 1H), 7.83 (dd, J = 8.6, 2.5 Hz, 1H), 7.64 (d, J = 1.5 Hz, 1H), 7.33 (d, J = 1.4 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H), 4.29 (t, J = 6.4 Hz, 2H), 4.08 (q, J = 7.1 Hz, 2H), 3.94 (s, 3H), 2.89 (s, 3H), 2.56 (t, J = 7.1 Hz, 2H), 2.27 (p, J = 6.7 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H). MS (ESI +) m/z: 382.2 [M + H]+. 4-Methyl-6-(2-methoxy-3-(2,4-difluorobenzenesulfonamido)-5pyridinyl)-8-(ethyl butyrate-4-oxy)quinazoline (57c). Compound 57c was prepared from compound 56b and N-(2-methoxy-5-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)-2,4-difluorobenzenesulfonamide (light yellow solid, 21% yield). 1H NMR (400 MHz, CDCl3): δ 9.21 (s, 1H), 8.17 (d, J = 2.2 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.90−7.81 (m, 1H), 7.67 (d, J = 1.2 Hz, 1H), 7.30−7.23 (d, J = 1.2 Hz, 1H), 6.98−6.92 (m, 2H), 4.35−4.29 (m, 2H), 4.11 (q, J = 7.1 Hz, 2H), 3.95 (s, 3H), 2.99 (s, 3H), 2.63 (t, J = 6.8 Hz, 2H), 2.43− 2.20 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 173.3, 168.2, 166.4 (dd, JC−F = 259, 11.4 Hz), 160.1 (dd, JC−F = 259, 12.8 Hz), 155.2, 154.7, 154.0, 141.6, 141.1, 136.9, 132.7 (d, JC−F = 10.4 Hz), 130.4, 127.2, 125.7, 123.6 (d, JC−F = 13.9 Hz), 120.7, 114.3, 112.2 (dd, JC−F = 22.0, 2.8 Hz), 112.0, 106.0 (t, JC−F = 25.3 Hz), 68.5, 60.8, 54.4, 30.8, 24.4, 22.6, 14.4. MS (ESI+) m/z: 572.9 [M + H]+. 4-Methyl-6-(2-methoxy-5-pyridinyl)-8-(ethyl pentanoate-5-oxy)quinazoline (57d). Compound 57d was prepared from compound 56c and (6-methoxypyridin-3-yl)boronic acid (light yellow solid, 45% yield). 1H NMR (400 MHz, CDCl3): δ 9.20 (s, 1H), 8.49 (d, J = 1.7 Hz, 1H), 7.89 (dd, J = 8.4, 2.0 Hz, 1H), 7.71 (d, J = 1.5 Hz, 1H), 7.36 (d, J = 1.5 Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H), 4.30 (t, J = 6.5 Hz, 2H), 4.12 (q, J = 7.1 Hz, 2H), 4.01 (s, 3H), 2.98 (s, 3H), 2.43 (t, J = 7.3 Hz, 2H), 2.08 (m, 2H), 1.96−1.86 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, CDCl3): δ 173.5, 168.0, 164.3, 155.2, 153.7, 145.7, 141.4, 137.8, 137.7, 129.6, 125.7, 113.8, 111.9, 111.3, 69.1, 60.5, 53.9, 34.1, 28.5, 22.5, 21.7, 14.4. MS (ESI+) m/z: 396.1 [M + H]+. 4-Methyl-6-(2-methoxy-3-(2,4-difluorobenzenesulfonamido)-5pyridinyl)-8-(ethyl pentanoate-5-oxy)quinazoline (57e). Compound 57e was prepared from compound 56c and N-(2-methoxy-5(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)-2,4-difluorobenzenesulfonamide (light yellow solid, 41% yield). 1H NMR (400 MHz, CDCl3): δ 9.23 (s, 1H), 8.19 (d, J = 2.2 Hz, 1H), 8.05 (d, J = 2.2 Hz, 1H), 7.96−7.82 (m, 1H), 7.67 (d, J = 1.2 Hz, 1H), 7.38−7.28 (d, J = 1.2 Hz, 1H), 7.04−6.86 (m, 2H), 4.38−4.27 (m, 2H), 4.13 (q, J = 7.1 Hz, 2H), 3.98 (s, 3H), 3.49 (s, 1H), 3.04 (s, 3H), 2.45 (t, J = 6.8 Hz, 2H), 2.30−2.10 (m, 2H), 1.98−1.88 (m, 2H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 173.5, 168.1, 166.5 (dd, JC−F = 260, 11.1 Hz), 160.2 (dd, JC−F = 260, 13.1 Hz), 155.3, 154.8, 153.7, 141.6, 141.2, 137.1, 132.7 (d, JC−F = 10.1 Hz), 130.5, 127.3, 125.7, 123.6 (dd, JC−F = 13.8, 3.9 Hz), 120.7, 114.3, 112.2 (dd, JC−F = 22.6, 3.4 Hz), 112.0, 106.0 (t, JC−F = 25.4 Hz), 69.2, 60.6, 54.4, 34.2, 28.6, 22.5, 21.8, 14.4. MS (ESI+) m/z: 587.1 [M + H]+. 4-Methyl-6-(2-methoxy-5-pyridinyl)-8-(ethyl hexanoate-6-oxy)quinazoline (57f). Compound 57f was prepared from compound 56d and (6-methoxypyridin-3-yl)boronic acid (light yellow solid, 47% yield). 1H NMR (400 MHz, CDCl3): δ 9.20 (s, 1H), 8.48 (d, J = 1.7 Hz, 1H), 7.89 (dd, J = 8.4, 2.0 Hz, 1H), 7.70 (d, J = 1.5 Hz, 1H), 7.35 (d, J = 1.5 Hz, 1H), 6.89 (d, J = 8.2 Hz, 1H), 4.28 (t, J = 6.0 Hz, 2H), 4.12 (q, J = 7.1 Hz, 2H), 4.01 (s, 3H), 2.98 (s, 3H), 2.35 (t, J = 7.3 Hz, 2H), 2.13−2.01 (m, 2H), 1.84−1.70 (m, 2H), 1.66−1.55 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 154.5, 149.9, 147.0, 139.7, 138.3, 132.0, 128.6, 125.8, 125.5, 119.1, 116.1, 106.5, 105.1, 104.6, 71.0, 63.8, 58.6, 43.0, 38.5, 36.1, 35.4, 33.4, 27.0. MS (ESI+) m/z: 409.9 [M + H]+. 4-Methyl-6-(2-methoxy-3-fluoro-5-pyridinyl)-8-(ethyl hexanoate-6-oxy)quinazoline (57g). Compound 57g was prepared from compound 56d and (5-fluoro-6-methoxypyridin-3-yl)boronic acid (light yellow solid, 46% yield). 1H NMR (400 MHz, CDCl3): δ 9.18 P



Article

Hz, 2H), 2.15−1.93 (m, 2H), 1.77−1.63 (m, 2H), 1.63−1.51 (m, 2H), 1.49−1.40 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 173.7, 167.9, 155.1, 154.7, 153.4, 141.4, 140.8, 136.9, 130.5, 127.5, 125.4, 121.4, 113.9, 111.7, 69.3, 60.2, 54.3, 40.0, 34.2, 28.8, 28.7, 25.7, 24.8, 22.1, 14.2. MS (ESI+) m/z: 517.0 [M + H]+. 4-Methyl-6-(2-methoxy-3-(2,4-difluorobenzenesulfonamido)-5pyridinyl)-8-(ethyl heptanoate-7-oxy)quinazoline (57m). Compound 57m was prepared from compound 56e and N-(2-methoxy5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)-2,4-difluorobenzenesulfonamide (light yellow solid, 40% yield). 1H NMR (400 MHz, CDCl3): δ 9.20 (s, 1H), 8.16 (s, 1H), 8.02 (s, 1H), 7.85 (d, J = 5.5 Hz, 1H), 7.62 (s, 1H), 7.35−7.30 (m, 1H), 7.28−7.25 (m, 1H), 7.01−6.86 (m, 2H), 4.30−4.22 (m, 2H), 4.09 (q, J = 7.1 Hz, 2H), 3.94 (d, J = 1.2 Hz, 3H), 2.96 (s, 3H), 2.29 (t, J = 7.3 Hz, 2H), 2.09−2.00 (m, 2H), 1.73−1.62 (m, 2H), 1.59−1.50 (m, 2H), 1.47− 1.38 (m, 2H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 173.9, 168.1, 166.4 (dd, JC−F = 259, 11.6 Hz), 160.1 (dd, JC−F = 259.0, 13.0 Hz), 155.4, 154.8, 153.8, 141.6, 141.2, 137.0, 132.6 (d, JC−F = 10.5 Hz), 130.5, 127.4, 125.7, 123.6 (dd, JC−F = 13.7, 3.6 Hz), 120.7, 114.1, 112.1 (dd, JC−F = 21.9, 3.3 Hz), 111.8, 106.0 (t, JC−F = 25.4 Hz), 69.5, 60.4, 54.4, 34.4, 29.1, 28.9, 26.0, 25.0, 22.4, 14.4. MS (ESI+) m/z: 615.1 [M + H]+. 4-Methyl-6-(2-methoxy-5-pyridinyl)-8-(ethyl octanoate-8-oxy)quinazoline (57n). Compound 57n was prepared from compound 56f and (6-methoxypyridin-3-yl)boronic acid (light yellow solid, 53% yield). 1H NMR (400 MHz, CDCl3): δ 9.18 (s, 1H), 8.46 (s, 1H), 7.87 (d, J = 7.7 Hz, 1H), 7.67 (s, 1H), 7.33 (s, 1H), 6.86 (d, J = 8.1 Hz, 1H), 4.24 (t, J = 5.9 Hz, 2H), 4.08 (q, J = 7.1 Hz, 2H), 3.98 (s, 3H), 2.96 (s, 3H), 2.26 (t, J = 7.4 Hz, 2H), 2.09−1.95 (m, 2H), 1.66−1.56 (m, 2H), 1.56−1.46 (m, 2H), 1.44−1.31 (m, 4H), 1.21 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 174.0, 167.9, 164.4, 155.3, 153.5, 145.7, 141.5, 137.9, 129.6, 125.7, 113.7, 112.0, 111.4, 69.6, 60.4, 53.9, 34.5, 29.3, 29.2, 29.0, 26.1, 25.1, 22.4, 14.4. MS (ESI +) m/z: 437.9 [M + H]+. 4-Methyl-6-(2-methoxy-3-fluoro-5-pyridinyl)-8-(ethyl octanoate8-oxy)quinazoline (57o). Compound 57o was prepared from compound 56f and (5-fluoro-6-methoxypyridin-3-yl)boronic acid (light yellow solid, 51% yield). 1H NMR (400 MHz, CDCl3): δ 9.20 (s, 1H), 8.24 (d, J = 1.8 Hz, 1H), 7.67 (s, 1H), 7.64 (dd, J = 10.8, 1.9 Hz, 1H), 7.30 (d, J = 0.5 Hz, 1H), 4.25 (t, J = 6.8 Hz, 2H), 4.15− 4.04 (m, 5H), 2.97 (s, 3H), 2.27 (t, J = 7.5 Hz, 2H), 2.09−1.97 (m, 2H), 1.69−1.58 (m, 2H), 1.58−1.48 (m, 2H), 1.46−1.29 (m, 4H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 174.0, 168.0, 155.5, 153.5 (d, JC−F = 3.3 Hz), 153.4, 147.7 (d, JC−F = 260 Hz), 141.7, 139.8 (d, JC−F = 5.6 Hz), 136.7, 130.5, 125.7, 122.3 (d, JC−F = 16.0 Hz), 114.0, 111.8, 69.7, 60.4, 54.3, 34.5, 29.9, 29.2, 29.0, 26.1, 25.1, 22.3, 14.5. MS (ESI+) m/z: 456.1 [M + H]+. 4-Methyl-6-(2-methoxy-3-methanesulfonamido-5-pyridinyl)-8(ethyl octanoate-8-oxy)quinazoline (57p). Compound 57p was prepared from compound 56f and N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)-methanesulfonamide (light yellow solid, 47% yield). 1H NMR (400 MHz, CDCl3): δ 9.22 (s, 1H), 8.27 (d, J = 1.4 Hz, 1H), 8.09 (d, J = 1.4 Hz, 1H), 7.71 (s, 1H), 7.35 (s, 1H), 6.85 (s, 1H), 4.27 (t, J = 6.7 Hz, 2H), 4.16−4.07 (m, 5H), 3.06 (s, 3H), 3.01 (s, 3H), 2.29 (t, J = 7.5 Hz, 2H), 2.10− 1.98 (m, 2H), 1.72−1.59 (m, 2H), 1.59−1.51 (m, 2H), 1.50−1.33 (m, 4H), 1.24 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 173.5, 167.7, 154.9, 154.8, 153.3, 141.1, 140.6, 136.5, 130.1, 127.8, 125.1, 121.2, 113.5, 111.3, 69.1, 59.9, 54.0, 39.8, 34.0, 28.8, 28.7, 28.6, 25.6, 24.6, 21.9, 14.0. MS (ESI+) m/z: 531.0 [M + H]+. 4-Methyl-6-(2-methoxy-3-(2,4-difluorobenzenesulfonamido)-5pyridinyl)-8-(ethyl octanoate-8-oxy)quinazoline (57q). Compound 57q was prepared from compound 56f and N-(2-methoxy-5-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)-2,4-difluorobenzenesulfonamide (light yellow solid, 44% yield). 1H NMR (400 MHz, CDCl3): δ 9.24 (s, 1H), 8.20 (d, J = 2.1 Hz, 1H), 8.08−8.01 (d, J = 2.1 Hz, 1H), 7.93−7.84 (m, 1H), 7.68 (d, J = 1.1 Hz, 1H), 7.33 (d, J = 1.1 Hz, 1H), 7.03−6.89 (m, 2H), 4.35−4.23 (m, 2H), 4.12 (q, J = 7.1 Hz, 2H), 3.98 (s, 3H), 3.06 (s, 3H), 2.30 (t, J = 7.5 Hz, 2H),

2.14−2.02 (m, 2H), 1.71−1.53 (m, 4H), 1.49−1.36 (m, 4H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 174.0, 168.0, 166.4 (dd, JC−F = 260, 11.5 Hz), 160.1 (dd, JC−F = 260, 12.8 Hz), 155.4, 154.8, 153.3, 141.6, 141.2, 137.2, 132.6 (d, JC−F = 10.5 Hz), 130.5, 127.4, 125.6, 123.7 (dd, JC−F = 13.9, 3.6 Hz), 120.7, 114.1, 112.1 (dd, JC−F = 22.6, 3.2 Hz), 112.0, 106.0 (t, JC−F = 25.8 Hz), 69.7, 60.4, 54.4, 34.5, 29.3, 29.2, 29.0, 26.1, 25.1, 22.3, 14.4. MS (ESI+) m/z: 629.0 [M + H]+. 4-Methyl-6-(2-methoxy-5-pyridinyl)-8-(ethyl nonanoate-9-oxy)quinazoline (57r). Compound 57r was prepared from compound 56g and (6-methoxypyridin-3-yl)boronic acid (light yellow solid, 56% yield). 1H NMR (400 MHz, CDCl3): δ 9.21 (s, 1H), 8.25 (d, J = 2.1 Hz, 1H), 7.68 (d, J = 1.5 Hz, 1H), 7.65 (dd, J = 10.8, 2.1 Hz, 1H), 7.31 (d, J = 1.4 Hz, 1H), 4.26 (t, J = 6.9 Hz, 2H), 4.15−4.06 (m, 5H), 2.97 (s, 3H), 2.27 (t, J = 7.5 Hz, 2H), 2.09−1.97 (m, 2H), 1.67−1.57 (m, 2H), 1.56−1.48 (m, 2H), 1.44−1.30 (m, 6H), 1.23 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 451.9 [M + H]+. 4-Methyl-6-(2-methoxy-3-fluoro-5-pyridinyl)-8-(ethyl Nonanoate-9-oxy)quinazoline (57s). Compound 57s was prepared from compound 56g and (5-fluoro-6-methoxypyridin-3-yl)boronic acid (light yellow solid, 48% yield). 1H NMR (400 MHz, CDCl3): δ 9.21 (s, 1H), 8.25 (d, J = 2.1 Hz, 1H), 7.68 (d, J = 1.5 Hz, 1H), 7.65 (dd, J = 10.8, 2.1 Hz, 1H), 7.31 (d, J = 1.4 Hz, 1H), 4.26 (t, J = 6.9 Hz, 2H), 4.15−4.06 (m, 5H), 2.97 (s, 3H), 2.27 (t, J = 7.5 Hz, 2H), 2.09−1.97 (m, 2H), 1.67−1.57 (m, 2H), 1.56−1.48 (m, 2H), 1.44−1.30 (m, 6H), 1.23 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 174.0, 168.1, 155.5, 153.8, 153.4 (d, JC−F = 11.3 Hz), 147.7 (d, JC−F = 260 Hz), 141.7, 139.8 (d, JC−F = 5.8 Hz), 136.5, 130.6 (d, JC−F = 1.6 Hz), 125.7, 122.3 (d, JC−F = 16.1 Hz), 114.0, 111.7, 69.7, 60.4, 54.3, 34.5, 29.4, 29.3, 29.2, 29.1, 26.2, 25.1, 22.4, 14.4. MS (ESI+) m/z: 470.0 [M + H]+. 4-Methyl-6-(2-methoxy-3-methanesulfonamido-5-pyridinyl)-8(ethyl nonanoate-9-oxy)quinazoline (57t). Compound 57t was prepared from compound 56g and N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)-methanesulfonamide (light yellow solid, 45% yield). 1H NMR (400 MHz, CDCl3): δ 9.15 (s, 1H), 8.22 (s, 1H), 8.05 (s, 1H), 7.65 (s, 1H), 7.29 (s, 1H), 7.12 (s, 1H), 4.22 (t, J = 5.7 Hz, 2H), 4.13−4.00 (m, 5H), 3.03 (s, 3H), 2.92 (s, 3H), 2.23 (t, J = 7.5 Hz, 2H), 2.06−1.88 (m, 2H), 1.64−1.48 (m, 4H), 1.42−1.29 (m, 6H), 1.19 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 173.9, 168.0, 155.3, 154.7, 153.7, 141.5, 140.9, 136.9, 130.7, 127.4, 125.5, 121.5, 114.0, 111.7, 69.5, 60.2, 54.4, 40.0, 34.4, 29.3, 29.2, 29.1, 28.9, 26.0, 25.0, 22.3, 14.3. MS (ESI+) m/z: 544.9 [M + H]+. 4-Methyl-6-(2-methoxy-3-(2,4-difluorobenzenesulfonamido)-5pyridinyl)-8-(ethyl Nonanoate-9-oxy)quinazoline (57u). Compound 57u was prepared from compound 56g and N-(2-methoxy-5-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)-2,4-difluorobenzenesulfonamide (light yellow solid, 43% yield). 1H NMR (400 MHz, CDCl3): δ 9.24 (s, 1H), 8.19 (d, J = 2.1 Hz, 1H), 8.05 (d, J = 2.1 Hz, 1H), 7.92−7.84 (m, 1H), 7.67 (d, J = 1.0 Hz, 1H), 7.32 (d, J = 1.0 Hz, 1H), 7.01−6.90 (m, 2H), 4.34−4.23 (m, 2H), 4.12 (q, J = 7.1 Hz, 2H), 3.98 (s, 3H), 3.05 (s, 3H), 2.28 (t, J = 7.5 Hz, 2H), 2.11−2.00 (m, 2H), 1.66−1.51 (m, 4H), 1.48−1.30 (m, 6H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 174.1, 168.0, 166.4 (dd, JC−F = 259, 11.7 Hz), 160.1 (dd, JC−F = 259, 12.9 Hz), 141.6, 141.2, 137.3, 132.6 (d, JC−F = 10.5 Hz), 130.5, 127.4, 125.6, 123.7 (dd, JC−F = 13.7, 3.8 Hz), 120.8, 114.0, 112.1 (dd, JC−F = 21.6, 3.5 Hz), 112.0, 106.0 (t, JC−F = 25.4 Hz), 69.7, 60.4, 54.4, 34.5, 29.5, 29.4, 29.2, 29.1, 26.2, 25.1, 14.5. MS (ESI+) m/z: 643.0 [M + H]+. General Procedure for the Synthesis of 58a−h. To a solution of compound 56 (1.0 equiv), triphenylphosphine (1.5 equiv), and Boc-protected amino alcohol (1.5 equiv) in anhydrous THF was added diethyl azodicarboxylate (DEAD, 1.5 equiv) dropwise. The reaction mixture was stirred at room temperature under argon for 16 h. The volatiles were removed under reduced pressure. The resulting residue was purified by column chromatography (silica gel, PE/EA = 1:1) to afford the desired product as yellow oil. 4-Methyl-6-bromo-8-(N-Boc-N-methyl-2-aminoethoxy)quinazoline (58a). Compound 58a was prepared from compound 55 Q



Article

and N-Boc-N-methyl-aminoethanol (yellow oil, 68% yield). 1H NMR (400 MHz, CDCl3): δ 9.18 (s, 1H), 7.82 (d, J = 1.8 Hz, 1H), 7.30 (d, J = 1.8 Hz, 1H), 4.30 (t, J = 5.9 Hz, 2H), 3.77 (t, J = 5.9 Hz, 2H), 3.04 (s, 3H), 2.89 (s, 3H), 1.45 (s, 9H). MS (ESI+) m/z: 396.1, 398.1 [M + H]+. 4-Methyl-6-bromo-8-(N-Boc-N-methyl-3-aminopropoxy)quinazoline (58b). Compound 58b was prepared from compound 55 and N-Boc-3-methylamino-1-propanol (yellow oil, 72% yield). 1H NMR (400 MHz, CDCl3): δ 9.21 (s, 1H), 7.81 (d, J = 1.9 Hz, 1H), 7.29 (d, J = 1.9 Hz, 1H), 4.31 (t, J = 6.1 Hz, 2H), 3.67 (t, J = 5.9 Hz, 2H), 3.12 (s, 3H), 2.89 (s, 3H), 2.41−2.36 (m, 2H), 1.45 (s, 9H). MS (ESI+) m/z: 410.0, 412.1 [M + H]+. 4-Methyl-6-bromo-8-(N-Boc-N-methyl-4-aminobutoxy)quinazoline (58c). Compound 58c was prepared from compound 55 and N-Boc-4-methylamino-1-butanol (yellow oil, 76% yield). 1H NMR (400 MHz, CDCl3): δ 9.18 (s, 1H), 7.78 (d, J = 1.7 Hz, 1H), 7.29 (d, J = 1.7 Hz, 1H), 4.21 (t, J = 6.5 Hz, 2H), 3.29 (t, J = 6.8 Hz, 2H), 2.92 (s, 3H), 2.83 (s, 3H), 2.03−1.91 (m, 2H), 1.82−1.70 (m, 2H), 1.41 (s, 9H). MS (ESI+) m/z: 424.0, 426.1 [M + H]+. 4-Methyl-6-bromo-8-(N-Boc-N-ethyl-2-aminoethoxy)quinazoline (58d). Compound 58d was prepared from compound 55 and N-Boc-2-ethylamino-1-ethanol (yellow oil, 70% yield). 1H NMR (400 MHz, CDCl3): δ 9.19 (s, 1H), 7.82 (d, J = 1.3 Hz, 1H), 7.46 (d, J = 1.3 Hz, 1H), 4.36 (t, J = 6.1 Hz, 2H), 3.75 (t, J = 6.1 Hz, 2H), 3.39 (q, J = 6.0 Hz, 2H), 2.94 (s, 3H), 1.47 (s, 9H), 1.14 (t, J = 6.0 Hz, 3H). MS (ESI+) m/z: 410.1, 412.2 [M + H]+. 4-Methyl-6-bromo-8-(N-Boc-N-isopropyl-2-aminoethoxy)quinazoline (58e). Compound 58e was prepared from compound 55 and N-Boc-2-isopropylamino-1-ethanol (yellow oil, 72% yield). 1H NMR (400 MHz, CDCl3): δ 9.18 (s, 1H), 7.79 (s, 1H), 7.24 (s, 1H), 4.42−4.26 (m, 3H), 3.63 (t, J = 6.8 Hz, 2H), 2.90 (s, 3H), 1.47 (s, 9H), 1.15 (d, J = 6.8 Hz, 6H). MS (ESI+) m/z: 424.1, 426.1 [M + H]+. 4-Methyl-6-bromo-8-(N-Boc-2-aminoethoxy)quinazoline (58f). Compound 58f was prepared from compound 55 and N-Boc-2bromoethylamine (yellow oil, 74% yield). 1H NMR (400 MHz, CDCl3): δ 9.16 (s, 1H), 7.80 (d, J = 1.8 Hz, 1H), 7.30 (d, J = 1.8 Hz, 1H), 5.71 (s, 1H), 4.23 (t, J = 3.8 Hz, 2H), 3.69 (t, J = 3.8 Hz, 2H), 2.89 (s, 3H), 1.42 (s, 9H). MS (ESI+) m/z: 382.1, 384.1 [M + H]+. 4-Methyl-6-bromo-8-(N-Boc-3-aminopropoxy)quinazoline (58g). Compound 58g was prepared from compound 55 and N-Boc3-bromopropylamine (yellow oil, 77% yield). 1H NMR (400 MHz, CDCl3): δ 9.19 (s, 1H), 7.80 (d, J = 1.8 Hz, 1H), 7.28 (d, J = 1.7 Hz, 1H), 6.05 (s, 1H), 4.28 (t, J = 5.8 Hz, 2H), 3.44 (t, J = 4.9 Hz, 2H), 2.90 (s, 3H), 2.22−2.12 (m, 2H), 1.45 (s, 9H). MS (ESI+) m/z: 396.1, 398.1 [M + H]+. 4-Methyl-6-bromo-8-(N-Boc-4-aminobutoxy)quinazoline (58h). Compound 58h was prepared from compound 55 and 4-(Bocamino)-1-butanol (yellow oil, 65% yield). 1H NMR (400 MHz, CDCl3): δ 9.20 (s, 1H), 7.79 (d, J = 1.8 Hz, 1H), 7.28 (d, J = 1.8 Hz, 1H), 4.21 (t, J = 6.2 Hz, 2H), 3.23 (t, J = 6.4 Hz, 2H), 2.92 (s, 3H), 2.09−2.00 (m, 2H), 1.81−1.72 (m, 2H), 1.43 (s, 9H). MS (ESI+) m/ z: 410.1, 412.1 [M + H]+. General Procedure for the Synthesis of 59a−h. To a solution of compound 58a−h (4.0 mmol) in DCM (30 mL) was added TFA (10 mL). The reaction mixture was stirred at room temperature for 1.5 h. The volatiles were removed under reduced pressure. The residue was used in the next step without further purification. Compound 59a−h was prepared from compound 58a−h, respectively. General Procedure for the Synthesis of Compound 60a−h. To a solution of compound 59a−h (1.0 equiv) and DIPEA (3.0 equiv) in anhydrous MeCN was added ethyl 2-chloropyrimidine-5carboxylate (1.5 equiv). The resulting mixture was stirred at room temperature for 16 h. The volatiles were removed under reduced pressure. The residue was dissolved in DCM, washed with water and brine, dried over anhydrous NaSO4, and concentrated. The obtained residue was then purified by column chromatography (silica gel, PE/ EA = 2:1) to afford the desired product as a light yellow solid.

Ethyl 2-((2-((6-bromo-4-methylquinazolin-8-yl)oxy)ethyl)(methyl)amino)pyrimidine-5-carboxylate (60a). Compound 60a was prepared from compound 59a (light yellow solid, 65% yield). 1 H NMR (400 MHz, CDCl3): δ 9.19 (s, 1H), 8.92 (s, 2H), 7.84 (d, J = 1.8 Hz, 1H), 7.61 (d, J = 1.8 Hz, 1H), 4.51 (t, J = 6.2 Hz, 2H), 4.36 (d, J = 7.1 Hz, 2H), 4.25 (t, J = 6.2 Hz, 2H), 3.45 (s, 3H), 2.97 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 446.1, 448.1 [M + H]+. Ethyl 2-((3-((6-bromo-4-methylquinazolin-8-yl)oxy)propyl)(methyl)amino)pyrimidine-5-carboxylate (60b). Compound 60b was prepared from compound 59b (light yellow solid, 63% yield). 1 H NMR (400 MHz, CDCl3): δ 9.22 (s, 1H), 8.81 (s, 2H), 7.81 (d, J = 1.5 Hz, 1H), 7.28 (d, J = 1.5 Hz, 1H), 4.34 (q, J = 7.1 Hz, 2H), 4.29 (t, J = 6.3 Hz, 2H), 4.01 (t, J = 6.8 Hz, 2H), 3.29 (s, 3H), 2.93 (s, 3H), 2.38 (p, J = 6.5 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H). MS (ESI+) m/ z: 460.0, 462.0 [M + H]+. Ethyl 2-((4-((6-bromo-4-methylquinazolin-8-yl)oxy)butyl)(methyl)amino)pyrimidine-5-carboxylate (60c). Compound 60c was prepared from compound 59c (light yellow solid, 63% yield). 1 H NMR (400 MHz, CDCl3): δ 9.17 (s, 1H), 8.81 (s, 2H), 7.77 (d, J = 1.8 Hz, 1H), 7.27 (d, J = 1.7 Hz, 1H), 4.30 (q, J = 7.1 Hz, 2H), 4.23 (t, J = 6.5 Hz, 2H), 3.82 (t, J = 7.2 Hz, 2H), 3.23 (s, 3H), 2.89 (s, 3H), 2.08−1.98 (m, 2H), 1.94−1.85 (m, 2H), 1.33 (t, J = 7.2 Hz, 3H). MS (ESI+) m/z: 474.1, 476.1 [M + H]+. Ethyl 2-((2-((6-Bromo-4-methylquinazolin-8-yl)oxy)ethyl)(ethyl)amino)pyrimidine-5-carboxylate (60d). Compound 60d was prepared from compound 59d (light yellow solid, 65% yield). 1H NMR (400 MHz, CDCl3): δ 9.18 (s, 1H), 8.94 (s, 1H), 8.89 (s, 1H), 7.80 (d, J = 1.8, 1H), 7.71 (d, J = 1.8, 1H), 4.49 (t, J = 6.7 Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 4.15 (t, J = 6.7 Hz, 2H), 3.85 (q, J = 7.1 Hz, 2H), 2.91 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H), 1.25 (t, J = 7.0 Hz, 3H). MS (ESI+) m/z: 460.0, 462.1 [M + H]+. Ethyl 2-((2-((6-Bromo-4-methylquinazolin-8-yl)oxy)ethyl)(isopropyl)amino)pyrimidine-5-carboxylate (60e). Compound 60e was prepared from compound 59e (light yellow solid, 67% yield). 1H NMR (400 MHz, CDCl3): δ 9.18 (s, 1H), 9.04 (s, 1H), 8.88 (s, 1H), 8.09 (d, J = 1.8 Hz, 1H), 7.80 (d, J = 1.8 Hz, 1H), 5.23−5.10 (m, 1H), 4.48 (t, J = 7.2 Hz, 2H), 4.34 (q, J = 7.1 Hz, 3H), 3.97 (t, J = 7.2 Hz, 2H), 2.90 (s, 3H), 1.36 (t, J = 7.1 Hz, 3H), 1.25 (d, J = 6.6 Hz, 6H). MS (ESI+) m/z: 474.0, 476.1 [M + H]+. Ethyl 2-((2-((6-Bromo-4-methylquinazolin-8-yl)oxy)ethyl)amino)pyrimidine-5-carboxylate (60f). Compound 60f was prepared from compound 59f (light yellow solid, 57% yield). 1H NMR (400 MHz, CDCl3): δ 9.18 (s, 1H), 8.92 (s, 1H), 8.77 (s, 1H), 7.80 (d, J = 1.8 Hz, 1H), 7.43 (d, J = 1.8 Hz, 1H), 6.68 (t, J = 5.7 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H), 4.13−4.01 (m, 4H), 2.88 (s, 3H), 1.23 (t, J = 7.2 Hz, 3H). MS (ESI+) m/z: 432.0, 434.0 [M + H]+. Ethyl 2-((3-((6-Bromo-4-methylquinazolin-8-yl)oxy)propyl)amino)pyrimidine-5-carboxylate (60g). Compound 60g was prepared from compound 59g (light yellow solid, 68% yield). 1H NMR (400 MHz, CDCl3): δ 9.27 (s, 1H), 8.88−8.79 (m, 2H), 7.82 (d, J = 1.9 Hz, 1H), 7.31 (d, J = 1.8 Hz, 1H), 4.41−4.29 (m, 4H), 3.90−3.81 (m, 2H), 2.91 (s, 3H), 2.40−2.27 (m, 2H), 1.37 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 446.0, 448.1 [M + H]+. Ethyl 2-((4-((6-Bromo-4-methylquinazolin-8-yl)oxy)butyl)amino)pyrimidine-5-carboxylate (60h). Compound 60h was prepared from compound 59h (light yellow solid, 70% yield). 1H NMR (400 MHz, CDCl3): δ 9.20 (s, 1H), 8.84 (s, 1H), 8.78 (s, 1H), 7.77 (d, J = 1.8 Hz, 1H), 7.23 (d, J = 1.8 Hz, 0H), 6.75 (t, J = 4.6 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H), 4.23 (t, J = 6.0 Hz, 2H), 3.61 (q, J = 6.6 Hz, 2H), 2.88 (s, 3H), 2.18−2.03 (m, 2H), 1.91 (p, J = 6.9 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 460.1, 462.1 [M + H]+. Ethyl 2-((2-((6-(6-Methoxypyridin-3-yl)-4-methylquinazolin-8yl)oxy)ethyl)(methyl)amino)pyrimidine-5-carboxylate (61a). Compound 61a was prepared from compound 60a and (6-methoxypyridin-3-yl)boronic acid (light yellow solid, 70% yield). 1H NMR (400 MHz, CDCl3): δ 9.18 (s, 1H), 8.86 (s, 2H), 8.51 (d, J = 2.3 Hz, 1H), 7.88 (dd, J = 8.6, 2.5 Hz, 1H), 7.73 (d, J = 1.4 Hz, 1H), 7.65 (d, J = 1.3 Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H), 4.59 (t, J = 6.5 Hz, 2H), 4.34 (q, J = 7.1 Hz, 2H), 4.25 (t, J = 6.4 Hz, 2H), 4.02 (s, 3H), 3.42 (s, R



Article

3H), 2.99 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 168.1, 165.0, 164.4, 162.8, 154.8, 145.9, 141.3, 138.0, 137.8, 131.1, 129.4, 129.0, 125.8, 114.0, 113.1, 112.5, 111.4, 66.2, 60.8, 53.9, 48.8, 37.5, 22.4, 19.4. MS (ESI+) m/z: 475.1 [M + H]+. Ethyl 2-((2-((6-(5-Fluoro-6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)ethyl)(methyl)amino)pyrimidine-5-carboxylate (61b). Compound 61b was prepared from compound 60a and (5fluoro-6-methoxypyridin-3-yl)boronic acid (light yellow solid, 67% yield). 1H NMR (400 MHz, CDCl3): δ 9.19 (s, 1H), 8.87 (s, 2H), 8.28 (d, J = 1.5 Hz, 1H), 7.71 (s, 1H), 7.65 (dd, J = 10.8, 1.5 Hz, 1H), 7.61 (s, 1H), 4.59 (t, J = 6.4 Hz, 2H), 4.33 (q, J = 7.1 Hz, 2H), 4.24 (t, J = 6.3 Hz, 2H), 4.10 (s, 3H), 3.40 (s, 3H), 2.98 (s, 3H), 1.36 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 493.1 [M + H]+. Ethyl 2-((2-((6-(5-((2,4-Difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)ethyl)(methyl)amino)pyrimidine-5-carboxylate (61c). Compound 61c was prepared from compound 60a and N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)-2,4-difluorobenzenesulfonamide (light yellow solid, 56% yield). 1H NMR (400 MHz, DMSO-d6): δ 11.09 (s, 1H), 10.33 (s, 1H), 9.07 (s, 1H), 8.99 (s, 1H), 8.69 (s, 2H), 8.53 (s, 1H), 8.09 (d, J = 1.8 Hz, 1H), 7.91 (d, J = 1.0 Hz, 1H), 7.76 (td, J = 8.5, 6.5 Hz, 1H), 7.69 (s, 1H), 7.60−7.52 (m, 1H), 7.20 (td, J = 8.4, 2.1 Hz, 1H), 4.52 (t, J = 5.7 Hz, 2H), 4.17 (t, J = 5.6 Hz, 2H), 3.66 (s, 3H), 3.38 (s, 3H), 2.95 (s, 3H). MS (ESI+) m/z: 666.1 [M + H]+. Ethyl 2-((3-((6-(6-Methoxypyridin-3-yl)-4-methylquinazolin-8yl)oxy)propyl)(methyl)amino)pyrimidine-5-carboxylate (61d). Compound 61d was prepared from compound 60b and (6methoxypyridin-3-yl)boronic acid (light yellow solid, 69% yield). 1H NMR (400 MHz, CDCl3): δ 9.19 (s, 1H), 8.77 (s, 2H), 8.45 (d, J = 2.2 Hz, 1H), 7.84 (dd, J = 8.6, 2.4 Hz, 1H), 7.70 (s, 1H), 7.31 (s, 1H), 6.87 (d, J = 8.5 Hz, 1H), 4.39−4.25 (m, 4H), 4.04−3.97 (m, 5H), 3.26 (s, 3H), 2.97 (s, 3H), 2.45−2.33 (m, 2H), 1.33 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 168.1, 165.2, 164.4, 162.8, 159.9, 159.8, 155.1, 153.7, 145.7, 141.4, 137.8, 129.5, 125.8, 114.0, 112.5, 112.1, 111.4, 67.1, 60.7, 53.9, 47.2, 36.4, 27.2, 22.4, 14.5. MS (ESI+) m/z: 489.1 [M + H]+. Ethyl 2-((3-((6-(5-Fluoro-6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)propyl)(methyl)amino)pyrimidine-5-carboxylate (61e). Compound 61e was prepared from compound 60b and (5fluoro-6-methoxypyridin-3-yl)boronic acid (light yellow solid, 71% yield). 1H NMR (400 MHz, CDCl3): δ 9.20 (s, 1H), 8.76 (s, 2H), 8.22 (d, J = 1.2 Hz, 1H), 7.69 (s, 1H), 7.61 (dd, J = 10.8, 1.2 Hz, 1H), 7.28 (d, J = 0.4 Hz, 1H), 4.39−4.24 (m, 4H), 4.08 (s, 3H), 3.99 (t, J = 6.6 Hz, 2H), 3.26 (s, 3H), 2.97 (s, 3H), 2.45−2.32 (m, 2H), 1.33 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 168.3, 165.1, 162.8, 159.8, 155.2, 154.0, 153.5 (d, JC−F = 11.3 Hz), 147.7 (d, JC−F = 260.5 Hz), 141.6, 139.8 (d, JC−F = 5.8 Hz), 136.5, 130.4, 125.7, 122.2 (d, JC−F = 16.1 Hz), 114.3, 112.5, 111.9, 67.2, 60.7, 54.3, 47.2, 36.4, 27.1, 22.5, 14.5. MS (ESI+) m/z: 507.1 [M + H]+. Ethyl 2-((3-((6-(5-((2,4-Difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)propyl)(methyl)amino)pyrimidine-5-carboxylate (61f). Compound 61f was prepared from compound 60b and N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)-2,4-difluorobenzenesulfonamide (light yellow solid, 53% yield). 1H NMR (400 MHz, CDCl3): δ 9.21 (s, 1H), 8.77 (s, 2H), 8.16 (d, J = 2.2 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.86 (dd, J = 14.6, 8.5 Hz, 1H), 7.65 (d, J = 1.3 Hz, 1H), 7.35 (s, 1H), 7.03−6.89 (m, 2H), 4.35−4.27 (m, 4H), 4.01 (t, J = 6.9 Hz, 2H), 3.95 (s, 3H), 3.28 (s, 3H), 2.98 (s, 3H), 2.44−2.36 (m, 2H), 1.33 (t, J = 7.1 Hz, 3H). MS (ESI+) m/z: 680.1 [M + H]+. Ethyl 2-((4-((6-(5-Fluoro-6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)butyl)(methyl)amino)pyrimidine-5-carboxylate (61g). Compound 61g was prepared from compound 60c and (5fluoro-6-methoxypyridin-3-yl)boronic acid (light yellow solid, 69% yield). 1H NMR (400 MHz, CDCl3): δ 9.21 (s, 1H), 8.81 (s, 2H), 8.25 (d, J = 1.7 Hz, 1H), 7.70 (s, 1H), 7.65 (dd, J = 10.7, 1.7 Hz, 1H), 7.33 (d, J = 1.7 Hz, 1H), 4.38−4.28 (m, 4H), 4.10 (s, 3H), 3.84 (t, J = 6.8 Hz, 2H), 3.24 (s, 3H), 3.00 (s, 3H), 2.14−2.03 (m, 2H), 1.99− 1.89 (m, 2H), 1.35 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 168.2, 165.2, 162.6, 159.8, 155.4, 153.6, 153.5 (d, JC−F = 11.2 Hz), 147.8 (d, JC−F = 261 Hz), 141.6, 139.8 (d, JC−F = 5.7 Hz), 136.7,

130.4, 125.7, 122.3 (d, JC−F = 16.0 Hz), 114.3, 112.4, 112.0, 69.4, 60.7, 54.3, 49.3, 35.9, 26.4, 24.2, 22.4, 14.5. MS (ESI+) m/z: 521.1 [M + H]+. Ethyl 2-(Ethyl(2-((6-(6-methoxypyridin-3-yl)-4-methylquinazolin8-yl)oxy)ethyl)amino)pyrimidine-5-carboxylate (61h). Compound 61h was prepared from compound 60d and (6-methoxypyridin-3yl)boronic acid (light yellow solid, 67% yield). 1H NMR (400 MHz, CDCl3): δ 9.20 (s, 1H), 8.87 (s, 2H), 8.53 (s, 1H), 7.91 (d, J = 7.7 Hz, 1H), 7.78 (s, 1H), 7.75 (s, 1H), 6.91 (d, J = 8.2 Hz, 1H), 4.60 (t, J = 4.8 Hz, 2H), 4.34 (q, J = 7.1 Hz, 2H), 4.20 (t, J = 4.8 Hz, 2H), 3.86 (q, J = 6.4 Hz, 2H), 3.03 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H), 1.25 (t, J = 6.4 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 168.1, 165.1, 164.5, 162.4, 160.0, 154.8, 153.0, 145.9, 141.2, 138.3, 137.9, 129.4, 125.8, 114.0, 113.1, 112.9, 111.4, 66.1, 60.8, 54.0, 46.8, 44.7, 29.9, 14.5, 13.1. MS (ESI+) m/z: 489.2 [M + H]+. Ethyl 2-(Ethyl(2-((6-(5-fluoro-6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)ethyl)amino)pyrimidine-5-carboxylate (61i). Compound 61i was prepared from compound 60d and (5-fluoro-6methoxypyridin-3-yl)boronic acid (light yellow solid, 68% yield). 1H NMR (400 MHz, CDCl3): δ 9.21 (s, 1H), 8.88 (s, 2H), 8.31 (s, 1H), 7.76 (s, 1H), 7.75 (s, 1H), 7.68 (d, J = 10.3 Hz, 1H), 4.61 (t, J = 4.8 Hz, 2H), 4.34 (q, J = 7.1 Hz, 2H), 4.20 (t, J = 4.8 Hz, 2H), 4.12 (s, 3H), 3.85 (q, J = 7.1 Hz, 2H), 3.03 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H), 1.25 (t, J = 4.8 Hz, 4H). 13C NMR (101 MHz, CDCl3): δ 168.2, 165.0, 162.3, 160.0, 154.9, 153.6 (d, JC−F = 11.2 Hz), 153.1, 147.8 (d, JC−F = 261 Hz), 141.4, 140.1, 137.1, 130.2, 125.7, 122.2 (d, JC−F = 16.0 Hz), 114.3, 113.2, 112.7, 66.1, 60.9, 54.3, 46.7, 44.7, 22.4, 14.5, 13.1. MS (ESI+) m/z: 507.2 [M + H]+. Ethyl 2-(Isopropyl(2-((6-(6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)ethyl)amino)pyrimidine-5-carboxylate (61j). Compound 61j was prepared from compound 60e and (6-methoxypyridin-3-yl)boronic acid (light yellow solid, 68% yield). 1H NMR (400 MHz, CDCl3): δ 9.21 (s, 1H), 8.88 (s, 2H), 8.57 (d, J = 2.2 Hz, 1H), 8.07 (s, 1H), 7.97 (dd, J = 8.6, 2.4 Hz, 1H), 7.74 (s, 1H), 6.93 (d, J = 8.5 Hz, 1H), 5.26−5.14 (m, 1H), 4.65−4.55 (m, 2H), 4.35 (q, J = 7.1 Hz, 2H), 4.10−4.00 (m, 5H), 3.01 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H), 1.28 (d, J = 6.8 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ 168.1, 165.0, 164.5, 162.7, 160.0, 154.8, 153.6, 146.0, 141.2, 138.1, 137.9, 129.7, 125.8, 113.8, 113.3, 112.8, 111.4, 65.9, 60.8, 53.9, 47.1, 40.5, 22.6, 20.5, 14.5. MS (ESI+) m/z: 503.1 [M + H]+. Ethyl 2-((2-((6-(5-Fluoro-6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)ethyl)(isopropyl)amino)pyrimidine-5-carboxylate (61k). Compound 61k was prepared from compound 60e and (5fluoro-6-methoxypyridin-3-yl)boronic acid (light yellow solid, 64% yield). 1H NMR (400 MHz, CDCl3): δ 9.22 (t, 1H), 8.89 (s, 2H), 8.36 (s, 1H), 8.05 (s, 1H), 7.79−7.70 (m, 2H), 5.27−5.14 (m, 1H), 4.60 (t, J = 7.6 Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 4.13 (s, 3H), 4.06 (t, J = 7.6 Hz, 2H), 3.03 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H), 1.29 (d, J = 6.8 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ 168.2, 164.9, 162.7, 160.0, 154.9, 154.0, 153.5 (d, JC−F = 11.3 Hz), 147.8 (d, JC−F = 260.5 Hz), 141.4, 140.0 (d, JC−F = 5.6 Hz), 136.8, 130.6, 125.8, 122.2 (d, JC−F = 15.9 Hz), 114.1, 113.4, 112.4, 65.9, 60.8, 54.3, 47.1, 40.4, 22.6, 20.5, 14.5. MS (ESI+) m/z: 521.1 [M + H]+. Ethyl 2-((2-((6-(5-Fluoro-6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)ethyl)amino)pyrimidine-5-carboxylate (61l). Compound 61l was prepared from compound 60f and (5-fluoro-6methoxypyridin-3-yl)boronic acid (light yellow solid, 57% yield). 1H NMR (400 MHz, CDCl3): δ 9.23 (s, 1H), 8.91 (s, 1H), 8.83 (s, 1H), 8.27 (d, J = 2.1 Hz, 1H), 7.76 (d, J = 2.1 Hz, 1H), 7.66 (dd, J = 10.7, 2.1 Hz, 1H), 7.41 (d, J = 1.5 Hz, 1H), 4.50 (t, J = 3.8 Hz, 2H), 4.34 (q, J = 7.1 Hz, 2H), 4.16−4.10 (m, 5H), 3.02 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 168.9, 164.7, 163.4, 160.5, 154.8, 153.9, 153.6 (JC−F = 11.1 Hz), 147.8 (JC−F = 262 Hz), 141.1, 139.9 (JC−F = 6.1 Hz), 136.8, 130.2, 125.8, 122.2 (JC−F = 16.2 Hz), 114.7, 114.3, 112.8, 68.2, 61.0, 54.3, 40.7, 22.6, 14.5. MS (ESI+) m/z: 479.1 [M + H]+. Ethyl 2-((3-((6-(5-Fluoro-6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)propyl)amino)pyrimidine-5-carboxylate (61m). Compound 61m was prepared from compound 60g and (5-fluoro6-methoxypyridin-3-yl)boronic acid (light yellow solid, 58% yield). S



Article

H NMR (400 MHz, CDCl3): δ 9.30 (s, 1H), 8.90−8.79 (m, 2H), 8.25 (s, 1H), 7.75 (s, 1H), 7.66 (dd, J = 10.5, 0.5 Hz, 1H), 7.38 (s, 1H), 4.50−4.40 (m, 2H), 4.35 (q, J = 7.1 Hz, 2H), 4.11 (s, 3H), 3.95−3.86 (m, 2H), 3.02 (s, 3H), 2.42−2.32 (m, 2H), 1.37 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 168.4, 165.0, 163.7, 160.3, 155.3, 154.3, 153.5 (JC−F = 12.1 Hz), 147.8 (JC−F = 261 Hz), 141.9, 139.7 (JC−F = 6.1 Hz), 136.5, 130.4, 125.8, 122.2 (JC−F = 16.2 Hz), 114.8, 113.8, 112.7, 68.7, 60.9, 54.3, 39.6, 28.9, 22.5, 14.6. MS (ESI+) m/z: 493.2 [M + H]+. Ethyl 2-((4-((6-(5-Fluoro-6-methoxypyridin-3-yl)-4-methylquinazolin-8-yl)oxy)butyl)amino)pyrimidine-5-carboxylate (61n). Compound 61n was prepared from compound 60h and (5-fluoro-6methoxypyridin-3-yl)boronic acid (light yellow solid, 61% yield). 1H NMR (400 MHz, CDCl3): δ 9.24 (s, 1H), 8.89 (s, 1H), 8.80 (s, 1H), 8.26 (d, J = 1.8 Hz, 1H),7.71 (d, J = 1.8 Hz, 1H), 7.66 (dd, J = 10.8, 1.9 Hz, 1H), 7.31 (d,J = 1.8 Hz, 1H), 4.35 (q, J = 7.0 Hz, 4H), 4.11 (s, 3H), 3.68 (q, J = 5.9 Hz, 2H), 3.00 (s, 3H), 2.21−2.09 (m, 2H), 2.02−1.92 (m, 2H),1.37 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 168.3, 164.7, 163.0, 160.7, 155.2, 154.0, 153.5 (d, JC−F = 11.3 Hz), 147.8 (d, JC−F = 261 Hz), 141.6, 139.8 (d, JC−F = 5.8 Hz), 136.6, 130.5, 125.7, 122.3 (d, JC−F = 16.0 Hz), 114.2, 113.6, 111.5, 69.3, 61.0, 54.3, 41.2, 26.5, 26.1, 22.5, 14.5. MS (ESI+) m/z: 507.1 [M + H]+. Enzymatic Inhibitory Activity Assay. The in vitro inhibition assays of all final compounds against PI3K, HDAC, and mTOR (compounds 23 and 36) were performed by Shanghai ChemPartner Co., Ltd (China). The inhibitory activity assay of PI3Kα was conducted with the Kinase-Glo assay (Promega, cat. no. V3771), while those of PI3Kβ, -γ, and -δ were conducted with the ADP-Glo assay (Promega, cat. no. V9102/3). Briefly, compounds were serially diluted to certain concentrations. Then, the compound solution, PI3K enzymes (purchased from Invitrogen), PIP2 substrate, and ATP were dissolved in the kinase buffer, which was then added to a multiwell plate. The plate was incubated in a dark place at room temperature for 1 h, to which was then added Kinase-Glo or ADP-Glo reagents to stop the kinase reaction. After the addition of the kinase detection reagent, the plate was placed in a PerkinElmer Envision plate reader for data collection. The measurement of inhibitory activities of compounds 23 and 36 against mTOR was conducted with the LANCE Ultra assay. Briefly, the compound solution, the mTOR protein (Millipore, cat. no. 14770), the ULight-4E-BP1 (Thr37/46) peptide substrate (PE, cat. no. TRF0128-M), and ATP were diluted in the kinase buffer, which was then transferred to a multiwell plate and incubated at room temperature for 30 min. The plate was allowed to equilibrate for 1 h at room temperature after the addition of the LANCE detection buffer containing EDTA and Eu-anti-phospho-4E-BP1 (Thr37/46) antibody (PE, cat. no. TRF0216-M). The PerkinElmer Envision plate reader was used for data collection. All six subtypes of HDACs were purchased from BPS. Briefly, the compound and the HDAC protein were mixed in the assay buffer and added to a multiwell plate. After incubation at room temperature for 15 min, the Ac-peptide substrate solution was added, followed by incubation at room temperature for 1 h. The plate was then placed into a Synergy MX plate reader for data collection. For all enzymatic inhibitory activity assays, the GraphPad Prism 8.0 was used for curve fitting and the calculation of IC50 values. Cell Viability Assay. HCT116, THP-1, K562, MCF-7, MDA-MB453, HCT-8, U87, NCI-H460, NCI-H1299, Capan2, SW1990, DU145, HGC-27, HepG2, Huh7, and BEL-7402 cell lines were purchased from National Infrastructure of Cell Line Resource, China. PBMC cells were purchased from HemaCare Corporation, USA. LO2 cells were kindly provided by the Academy of Military Medical Sciences, China. The in vitro inhibitory effects of the compounds were measured by MTT assay. Briefly, different kinds of cells were plated in 96-well plates at a density of 2000/well. After attachment for overnight at 37 °C, the cells were treated with the compounds at various concentrations. After 96 h culture, the MTT reagent was added to each well, and plates were incubated for another 4 h at 37 °C. Then, the blue-purple crystal formamidine was dissolved in 1

DMSO after discarding the supernatant. Samples were measured using a microplate reader at a wavelength of 570 nm (BioTek Instruments, Inc. USA). IC50 values were calculated with GraphPad Prism 8.0 software, and IC90 values were calculated with QuickCalcs of GraphPad.66 Flow Cytometry Assay (Cell Cycle and Apoptosis). HCT116 cells were cultured in 6-well plates. After culture overnight, cells were incubated with various concentrations of compounds for 24 h (cell cycle) or 48 h (apoptosis). The effects of the compounds on cell cycle progression and apoptosis were determined by fluorescence-activated cell sorting analysis. The procedures were conducted according to the manufacturer’s recommended procedures. FxCycle PI/RNase Staining Solution for the cell cycle was purchased from Thermo Fisher Scientific Inc. The Annexin V-FITC/PI apoptosis detection kit was purchased from Beijing Kang Run Cheng Ye Biotech Co., Ltd. Western Blot. HCT116 cells were collected and washed twice with cold PBS, and then proteins were obtained by cell lysis with RIPA incubation. Protein samples (40 μg) were separated by 10−12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then were transferred to a nitrocellulose membrane using semi-wet electrophoresis. Membranes were blocked with 5% milk in TBSTween 20 (TBST) for 0.5 h and then were incubated overnight with primary antibodies (1:1000 dilution) at 4 °C. After washing for 3 times with TBST, the membranes were incubated with secondary antibodies (1:1000) for 1 h. The bands were visualized using an enhanced ECL detection kit by ImageQuant LAS 4000 (GE Healthcare, Piscataway, NJ, USA). In Vivo Studies. The HCT116 cells and HGC-27 cells were cultured and harvested. Then, the cells were resuspended in saline at 2 × 106 cells/0.2 mL volume. The animal protocol was both followed and approved by the Experimental Animal Management and Welfare Committee at the Institute of Materia Medica, Peking Union Medical College. Athymic nude mice (BALB/c-nu/nu females, 6−8 weeks old) were subcutaneously injected with 0.2 mL cells on the right flanks. When the tumors reached to 100−200 mm3, the mice were randomly divided into four groups of the HCT116 injected mice and five groups of the HGC-27 injected mice. For the control group, 0.5% critical micelle concentration (cmc) with Tween 80 was orally administered every day. Compound 23 was dissolved in 0.5% cmc with Tween 80 for oral treatment or dissolved in saline for intraperitoneal treatment. The mice body weights and the TVs were measured 2 times every week. When the mice were sacrificed, the tumors were stripped and weighted. The TV was calculated as TV = 1/2 × L × W2, in which L is the maximum length of the tumor and W is the maximum width of the tumor. The relative TV (RTV) was calculated as RTV = TVt/TV0, where TVt is the TV at each time point and TV0 is the TV at day 0. The tumor growth inhibition (TGI) was calculated as TGI = (1 − RTVtreatment/RTVvehicle) × 100%, in which RTVtreatment is the relative TV of the treatment groups, while RTVvehicle is the RTV of the vehicle group. The statistical analysis was performed with GraphPad Prism 8.0 software, and the significance level was evaluated with one-way ANOVA model. Immunohistochemistry. The p-AKT, Ac-H3, and Ki-67 expression levels of the tumor samples were detected using a 3,3′diaminobenzidine-based PV-9000-D kit (ZSGB-BIO, Zhong Shan Golden Bridge Biotechnology Co., Ltd, People’s Republic of China), following the manufacturer’s instructions. The primary antibodies were obtained from Cell Signaling Technology, Inc. The optical density was determined by ImagePRO Plus 6.0, and the statistic analysis was performed with GraphPad Prism 8.0 software. PK Studies. The ICR mice were used in the PK studies of compound 23 and 36. Five mice were in each group for oral administration, while three were in each group for intravenous administration. The formulation for oral administration was 0.5% cmc suspension/0.3% Tween 80 (30 mg/kg) and that for intravenous administration was 10% DMSO in saline (3 mg/kg). The time points for blood sample collection were 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h after dosing for oral administration, and 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h for intravenous injection. After centrifugation, the plasma samples were extracted with acetonitrile and analyzed by high T



Article

pressure liquid chromatography/tandem mass spectrometry (LC/ MS/MS) with a Zobax C18 column (50 mm × 2.1 mm, 3.5 μm). The tumor tissues were homogenized with saline, extracted with acetonitrile, and analyzed also with LC/MS/MS. Compound detection was performed with the mass spectrometer in MRM (multiple reaction monitoring) positive/negative ionization mode. The selected reaction monitoring transitions were m/z 429 → 286 (positive) for compound 23 and m/z 460 → 266 (negative) for compound 36. The PK parameters were calculated with WinNonlin software V6.3 using noncompartmental analysis (Pharsight Corporation, Mountain Vew, USA). AMES Test. The mutagenic potentials of compounds 23 and 36 were evaluated with S. typhimurium strains TA97a, TA98, TA100, TA102, and TA1535 with the plate incorporation method. Compounds 23 and 36 were tested at three doses, which were 5, 50, and 500 μg/plate. At least three plates for each dose were assessed with and without metabolic activation (S9). Plates were incubated at 37 °C for 72 h before scoring for revertants. The mean value of revertant frequency was calculated as the average of the duplicate plates in the test. The mean number of revertants of the compound treated and the positive control groups was compared with that of revertants of the vehicle controls. hERG Inhibition Assay. A stable cell line of Chinese hamster ovary cells expressing hERG potassium channels (CHO/hERG) was used in the hERG inhibition assay for compounds 23 and 36. The culture medium for the cells was F-12 (Ham)/GlutaMAX supplemented with 10% fetal bovine serum containing 100 μg/mL of penicillin G, 30 μg/mL of blasticidin S HCl and 400 μg/mL of hygromycin B. Transfected cells were selected by the addition of 1 μg/mL of doxycycline. Cells were cultured for 24 h before being used for the whole cell patch clamp recordings. A PP-830 patch-pipette puller (Narishige, Tokyo, Japan) was used to pull the recording pipettes. The currents were recorded with Pulse software (V8.74, Heka Electronic, Lambrecht, Pfalz, Germany) through an EPC-10 patch clamp amplifier (Heka Electronic, Lambrecht, Pfalz, Germany). Itail was used to indicate the hERG current. All experiments were conducted at room temperature. The PulseFit software (V8.74, Heka Electronic, Lambrecht, Pfalz, Germany) was used for data analysis. Molecular Docking Studies. The molecular docking was performed with the GLIDE module of the Schrodinger software with the Maestro interface. Briefly, the crystal structures of PI3Kα (PDB ID: 4JPS) and HDAC1 (PDB ID: 1C3S) were downloaded from the Protein Data Bank and prepared using the Protein Preparation Wizzard. The binding pockets of the proteins were generated with the Grid Generation tool. The molecular docking between the target proteins and prepared molecules was conducted using the GLIDE docking wizard. The binding modes were analyzed with Pymol. Single Dose Toxicity. ICR mice, male, 6−8 weeks old, were used for single dose toxicity study. Compounds 23 and 36 were dissolved in 0.5% cmc with Tween 80 for oral treatment or dissolved in saline for intraperitoneal treatment. Once treated with the compounds, clinical signs of the mice were observed and body weights were recorded for 7 consecutive days. Intracellular Drug Concentration Detection. HCT116 cells were seeded into 6-well plates and cultured overnight in a cell culture incubator. After 1 hour treatment with compounds 23 and 36 of 25 μM for 4 repeated wells, the cells were washed three times with PBS solution. The cells in one well were digested with trypsin and the cell number was counted. The cells in the other 3 wells were scraped off and collected respectively into 1.5 mL EP tubes with total volume of 120 μL. After breaking the cells by ultrasound, all the samples were extracted with acetonitrile and analyzed with LC/MS/MS as described in PK studies. The intracellular concentration in nmol per million cells was calculated as intracellular concentration = (the concentration of test compound × total volume for analysis)/number of cells. Plasma Stability. The stability of compound 23 in mouse plasma was evaluated by Shanghai ChemPartner Co., Ltd (China). Briefly, the spiking solution containing 0.02 mM of compound 23, 0.05 mM

of sodium phosphate, and 0.5% BSA was prepared and pre-warmed at 37 °C for 5 min, which was transferred to a multiwell plate. The prewarmed plasma was then added to each well. At certain time points, acetonitrile was added to corresponding wells to quench the reaction. The multiwell plate was then shaked with the vibrator (IKA, MTS 2/ 4) and centrifuged with Thermo Multifuge × 3R. The supernatant of each well was diluted for LC/MS analysis. The T1/2 value was calculated as T1/2 = 0.693/K (K is the rate constant from a plot of ln [concentration] vs incubation time). Metabolic Stability. The metabolic stability of compound 23 was evaluated with mouse liver microsomes by Shanghai ChemPartner Co., Ltd (China). Briefly, the liver microsome solution along with the spiking solution containing compound 23 was added to the assay plate designated for different time points. The plate was then preincubated at 37 °C for 5 min, to which was then added the solution of NADPH. At certain time points, acetonitrile was added to corresponding wells. The plate was shaken with the vibrator (IKA, MTS 2/4) and centrifuged with Thermo Multifuge × 3R. The supernatant of each well was diluted for LC/MS analysis. The T1/2 and Clint values were calculated as T1/2 = 0.693/K (K is the rate constant from a plot of ln [concentration] vs incubation time) and Clint = (0.693/T1/2) × (1/(microsomal protein concentration (0.5 mg/mL))) × scaling factor. The scaling factor for mouse was 3937.5.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.9b00390. 1 H NMR and 13C NMR spectra of compound 23; 1H NMR and 13C NMR spectra of compound 36; X-ray crystal structure of compound 36; KINOMEscan Profiling of compound 23; molecular modeling; the tentative identification of metabolites of 23; and LCMS traces of final compounds 15−49 (PDF) Molecular formula strings (CSV)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +86-10-63165207 (X.C.). *E-mail: [email protected]. Phone: +86-10-83161089 (H.X.). ORCID

Heng Xu: 0000-0002-1720-5286 Author Contributions ∥

K.Z. and F.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Financial support from the Drug Innovation Major Project (2018ZX09711-001-005), the CAMS Innovation Fund for Medical Sciences (2017-I2M-3-011 and 2016-I2M-3-008), the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2018PT35003), China Postdoctoral Science Foundation (2017M620686) and National Postdoctoral Program for Innovative Talents (BX201700037) is gratefully acknowledged. We thank Dr. Xiaobing Deng for his assistance on molecular modeling.

■

ABBREVIATIONS PI3K, phosphoinositide 3-kinase; PIP2, phosphatidylinositol 4,5-diphosphate; PIP3, phosphatidylinositol 3,4,5-triphosU



Article

(17) Antolin, A. A.; Workman, P.; Mestres, J.; Al-Lazikani, B. Polypharmacology in precision oncology: current applications and future prospects. Curr Pharm Des 2017, 22, 6935−6945. (18) Luan, Y.; Li, J.; Bernatchez, J. A.; Li, R. Kinase and histone deacetylase hybrid inhibitors for cancer therapy. J. Med. Chem. 2019, 62, 3171−3183. (19) Yao, L.; Mustafa, N.; Tan, E. C.; Poulsen, A.; Singh, P.; DuongThi, M.-D.; Lee, J. X. T.; Ramanujulu, P. M.; Chng, W. J.; Yen, J. J. Y.; Ohlson, S.; Dymock, B. W. Design and synthesis of ligand efficient dual inhibitors of Janus kinase (JAK) and histone deacetylase (HDAC) based on ruxolitinib and vorinostat. J. Med. Chem. 2017, 60, 8336−8357. (20) Bhatia, S.; Krieger, V.; Groll, M.; Osko, J. D.; Reßing, N.; Ahlert, H.; Borkhardt, A.; Kurz, T.; Christianson, D. W.; Hauer, J.; Hansen, F. K. Discovery of the first-in-class dual histone deacetylase− proteasome Inhibitor. J. Med. Chem. 2018, 61, 10299−10309. (21) Chen, Y.; Yuan, X.; Zhang, W.; Tang, M.; Zheng, L.; Wang, F.; Yan, W.; Yang, S.; Wei, Y.; He, J.; Chen, L. Discovery of novel dual histone deacetylase and mammalian target of rapamycin target inhibitors as a promising strategy for cancer therapy. J. Med. Chem. 2019, 62, 1577−1592. (22) Dong, G.; Chen, W.; Wang, X.; Yang, X.; Xu, T.; Wang, P.; Zhang, W.; Rao, Y.; Miao, C.; Sheng, C. Small molecule inhibitors simultaneously targeting cancer metabolism and epigenetics: discovery of novel nicotinamide phosphoribosyltransferase (NAMPT) and histone deacetylase (HDAC) dual inhibitors. J. Med. Chem. 2017, 60, 7965−7983. (23) Huang, Y.; Dong, G.; Li, H.; Liu, N.; Zhang, W.; Sheng, C. Discovery of Janus kinase 2 (JAK2) and histone deacetylase (HDAC) dual inhibitors as a novel strategy for the combinational treatment of leukemia and invasive fungal infections. J. Med. Chem. 2018, 61, 6056−6074. (24) He, S.; Dong, G.; Wu, S.; Fang, K.; Miao, Z.; Wang, W.; Sheng, C. Small molecules simultaneously inhibiting p53-murine double minute 2 (MDM2) interaction and histone deacetylases (HDACs): discovery of novel multitargeting antitumor agents. J. Med. Chem. 2018, 61, 7245−7260. (25) Wilhelm, S.; Carter, C.; Lynch, M.; Lowinger, T.; Dumas, J.; Smith, R. A.; Schwartz, B.; Simantov, R.; Kelley, S. Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat. Rev. Drug Discovery 2006, 5, 835−844. (26) Liu, P.; Cheng, H.; Roberts, T. M.; Zhao, J. J. Targeting the phosphoinositide 3-kinase pathway in cancer. Nat. Rev. Drug Discovery 2009, 8, 627−644. (27) Janku, F.; Yap, T. A.; Meric-Bernstam, F. Targeting the PI3K pathway in cancer: are we making headway? Nat. Rev. Clin. Oncol. 2018, 15, 273−291. (28) Cantley, L. C. The phosphoinositide 3-kinase pathway. Science 2002, 296, 1655−1657. (29) Vivanco, I.; Sawyers, C. L. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat. Rev. Cancer 2002, 2, 489−501. (30) Millis, S. Z.; Ikeda, S.; Reddy, S.; Gatalica, Z.; Kurzrock, R. Landscape of phosphatidylinositol-3-kinase pathway alterations across 19 784 diverse solid tumors. JAMA Oncol. 2016, 2, 1565−1573. (31) Garces, A. E.; Stocks, M. J. Class 1 PI3K clinical candidates and recent inhibitor design strategies: a medicinal chemistry perspective. J. Med. Chem. 2019, 62, 4815−4850. (32) Perry, M. W. D.; Abdulai, R.; Mogemark, M.; Petersen, J.; Thomas, M. J.; Valastro, B.; Westin Eriksson, A. Evolution of PI3Kγ and δ Inhibitors for Inflammatory and Autoimmune Diseases. J. Med. Chem. 2019, 62, 4783−4814. (33) U. S. Food & Drug Administration, https://www.accessdata. fda.gov/drugsatfda_docs/appletter/2014/205858orig1s000ltr.pdf (accessed Feb 13, 2019). (34) U. S. Food & Drug Administration, https://www.accessdata. fda.gov/drugsatfda_docs/appletter/2017/209936orig1s000ltr.pdf (accessed Feb 13, 2019).

phate; RTK, receptor tyrosine kinase; GPCR, G proteincoupled receptor; HDAC, histone deacetylase; AKT, known as protein kinase B or PKB; mTOR, mammalian target of rapamycin; NSCLC, non-small cell lung carcinoma; TGI, tumor growth inhibition; hERG, human ether-a-go-go-related gene; AML, acute monocytic leukemia; CML, chronic myelogenous leukemia; HCC, hepatocellular carcinoma; PARP, poly(ADP-ribose) polymerase; DLBCL, diffuse large B-cell lymphoma; NBS, N-bromosuccinimide; PdCl2(dppf), [1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II); DMF, N,N-dimethylformamide; DEAD, ethyl azodicarboxylate; DCE, 1,2-dichloroethane; DMSO, dimethyl sulfoxide; THF, tetrahydrofuran; TFA, trifluoroacetic acid; DIPEA, N,N-diisopropylethylamine; Boc, tert-butyloxycarbonyl; rt, room temperature; SAHA, suberanilohydroxamic acid or vorinosta; NHL, non-hodgkin lymphoma; ATP, adenosine 5′triphosphate; PBMC, peripheral blood mononuclear cell; mpk, milligram per kilogram

■

REFERENCES

(1) Gotwals, P.; Cameron, S.; Cipolletta, D.; Cremasco, V.; Crystal, A.; Hewes, B.; Mueller, B.; Quaratino, S.; Sabatos-Peyton, C.; Petruzzelli, L.; Engelman, J. A.; Dranoff, G. Prospects for combining targeted and conventional cancer therapy with immunotherapy. Nat. Rev. Cancer 2017, 17, 286−301. (2) Colli, L. M.; Machiela, M. J.; Zhang, H.; Myers, T. A.; Jessop, L.; Delattre, O.; Yu, K.; Chanock, S. J. Landscape of combination immunotherapy and targeted therapy to improve cancer management. Cancer Res. 2017, 77, 3666−3671. (3) Doroshow, J. H.; Simon, R. M. On the design of combination cancer therapy. Cell 2017, 171, 1476−1478. (4) Yap, T. A.; Omlin, A.; de Bono, J. S. Development of therapeutic combinations targeting major cancer signaling pathways. J. Clin. Oncol. 2013, 31, 1592−1605. (5) Mokhtari, B. R.; Homayouni, T. S.; Baluch, N.; Morgatskaya, E.; Kumar, S.; Das, B.; Yeger, H. Combination therapy in combating cancer. Oncotarget 2017, 8, 38022−38043. (6) Basanta, D.; Gatenby, R. A.; Anderson, A. R. A. Exploiting evolution to treat drug resistance: combination therapy and the double bind. Mol. Pharm. 2012, 9, 914−921. (7) Anighoro, A.; Bajorath, J.; Rastelli, G. Polypharmacology: challenges and opportunities in drug discovery. J. Med. Chem. 2014, 57, 7874−7887. (8) Knight, Z. A.; Lin, H.; Shokat, K. M. Targeting the cancer kinome through polypharmacology. Nat. Rev. Cancer 2010, 10, 130− 137. (9) Proschak, E.; Stark, H.; Merk, D. Polypharmacology by design: a medicinal chemist’s perspective on multitargeting compounds. J. Med. Chem. 2019, 62, 420−444. (10) Peters, J.-U. Polypharmacology − foe or friend? J. Med. Chem. 2013, 56, 8955−8971. (11) Hopkins, A.; Mason, J.; Overington, J. Can we rationally design promiscuous drugs? Curr. Opin. Struct. Biol. 2006, 16, 127−136. (12) Hopkins, A. L. Network pharmacology: the next paradigm in drug discovery. Nat. Chem. Biol. 2008, 4, 682−690. (13) Bolognesi, M. L. Polypharmacology in a single drug: multitarget drugs. Curr. Med. Chem. 2013, 20, 1639−1645. (14) Ramsay, R. R.; Popovic-Nikolic, M. R.; Nikolic, K.; Uliassi, E.; Bolognesi, M. L. A perspective on multi-target drug discovery and design for complex diseases. Clin. Transl. Med. 2018, 7, 3. (15) Bolognesi, M. L. Harnessing polypharmacology with medicinal chemistry. ACS Med. Chem. Lett. 2019, 10, 273−275. (16) Kummar, S.; Chen, H. X.; Wright, J.; Holbeck, S.; Millin, M. D.; Tomaszewski, J.; Zweibel, J.; Collins, J.; Doroshow, J. H. Utilizing targeted cancer therapeutic agents in combination: novel approaches and urgent requirements. Nat. Rev. Drug Discovery 2010, 9, 843−856. V



Article

(35) U. S. Food & Drug Administration, https://www.accessdata. fda.gov/drugsatfda_docs/label/2018/211155s000lbl.pdf (accessed Feb 13, 2019). (36) Rodon, J.; Dienstmann, R.; Serra, V.; Tabernero, J. Development of PI3K inhibitors: lessons learned from early clinical trials. Nat. Rev. Clin. Oncol. 2013, 10, 143−153. (37) Fruman, D. A.; Rommel, C. PI3K and cancer: lessons, challenges and opportunities. Nat. Rev. Drug Discovery 2014, 13, 140− 156. (38) West, A. C.; Johnstone, R. W. New and emerging HDAC inhibitors for cancer treatment. J. Clin. Invest. 2014, 124, 30−39. (39) Witt, O.; Deubzer, H. E.; Milde, T.; Oehme, I. HDAC family: what are the cancer relevant targets? Cancer Lett. 2009, 277, 8−21. (40) Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. Histone deacetylase inhibitors: from bench to clinic. J. Med. Chem. 2008, 51, 1505−1529. (41) Falkenberg, K. J.; Johnstone, R. W. Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat. Rev. Drug Discovery 2014, 13, 673−691. (42) Haberland, M.; Montgomery, R. L.; Olson, E. N. The many roles of histone deacetylases in development and physiology: implications for disease and therapy. Nat. Rev. Genet. 2009, 10, 32− 42. (43) Minucci, S.; Pelicci, P. G. Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat. Rev. Cancer 2006, 6, 38−51. (44) Manal, M.; Chandrasekar, M. J. N.; Gomathi Priya, J.; Nanjan, M. J. Inhibitors of histone deacetylase as antitumor agents: a critical review. Bioorg. Chem. 2016, 67, 18−42. (45) Shi, Y.; Dong, M.; Hong, X.; Zhang, W.; Feng, J.; Zhu, J.; Yu, L.; Ke, X.; Huang, H.; Shen, Z.; Fan, Y.; Li, W.; Zhao, X.; Qi, J.; Huang, H.; Zhou, D.; Ning, Z.; Lu, X. Results from a multicenter, open-label, pivotal phase II study of chidamide in relapsed or refractory peripheral T-cell lymphoma. Ann. Oncol. 2015, 26, 1766− 1771. (46) Rahmani, M.; Aust, M. M.; Benson, E. C.; Wallace, L.; Friedberg, J.; Grant, S. PI3K/mTOR inhibition markedly potentiates HDAC inhibitor activity in NHL cells through BIM- and MCL-1dependent mechanisms in vitro and in vivo. Clin. Cancer Res. 2014, 20, 4849−4860. (47) Yoshioka, T.; Yogosawa, S.; Yamada, T.; Kitawaki, J.; Sakai, T. Combination of a novel HDAC inhibitor OBP-801/YM753 and a PI3K inhibitor LY294002 synergistically induces apoptosis in human endometrial carcinoma cells due to increase of Bim with accumulation of ROS. Gynecol. Oncol. 2013, 129, 425−432. (48) Qian, C.; Lai, C.-J.; Bao, R.; Wang, D.-G.; Wang, J.; Xu, G.-X.; Atoyan, R.; Qu, H.; Yin, L.; Samson, M.; Zifcak, B.; Ma, A. W. S.; DellaRocca, S.; Borek, M.; Zhai, H.-X.; Cai, X.; Voi, M. Cancer network disruption by a single molecule inhibitor targeting both histone deacetylase activity and phosphatidylinositol 3-kinase signaling. Clin. Cancer Res. 2012, 18, 4104−4113. (49) Younes, A.; Berdeja, J. G.; Patel, M. R.; Flinn, I.; Gerecitano, J. F.; Neelapu, S. S.; Kelly, K. R.; Copeland, A. R.; Akins, A.; Clancy, M. S.; Gong, L.; Wang, J.; Ma, A.; Viner, J. L.; Oki, Y. Safety, tolerability, and preliminary activity of CUDC-907, a first-in-class, oral, dual inhibitor of HDAC and PI3K, in patients with relapsed or refractory lymphoma or multiple myeloma: an open-label, dose-escalation, phase 1 trial. Lancet Oncol. 2016, 17, 622−631. (50) Kotian, S.; Zhang, L.; Boufraqech, M.; Gaskins, K.; Gara, S. K.; Quezado, M.; Nilubol, N.; Kebebew, E. Dual Inhibition of HDAC and tyrosine kinase signaling pathways with CUDC-907 inhibits thyroid cancer growth and metastases. Clin. Cancer Res. 2017, 23, 5044−5054. (51) Curis, Inc. http://www.curis.com/pipeline/fimepinostat (accessed Mar 3, 2019). (52) Chen, Y.; Wang, X.; Xiang, W.; He, L.; Tang, M.; Wang, F.; Wang, T.; Yang, Z.; Yi, Y.; Wang, H.; Niu, T.; Zheng, L.; Lei, L.; Li, X.; Song, H.; Chen, L. Development of purine-based hydroxamic acid derivatives: potent histone deacetylase inhibitors with marked in vitro and in vivo antitumor activities. J. Med. Chem. 2016, 59, 5488−5504.

(53) Chen, D.; Soh, C. K.; Goh, W. H.; Wang, H. Design, synthesis, and preclinical evaluation of fused pyrimidine-based hydroxamates for the treatment of hepatocellular carcinoma. J. Med. Chem. 2018, 61, 1552−1575. (54) Lin, S.; Wang, C.; Ji, M.; Wu, D.; Lv, Y.; Zhang, K.; Dong, Y.; Jin, J.; Chen, J.; Zhang, J.; Sheng, L.; Li, Y.; Chen, X.; Xu, H. Discovery and optimization of 2-amino-4-methylquinazoline derivatives as highly potent phosphatidylinositol 3-kinase inhibitors for cancer treatment. J. Med. Chem. 2018, 61, 6087−6109. (55) Marks, P. A.; Richon, V. M.; Rifkind, R. A. Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells. J. Natl. Cancer Inst. 2000, 92, 1210−1216. (56) Cheng, H.; Hoffman, J. E.; Le, P. T.; Pairish, M.; Kania, R.; Farrell, W.; Bagrodia, S.; Yuan, J.; Sun, S.; Zhang, E.; Xiang, C.; Dalvie, D.; Rahavendran, S. V. Structure-based design, SAR analysis and antitumor activity of PI3K/mTOR dual inhibitors from 4methylpyridopyrimidinone series. Bioorg. Med. Chem. Lett. 2013, 23, 2787−2792. (57) Fong, P. C.; Settatree, S.; Sinha, R.; Hardcastle, A.; Hellemans, P. W.; Arts, J.; Brown, K. H.; Janicot, M.; Aherne, W.; De Bono, J. S. A first-in-man phase I study of R306465, a histone deacetylase (HDAC) inhibitor exploring pharmacokinetics (PK) and pharmacodynamics (PD) utilizing an electrochemiluminescent immunoassay in patients (p) with advanced tumours. J. Clin. Oncol. 2007, 25, 3578. (58) Arts, J.; King, P.; Marien, A.; Floren, W.; Belien, A.; Janssen, L.; Pilatte, I.; Roux, B.; Decrane, L.; Gilissen, R.; Hickson, I.; Vreys, V.; Cox, E.; Bol, K.; Talloen, W.; Goris, I.; Andries, L.; Du Jardin, M.; Janicot, M.; Page, M.; van Emelen, K.; Angibaud, P. JNJ-26481585, a Novel ″Second-Generation″ Oral Histone Deacetylase Inhibitor, Shows Broad-Spectrum Preclinical Antitumoral Activity. Clin. Cancer Res. 2009, 15, 6841−6851. (59) Du, L.; Musson, D. G.; Wang, A. Q. Stability studies of vorinostat and its two metabolites in human plasma, serum and urine. J. Pharm. Biomed. Anal. 2006, 42, 556−564. (60) Wang, Y.; Stowe, R. L.; Pinello, C. E.; Tian, G.; Madoux, F.; Li, D.; Zhao, L. Y.; Li, J.-L.; Wang, Y.; Wang, Y.; Ma, H.; Hodder, P.; Roush, W. R.; Liao, D. Identification of histone deacetylase inhibitors with benzoylhydrazide scaffold that selectively inhibit class I histone deacetylases. Chem. Biol. 2015, 22, 273−284. (61) McClure, J. J.; Zhang, C.; Inks, E. S.; Peterson, Y. K.; Li, J.; Chou, C. J. Development of allosteric hydrazide-containing class I histone deacetylase inhibitors for use in acute myeloid leukemia. J. Med. Chem. 2016, 59, 9942−9959. (62) Li, X.; Peterson, Y. K.; Inks, E. S.; Himes, R. A.; Li, J.; Zhang, Y.; Kong, X.; Chou, C. J. Class I HDAC inhibitors display different antitumor mechanism in leukemia and prostatic cancer cells depending on their p53 status. J. Med. Chem. 2018, 61, 2589−2603. (63) Wang, H.; Yu, N.; Chen, D.; Lee, K. C. L.; Lye, P. L.; Chang, J. W. W.; Deng, W.; Ng, M. C. Y.; Lu, T.; Khoo, M. L.; Poulsen, A.; Sangthongpitag, K.; Wu, X.; Hu, C.; Goh, K. C.; Wang, X.; Fang, L.; Goh, K. L.; Khng, H. H.; Goh, S. K.; Yeo, P.; Liu, X.; Bonday, Z.; Wood, J. M.; Dymock, B. W.; Kantharaj, E.; Sun, E. T. Discovery of (2E)-3-{2-butyl-1-[2-(diethylamino)ethyl]-1H-benzimidazol-5-yl}-Nhydroxyacrylamide (SB939), an orally active histone deacetylase inhibitor with a superior preclinical profile. J. Med. Chem. 2011, 54, 4694−4720. (64) Baell, J. B.; Holloway, G. A. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J. Med. Chem. 2010, 53, 2719−2740. (65) False Positive Remover, https://www.cbligand.org/PAINS/ search_struct.php (accessed Dec 6, 2018). (66) QuickCalcs, https://www.graphpad.com/quickcalcs/ Ecanything1/ (accessed Apr 5, 2019).

W


Design, Synthesis, and Biological Evaluation of 4-Methyl Quinazoline

Recommend Documents