First SAR Study for Overriding NRAS Mutant Driven Acute Myeloid

Article Cite This: J. Med. Chem. 2018, 61, 8353−8373

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First SAR Study for Overriding NRAS Mutant Driven Acute Myeloid Leukemia Hanna Cho,† Injae Shin,† Eunhye Ju,† Seunghye Choi,† Wooyoung Hur,‡ Haelee Kim,§ Eunmi Hong,§ Nam Doo Kim,§,∥ Hwan Geun Choi,§ Nathanael S. Gray,⊥,# and Taebo Sim*,†,‡

J. Med. Chem. 2018.61:8353-8373. Downloaded from pubs.acs.org by MOUNT ALLISON UNIV on 09/30/18. For personal use only.

†

KU-KIST Graduate School of Converging Science and Technology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea ‡ Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), 5 Hwarangro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea § Daegu-Gyeongbuk Medical Innovation Foundation, 2387 dalgubeol-daero, Suseong-gu, Daegu 42019, Republic of Korea ∥ NDBio Therapeutics Inc., 32 Songdogwahak-ro, Yeonsu-gu, Incheon 21984, Republic of Korea ⊥ Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, United States # Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, United States S Supporting Information *

ABSTRACT: GNF-7, a multitargeted kinase inhibitor, served as a dual kinase inhibitor of ACK1 and GCK, which provided a novel therapeutic strategy for overriding AML expressing NRAS mutation. This SAR study with GNF-7 derivatives, designed to target NRAS mutant-driven AML, led to identification of the extremely potent inhibitors, 10d, 10g, and 11i, which possess single-digit nanomolar inhibitory activity against both ACK1 and GCK. These substances strongly suppress proliferation of mutant NRAS expressing AML cells via apoptosis and AKT/mTOR signaling blockade. Compound 11i is superior to GNF-7 in terms of kinase inhibitory activity, cellular activity, and differential cytotoxicity. Moreover, 10k possessing a favorable mouse pharmacokinetic profile prolonged life-span of Ba/F3-NRAS-G12D injected mice and significantly delayed tumor growth of OCI-AML3 xenograft model without causing the prominent level of toxicity found with GNF-7. Taken together, this study provides insight into the design of novel ACK1 and GCK dual inhibitors for overriding NRAS mutant-driven AML.

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poor prognosis,4 especially for patients 65 years or older whose median overall survival is less than one year.5 Therefore, there exists an unmet medical need to create further innovative therapeutic strategies for overcoming AML even though enormous efforts6 have been devoted to developing targeted therapies for AML treatment including FLT3 inhibitors, PLK inhibitors, IDH inhibitors, DNMT inhibitor, LSD1 inhibitor, DOT1L inhibitors, HDAC inhibitors, BET inhibitors, and BCL-2 inhibitors.

INTRODUCTION The small GTPase, RAS, plays the role of a molecular switch that regulates signal transduction in response to extracellular stimuli. In this role, it participates in diverse cellular events such as cell cycle, differentiation, and survival. Malfunctioning RAS is strongly related to tumorigenesis. Mutations in the RAS gene, which encode overactive RAS proteins, are found in 30% of all human tumors. Among the three mutants, HRAS, KRAS, and NRAS, KRAS is most frequently present in various types of cancers including lung (35%), colon (45%), and pancreatic (90%).1 NRAS mutations are also common in acute myeloid leukemia (AML),2 appearing in 10% of AML patients.3 AML has remained a devastating and life-threatening disease with © 2018 American Chemical Society

Received: June 4, 2018 Published: August 28, 2018 8353

DOI: 10.1021/acs.jmedchem.8b00882 J. Med. Chem. 2018, 61, 8353−8373

Journal of Medicinal Chemistry

Article

Figure 1. Structure of GNF-7 (left) and representative GNF-7 analogues (right).

Scheme 1. Synthesis of GNF-7, 10a−k, and 11a−ia

Reagent and conditions: (A) POCl3, DIPEA, toluene, 110 °C, 77%; (B) 2-methyl-5-nitroaniline, NaI, K2CO3, acetone, 50 °C, 74%; (C) MeNH2 (2 M in MeOH solution), DIPEA, 1,4-dioxane, 60 °C, 77%; (D) triphosgene, TEA, THF, 70 °C, 63%; (E) Fe powder, NH4Cl, THF/MeOH/H2O, 80 °C, 87%; (F) 3-(trifluoromethyl)benzoyl chloride, K2CO3, DCM, rt, 73%; (G) various amines, K2CO3, Xphos, Pd2(dba)3, 2-butanol, 100 °C, 13−84% or ammonia solution, DMSO, 150 °C, 28%; (H) 6-methylpyridin-3-amine, K2CO3, Xphos, Pd2(dba)3, 2-butanol, 100 °C, 65%; (I) Fe powder, NH4Cl, THF/MeOH/H2O, 80 °C, 81%; (J) various carboxylic acids, HATU, DIPEA, DMF, rt, 30−61%. a

ACK1 and GCK are noncanonical effectors of the RAS pathway. Activated cdc42-associated kinase 1 (ACK1 or TNK2) is a nonreceptor tyrosine kinase originally identified as an inhibitory regulator of cdc42. Recent studies have highlighted the oncogenic properties of ACK118 by showing that it directly phosphorylates Tyr176 of AKT and thus activates AKT in a PI3K-independent manner.19 Germinal center kinase (GCK/MAP4K2) belongs to the STE20 family of serine/threonine kinases associated with the JNK pathway and inflammatory process.20 In a previous study, we showed that the multikinase inhibitor GNF-721,22 effectively and selectively suppresses proliferation of primary AML patient-derived cells harboring mutant NRAS as well as AML cell lines expressing mutant NRAS. Through mechanistic analysis using small molecule kinase inhibitors and RNA interference studies and in situ kinase profiling (KiNativ method), we ascertained that the effect of GNF-7 on NRAS mutant AML cells is associated with a synthetic lethal interaction between ACK1 and GCK. This synthetically lethal effect of GNF-7 significantly reduces the disease burden and prolongs overall survival in an AML xenotransplantation mice model.3 The current study was designed to discover new inhibitors of ACK1 and GCK, which are more potent and/or less toxic than GNF-7, in order to overcome AML caused by the NRAS mutant. In the effort described below, a structure−activity relationship (SAR) study was carried out exploring the effects on NRAS mutant AML cells as well as ACK1 and GCK enzymes of GNF-7 derivatives, which possess the

The high relevance and crucial role of its mutations in cancers has made RAS an attractive therapeutic target for cancer treatment.7 However, despite being subjected to intensive efforts over several decades, no efficacious pharmacological RAS inhibitors have been identified. This is caused by the extraordinarily high affinity of RAS for its substrates, GTP and GDP, as well as the presence of a surface in this protein that does not participate in high-affinity interactions with small molecules.8 Thus, other strategies have been explored to inhibit the function of hyperactivated RAS.9 One involves targeting effectors such as RAF,10 MEK,11 and ERK12 in canonical RAS13 and PI3K/AKT14 pathways. Although many substances which function in this manner are under clinical evaluation, rapid drug resistance and complex feedback mechanisms are major hurdles for this approach to drug development.15 Blocking RAS localization by preventing farnesylation of RAS has also been investigated. In spite of displaying in vitro anticancer effects, farnesyltransferase inhibitors (FTIs)16 generally have poor clinical outcomes. Furthermore, genetic approaches have been employed to identify new target genes that interact with oncogenic RAS in a lethal manner. Although this strategy led to identification of STK33 as an effective target, more significant effort is required to discover active candidates.17 As part of a program to explore a promising anticancer strategy that focuses on blocking signaling by the oncogenic NRAS mutation, we recently identified dual inhibition of ACK1 and GCK as a novel therapeutic target to overcome NRAS mutant-driven acute myeloid leukemia (AML).3 Both 8354



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Scheme 2. Synthesis of 15a−ea

Reagent and conditions: (A) N-(3-amino-4-methylphenyl)-3-(trifluoromethyl)benzamide, NaI, K2CO3, acetone, 50 °C, 69%; (B) various amines, THF, 60 °C or aniline, TEA, n-butanol, 120 °C; (C) triphosgene, TEA, THF, 70 °C; (D) 6-methylpyridin-3-amine, K2CO3, Xphos, Pd2(dba)3, 2butanol, 100 °C, 12−27% over 3 steps. a

Table 1. Enzymatic Inhibitory Activities against ACK1 and GCK and Antiproliferative Activities on Ba/F3-NRAS-G12D Cells

a Radiometric biochemical kinase assay results. bGI50 represents the concentration at which a compound causes half-maximal growth inhibition. NRAS-G12D-transformed Ba/F3 cells and parental Ba/F3 cells were treated with inhibitors for 72 h in a dose escalation. Average GI50 values with SD (n = 3, duplicate) are shown.

dihydropyrimido[4,5-d]pyrimidin-2(1H)-one scaffold. We observed that among the 29 synthetic GNF-7 derivatives

prepared in this study, 10d and 11i exhibit much higher enzymatic inhibition and cellular activities compared with 8355



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ethanone (10d) and ethylpiperazinyl methanone groups (10e) led to a significant increase in kinase inhibitory activity (10d, GCK IC50 = 1 nM, ACK1 IC50 = 8 nM; 10e, GCK IC50 = 2 nM, ACK1 IC50 = 13 nM). Taken together, these results demonstrate that extended head groups in GNF-7 analogues may enable additional interaction with both ACK1 and GCK and thus increase binding affinity. Compared with that of GNF-7, the antiproliferative activities of 10d and 10e on Ba/F3-NRAS-G12D cells were moderately greater or the same as GNF-7 (GI50s of 0.059 and 0.107 μM, respectively) in spite of their higher enzymatic inhibition activities. Compared to GNF-7, 10a−e exhibited lower differential cytotoxicities against Ba/F3-NRAS-G12D cells relative to parental Ba/F3 cells which serve as a control. It is of note that compared with GNF-7, 10d possesses increased kinase inhibitory activity against both ACK1 (8 nM of IC50) and GCK (1 nM of IC50) and a higher cellular activity on Ba/ F3-NRAS-G12D cells. We also explored analogues that contain an aminopyrazole R1 substituent that is present in NG25, a dual TAK1 and GCK inhibitor.25 The kinase inhibitory and cellular activities of 10f, bearing a 1-methyl-pyrazole group, are comparable to those of GNF-7 and to other substances (10b−e) containing a piperazinyl group. Notably, 10g exhibited a slightly higher enzymatic inhibitory activity (GCK IC50 = 5 nM, ACK1 IC50 = 9 nM) relative to GNF-7, which indicates that 1,3-dimethyl pyrazole group may fit in the hydrophobic pocket adjacent to the hinge region in the kinases. Also, the cellular activity and differential cytotoxicity of 10g on Ba/F3 cells are comparable to those of GNF-7. In contrast to analogues with aromatic groups at the R1 position, derivatives containing aliphatic amine groups (10h, 10i, and 10j) generally displayed lower kinase inhibitory activities and antiproliferative activities on Ba/F3-NRAS-G12D cells, whereas 10h bearing a cyclopropylamine group has similar IC50 and GI50 values to those of aniline bearing 10a. This observation suggests that the cyclopropyl moiety, which has pseudo double-bond character,26 can participate in π−π stacking interactions in the hydrophobic pocket near the hinge region. In contrast, 10i, having a bulky and flexible cyclohexyl group, has substantially diminished potency compared with those of 10h and 10j. Compared with GNF-7, 10k exhibited a comparable inhibitory activity against ACK1 and a 3-fold less potency against GCK (IC50 = 28 nM). Overall, the data gained from the SAR study with analogues containing different R1 groups suggest that aromatic amine groups containing an additional hydrogen bonding acceptor have enhanced kinase inhibitory activities against both ACK1 and GCK. Our attention next focused on optimization of the R2 group (Table 2). The inhibitory effect of the aryl-CF3 group in GNF7 was assessed using the unsubstituted analogue 11a. Interestingly, the inhibitory potency of 11a against GCK (IC50 = 1080 nM) is greatly less than that of GNF-7 while its activity against ACK1 (IC50 = 21 nM) is the same as that of GNF-7, indicating that CF3 group is essential for kinase inhibitory activity on GCK but not ACK1. The 4-methyl aryl group containing analogue, 11b, has a 5-fold reduced kinaseinhibitory activity against GCK (IC50 = 39 nM) and antiproliferative activity (GI50 = 0.387 μM) on Ba/F3NRAS-G12D cells. In contrast, the in vitro potencies of 11b against ACK1 (IC50 = 20 nM) and parental Ba/F3 cells (GI50 = 0.870 μM) were retained.

GNF-7. Moreover, 11i clearly exceeds GNF-7 in terms of kinase-inhibitory activity, cellular potency, and differential cytotoxicity. We observed that another derivative, 10k, having a favorable mouse PK profile, significantly prolonged the lifespan of Ba/F3-NRAS-G12D injected mice without prominent toxicity found in GNF-7, which makes 10k superior to GNF-7.

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RESULTS AND DISCUSSION General Procedure for Synthesis of GNF-7 Derivatives. By employing a more efficient synthetic route for the preparation GNF-7 than the one previously reported,21 we were able to generate derivatives that contain a variety of different moieties at the R1, R2, and R3 positions identified in Figure 1, Schemes 1−3, and Tables 1−3. The synthetic route developed to prepare GNF-7 along with 10a−k and 11a−i, given in Scheme 1, begins with chlorination of the commercially available pyrimidine derivative 1 using POCl3 and DIPEA to yield the corresponding chloride 2.23 Reaction of 2 with 2-methyl-5-nitroaniline produced aniline derivative 3, which upon reaction with methylamine provided 4. Cyclic urea formation promoted by reaction of 4 with triphosgene formed the key intermediate 5, which was utilized in routes to generate GNF-7 and derivatives containing various R 1 and R 2 replacements. Reduction of the nitro group in 5 to give 6 followed by acylation with 3-(trifluoromethyl)benzoyl chloride provided amide 7, which through Buckwald amination with various aniline derivatives is transformed to GNF-7 and R1substituted analogues 10a−k. Similarly, a sequence involving reaction of 5 with 6-methylpyridin-3-amine (giving 8), nitro group reduction (giving 9), and amide coupling with various carboxylic acids produced R2-substituted analogues 11a−i. A modification of the routes shown in Scheme 1 was used to prepare R3-substituted analogues. As shown in Scheme 2, pyrimidine trichloride 2 was first converted to 12, which contains N-(3-amino-4-methylphenyl)-3-(trifluoromethyl)benzamide group present in GNF-7 and a handle to introduce various R3 substituents to create analogues 13a−e. In addition, cyclic urea forming reactions of 13a−e produced 14a−e, which were then transformed to 15a−e by using Buchwald amination with 6-methylpyridin-3-amine. Structure−Activity Relationships. IC50 values for all synthetic GNF-7 analogues against both ACK1 and GCK kinases were determined using an in vitro biochemical kinase assay. The growth inhibitory activities (GI50s) of these substances in the NRAS-G12D transformed Ba/F3 cell line were also estimated. The first phase of this effort, focusing on optimization of the R1 substituent (Table1), showed that 10a, containing an aniline head group, displays a 6-fold lower activity (IC50 = 47 nM) against GCK compared with that of GNF-7 (IC50 = 8 nM) while its potency against ACK1 (IC50 = 37 nM) is nearly equal to that of GNF-7. This finding is consistent with information gained from a previous docking study,3 which suggested that the pyridine nitrogen in GNF-7 contributes to binding with GCK through hydrogen bonding with Tyr27. ACK1 lacks a residue analogous to Tyr27 in GCK. Both 10b containing the 4-(4-ethylpiperazin-1-yl)-phenylamine head group that is present in the pan-FGFR inhibitor BGJ39824 and 10c containing a 6-(4-ethylpiperazin-1-yl)pyridin-3-amine group were found to have a kinase inhibitory activity against GCK that is in the range of that of the parent GNF-7, indicating that the nitrogen on the ethylpiperazine ring could form a hydrogen bond with Tyr27 of GCK. Similarly, replacement of ethylpiperazine group in 10c with piperazinyl 8356



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μM). Moreover, introduction of a meta-4-methylimidazole group (11e) also brought about a slight reduction in kinase inhibitory activities (GCK IC50 = 27 nM, ACK1, IC50 = 53 nM) and cellular Ba/F3-NRAS-G12D cellular activity (GI50 = 0.238 μM) compared with that of GNF-7. In exploring the effect para-aryl substituents, we found that 11f possessing para-pyrrole moiety has a marginally increased activity against ACK1 (IC50 = 10 nM) but not against GCK (IC50 = 24 nM), while 11g containing a para-piperazine moiety has retained kinase inhibitory activity against both ACK1 and GCK (ACK1 IC50 = 28 nM, GCK IC50 = 10 nM). On the basis of these results, we conclude that para- rather than meta-substitution is more suitable for altering inhibitory activities against both ACK1 and GCK. Indeed, 11h, having a piperazinylmethylphenyl substituent, has a 2-fold increased potency (IC50 = 5 nM) against GCK kinase compared to that of 11g. Furthermore, 11i, containing a 3-(dimethylamino)pyrrolidinemethylphenyl group, has enhanced kinase inhibitory activities against both ACK1 (IC50 = 6 nM) and GCK (IC50 = 2 nM) compared with GNF-7. Also, 11i has a significantly increased anticellular proliferation potency (GI50 = 0.023 μM) on Ba/F3-NRAS-G12D relative to GNF-7. It should be emphasized that 11i compared to GNF-7 possesses a higher differential cytotoxicity against Ba/F3-NRAS-G12D cells relative to parental Ba/F3 cells. The effects of R3 groups on inhibitory activities were explored by replacing the methyl group in GNF-7 with more sterically bulky aliphatic groups, phenyl groups, and benzyl group. The results show that as the size of the R3 substituent increases (15a−c, 15e), both enzymatic activities and cellular activities decrease. Although the phenyl group in 15d causes a slightly increased kinase inhibitory activity against ACK1 (IC50 = 15 nM), it causes a decreased activity against GCK and cellular activity compared with GNF-7 in which R3 substituent is methyl group. The observations made in the SAR study suggest that 3(dimethylamino)pyrrolidinemethylphenyl and methyl are optimal R2 and R3 groups, respectively. Moreover, the results indicate that that the R1 substituents in 10d−g are superior to that in GNF-7 in terms of enzymatic inhibitory activities. As a result, the activities of analogues 17a−d (Scheme 3), containing a combination of the optimal R2/R3 substituent and the R1 substituents present 10d−g, were assessed. Contrary to expectations, compared with those of 11i, none of these compounds exhibited enhanced enzymatic inhibitory activities against ACK1 or GCK (Table 3). Compared with GNF-7, 17a−d have lower differential cytotoxicities against Ba/F3-NRAS-G12D cells relative to parental Ba/F3 cells even though they displayed comparable cellular activities against BaF3-NRAS-G12D cells.

Table 2. Enzymatic Inhibitory Activities against ACK1 and GCK and Antiproliferative Activities on Ba/F3-NRASG12D Cells

a Radiometric biochemical kinase assay results. bGI50 represents the concentration at which a compound causes half-maximal growth inhibition. NRAS-G12D-transformed Ba/F3 cells and parental Ba/F3 cells were treated with inhibitors for 72 h in a dose escalation. Average GI50 values with SD (n = 3, duplicate) are shown.

The effects of additional substituents at the meta or para positions of the tail aryl ring in GNF-7 were explored using 11c−i. The results showed that addition of meta-morpholine (11c) and 4-piperidinol moiety (11d) resulted in slight decreases in inhibitory activity against GCK (11c, IC50 = 45 nM; 11d, IC50 = 72 nM) and antiproliferation of Ba/F3NRAS-G12D cells (11c, GI50 = 0.284 μM; 11d, GI50 = 0.118 Scheme 3. Synthesis of 17a−ea

a

Reagent and condition: (A) 4-((3-(dimethylamino)pyrrolidin-1-yl)methyl)-3-(trifluoromethyl)benzoic acid, HATU, DIPEA,DMF, rt, 65%; (B) various amines, K2CO3, Xphos, Pd2(dba)3, 2-butanol, 100 °C, 34−62%. 8357



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Table 3. Enzymatic Inhibitory Activities against ACK1 and GCK and Antiproliferative Activities on Ba/F3-NRAS-G12D Cells

a

Radiometric biochemical kinase assay results. bGI50 represents the concentration at which a compound causes half-maximal growth inhibition. NRAS-G12D-transformed Ba/F3 cells and parental Ba/F3 cells were treated with inhibitors for 72 h in a dose escalation. Average GI50 values with SD (n = 3, duplicate) are shown.

against OCI-AML3 cells over U937 cells. It is worth recalling that 11i exhibits the highest differential cytotoxicity against Ba/F3-NRAS-G12D cells relative to parental Ba/F3 cells. Thus, this effort has led to the identification of promising new GNF-7 analogues that have strong antiproliferative activities and high cellular selectivities against NRAS mutant expressing AML cell lines. It should be noted that 11i clearly surpasses GNF-7 in terms of cellular potency and selectivity on NRAS mutation cells (Ba/F3-NRAS-G12D and OCI-AML3) as well as enzymatic inhibitory activities against ACK1 and GCK. Microsomal Stability and Mouse PK Profiling. With selected compounds (10b, 10c, 10d, 10e, 10f, 10g, 10k, and 11i) displaying distinctive in vitro potency, we evaluated their microsomal stability to estimate metabolic clearance in liver. The remaining amount of each compound after 30 min incubation in liver microsomes of human, dog, rat, and mouse

On the basis of the results of the SAR study presented above, the antiproliferative activities of selected analogues in the human AML cell lines, OCI-AML3 (NRAS-Q61L) and U937 (NRAS wt), were assessed (Table 4). In this assessment, a higher differential cytotoxic effect on OCI-AML3 (NRAS mt) cell lines relative to U937 (NRAS wt) is a positive finding. GI50s on OCI-AML3 cells were found to be in the range of 0.030−0.466 μM. The four GNF-7 derivatives, 10b, 10c, 10f, and 11i, have remarkable potencies against OCI-AML3 cells (GI50s range from 0.030 to 0.042 μM) and reasonable antiproliferative activities against U937 cells (GI50 = ca. 1 μM). On the basis of their cellular selectivities against OCIAML3 cells (NRAS mt) over U937 cells (NRAS wt), five derivatives, 10c, 10e, 11b, 11f, and 11i, were found to be superior to GNF-7. This is especially true for 11f, which displayed an exceptional cellular selectivity (ca. 100-fold) 8358



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Table 6. Mouse Oral PK Property of 10ka

Table 4. Antiproliferative Activities of the Selected GNF-7 Analogues against OCI-AML3 and U937 Cells entry GNF-7 10b 10c 10d 10e 10f 10g 10j 10k 11b 11c 11d 11f 11i

OCI-AML3 (NRAS Q61L) GI50 (μM)a

U937 (NRAS wt) GI50 (μM)a

± ± ± ± ± ± ± ± ± ± ± ± ± ±

4.377 ± 0.081 0.839 ± 0.317 1.412 ± 0.276 2.483 ± 0.499 6.479 ± 0.383 1.125 ± 0.129 2.463 ± 1.679 8.531 ± 0.039 4.967 ± 1.052 19.375 ± 1.775 2.805 ± 0.134 2.180 ± 0.785 >25 1.973 ± 0.182

0.219 0.040 0.030 0.179 0.122 0.042 0.208 0.367 0.466 0.394 0.260 0.143 0.267 0.037

0.058 0.002 0.001 0.017 0.040 0.031 0.034 0.065 0.120 0.025 0.064 0.002 0.092 0.005

parameter

10k

dose (mg/kg) AUCinf (ng·h/mL) AUClast (ng·h/mL) Cmax (ng/mL) t1/2 (h) tmax (h) MRTinf (h)

10 52464.8 ± 20913.0 36363.7 ± 5340.0 2406.7 ± 434.7 12.4 ± 7.9 2.7 ± 1.2 18.1 ± 11.3

a

10k was formulated as a solution in 10% DMAC, 10% Tween80, 80% HP-β-CD (20% in water).

GCK, respectively. The amide linker in these compounds also participates in interactions with ACK1 and GCK through a pair of H-bonds, which is a typical binding mode of type II kinase inhibitors. While the pyridine nitrogen of GNF-7 forms a hydrogen bond with the hydroxyl group of Tyr27 in GCK, no such interaction exists with Phe137 of ACK1, which might be responsible for the higher potency of GNF-7 against GCK than against ACK1 (Figure 2a). The nitrogen on piperazinylethanone moiety of 10d contributes additional hydrogen bonding interactions with Asp215 of ACK1 and Glu100 of GCK, respectively (Figure 2b). This interaction might serve as a driving force for improved binding affinity to each of the kinases. Especially, the four hydrogen bonds existing between 10d and Cys93/Tyr27/Glu100 in GCK could be the source of its exceptional inhibitory activity against GCK (IC50 = 1 nM). It is of note that 10k possesses high inhibitory activities against both ACK1 and GCK even though it does not participate in hydrophobic interactions in the hinge region besides a pair of hinge contact (Figure 2c). The trifluoromethylphenyl group in the tail parts of GNF-7, 10d, and 10k is likely ideal for binding in the hydrophobic pocket of GCK. The results of a previous docking study of NG25 on GCK also demonstrated that the hydrophobic pocket composed of Ile64 and Gln60 provides a suitable binding site for the trifluoromethylphenyl group.25 In this regard, the unsubstituted phenyl tail of 11a, lacking the CF3 moiety, might be too small to tightly interact with GCK (Figure 2d). Accordingly, 11a displays a greatly decreased activity against GCK (IC50 = 1080 nM). Unlike GCK, no such binding site exists in ACK1 for interaction with the CF3. Therefore, 11a, which lacks the CF3 group, has a similar level of inhibitory activity against ACK1 compared with GNF-7. X-ray Cocrystal Structure of ACK1-10d Complex. We determined X-ray cocrystal structure of 10d bound to the kinase domain of ACK1 (Table 7 and Figure 3). Unfortu-

a

GI50 represents the concentration at which a compound causes halfmaximal growth inhibition. OCI-AML3 and U937 cells were treated with inhibitors for 72 h. Average GI50 values with SD (n = 3, duplicate) are shown.

was measured (Table 5). Briefly, the microsomal stabilities of compounds with five-membered aromatic pyrazole rings (10f, 10g) or the primary amine (10k) as head group are comparable to those of GNF-7. The presence of piperazine moiety in a head group (10b, 10c, 10d, and 10e) caused poor microsomal stability. 11i, possessing additional substituents at the meta position of the GNF-7 tail, showed the little reduced stability compared with GNF-7. We next assessed inhibitory activities of the compounds against four cytochromes P450 (CYPs) and found that 10k had little or no inhibition effects on CYP1A2, CYP2C9, CYP2D6, and CYP3A4 at 10 μM. Especially, 10k is far superior to GNF-7 in terms of CYP2C9 inhibition profile. We also investigated the mouse pharmacokinetic property (PO, 10 mg/kg) of 10k (Table 6). Compound 10k exhibited excellent oral systemic exposure (AUCinf = 52464.8 ng h/mL, AUClast = 36363.7 ng h/mL, Cmax = 2406.7 ng/mL), thus we chose this compound for in vivo efficacy study using mouse leukemia model. Docking Study of 10d, 10k, and 11a. To gain an insight into the binding mode of the GNF-7 analogues, molecular docking studies were performed using GNF-7, 10d, 10k, and 11a and the kinases ACK1 and GCK (Figure 2). The results suggest that these four inhibitors make a hinge contact through a pair of hydrogen bonds with Ala208 of ACK1 and Cys93 of Table 5. Microsomal Stabilitya and CYP Inhibitionb Profile microsomal stability (%)

CYP remaining activity (%)

entry

human

dog

rat

mouse

1A2

2C9

2D6

3A4

GNF-7 10b 10c 10d 10e 10f 10g 10k 11i

78.6 46.7 29.8 47.3 24.6 78.8 69.2 84.4 57.5

55.3 21.3 21.4 38.0 23.3 71.3 69.9 67.9 43.1

84.8 19.7 19.2 75.4 38.3 98.4 82.3 81.3 76.5

80.5 20.9 21.2 49.1 24.2 73.3 74.7 80.0 58.7

93.2 92.1 89.7 87.2 81.8 88.0 88.2 89.2 67.5

27.0 34.3 23.0 15.6 29.0 42.9 38.3 68.1 55.1

68.4 79.8 68.0 83.1 32.8 78.8 72.0 82.2 4.9

>100 >100 >100 88.5 94.2 >100 >100 >100 21.6

Liver microsomal stability (% remaining during 30 min at 1 μM). b% of control activity at 10 μM.

a

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Figure 2. Binding models of inhibitors on ACK1 (left) and GCK (right). (a) GNF-7, (b) 10d, (c) 10k, and (d) 11a.

pyrimidine and the pyridine make a pair of hydrogen bonds with the A208 in the hinge region at a distance of 2.6 and 2.8 Å. The pyrimidine ring orients for π−π interaction with F271. The side chains of the L184, L189, L243, F248, I190, and L259 improve binding affinity of 10d through hydrophobic interaction. In contrast to molecular docking analysis (Figure 2b) suggesting a hydrogen bond interaction between the piperazine moiety of 10d and D215-ACK1, the X-ray cocrystal structure reveals that a water molecule forms a hydrogen bonding with the nitrogen on piperazine of 10d at a distance of 3.3 Å as well as with the backbone amino group of S212 at a distance of 2.5 Å. This water-mediated hydrogen bond network contributes to the affinity of 10d on ACK1 and leads to the higher potency of 10d against ACK1 compared with GNF-7. Kinase Selectivity Profile of 10k. To assess the kinase selectivity of 10k, kinome-wide inhibition profiling was performed at 1 μM (Figure 4 and Supporting Information, Table S1). Among a total of 351 kinases, 33 kinases were inhibited more than 90%, including a number of tyrosine kinases (c-Src, YES, LCK, BMX, LYN, FYN, FMS, FGR, CSK, HCK, RET, ABL2, PDGFRβ, DDR2, DDR1, EphB1, KDR, BLK, ABL1, PDGFRα, JAK1, JAK2, FGFR2, FGFR1, EphA1), SIK1, RAF family (ARAF, BRAF, RAF1), and p38 family (p38α, 38β) as well as ACK1 and GCK. This indicates that 10k is a multitargeted kinase inhibitor, like GNF-7. 10k is capable of inhibiting several kinases besides ACK1 and GCK, suggesting that off-target effects are associated with the antiproliferative activity of 10k. To investigate whether off-targets also contribute to the antiproliferative activities of nonselective GNF7 analogues, we knocked down both ACK1 and GCK in OCI-AML3 cells using electroporation method and assessed the antiproliferative activities of several analogues (Supporting Information, Figure S1). The double siRNA-mediated ACK1/GCK knockdown in OCI-AML3 cells resulted in about 40% inhibition of cell growth. Treatment of ACK1/GCK dual inhibitors to the double-knockdown OCI-AML3 cells further suppressed the cell growth. The knockdown experiment results suggest that other targets besides ACK1 and GCK are involved in the antiproliferative activities of these compounds.

Table 7. Data Collection and Refinement Statistics for ACK1 in Complex with 10da ACK1-10d wavelength (Å) resolution range (Å) space group unit cell a, b, c α, β, γ multiplicity completeness (%) mean I/σ (I) Wilson B-factor R-merge R-work R-free no. of atoms macromolecules ligands water protein residues RMS (bonds) RMS (angles) Ramachandran favored (%) Ramachandran outlier (%) clash score average B-factor macromolecules ligands solvent

0.97950 50.00−2.20 (2.24−2.20) P1 21 1 71.18, 43.12, 93.890 90, 98.34, 9 3.8 (2.9) 98.2 (87.0) 18.64 (1.36) 40.26 0.147 (0.906) 0.2060 (0.2996) 0.2566 (0.3242) 4074 3885 96 93 481 0.009 1.44 96.76 0.22 4.57 50.69 50.78 50.62 46.99

a

Statistics for the highest-resolution shell is shown in parentheses.

nately, X-ray cocrystal structure of GCK-10d complex could not be obtained. The X-ray cocrystal structure reveals that 10d is bound on ACK1 as a typical type II kinase inhibitor. The amide linker of 10d forms hydrogen bonds with the K158E177 salt bridge as well as with the backbone amino group of D270 at a distance of 3.1 and 2.9 Å. The trifluoromethylphenyl group of 10d binds to the hydrophobic pocket, thereby forcing the DFG F271 out of the ATP binding pocket. The nitrogen on the pyrimidine ring and the NH group between the 8360



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Figure 3. X-ray crystal structure of 10d bound to ACK1 (PDB 5ZXB). (a) Overall fold of the ACK1-10d complex structure. Some residues (135− 137, 161−169, and 272−292) are missing in the structure due to a lack of electron density. (b) Protein residues are represented by white, and 10d is designated by gold. Sulfur is displayed in yellow, nitrogen in blue, oxygen in red, and fluoride in cyan. Lys158 is eliminated for clarity.

Figure 4. (a) Kinome-wide selectivity profiling of 10k at 1 μM. Kinases showing >90% inhibition are indicated with red circles. Illustration is reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com). (b) The list of 33 kinases out of 351 kinases showing >90% inhibition.

Effects on AKT/mTOR and GCK Signalings. To validate the results of the SAR study in a cellular context, Western blot analysis in Ba/F3-NRAS-G12D and OCI-AML3 cell lines was performed. Considering both in vitro potency and metabolic stability, we chose derivatives 10d, 10e, 10g, 10k, and 11i. After 2 h treatment with each compound, the cells were subjected to Western blot analysis. As a consequence of our previous observation that GNF-7 suppresses AKT/mTOR signaling and GCK downstream in Ba/F3-NRAS-G12D cells,3 we examined the phosphorylation level of p70S6K1, AKT, JNK, and p38 in both Ba/F3-NRAS-G12D and OCI-AML3 cell lines (Figure 5a,b). All five compounds at 1 μM were

found to attenuate phosphorylation of p70S6K1, JNK, and p38 and moderately suppressed phosphorylation of AKT (S473). Analogue 10d, which possesses an excellent in vitro potency, dramatically abolished phosphorylation of downstream molecules, which is consistent to our SAR study. Similar results were observed using OCI-AML3 cells. All compounds (10d, 10e, 10g, 10k, 11i, and GNF-7) caused a decreased level of phosphorylation of p70S6K1, AKT (S473), JNK, and p38 (Figure 5b). Compound 10k displayed a moderate suppression of AKT/mTOR signaling while it completely abolished GCK signaling at 1 μM. 8361



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Figure 5. Effect on downstream signaling inhibition. The phospho-p70S6K1, phospho-AKT (S473), phospho-JNK, and phospho-p38 levels in Ba/ F3-NRAS-G12D (a) and OCI-AML3 (b) treated with indicated compounds for 2 h. Compounds at 1 μM attenuated phosphorylation of each molecule. Actin was used for loading control. (c) 10d and 10k block both AKT/mTOR signaling and GCK signaling in a dose-dependent manner in OCI-AML3.

Figure 6. Effect on the cell apoptosis. (a) Western blot for apoptotic molecules in Ba/F3-NRAS-G12D (upper panel) and OCI-AML3 (low panel) treated with indicated compounds for 24 h. Cleaved PARP form was induced by each compound at 1 μM in OCI-AML3. Cleaved caspase 3 was increased by 1 μM of 10d and GNF-7 in Ba/F3-NRAS-G12D cells (upper panel). Actin was used for loading control. (b) Percent of apoptotic cells detected by FACS analysis in Ba/F3-NRAS-G12D (upper panel) and OCI-AML3 (low panel). Apoptotic cells were determined via dual-staining of FITC-conjugated annexin V and propidium iodide after 24 h treatment of indicated compounds at 1 μM. All experiments were performed three times, and the bar graph represents the average (n = 3) and SD. One-Way ANOVA; ** P < 0.05. (c) Dose dependent increase of apoptotic cells by compound 10k in Ba/F3-NRAS-G12D (upper panel) and OCI-AML3 (low panel). Each cell line was treated with indicated concentrations of 10k for 24 h and subjected to flow cytometry analysis. All experiments were performed in three times, and bar graph represents the average (n = 3) and SD. One-Way ANOVA; ** P < 0.05.

cleaved PARP and cleaved caspase 3 and diminished bcl-2 and MCL1 in a similar manner as does GNF-7. Compound 10k increased apoptotic markers in OCI-AML3 at 1 μM and significantly increases the level of apoptotic markers in Ba/F3NRAS-G12D cells at 5 μM (Supporting Information, Figure S2b). Flow cytometry analysis was carried out next using 1 μM of each analogue and by staining apoptotic cells with annexin V and propidium iodide (Figure 6b and Supporting Information, Figure S3). Treatment with 10d led to a noticeable increase of apoptotic cells, while treatment with 10k slightly increases the number of apoptotic cells in Ba/F3-NRAS-G12D cells, a finding that is consistent with the Western blot results. In OCIAML3, apoptotic cells were increased up to 15−30% by treatment with 10k and 10d. It is noteworthy that 10d displays better or similar apoptotic induction effect in Ba/F3-NRASG12D and OCI-AML3 cells compared to GNF-7. We also observed dose-dependent apoptosis occurs by treatment with 10k in both cell lines (Figure 6c and Supporting Information, Figure S2a).

We further examined dose dependent inhibition of compound 10d and 10k in OCI-AML3 (Figure 5c). 10d and 10k showed dose-dependent diminish of p70S6K1, AKT, and p38 phosphorylation. This finding indicates that the kinase inhibition data gained in the SAR study correlates with the activities in Ba/F3 cells transformed with the NRAS mutation and human OCI-AML3 cell line expressing the NRAS mutation. Induction of Apoptosis and Cell Cycle Arrest in NRAS Mutant Cell Lines. To determine if the antiproliferative effect of the new GNF-7 analogues is mainly responsible for apoptosis and cell cycle arrest, we examined the level of proapoptotic markers (cleaved PARP and cleaved caspase 3) and antiapoptotic markers (bcl-2 and MCL1) by using Western blot analysis. Analogues 10d and 10k were explored to assess their ability to induce apoptosis in both Ba/F3-NRAS-G12D and OCI-AML3 cells. For this purpose, cells were treated for 24 h with indicated concentrations of compounds (Figure 6a). In both Ba/F3-NRAS-G12D and OCI-AML3 cells, 10d at 1 μM was found to dramatically increase the levels of both 8362



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Figure 7. Induction of G0−G1 arrest after compound treatment. (a,b) OCI-AML3 were treated with indicated concentration of each compound for 24 h, and the percentage of each cell cycle phase were analyzed by flow cytometry after propidium iodide staining; 1 μM of each compound significantly increases population of G0−G1 phase.

Figure 8. Anchorage independent growth inhibition of 10d, 10k, and 11i in OCI-AML3. (a) Cells embedded in 0.7% low melting agar were incubated with the indicated concentrations of compounds for 2 weeks, and colonies were observed (n = 3 experiment per condition). (b) Average number of colonies per well was automatically counted using ImageJ software and shown in the bar graph (± SD, One-Way ANOVA; **** p < 0.0001, *** p < 0.0005, ** p < 0.05). (c) Average size of colonies per well was automatically measured using ImageJ software and shown in the bar graph (± SD, One-Way ANOVA; **** p < 0.0001, *** p < 0.0005, ** p < 0.05).

diminished colony number and size at 1 μM while they moderately decreased colony formation at 0.1 μM concentration, indicating that blockade of ACK1 and GCK activity prevents tumorigenesis of NRAS mutant driven AML cell line. In Vivo Efficacy Study. The antileukemic efficacies of 10k (15 mg/kg/day) and GNF-7 (8 mg/kg/day) compared with a vehicle control were evaluated using the Ba/F3-NRAS-G12D/ luciferase-bearing blood circulating mouse model. Analogue 10k was found to display a desirable mouse pharmacokinetic profile (data not shown) for oral administration. Differences in tumor engraftment and progression among the different treatment groups (po, qd) were monitored by using bioluminescence imaging (Figure 9a) and quantitative biophotonic imaging analysis (Figure 9b). Derivative 10k (15 mg/kg/ day) diminished more than 50% of leukemic cells at day 20, which is comparable to the efficacy of GNF-7 (8 mg/kg/day). The incidence of death due to leukemia progression was also evaluated following administration of 10k (po, qd) (Figure 9c). Similar to the results arising from the blood circulating

Next, we analyzed cell cycle phase after treatment of each compounds (Figure 7). G0−G1 arrest was mainly induced by 10d at 1 μM. It was compatible with that of GNF-7. 10k at 1 μM also increased G0−G1 population. Taken together, these compounds suppressed cell proliferation via apoptosis and cell cycle arrest. Suppression of Anchorage Independent Growth. To investigate if 10d, 10k, and 11i block tumorigenesis of NRAS mutated AML cell line, we carried out soft agar assay in OCIAML3 (Figure 8). Incubation with each compound at two different concentrations (0.1 and 1 μM) for 14 days suppressed anchorage-independent growth in terms of both colony number and colony size of OCI-AML3 (Figure 8b,c). Especially, it is noteworthy that 1 μM of 10d completely reduced both colony number and colony size compared to the DMSO treated cells. Furthermore, the effect caused by 1 μM of 10d is superior to that promoted by GNF-7, indicating that 10d is more effective than GNF-7 in inhibiting anchorageindependent growth of OCI-AML3. 10k and 11i also 8363



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Figure 9. Antileukemic efficacy of vehicle alone, GNF-7, and 10k in Ba/F3-NRAS-G12D blood circulating model and OCI-AML3 xenograft model. (a) Bioluminescence images of mice administered with vehicle only, GNF-7, or 10k once daily (n = 4 per cohort). Immunodeficient mice were tail intravenously injected with Ba/F3-NRAS-G12D/Luc cells and treated with vehicle, GNF-7 (8 mg/kg/day, po, qd), or 10k (15 mg/kg/day, po, qd) for 3 weeks. Bioluminescence images were obtained once every 2 days using IVIS spectrum (PerkinElmer) after administration of luciferin (150 mg/kg). (b) Ba/F3-NRAS-G12D/Luc cell progression was monitored by quantitative biophotonic imaging analysis of control and compoundtreatment groups. Quantitation from the in vivo imaging using Opti-view software (version 2.02, ART Inc.). Results represent mean ± SEM, n = 4 per group, **p < 0.05. The mean values and their 95% confidence intervals (error bars) are given. (c) Comparison of lifespan of mice orally administered with vehicle, GNF-7, and 10k in AML mouse model bearing Ba/F3-NRAS-G12D. Immunodeficient mice were injected with BaF3NRAS-G12D cells via the lateral tail vein, and compound administration was initiated 6 d after implantation. Mice were treated with vehicle, GNF7 (8 mg/kg/day, qd), or 10k (15 or 25 mg/kg/day, qd) for 8 weeks. Survival of the mice is represented by a Kaplan−Meier plot. P = 0.05 for mice treated 10k oral administrated group with the vehicle control mice. (d) Body weight change during the lifespan measurement. (e) Mouse xenograft study. Immuno-deficient mice were subcutaneously injected with OCI-AML3 cells with 50% Matrigel and treated with vehicle, GNF-7, or 10k (15 or 25 mg/kg/day, po, qd) for 3 w (n = 5 per group). Mouse tumor size was measured once every 2 days.

and GCK. To the best of our knowledge, this is the first SAR study for targeting NRAS mutant driven AML cell lines. In these current effort, we synthesized 29 derivatives of GNF-7 and investigated their inhibition capabilities of both kinase activities of GCK and ACK1 and cellular proliferation against AML cells bearing NRAS mutation. Analogue 10d was found to exhibit exceptional kinase inhibitory activities against both ACK1 (IC50 of 8 nM) and GCK (IC50 of 1 nM). We solved the X-ray cocrystal structure of ACK1 complexed with 10d, enabling an accurate determination of the binding mode of 10d on ACK1. Compound 10d binds to the kinase domain of ACK1 as a typical type II inhibitor, and there are unexpected interactions mediated by water molecules, which contributes to stronger activity of 10d compared with GNF-7. The results of docking study with 10d, which possesses a piperazinylethanone moiety in the head group, led to the suggestion that an additional binding site exists in GCK, in which Glu100-GCK participates in hydrogen bonding interactions with 10d, contributing to an improved affinity. Thus, knowledge about the existence of these residues could aid the rationale design of substances that have improved selectivities and activities against ACK1 and GCK. Compared with GNF-7, 11i possesses higher kinase inhibitory activity against both ACK (IC50 = 6 nM) and GCK1 (IC50 = 2 nM) and more significantly blocks proliferation of both Ba/F3NRAS-G12D (GI50 23 nM) and OCI-AML3 (GI50 37 nM) cells. Furthermore, 11i possesses a higher differential cytotoxicity than GNF-7 on Ba/F3-NRAS-G12D cells (28-

model, 10k (15 mg/kg/day) has a level of lifespan extension that is similar to that of GNF-7 (8 mg/kg/day). Moreover, the 10k (25 mg/kg/day)-treated group shows a median survival of 35 days, which is almost 50% longer compared with the vehicle control group. In the GNF-7 (8 mg/kg/day)-treated group, noticeable body weight loss (>15%) was observed, which is a common symptom of in vivo toxicity. However, the 10k (15 mg/kg/day)-treated group exhibited a very small weight change (Figure 9d). It should be noted that 10k is superior to GNF-7 as prominent toxicity was found in mice treated with GNF-7. The antileukemic efficacy of 10k was also assessed in AML xenograft model of OCI-AML3 cells. The tumor volumes of the group treated with vehicle, GNF-7 (8 mg/kg/day), 10k (15 mg/kg/day), and 10k (25 mg/kg/day) were 100%, 53.8%, 41.9%, and 14.9%, respectively. Overall, the dose-dependent antileukemia effect of 10k has been demonstrated using lifespan extension and OCI-AML3 xenograft AML models.

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CONCLUSIONS Hyperactive RAS is one of most attractive cancer therapeutic targets. As part of a study aimed at developing a novel strategy to target mutant NRAS expressing AML, we recently reported that dual inhibition of the kinases ACK1 and GCK by GNF-7 effectively suppresses proliferation of NRAS mutant driven AML cells. A SAR study was carried out to identify GNF-7 analogues that display superior inhibitory effects against ACK1 8364



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12.2 mmol) and 2-methyl-5-nitroaniline (1.48 g, 9.8 mmol) in acetone (12 mL) was added NaI (2.2 g, 14.6 mmol) and K2CO3 (2.5 g, 18.3 mmol). The mixture was stirred at 50 °C for 10 h. The reaction was cooled to rt, filtered, and extracted with DCM. The organic layers were dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography (20% EtOAc/hexane) to afford the title compound as a yellow solid (2.83 g, 74%). 1H NMR (400 MHz, CDCl3) δ 8.50 (s, 1H), 7.61 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.29 (d, J = 2.0 Hz, 1H), 7.23 (dd, J = 8.0 Hz, 0.4 Hz, 1H), 4.57 (d, J = 6.0 Hz), 4.29 (t, J = 6.0 Hz), 2.29 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 161.06, 160.50, 157.63, 147.10, 146.16, 130.80, 130.42, 130.13, 111.42, 102.51, 41.31, 17.92. LRMS (ESI) m/z 313 [M + H]+. 2-Chloro-N-methyl-5-(((2-methyl-5-nitrophenyl)amino)methyl)pyrimidin-4-amine (4). N-((2,4-Dichloropyrimidin-5-yl)methyl)-2methyl-5-nitroaniline (2.83 g, 9.0 mmol) is dissolved in 1,4-dioxane (30 mL). The solution was treated with methylamine (2 M in MeOH solution, 4.52 mL, 18.1 mmol) and DIPEA (4.7 mL, 27.1 mmol). The mixture was stirred at 60 °C for 1.5 h, then cooled to room temperature, quenched with water, and extracted with DCM. The organic layers were dried over magnesium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (35% EtOAc/hexane) to afford the title compound as a yellow solid (2.15 g, 77%). 1H NMR (400 MHz, DMSO-d6) δ 7.90 (s, 1H), 7.54−7.53 (m, 1H), 7.39 (dd, J = 8.0 Hz, J = 2.0 Hz, 1H), 7.24 (s, 1H), 7.22 (s, 1H), 7.18 (d, J = 2.0 Hz, 1H), 6.00 (t, J = 5.6 Hz, 1H), 4.21 (d, J = 5.6 Hz, 2H), 2.89 (d, J = 4.4 Hz, 3H), 2.22 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 161.88, 158.57, 153.79, 146.95, 146.54, 130.73, 130.24 112.99, 110.97, 102.76, 27.64, 17.91. LRMS (ESI) m/z 308 [M + H]+. 7-Chloro-1-methyl-3-(2-methyl-5-nitrophenyl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (5). To a solution of 2chloro-N-methyl-5-(((2-methyl-5-nitrophenyl)amino)methyl)pyrimidin-4-amine (5.12 g, 16.6 mmol) in THF (50 mL) was slowly added triphosgene (2.5 g, 8.2 mmol) at 0 °C under nitrogen atmosphere. The mixture was slowly treated with TEA (12 mL, 83.0 mmol) and stirred at 70 °C for 1 h. The reaction mixture was quenched with water and extracted with DCM. The organic phase was washed with brine, dried over MgSO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (30% EtOAc/hexane) to get the title compound (3.46 g, 63%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.35 (d, J = 2.4 Hz, 1H), 8.31 (t, J = 0.08 Hz, 1H), 8.15 (dd, J = 8.4 Hz, J = 2.8 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 4.94 (d, J = 15.2 Hz, 1H), 4.65 (d, J = 15.2, 1H), 3.28 (s, 3H), 2.27 (s, 3H). 13C NMR (100 MHz, DMSOd6) δ 158.39, 158.16, 154.07, 151.28, 146.33, 144.58, 141.44, 131.85, 122.77, 122.52, 111.64, 46.03, 28.36, 17.43. LRMS (ESI) m/z 334 [M + H]+. 3 - ( 5 - A m i n o - 2 - m e t h y l p h e n y l )- 7 - c h l o r o -1 - m e t h y l - 3 , 4 dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (6). A mixture of 7chloro-1-methyl-3-(2-methyl-5-nitrophenyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (1.4 g, 4.2 mmol), Fe powder (2.3 g, 42 mmol), and NH4Cl (4.5 g, 84 mmol) in THF (10 mL), MeOH (5 mL), and H2O (2.5 mL) was stirred at 80 °C for 2 h. TLC showed the reaction was completed. The mixture was cooled to room temperature, and the resulting solid was filtered off. The filtrate was basified with NaHCO3 and extracted by DCM. The organic layer dried over MgSO4, filtered, and concentrated to afford the title compound as a pale-yellow solid (1.1 g, 87%). 1H NMR (400 MHz, DMSO-d6) δ 8.29 (s, 1H), 6.93−6.91 (m, 1H), 6.51−6.48 (m, 2H), 5.04 (bs, 2H), 4.72 (d, J = 15.6 Hz, 1H), 4.55 (d, J = 15.6 Hz, 1H), 3.26 (s, 3H), 1.95 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 158.51, 158.09, 153.81, 150.83 147.60, 140.90, 130.85, 121.31, 113.66, 112.21, 111.54, 46.14, 28.23, 16.07. LRMS (ESI) m/z 304 [M + H]+. N-(3-(7-Chloro-1-methyl-2-oxo-1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-3-(trifluoromethyl)benzamide (7). To a solution of 3-(5-amino-2-methylphenyl)-7-chloro-1-methyl3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (230 mg, 0.8 mmol) in DCM (2.52 mL) was added K2CO3 (209.3 mg, 1.5

fold) relative to parental Ba/F3 and on OCI-AML3 (53-fold) relative to U937. Thus, 11i is obviously superior to GNF-7 in terms of enzymatic activity inhibition and cellular selectivity as well as cellular potency. The results of Western blot analysis showed that this series of compounds dose-dependently attenuates phosphorylation of p70S6K1, AKT, and p38 in OCI-AML3. It was also revealed that they exert their antiproliferative effects by inducing apoptosis and G0−G1 arrest and significantly induce apoptotic markers (cleave PARP and caspase3) in both Ba/F3-NRAS-G12D and OCI-AML3 cells. We also observed that this series of compounds suppressed anchorage independent growth of OCI-AML3 in soft agar assay. Analogue 10k having a desirable mouse PK profile exhibited a significant efficacy in a Ba/F3-NRASG12D/Luc-bearing blood circulating mouse model, remarkably prolonging the lifespan of Ba/F3-NRAS-G12D injected mice and significantly delaying tumor growth of OCI-AML3 xenograft model without prominent toxicity found in mice treated with GNF-7, which makes 10k superior to GNF-7. It is worth recalling that 10k displayed more favorable profiles with respect to CYPs inhibition and especially induced evident improvement on CYP2C9 compared with GNF-7. Overall, in this investigation, we have established structure− activity relationship of GNF-7 derivatives against AML expressing NRAS mutation, which is the first SAR study for overcoming AML caused by NRAS mutation. Therefore, this study has, for the first time, provided insight into the design of novel and potent inhibitors that operate by ACK1 and GCK dual inhibition and, as a result, approaches for overriding NRAS mutant driven AML.

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EXPERIMENTAL PROCEDURES

Chemistry. General Information. Unless otherwise described, all commercial reagents and solvents were purchased from commercial suppliers and used without further purification. All reactions were performed under N2 atmosphere in flame-dried glassware. Reactions were monitored by TLC with 0.25 mm E. Merck precoated silica gel plates (60 F254). Reaction progress was monitored by TLC analysis using a UV lamp, ninhydrin, or p-anisaldehyde stain for detection purposes. All solvents were purified by standard techniques. Purification of reaction products was carried out by silica gel column chromatography using Kieselgel 60 Art. 9385 (230−400 mesh). The purity and of all compounds was over 95%, and mass spectra and purity of all compounds were analyzed by using a Waters LCMS system (Waters 2998 photodiode array detector, a Waters 3100 mass detector, a Waters SFO system fluidics organizer, a Water 2545 binary gradient module, a Waters reagent manager, and a Waters 2767 sample manager) using a SunFire C18 column (4.6 mm × 50 mm, 5 μm particle size): solvent gradient = 60% (or 95%) A at 0 min, 1% A at 5 min. Solvent A = 0.035% TFA in H2O; solvent B = 0.035% TFA in MeOH; flow rate 3.0 (or 2.5) mL/min. 1H and 13C NMR spectra were obtained by using a Bruker 400 MHz FT-NMR (400 MHz for 1 H, and 100 MHz for 13C) spectrometer. Standard abbreviations are used for denoting the signal multiplicities. 2,4-Dichloro-5-(chloromethyl)pyrimidine (2). To a solution of 5(hydroxymethyl)pyrimidine-2,4-diol (3.0 g, 21.1 mmol) in toluene was added DIPEA (11 mL, 63.3 mmol) and equipped with nitrogeninlet bubbler. POCl3 (9.8 mL, 105.5 mmol) was added dropwise at 0 °C. The mixture was stirred at 110 °C for 1 h and then concentrated. The resulting residue was purified by silica gel column chromatography (15% EtOAc/hexane) to obtain the title compound (3.20 g, 77%) as colorless liquid. 1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 4.64 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 161.62, 160.03, 128.37, 38.66. LRMS (ESI) m/z 196 [M + H]+. N-((2,4-Dichloropyrimidin-5-yl)methyl)-2-methyl-5-nitroaniline (3). To a solution of 2,4-dichloro-5-(chloromethyl)pyrimidine (2.4 g, 8365



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= 8.0 Hz, J = 2.4 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 4.71 (d, J = 14.4 Hz, 1H), 4.53 (d, J = 14.4 Hz, 1H), 3.34 (s, 3H), 2.41 (s, 3H), 2.14 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.65, 158.74, 156.81, 153.01, 151.91, 149.99, 140.96, 139.82, 137.40, 135.41, 134.50, 131.60, 130.73, 130.55, 129.55, 129.47, 129.15, 128.83, 128.52, 127.98, 127.94, 127.83, 126.01, 125.12, 123.98, 123.94, 123.90, 122.42, 122.23, 119.71, 119.62, 119.15, 102.73, 46.44, 28.02, 23.00, 16.58. LRMS (ESI) m/z 548 [M + H]+. HRMS (ESI) m/z calculated for C28H25F3N7O2+ [M + H]+ 548.20, found 548.2014. N-(4-Methyl-3-(1-methyl-2-oxo-7-(phenylamino)-1,4dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)phenyl)-3(trifluoromethyl)benzamide (10a). Compound 7 (100 mg, 0.2 mmol) was converted to the target compound using general procedure A. The resulting crude product was purified by flash column chromatography on silica gel (40% EtOAc/hexane) to afford 10a (22.9 mg, 22%) as a brown solid. 1H NMR (400 MHz, DMSOd6) δ 10.52 (s, 1H), 9.56 (s, 1H), 8.30 (s, 1H), 8.26 (d, J = 7.6 Hz, 1H), 8.15 (s, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.80−7.75 (m, 4H), 7.64 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 7.32−7.26 (m, 3H), 6.93 (t, J = 7.6, 1H), 4.70 (d, J = 14.4 Hz, 1H), 4.52 (d, J = 14.4 Hz, 1H), 3.35 (s, 3H), 2.13 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.65, 158.81, 156.73, 152.97, 151.98, 141.01, 140.36, 137.39, 135.42, 131.61, 130.75, 130.54, 129.56, 129.47, 129.15, 128.83, 128.52, 128.27, 127.99, 127.95, 127.84, 125.13, 123.98, 123.94, 123.90, 122.42, 121.07, 119.71, 119.60, 119.16, 118.60, 102.45, 46.47, 28.02, 16.59. LRMS (ESI) m/z 533 [M + H]+. HRMS (ESI) m/z calculated for C28H23F3N6NaO2+ [M + Na]+ 555.17, found 555.1729. N-(3-(7-((4-(4-Ethylpiperazin-1-yl)phenyl)amino)-1-methyl-2oxo-1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-3-(trifluoromethyl)benzamide (10b). Compound 7 (100 mg, 0.2 mmol) was converted to the target compound using general procedure A. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 10b (53.9 mg, 42%) as a gray solid. 1H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 9.29 (s, 1H), 8.29 (s, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.08 (s, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.80−7.77 (m, 2H), 7.63 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 8.8 Hz, 2 H), 7.30 (d, J = 8.4 Hz, 1H), 6.87 (d, J = 8.8 Hz, 2 H), 4.66 (d, J = 14.0 Hz, 1H), 4.48 (d, J = 14.0 Hz, 1H), 3.04 (s, 4H), 2.34 (q, J = 7.2 Hz, 2H), 2.12 (s, 3H), 1.01 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.63, 158.96, 156.64, 152.99, 152.05, 145.91, 141.05, 137.39, 135.41, 132.42, 131.61, 130.73, 130.53, 129.56, 129.46, 129.14, 128.83, 128.51, 127.96, 127.84, 125.13, 123.98, 123.94, 122.42, 119.92, 119.55, 119.13, 115.59, 101.56, 52.23, 51.44, 48.76, 46.48, 27.95, 16.60, 11.78. LRMS (ESI) m/z 645 [M + H]+. HRMS (ESI) m/z calculated for C34H36F3N8O2+ [M + H]+ 645.29, found 645.2903. N-(3-(7-((6-(4-Ethylpiperazin-1-yl)pyridin-3-yl)amino)-1-methyl2-oxo-1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-3-(trifluoromethyl)benzamide (10c). Compound 7 (100 mg, 0.2 mmol) was converted to the target compound using general procedure A. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 10c (77.4 mg, 60%) as a brown solid. 1H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 9.32 (s, 1H), 8.46 (d, J = 2.4 Hz, 1H), 8.30 (s, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.09 (s, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.88 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 7.81−7.78 (m, 2H), 7.64 (dd, J = 8.0 Hz, J = 2.0 Hz, 1H), 7.31 (d, J = 8.8 H, 1H), 6.82 (d, J = 9.2 Hz, 1H), 4.68 (d, J = 14.0, 1H), 4.50 (d, J = 14.0, 1H), 3.39 (s, 4H), 3.33 (s, 3H), 2.45 (s, 4H), 2.36 (q, J = 7.2 Hz, 2H), 2.13 (s, 3H), 1.03 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.64, 159.08, 156.74, 154.83, 153.05, 152.00, 141.02, 138.95, 137.39, 135.41, 131.60, 130.72, 130.53, 129.80, 129.55, 129.15, 128.83, 128.51, 128.04, 125.12, 123.98, 123.94, 122.42, 119.57, 119.13, 106.60, 101.83, 51.99, 51.50, 46.46, 45.17, 27.94, 16.58, 11.75. LRMS (ESI) m/z 646. [M + H]+. HRMS (ESI) m/z calculated for C33H35F3N9O2+ [M + H]+ 646.29, found 646.2876. N-(3-(7-((6-(4-Acetylpiperazin-1-yl)pyridin-3-yl)amino)-1-methyl-2-oxo-1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-3-(trifluoromethyl)benzamide (10d). Compound 7 (100 mg, 0.2 mmol) was converted to the target compound using general

mmol) and 3-(trifluoromethyl)benzoyl chloride (0.12 mL, 0.8 mmol). The reaction mixture was stirred at room temperature for 1 h, quenched with water, and extracted by DCM. The organic layers were dried over MgSO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (35% EtOAc/ hexane) to afford the title compound (262.1 mg, 73%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.54 (s, 1H), 8.33 (s, 1H), 8.30 (s, 1H), 8.26 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.83 (d, J = 2.0 Hz, 1H), 7.79 (t, J = 8.0 Hz, 1H), 7.63 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 4.83 (d, J = 15.2 Hz, 1H), 4.66 (d, J = 15.2 Hz, 1H), 3.29 (s, 1H), 2.14 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 163.87, 158.59, 158.20, 154.03, 151.12, 140.60, 137.59, 135.56, 131.79, 130.96, 130.82, 129.76, 129.33, 129.01, 128.19, 128.16, 125.29, 124.14, 124.10, 122.59, 120.07, 119.31, 111.66, 46.23, 28.38, 16.68. LRMS (ESI) m/z 476 [M + H]+. 1-Methyl-3-(2-methyl-5-nitrophenyl)-7-((6-methylpyridin-3-yl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (8). A suspension of 7-chloro-1-methyl-3-(2-methyl-5-nitrophenyl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (1.1 g, 3.3 mmol), 6methylpyridin-3-amine (256.9 mg, 3.3 mmol), K2CO3 (2.2 g, 6.6 mmol), Xphos (314.6 mg, 0.66 mmol), and Pd2(dba)3 (604.3 mg, 0.66 mmol) in 2-butanol (16.5 mL) was stirred at 100 °C for 2 h. The reaction mixture was cooled to room temperature, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (5−10% MeOH/DCM) to afford the title compound (863.8 mg, 65%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H), 8.79 (d, J = 2.4 Hz, 1H), 8.34 (d, J = 2.4 Hz), 8.16−8.13 (m, 2H), 8.05 (dd, J = 8.4 Hz, J = 2.8 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 4.86 (d, J = 13.6 Hz, 1H), 4.51 (d, J = 13.6 Hz, 1H), 3.34 (s, 3H), 2.40 (s, 3H), 2.28 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 158.90, 156.82, 153.29, 152.24, 150.25, 146.38, 144.50, 142.03, 140.03, 134.63, 131.81, 126.25, 122.66, 122.43, 122.31, 102.94, 46.37, 28.22, 23.20, 17.55. LRMS (ESI) m/z 406 [M + H]+. 3-(5-Amino-2-methylphenyl)-1-methyl-7-((6-methylpyridin-3yl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (9). A mixture of methyl-5-nitrophenyl-7-((6-methylpyridin-3-yl)amino)3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (215.2 mg, 0.53 mmol), Fe powder (296 mg, 5.3 mmol), and NH4Cl (567 mg, 10.6 mmol) in THF (10 mL), MeOH (5 mL), and H2O (2.5 mL) was stirred at 80 °C for 2 h. TLC showed the reaction was completed. The reaction mixture was cooled to room temperature and filtered. The filtrate was basified with NaHCO3 and extracted by DCM. The organic layer was dried over MgSO4, filtered, and concentrated to afford the title compound as a pale-yellow solid (161.4 mg, 81%). 1H NMR (400 MHz, DMSO-d6) δ 9.60 (s, 1H), 8.78 (s, 1H), 8.12 (s, 1H), 8.04 (dd, J = 2.8 Hz, J = 8.4 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H), 6.46 (m, 2H), 4.99 (s, 2H), 4.60 (d, J = 14.4 Hz, 1H), 4.42 (d, J = 14.4 Hz, 1H), 3.30 (s, 3H), 2.40 (s, 3H), 1.95 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 158.74, 156.92, 152.93, 151.77, 149.98, 147.56, 141.39, 139.83, 134.56, 130.70, 126.02, 122.27, 121.22, 113.31, 112.26, 102.85, 46.47, 28.01, 23.05, 16.10. LRMS (ESI) m/z 376 [M + H]+. General Procedure A for the Synthesis of Compounds GNF-7 and 10a−10k. To a solution of 7 (1 equiv) in 2-butanol (0.15 M) was added various amines (1.1 equiv), K2CO3 (5 equiv), Xphos (0.2 equiv), and Pd2(dba)3 (0.2 equiv) at room temperature. The reaction mixture was then stirred for 1 h at 100 °C, cooled to room temperature, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography. N-(4-Methyl-3-(1-methyl-7-((6-methylpyridin-3-yl)amino)-2-oxo1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)phenyl)-3(trifluoromethyl)benzamide (GNF-7). Compound 7 (100 mg, 0.2 mmol) was converted to the target compound using general procedure A. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford GNF-7 (66.2 mg, 61%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 10.54 (s, 1H), 9.65 (s, 1H), 8.79 (d, J = 2.4 Hz, 1H), 8.31 (s, 1H), 8.27 (d, J = 7.6 Hz, 1H), 8.16 (s, 1H), 8.05 (dd, J = 8.4, J = 2.4, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.81−7.78 (m, 2H), 7.64 (dd, J 8366



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procedure A. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 10d (25.0 mg, 36%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 9.35 (s, 1H), 8.48 (d, J = 2.4 Hz, 1H), 8.30 (s, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.10 (s, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.92 (dd, J = 9.2 Hz, J = 2.8 Hz, 1H), 7.81−1.77 (m, 2H), 7.64 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 6.87 (d, J = 9.2 Hz, 1H), 4.68 (d, J = 13.6 Hz, 1H), 4.50 (d, J = 13.6 Hz, 1H), 3.54−3.53 (m, 4H), 3.46−3.45 (m, 2H), 3.39−3.38 (m, 2H), 3.32 (s, 3H), 2.13 (s, 3H), 2.04 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 168.23, 163.75, 159.15, 156.86, 154.49, 153.15, 152.09, 141.12, 138.97, 137.48, 135.51, 131.71, 130.83, 130.64, 129.95, 129.66, 129.57, 129.25, 128.93, 128.50, 128.09, 128.05, 125.22, 124.07, 124.03, 122.52, 119.68, 119.24, 107.05, 102.02, 46.55, 45.45, 45.19, 45.13, 28.05, 21.14, 16.68. LRMS (ESI) m/z 660 [M + H]+. HRMS (ESI) m/z calculated for C33H32F3N9NaO3+ [M + Na]+ 682.25, found 682.2476. N-(3-(7-((6-(4-Ethylpiperazine-1-carbonyl)pyridin-3-yl)amino)-1methyl-2-oxo-1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4methylphenyl)-3-(trifluoromethyl)benzamide (10e). Compound 7 (100 mg, 0.2 mmol) was converted to the target compound using general procedure A. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 10e (18.0 mg, 13%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 10.02 (s, 1H), 8.89 (d, J = 1.6 Hz, 1H), 8.34 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 8.29 (s, 1H), 8.25 (d, J = 7.6 Hz, 1H), 8.21 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.80−7.76 (m, 2H), 7.63 (dd, J = 8.4 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 4.73 (d, J = 14.4 Hz, 1H), 4.55 (d, J = 14.4 Hz, 1H), 3.61− 3.54 (m, 4H), 3.35 (s, 3H), 2.40 (s, 2H), 2.36−2.31 (m, 4H), 2.13 (s. 3H), 0.99 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 166.32, 163.73, 158.44, 157.02, 153.01, 151.88, 145.86, 140.96, 138.19, 137.86, 137.46, 135.47, 131.69, 130.80, 130.64, 129.65, 129.20, 128.88, 128.05, 125.16, 124.04, 124.00, 123.78, 122.48, 119.71, 119.20, 103.79, 52.64, 51.98, 51.38, 46.60, 46.47, 41.65, 28.21, 16.66, 11.73. LRMS (ESI) m/z 674 [M + H]+. HRMS (ESI) m/z calculated for C34H34F3N9NaO3+ [M + Na]+ 696.26, found 696.2632. N-(4-Methyl-3-(1-methyl-7-((1-methyl-1H-pyrazol-4-yl)amino)2-oxo-1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)phenyl)-3(trifluoromethyl)benzamide (10f). Compound 7 (100 mg, 0.2 mmol) was converted to the target compound using general procedure A. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 10f (20.0 mg, 14%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 9.40 (s, 1H), 8.29 (s, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.08 (s, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.83−7.77 (m, 3 H), 7.63 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 7.50 (s, 1H), 7.30 (d, J = 8.4 Hz, 1H), 4.66 (d, J = 14.0 Hz, 1H), 4.48 (d, J = 14.0 Hz, 1H), 3.80 (s, 3H), 3.34 (s, 3H), 2.12 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.87, 158.65, 157.16, 152.30, 141.28, 137.58, 135.63, 131.84, 130.94, 130.75, 129.80, 129.35, 129.03, 128.19, 125.34, 124.15, 123.29, 122.64, 120.24, 119.76, 119.34, 46.65, 28.32, 16.82. LRMS (ESI) m/z 537 [M + H]+. HRMS (ESI) m/z calculated for C26H23F3N8NaO2+ [M + Na]+ 559.18, found 559.1793. N-(3-(7-((1,3-Dimethyl-1H-pyrazol-5-yl)amino)-1-methyl-2-oxo1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-3(trifluoromethyl)benzamide (10g). Compound 7 (100 mg, 0.2 mmol) was converted to the target compound using general procedure A. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 10g (55.5 mg, 51%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 9.30 (s, 1H), 8.30 (s, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.10 (s, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.81−7.77 (m, 2H), 7.63 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 6.02 (s, 1H), 4.68 (d, J = 14.4 Hz, 1H), 4.51 (d, J = 14.4 Hz, 1H), 3.59 (s, 3H), 3.27 (s, 3H), 2.13 (s, 3H), 2.10 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.85, 159.26, 157.26, 153.33, 152.10, 145.06, 141.15, 138.35, 137.58, 135.61, 131.81, 130.93, 130.74, 129.76, 129.34, 129.02, 128.19, 125.32, 124.17, 124.13, 122.62, 119.81, 119.34, 103.09, 98.01, 46.56, 35.07, 28.04, 16.77, 13.78. LRMS (ESI)

m/z 551 [M + H]+. HRMS (ESI) m/z calculated C27H25F3N8NaO2+ [M + Na]+ 573.19, found 573.1942. N-(3-(7-(Cyclopropylamino)-1-methyl-2-oxo-1,4dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-3(trifluoromethyl)benzamide (10h). Compound 7 (100 mg, 0.2 mmol) was converted to the target compound using general procedure A. The resulting crude product was purified by flash column chromatography on silica gel (10% MeOH/DCM) to afford 10h (74.2 mg, 75%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.31 (s, 1H), 8.27 (d, J = 7.6 Hz, 1H), 8.00−7.97 (m, 2H), 7.82−7.77 (m, 2H), 7.65 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 7.32 (s, 1H), 7.31 (d, J = 4.8 Hz, 1H), 4.63 (d, J = 14.0 Hz, 1H), 4.44 (d, J = 13.6 Hz, 1H), 3.29 (s, 3H), 2.76−2.70 (m, 1H), 2.13 (s, 3H), 0.69−0.65 (m, 2H), 0.50−0.47 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 163.63, 162.33, 156.59, 153.01, 152.22, 141.14, 137.35, 135.42, 131.61, 130.71, 130.49, 130.20, 129.56, 129.14, 128.83, 127.98, 125.12, 123.97, 123.93, 122.42, 119.49, 119.12, 66.93, 46.44, 31.49, 30.47, 27.58, 23.66, 22.87, 16.59, 9.85, 6.11. LRMS (ESI) m/z 497 [M + H]+. HRMS (ESI) m/z calculated for C25H23F3N6NaO2+ [M + Na]+, 519.17, found 519.17.32. N-(3-(7-(Cyclohexylamino)-1-methyl-2-oxo-1,4dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-3(trifluoromethyl)benzamide (10i). Compound 7 (100 mg, 0.2 mmol) was converted to the target compound using general procedure A. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 10i (89.6 mg, 83%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 8.30 (s, 1H), 8.26 (d, J = 8.0 Hz, 1H), 7.97−7.95 (m, 2H), 7.81−7.75 (m, 2H), 7.64 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 6.94 (s, 1H), 4.60 (d, J = 14.0 Hz, 1H), 4.41 (d, J = 14.0 Hz, 1H), 3.70 (s, 1H), 3.3 (s, 3H), 2.12 (s, 3H), 1.90−1.57 (m, 5H), 1.34−1.09 (m, 5H). 13C NMR (100 MHz, DMSO-d6) δ 163.78, 160.80, 156.81, 152.39, 141.32, 137.50, 135.58, 131.75, 130.85, 130.64, 129.71, 129.30, 128.98, 128.13, 125.28, 124.12, 124.09, 122.57, 119.62, 119.27, 49.38, 46.60, 32.40, 27.69, 25.37, 24.82, 16.74. LRMS (ESI) m/z 539 [M + H]+. HRMS (ESI) m/z calculated for C28H29F3N6NaO2+ [M + Na]+ 561.22, found 561.2189. N-(3-(7-((2-Hydroxyethyl)amino)-1-methyl-2-oxo-1,4dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-3(trifluoromethyl)benzamide (10j). Compound 7 (100 mg, 0.2 mmol) was converted to the target compound using general procedure A. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 10j (83.8 mg, 84%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H), 8.30 (s, 1H), 8.26 (d, J = 7.6 Hz, 1H), 7.97−7.96 (m, 2H), 7.78 (t, J = 8.0 Hz, 1H), 7.75 (d, J = 2.0, 1H), 7.63 (dd, J = 8.4 Hz, J = 2.4 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 6.96 (t, J = 5.6 Hz, 1 H), 4.66 (t, J = 5.6, 1H), 4.61 (d, J = 13.6 Hz, 1H), 4.42 (d, J = 13.6 Hz, 1H), 3.53 (q, J = 6.0 Hz, 2H), 3.38−3.35 (m, 2H), 3.26 (s, 3H), 2.11 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.82, 161.56, 156.84, 152.38, 141.30, 137.51, 135.58, 131.77, 130.88, 130.67, 129.74, 129.31, 128.99, 128.12, 125.29, 124.14, 124.10, 122.59, 119.66, 119.29, 59.76, 46.58, 43.52, 27.75, 16.76. LRMS (ESI) m/z 501 [M + H]+. HRMS (ESI) m/z calculated for C24H24F3N6O3+ [M + H]+ 501.19, found 501.1863. N-(3-(7-Amino-1-methyl-2-oxo-1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-3-(trifluoromethyl)benzamide (10k). To a solution of compound 7 (100 mg, 0.2 mmol) in DMSO (5 mL) was added ammonia solution (28.0∼30.0%, 5 mL) at room temperature. The mixture was stirred at 150 °C for 15 h. The reaction mixture was cooled to room temperature, diluted with EtOAc, and quenched with H2O. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (5−10% MeOH/DCM) to afford the title compound (25.8 mg, 28%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 8.30 (s, 1H), 8.26 (d, J = 7.6 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.93 (s, 1H), 7.79 (t, J = 8.0 Hz, 1H), 7.75 (d, J = 2.0 Hz, 1H), 7.64 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 6.58 (s, 2H), 4.60 (d, J = 14.0 Hz, 1H), 4.41 (d, J = 14.0 Hz, 1H), 3.25 (s, 3H), 2.12 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.81, 162.72, 157.00, 153.42, 152.40, 141.30, 8367



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was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 11d (116.1 mg, 36%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.63 (s, 1H), 8.79 (d, J = 2.4 Hz, 1H), 8.16 (s, 1H), 8.05 (dd, J = 8.4 Hz, J = 2.4 Hz, 1H), 7.75 (s, 1H), 7.70 (s, 1H), 7.65 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 7.57 (s, 1H), 7.35 (s, 1H), 7.31 (d, J = 8.8 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 4.73 (d, J = 4.4 Hz, 1H), 4.70 (d, J = 14.4 Hz, 1H), 4.53 (d, J = 14.0 Hz, 1H), 3.72−3.69 (m, 4H), 3.04 (t, J = 10.0 Hz, 2H), 2.40 (s, 3H), 2.14 (s, 3H), 1.85−1.83 (m, 2H), 1.51−1.43 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 164.23, 158.80, 156.88, 153.08, 151.97, 150.99, 150.06, 140.98, 139.88, 137.49, 136.34, 134.55, 130.68, 130.56, 130.20, 129.89, 126.08, 125.38, 122.67, 122.31, 119.79, 119.35, 117.30, 113.41, 112.70, 102.81, 65.50, 46.48, 45.52, 33.31, 28.09, 23.08, 16.63. LRMS (ESI) m/z 647 [M + H]+. HRMS (ESI) m/z calculated for C33H34F3N8O3+ [M + H]+ 647.27, found 647.2714. 3-(4-Methyl-1H-imidazol-1-yl)-N-(4-methyl-3-(1-methyl-7-((6methylpyridin-3-yl)amino)-2-oxo-1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)phenyl)-5-(trifluoromethyl)benzamide (11d). Compound 9 (200 mg, 0.5 mmol) was converted to the target compound using general procedure B. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 11e (191.5 mg, 61%) as a pale-brown solid. 1 H NMR (400 MHz, DMSO-d6) δ 10.56 (s, 1H), 9.64 (s, 1H), 8.79 (m, 1H), 8.45 (s, 1H), 8.40 (s, 1H), 8.24 (s, 1H), 8.16 (s, 2H), 8.05 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 7.77 (s, 1H), 7.71 (s, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 4.70 (d, J = 14.0 Hz, 1H), 4.54 (d, J = 14.0 Hz, 1H), 3.34 (s, 3H), 2.40 (s, 3H), 2.19 (s, 3H), 2.15 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.06, 158.95, 157.02, 153.24, 152.13, 150.21, 141.22, 140.03, 138.97, 137.76, 137.42, 137.37, 135.25, 134.69, 131.34, 131.17, 131.01, 130.84, 130.68, 126.23, 124.80, 122.58, 122.45, 122.08, 121.78, 119.85, 119.43, 119.16, 114.24, 102.94, 46.63, 28.22, 23.22, 16.80, 13.57. LRMS (ESI) m/z 628 [M + H]+. HRMS (ESI) m/z calculated for C32H28F3N9NaO2+ [M + Na]+ 650.22, found 650.2266. N-(4-Methyl-3-(1-methyl-7-((6-methylpyridin-3-yl)amino)-2-oxo1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)phenyl)-4-(1H-pyrrol-1-yl)-3-(trifluoromethyl)benzamide (11f). Compound 9 (200 mg, 0.5 mmol) was converted to the target compound using general procedure B. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 11f (138.3 mg, 45%) as pale-gray solid. 1H NMR (400 MHz, DMSO-d6) δ 10.62 (s, 1H), 9.64 (s, 1H), 8.79 (d, J = 2.0 Hz, 1H), 8.44 (d, J = 1.2 Hz, 1H), 8.35 (d, J = 8.4 Hz, 1H), 8.16 (s, 1H), 8.05 (dd, J = 8.4 Hz, J = 2.4 Hz, 1H), 7.81 (d, J = 1.6 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.66 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 7.01 (s, 2H), 6.29 (t, J = 2.0 Hz, 2H), 4.71 (d, J = 14.0 Hz, 1H), 4.53 (d, J = 14.0 Hz, 1H), 3.34 (s, 3H), 2.40 (s, 3H), 2.14 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.04, 158.85, 156.93, 153.14, 152.03, 150.11, 141.19, 141.09, 139.94, 137.42, 134.60, 134.12, 132.70, 130.96, 130.69, 129.85, 127.01, 126.42, 126.37, 126.13, 124.53, 124.29, 124.23, 123.13, 122.35, 121.57, 119.74, 119.30, 109.67, 102.86, 46.54, 28.14, 23.12, 16.70. LRMS (ESI) m/z 613 [M + H]+. HRMS (ESI) m/z calculated for C32H27F3N8NaO2+ [M + Na]+ 635.21, found 635.2090. N-(4-Methyl-3-(1-methyl-7-((6-methylpyridin-3-yl)amino)-2-oxo1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)phenyl)-4-(4-methylpiperazin-1-yl)-3-(trifluoromethyl)benzamide (11g). Compound 9 (200 mg, 0.5 mmol) was converted to the target compound using general procedure B. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 11g (187.7 mg, 58%) as a pale-brown solid. 1H NMR (400 MHz, DMSO-d6) δ 10.41 (s, 1H), 9.65 (s, 1H), 8.79 (s, 1H), 8.24 (s, 1H), 8.21 (d, J = 8.4 Hz, 1H), 8.16 (s, 1H), 8.05 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 7.78 (s, 1H), 7.63−7.60 (m, 2H), 7.30 (d, J = 8.4 Hz, 1H), 7.18 (d, J = 8.8 Hz, 1H), 4.70 (d, J = 14.0 Hz, 1H), 4.52 (d, J = 14.0 Hz, 1H), 2.96 (t, J = 4.0 Hz, 4H), 2.47 (m, 4H), 2.40 (s, 3H), 2.23 (s, 3H), 2.13 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.69, 158.93, 157.02, 154.83, 153.22, 152.09, 150.19, 141.12, 140.02, 137.73, 134.68, 132.76, 130.69, 130.07, 126.88, 126.22, 123.62, 122.44, 119.72, 119.25, 102.95, 54.82, 52.68, 46.61, 45.75,

137.50, 135.58, 131.76, 130.88, 130.66, 129.73, 129.30, 128.99, 128.11, 125.28, 124.13, 124.09, 119.66, 119.31, 100.36, 46.58, 27.83, 16.75. LRMS (ESI) m/z 457 [M + H]+. HRMS (ESI) m/z calculated for C22H20F3N6O2+ [M + H]+ 457.16, found 457.1590. General Procedure B for the Synthesis of Compounda 11a−11i. To a solution of 9 (1 equiv) in DMF (0.1 M) was added different carboxylic acids (1.5 equiv), HATU (2 equiv), and DIPEA (3 equiv). The reaction mixture was then stirred for 1 h at room temperature, quenched with water, and diluted with EtOAc. The organic layer was washed with H2O, dried over MgSO4, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel. N-(4-Methyl-3-(1-methyl-7-((6-methylpyridin-3-yl)amino)-2-oxo1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)phenyl)benzamide (11a). Compound 9 (200 mg, 0.5 mmol) was converted to the target compound using general procedure B. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 11a (112.8 mg, 33%) as a pale-brown solid. 1 H NMR (400 MHz, DMSO-d6) δ 10.30 (s, 1H), 9.63 (s, 1H), 8.79 (d, J = 2.8 Hz, 1H), 8.15 (s, 1H), 8.05 (dd, J = 8.4 Hz, J = 2.8 Hz, 1H), 7.96−7.94 (m, 2H), 7.82 (d, J = 2.0 Hz, 1H), 7.63−7.51 (m, 4H), 7.28 (d, J = 8.4 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 4.70 (d, J = 14.4 Hz, 1H), 4.52 (d, J = 14.4 Hz, 1H), 3.33 (s, 3H), 2.40 (s, 3H), 2.12 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 165.21, 158.74, 156.79, 152.99, 151.89, 149.96, 140.90, 139.81, 137.82, 134.61, 134.52, 131.36, 130.45, 130.27, 128.18, 127.40, 125.97, 122.22, 119.42, 118.86, 102.71, 54.69, 46.45, 28.02, 23.01, 16.57. LRMS (ESI) m/z 480 [M + H]+. HRMS (ESI) m/z calculated for C27H26N7O2+ [M + H]+ 480.21, found 480.2145. 4-Methyl-N-(4-methyl-3-(1-methyl-7-((6-methylpyridin-3-yl)amino)-2-oxo-1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)phenyl)-3-(trifluoromethyl)benzamide (11b). Compound 9 (200 mg, 0.5 mmol) was converted to the target compound using general procedure B. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 11b (114.5 mg, 48%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 10.46 (s, 1H), 9.63 (s, 1H), 8.79 (d, J = 2.4 Hz, 1H) 8.25 (s, 1H), 8.16−8.15 (m. 2H), 8.05 (dd, J = 8.4 Hz, J = 2.4 Hz, 1H), 7.78 (d, J = 2.0 Hz, 1H), 7−65−7.62 (m, 2H), 7.31 (d, J = 8.4 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 4.70 (d, J = 14.0, 1H), 4.52 (d, J = 14.0, 1H),3.34 (s, 3H), 2.53 (s, 3H), 2.40 (s, 3H), 2.13 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.79, 158.93, 157.01, 153.20, 152.09, 150.18, 141.13, 140.01, 139.90, 137.65, 134.68, 132.68, 132.48, 131.48, 130.78, 130.71, 128.35, 127.88, 127.59, 127.29, 126.99, 126.20, 125.62, 124.86, 124.81, 124.75, 122.90, 122.42, 120.18, 119.77, 119.30, 102.94, 64.89, 46.62, 28.21, 23.20, 18.79, 18.77, 16.76. LRMS (ESI) m/z 562 [M + H]+. HRMS (ESI) m/z calculated for C29H26F3N7NaO2+ [M + Na]+ 584.20, found 584.1993. N-(4-Methyl-3-(1-methyl-7-((6-methylpyridin-3-yl)amino)-2-oxo1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)phenyl)-3-morpholino-5-(trifluoromethyl)benzamide (11c). Compound 9 (200 mg, 0.5 mmol) was converted to the target compound using general procedure B. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 11c (97.9 mg, 30%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.64 (s, 1H), 8.79 (d, J = 2.4 Hz, 1H), 8.16 (s, 1H), 8.05 (dd, J = 8.4 Hz, J = 2.4 Hz, 1H), 7.75 (d, J = 2.0 Hz, 1H), 7.72 (s, 1H), 7.65 (s, 1H), 7.63 (d, J = 2.4 Hz, 1H), 7.39 (s, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 4.70 (d, J = 14.0 Hz, 1H), 4.53 (d, J = 14.4 Hz, 1H), 3.78−3.76 (m, 4H), 3.34 (s, 3H), 3.31−3.28 (m, 4H), 2.40 (s, 3H), 2.14 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 164.14, 158.83, 156.91, 153.11, 152.00, 151.33, 150.09, 141.02, 139.91, 137.48, 136.37, 134.57, 130.74, 130.60, 130.20, 129.88, 126.11, 125.35, 122.64, 122.33, 119.80, 119.36, 117.00, 113.73, 113.69, 113.32, 102.83, 65.76, 47.55, 46.51, 28.11, 23.10, 16.66. LRMS (ESI) m/z 633 [M + H]+. HRMS (ESI) m/z calculated for C32H31F3N8NaO3+ [M + Na]+ 655.24, found 655.2336. 3-(4-Hydroxypiperidin-1-yl)-N-(4-methyl-3-(1-methyl-7-((6methylpyridin-3-yl)amino)-2-oxo-1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)phenyl)-5-(trifluoromethyl)benzamide (11d). Compound 9 (200 mg, 0.5 mmol) was converted to the target compound using general procedure B. The resulting crude product 8368



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28.22, 23.21, 16.76. LRMS (ESI) m/z 646 [M + H]+. HRMS (ESI) m/z calculated for C33H35F3N9O2+ [M + H]+ 646.29, found 646.2866. N-(4-Methyl-3-(1-methyl-7-((6-methylpyridin-3-yl)amino)-2-oxo1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)phenyl)-4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)benzamide (11h). Compound 9 (200 mg, 0.5 mmol) was converted to the target compound using general procedure B. The resulting crude product was purified by flash column chromatography on silica gel (10% MeOH/DCM) to afford 11h (122.8 mg, 37%) as a gray solid. 1H NMR (400 MHz, CDCl3) δ 8.71 (d, J = 1.6 Hz, 1H), 8.38 (s, 1H), 8.11 (s, 1H), 8.03−7.92 (m, 4H), 7.75 (s, 1H), 7.33 (d, J = 8.8 Hz, 1H), 7.18−7.15 (m, 2H), 7.07 (s, 1H), 4.70 (d, J = 14.4 Hz, 1H), 4.46 (d, J = 14.0 Hz, 1H), 3.70 (s, 2H), 3.48 (s, 3H), 2.54 (m, 11H), 2.32 (s, 3H), 1.99 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 164.30, 159.24, 157.59, 153.18, 152.93, 152.30, 141.89, 140.48, 137.29, 133.61, 133.50, 131.29, 130.70, 128.73, 127.11, 124.83, 122.99, 120.36, 119.64, 102.92, 57.94, 55.10, 53.03, 47.37, 45.92, 28.72, 23.69, 16.74. LRMS (ESI) m/z 660 [M + H]+. HRMS (ESI) m/z calculated for C34H37F3N9O2+ [M + H]+ 660.30, found 660.3019. 4-((3-(Dimethylamino)pyrrolidin-1-yl)methyl)-N-(4-methyl-3-(1methyl-7-((6-methylpyridin-3-yl)amino)-2-oxo-1,4dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)phenyl)-3(trifluoromethyl)benzamide (11i). Compound 9 (200 mg, 0.5 mmol) was converted to the target compound using general procedure B. The resulting crude product was purified by flash column chromatography on silica gel (10% MeOH/DCM) to afford 11i (138.9 mg, 41%) as a pale-gray solid. 1H NMR (400 MHz, CD3ODd4) δ 8.78 (d, J = 2.4 Hz, 1H), 8.27 (s, 1H), 8.18 (d, J = 8.4 Hz, 1H), 8.12−8.09 (m, 2H), 7.92 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 2.0 Hz, 1H), 7.60 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 8.4 Hz, 1H), 4.78 (d, J = 14.4 Hz, 1H), 4.59 (d, J = 14.4 Hz, 1H), 3.95−3.86 (m, 2H), 3.76 (s, 1H), 3.44 (s, 3H), 2.97−2.90 (m, 2H), 2.81−2.76 (m, 8H), 2.49 (s, 3H), 2.34−2.27 (m, 1H), 2.23 (s, 3H), 2.07−1.95 (m, 1H). 13C NMR (100 MHz, CD3OD-d4) δ 209.98, 166.69, 160.64, 158.75, 154.78, 154.26, 151.97, 142.47, 142.24, 140.60, 138.78, 136.56, 135.40, 133.28, 132.30, 132.15, 129.14, 126.34, 124.49, 121.82, 120.96, 104.53, 66.62, 56.57, 56.07, 53.56, 41.75, 30.57, 28.94, 27.64, 22.82, 17.11. LRMS (ESI) m/z 674 [M + H]+. HRMS (ESI) m/z calculated for C35H39F3N9O2+ [M + H]+ 674.32, found 674.3172. N-(3-(((2,4-Dichloropyrimidin-5-yl)methyl)amino)-4-methylphenyl)-3-(trifluoromethyl)benzamide (12). To a solution of 2,4dichloro-5-(chloromethyl)pyrimidine (5.0 g, 25.6 mmol) and N-(3amino-4-methylphenyl)-3-(trifluoromethyl)benzamide (7.5 g, 25.6 mmol) in acetone (30 mL) was added NaI (4.6 g, 30.72 mmol), K2CO3 (5.3 g, 38.40 mmol). The mixture was stirred at 50 °C for 14 h. The reaction was cooled to rt, filtered, and extracted with DCM. The organic layers were dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography (70% EtOAc/hexane) to afford the title compound as a brown solid (8.0 g, 69%). 1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 8.58 (s, 1H), 8.19−8.17 (m, 2H), 7.92 (d, J = 7.6 Hz, 1H), 7.74 (t, J = 7.6 Hz, 1H), 7.06 (d, J = 7.6 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.82 (s, 1H), 5.72 (t, J = 6.0 Hz, 1H), 4.40 (d, J = 5.2 Hz, 2H), 2.15 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 163.75, 160.85, 160.26, 157.43, 145.21, 137.75, 136.13, 131.67, 130.68, 129.93, 129.60, 129.24, 128.92, 127.82, 125.31, 124.15, 124.11, 122.61, 118.35, 109.12, 102.12, 41.77, 17.21. LRMS (ESI) m/z 455 [M + H]+. General Procedure C for the Synthesis of Compounds 15a−c, 15e. To a solution of 12 (1 equiv) in THF (0.1 M) was added different amine (1 equiv) at room temperature. The mixture was stirred at 60 °C for 2 h, then cooled to room temperature, quenched with water, and extracted with DCM. The organic layers were dried over magnesium sulfate, filtered, and concentrated. The residue was diluted with dry THF (5 mL) and treated slowly with triphosgene (0.5 equiv) at 0 °C under nitrogen atmosphere. The mixture was slowly treated with TEA (5 equiv) and stirred at 70 °C for 1 h. The mixture was quenched with water and extracted with DCM. The organic phase was washed with brine, dried over MgSO4, filtered, and concentrated. The resulting residue was diluted with 2-butanol (0.15

M), treated with 6-methylpyridin-3-amine (1.2 equiv), K2CO3 (5 equiv), Xphos (0.2 equiv), and Pd2(dba)3 (0.2 equiv) at room temperature. The mixture was stirred at 100 °C for 2 h, then cooled to room temperature, filtered, and concentrated. The residue was purified by flash column chromatography on silica gel. N-(3-(1-Ethyl-7-((6-methylpyridin-3-yl)amino)-2-oxo-1,4dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-3(trifluoromethyl)benzamide (15a). Compound 12 (0.5 g, 1.10 mmol) was converted to the target compound using general procedure C. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 15a (130.2 mg, 21%) as a pale-gray solid. 1H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 9.62 (s, 1H), 8.79 (d, J = 2.4 Hz, 1H), 8.31 (s, 1H), 8.27 (d, J = 7.6 Hz, 1H), 8.15 (s, 1H), 8.04 (dd, J = 8.4 Hz, J = 2.4 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.79 (t, J = 7.6 Hz, 2H), 7.65 (dd, J = 8.0 Hz, J = 2.0 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 4.72 (d, J = 14.0 Hz, 1H), 4.51 (d, J = 14.0 Hz, 1H), 4.02 (q, J = 6.8 Hz, 2H), 2.40 (s, 3H), 2.13 (s, 3H), 1.22 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.70, 158.85, 156.12, 153.41, 151.33, 150.09, 141.04, 139.81, 137.44, 135.47, 134.56, 131.67, 130.77, 130.60, 129.64, 129.21, 128.89, 128.06, 125.99, 125.19, 124.03, 123.99, 122.48, 122.27, 119.62, 119.05, 102.63, 46.47, 35.78, 23.07, 16.63, 13.16. LRMS (ESI) m/z 562 [M + H]+. HRMS (ESI) m/z calculated for C29H26F3N7NaO2+ [M + Na]+ 584.20, found 584.1996. N-(3-(1-Cyclopropyl-7-((6-methylpyridin-3-yl)amino)-2-oxo-1,4dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-3(trifluoromethyl)benzamide (15b). Compound 12 (0.5 g, 1.10 mmol) was converted to the target compound using general procedure C. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 15b (123.5 mg, 20%) as a pale-gray solid. 1H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 9.66 (s, 1H), 8.88 (s,1H), 8.31 (s, 1H), 8.27 (d, J = 8.0 Hz, 1H), 8.21−8.18 (m, 2H), 7.97 (d, J = 7.6 Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.74 (s, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 4.59 (d, J = 14.0 Hz, 1H), 4.44 (d, J = 14.0 Hz, 1H), 2.77−2.76 (m, 1H), 2.40 (s, 3H), 2.12 (s, 3H), 1.08−1.06 (m, 2H), 0.75−0.69 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 163.81, 159.03, 158.73, 153.07, 152.99, 149.98, 141.14, 139.84, 137.46, 135.56, 134.80, 131.78, 130.86, 130.67, 129.31, 128.99, 128.17, 125.29, 124.14, 124.10, 122.59, 122.35, 119.71, 119.38, 104.11, 46.39, 24.67, 23.18, 16.92, 9.14, 8.90. LRMS (ESI) m/z 574 [M + H]+. HRMS (ESI) m/z calculated for C30H26F3N7NaO2+ [M + Na]+ 596.20, found 596.1996. N-(3-(1-Cyclohexyl-7-((6-methylpyridin-3-yl)amino)-2-oxo-1,4dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-3(trifluoromethyl)benzamide (15c). Compound 12 (0.5 g, 1.10 mmol) was converted to the target compound using general procedure C. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 15c (109.1 mg, 16%) as a gray solid. 1H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H), 9.57 (s, 1H), 8.74 (d, J = 2.0 Hz, 1H), 8.29 (s, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.16 (s, H), 8.03 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.78 (t, J = 8.0 Hz, 1H), 7.74 (s, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 8.4 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 4.66−4.55 (m, 2H), 4.44 (d, J = 14.0 Hz, 1H), 2.40 (s, 3H), 2.09 (s, 3H), 1.82−1.63 (m, 6H), 1.36−1.14 (m, 4H). 13 C NMR (100 MHz, DMSO-d6) δ 163.81, 158.88, 157.42, 153.62, 151.98, 150.46, 141.29, 140.34, 137.48, 135.57, 134.56, 131.78, 130.85, 130.62, 129.77, 129.33, 129.01, 128.16, 126.44, 125.31, 124.15, 124.11, 122.60, 122.32, 119.54, 119.03, 103.80, 54.44, 46.34, 29.19, 28.90, 26.29, 26.19, 25.07, 23.24, 16.86. LRMS (ESI) m/z 616 [M + H]+. HRMS (ESI) m/z calculated for C33H32F3N7NaO2+ [M + Na]+ 638.25, found 638.2465. N-(4-Methyl-3-(7-((6-methylpyridin-3-yl)amino)-2-oxo-1-phenyl1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)phenyl)-3(trifluoromethyl)benzamide (15d). To a solution of 12 (0.5 g, 1.10 mmol) in n-butanol (10 mL) was added aniline (0.1 mL, 1.10 mmol) and TEA (0.32 mL, 2.20 mmol) at room temperature. The mixture was stirred at 120 °C for 3 h, then cooled to room temperature, quenched with water, and extracted with DCM. The organic layers 8369



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were dried over magnesium sulfate, filtered, and concentrated. The residue was dissolved in dry THF (5 mL) and treated slowly with triphosgene (162.8 mg, 0.55 mmol) at 0 °C under nitrogen atmosphere. The mixture was slowly treated with TEA (0.79 mL, 5.50 mmol) and stirred at 70 °C for 1 h. The mixture was quenched with water and extracted with DCM. The organic phase was washed with brine, dried over MgSO4, filtered, and concentrated. The resulting residue was dissolved in 2-butanol (8 mL) and treated with 6-methylpyridin-3-amine (142.6 mg, 1.32 mmol), K2CO3 (760 mg, 5.50 mmol), Xphos (104.8 mg, 0.22 mmol), and Pd2(dba)3 (101.4 mg, 0.22 mmol) at room temperature. The mixture was stirred at 100 °C for 2 h, then cooled to room temperature, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (5−10% MeOH/DCM) to afford the title compound (183.6 mg, 27%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 9.55 (s, 1H), 8.32−8.31 (m, 2H), 8.28− 8.25 (m, 2H), 7.97 (d, J = 7.6 Hz, 1H), 7.89 (s, 1H), 7.79 (t, J = 8 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.55−7.47 (m, 4H), 7.37 (d, J = 7.6 Hz, 2H), 7.32 (d, J = 8.4 Hz, 1H), 6.72(d, J = 8.4 Hz, 1H), 4.90 (d, J = 14.0 Hz, 1H), 4.67 (d, J = 14.0 Hz, 1H), 2.31 (s, 3H), 2.22 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 163.85, 158.51, 157.56, 154.20, 151.37, 149.57, 140.95, 139.37, 137.59, 136.97, 135.60, 134.58, 131.81, 130.99, 130.77, 130.01, 129.77, 129.34, 129.02, 128.87, 128.19, 125.32, 124.98, 124.16, 124.12, 122.61, 121.84, 119.82, 119.23, 102.30, 47.05, 23.09, 16.80. LRMS (ESI) m/z 610 [M + H]+. HRMS (ESI) m/z calculated for C33H26F3N7NaO2+ [M + Na]+ 632.20, found 632.1997. N-(3-(1-Benzyl-7-((6-methylpyridin-3-yl)amino)-2-oxo-1,4dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-3(trifluoromethyl)benzamide (15e). Compound 12 (0.5 g, 1.10 mmol) was converted to the target compound using general procedure C. The resulting crude product was purified by flash column chromatography on silica gel (5−10% MeOH/DCM) to afford 15e (80.9 mg, 12%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 9.58 (s, 1H), 8.59 (d, J = 2.4 Hz, 1H), 8.30 (s, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.19 (s, 1H), 7.97 (d, J = 8 Hz, 1H), 7.83−7.77 (m, 3H), 7.66 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 7.32− 7.31 (m, 5H), 7.25−7.19 (m, 1H), 7.04 (d, J = 8.8 Hz, 1H), 5.26− 5.18 (m, 2H), 4.82 (d, J = 14.4 Hz, 1H), 4.60 (d, J = 14.4 Hz, 1H), 2.38 (s, 3H), 2.15 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.84, 158.89, 156.21, 153.94, 151.71, 150.30, 141.06, 140.13, 138.09, 137.61, 135.59, 134.40, 131.81, 130.89, 130.79, 129.77, 129.65, 129.34, 129.02, 128.27, 128.16, 126.69, 126.59, 126.20, 125.32, 124.17, 124.13, 122.61, 122.29, 119.91, 119.28, 102.56, 46.74, 43.42, 23.21, 16.76. LRMS (ESI) m/z 624 [M + H]+. HRMS (ESI) m/z calculated for C34H28F3N7NaO2+ [M + Na]+ 646.22, found 646.2159. N-(3-(7-Chloro-1-methyl-2-oxo-1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-4-((3-(dimethylamino)pyrrolidin-1-yl)methyl)-3-(trifluoromethyl)benzamide (16). To a solution of 6 (2.0 g, 6.60 mmol) in DMF (13 mL) was added 4((3-(dimethylamino)pyrrolidin-1-yl)methyl)-3-(trifluoromethyl)benzoic acid (3.2 g, 9.90 mmol), HATU (5.0 g, 13.20 mmol), and DIPEA (3.4 mL, 19.80 mmol). The mixture was then stirred for 1 h at room temperature, quenched with water, and diluted with EtOAc. The organic layer was washed with H2O, dried over MgSO4, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (5−10% MeOH/DCM) to afford the title compound (2.6 g, 65%) as a brown solid. 1H NMR (400 MHz, CDCl3) δ 9.03 (s, 1H), 8.10 (s, 1H), 8.06 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.76 (dd, J = 6.4 Hz, J = 2.0 Hz, 1H), 7.23−7.19 (m,1H), 7.01 (d, J = 8.4 Hz, 1H), 4.71 (d, J = 15.6 Hz, 1H), 4.48 (d, J = 15.2 Hz, 1H), 3.77 (q, J = 15.2 Hz, 1H), 3.47 (s, 1H), 2.95−2.92 (m, 1H), 2.72−2.55 (m, 4H), 2.26 (s, 6H), 2.04− 1.93 (m. 1H), 1.83−1.76 (m, 1H), 1.73 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 164.46, 160.21, 158.64, 153.61, 152.56, 142.14, 139.61, 137.79, 133.39, 131.18, 131.11, 130.75, 130.55, 128.38, 128.07, 125.49, 125.09, 125.03, 122.76, 120.87, 119.74, 110.24, 65.36, 55.73, 53.51, 53.47, 46.95, 28.94, 28.67, 16.50. LRMS (ESI) m/z 602 [M + H]+.

N-(3-(7-((6-(4-Acetylpiperazin-1-yl)pyridin-3-yl)amino)-1-methyl-2-oxo-1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-4-((3-(dimethylamino)pyrrolidin-1-yl)methyl)-3(trifluoromethyl)benzamide (17a). Compound 16 (100 mg, 0.17 mmol) was converted to the target compound using general procedure A. The resulting crude product was purified by flash column chromatography on silica gel (10% MeOH/DCM) to afford 17a (74.6 mg, 56%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 9.35 (s, 1H), 8.48 (d, J = 2.4 Hz, 1H), 8.24−8.21 (d, J = 12 Hz, 2H), 8.09 (s, 1H), 7.93−7.89 (m, 2H), 7.78 (s, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 9.2 Hz, 1H), 4.68 (d, J = 14.0 Hz, 1H), 4.50 (d, J = 14.0 Hz, 1H), 3.84−3.73 (m, 2H), 3.54 (m, 4H), 3.46−3.45 (m, 2H), 3.37 (t, J = 5.2 Hz, 2H), 3.32 (s, 3H), 2.79−2.72 (m, 1H), 2.68−2.59 (m, 1H), 2.39−2.36 (m, 1H), 2.13 (s, 3H), 2.08 (s, 6H), 2.04 (s, 3H), 1.92−1.84 (m, 1H), 1.68− 1.57 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 168.28, 163.83, 159.20, 156.92, 154.54, 153.22, 152.14, 141.65, 141.16, 139.02, 137.61, 133.50, 131.55, 130.78, 130.68, 130.54, 130.01, 128.55, 126.86, 126.56, 124.93, 124.87, 119.69, 119.22, 107.12, 64.82, 57.62, 55.36, 53.09, 46.60, 45.51, 45.24, 45.19, 43.19, 28.34, 28.11, 21.22, 16.74. LRMS (ESI) m/z 786 [M + H]+. HRMS (ESI) m/z calculated for C40H46F3N11NaO3+ [M + Na]+ 808.36, found 808.3635. 4-((3-(Dimethylamino)pyrrolidin-1-yl)methyl)-N-(3-(7-((6-(4-ethylpiperazine-1-carbonyl)pyridin-3-yl)amino)-1-methyl-2-oxo-1,4dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-3(trifluoromethyl)benzamide (17b). Compound 16 (100 mg, 0.17 mmol) was converted to the target compound using general procedure A. The resulting crude product was purified by flash column chromatography on silica gel (10% MeOH/DCM) to afford 17b (46.5 mg, 34%) as a pale-yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 10.02 (s, 1H), 8.90 (d, J = 2.4 Hz, 1H), 8.35 (dd, J = 8.8 Hz, J = 2.0 Hz, 1H), 8.25−8.21 (m, 3H), 7.90 (d, J = 8.4 Hz, 1H), 7.80 (s, 1H), 7.63 (d, J = 6.8 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 4.73 (d, J = 14.4 Hz, 1H), 4.56 (d, J = 14.0 Hz, 1H), 3.79 (q, J = 15.2, 2H), 3.62−3.55 (m, 4H), 3.36 (s, 1H), 2.80−2.79 (m, 1H), 2.68−2.64 (m, 1H), 2.61−2.58 (m, 1H), 2.42−2.34 (m, 2H), 2.37−2.34 (m, 4H), 2.14 (s, 3H), 2.11 (s, 6H), 1.93−1.85 (m, 1H), 1.70−1.62 (m, 1H), 1.00 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 166.36, 163.78, 158.48, 157.05, 153.06, 151.91, 145.90, 141.58, 140.99, 138.23, 137.89, 137.56, 133.45, 131.51, 130.74, 130.66, 130.50, 126.51, 125.21, 123.81, 119.71, 119.18, 103.83, 64.74, 57.47, 55.29, 53.00, 52.68, 52.02, 51.41, 46.63, 46.50, 43.05, 28.24, 16.69, 11.76. LRMS (ESI) m/z 800 [M + H]+. HRMS (ESI) m/z calculated for C42H49F3N10NaO3+ [M + Na]+ 822.38, found 822.4006. 4-((3-(Dimethylamino)pyrrolidin-1-yl)methyl)-N-(4-methyl-3-(1methyl-7-((1-methyl-1H-pyrazol-4-yl)amino)-2-oxo-1,4dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)phenyl)-3(trifluoromethyl)benzamide (17c). Compound 16 (100 mg, 0.17 mmol) was converted to the target compound using general procedure A. The resulting crude product was purified by flash column chromatography on silica gel (10% MeOH/DCM) to afford 17c (55.3 mg, 49%) as a pale-gray solid. 1H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H), 9.41 (s, 1H), 8.25−8.21 (m, 2H), 8.09 (s, 1H), 7.90 (d, J = 8 Hz, 1H), 7.84 (s, 1H), 7.77 (s, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.50 (s, 1H), 7.30 (d, J = 8 Hz, 1H), 4.67 (d, J = 13.6 Hz, 1H), 4.49 (d, J = 14.0 Hz, 1H), 3.84−3.73 (m, 5H), 3.33 (d, J = 7.6 Hz, 3H), 2.80−2.73 (m, 1H), 2.68−2.64 (m, 1H), 2.62−2.58 (m, 1H), 2.40−2.36 (m, 1H), 2.13 (s, 3H), 2.09 (s, 6H), 1.92−1.84 (m, 1H), 1.69−1.61 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 163.82, 158.60, 157.09, 152.23, 141.64, 141.21, 137.60, 133.51, 131.55, 130.77, 130.67, 130.54, 129.72, 126.87, 126.57, 125.49, 124.93, 123.23, 122.76, 120.18, 119.65, 119.21, 64.82, 57.59, 55.36, 53.08, 46.60, 43.17, 28.31, 16.75. LRMS (ESI) m/z 663 [M + H]+. HRMS (ESI) m/z calculated for C33H37F3N10NaO2+ [M + Na]+ 685.30, found 685.2955. N-(3-(7-((1,3-Dimethyl-1H-pyrazol-5-yl)amino)-1-methyl-2-oxo1,4-dihydropyrimido[4,5-d]pyrimidin-3(2H)-yl)-4-methylphenyl)-4((3-(dimethylamino)pyrrolidin-1-yl)methyl)-3-(trifluoromethyl)benzamide (17d). Compound 16 (100 mg, 0.17 mmol) was converted to the target compound using general procedure A. The 8370



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resulting crude product was purified by flash column chromatography on silica gel (10% MeOH/DCM) to afford 17d (71.0 mg, 62%) as a pale-gray solid. 1H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H), 9.28 (s, 1H), 8.25 (s, 1H), 8.23 (d, J = 8.4 Hz, 1H), 8.10 (s, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.78 (s, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 6.02 (s, 1H), 4.68 (d, J = 14.0 Hz, 1H), 4.51 (d, J = 14.0 Hz, 1H), 3.79 (q, J = 14.8 Hz, 2H), 3.60 (s, 3H), 3.28 (s, 3H), 2.84−2.81 (m, 1H), 2.69−2.54 (m, 3H), 2.43−2.40 (m, 1H), 2.13 (s, 9H), 2.11 (s, 3H), 1.94−1.85 (m, 1H), 1.71−1.63 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 163.86, 159.25, 157.24, 153.32, 152.08, 145.05, 141.63, 141.12, 138.34, 137.63, 133.54, 131.58, 130.83, 130.72, 130.59, 126.90, 126.60, 125.51, 124.96, 124.91, 122.78, 119.75, 119.26, 103.08, 98.01, 64.81, 57.50, 55.35, 53.07, 46.54, 43.07, 35.06, 28.22, 28.03, 16.76, 13.77. LRMS (ESI) m/z 677 [M + H]+. HRMS (ESI) m/z calculated for C34H39F3N10NaO2+ [M + Na]+ 699.31, found 699.3107. Cell Culture. OCI-AML3 and U937 cells were purchased from DSMZ (Braunschweig, Germany) and Korean Cell Line Bank (Seoul, Korea), respectively. All cells used in this study were grown in RPMI media supplemented with 10% FBS and 1% antibiotics (Welgene, Korea). Parental Ba/F3 cells were cultured in the presence of 1 ng/ mL of IL-3 as an extra supplement. Ba/F3-NRAS-G12D cells were established following by previously described procedure.27 Antiproliferation Assay. In the case of suspension cells, 1 × 104 cells per well were seeded in a 96-well plate. After 4 h stabilization of cells, compound was treated to the cells with 1:3 serial dilution in DMSO. After 72 h, the viability of cells was determined by CellTiterGlo reagent (G7572, Promega, USA). Dose−response curve was fitted and GI50 values were calculated using Graphpad prism 5.0 software. All assays were performed in duplicate, and standard deviation (SD) was determined from three independent experiments. ACK1/GCK siRNA (200 nM of each) or 400 nM scramble siRNA (AccuTarget, Bioneer, Korea) were transfected into 5 × 106 OCIAML3 cells using Amaxa Nucleofector (Lonza, USA) following manufacturer’s instruction. After 10 h transfection, the cells were seeded in 96-well plates and treated with indicated compounds for 36 h. Cell proliferation was measured using CellTiter-Glo Reagent (Promega, USA). Molecular Docking Atudy. To establish the homology model, the type II structure of ABL1 (PDB 3IK3) was adopted for GCK modeling28 and ABL2 (PDB 3GVU) was utilized for ACK1 using BIOVIA Discovery Studio 4.5.29 The protein structures were minimized using the Protein Preparation Wizard by applying an OPLS force field. For the grid generation, the binding site of inhibitor was defined as the centroid of the ATP binding site. Docking study of a compound onto the ATP-binding site of the kinases was carried out using the Schrödinger docking program, Glide. The best-docked poses were selected as the lowest Glide score. Protein Expression and Purification. The ACK1 kinase domain encompassing residues 115−389 with an N-terminal tabacco etch virus cleavable His6 tag was subcloned into pFastBac1 vector. Recombinant baculoviruses were prepared by using the Bac-to-Bac system protocol (Invitrogen). The ACK1 viruses were coinfected in Sf9 cells with a baculovirus expressing PTPN1 tyrosine phosphatase in order to generate the nonphosphorylated protein. Infected cells were harvested 72 h postinfection by centrifugation and pellets were stored at −80 °C until purification. Purification was conducted at 4 °C. Cell pellets were lysed by resuspension in buffer containing 25 mM Hepes, pH 7.5, 300 mM NaCl, 0.5 mM TCEP, 10% glycerol, 20 mM imidazole, and EDTAfree complete protease inhibitor tablet (Roche) followed by centrifugation at 35000g for 1 h. The supernatant was loaded with 5 mL of Ni-NTA resin. The resin was washed with lysis buffer and the bound protein eluted using elution buffer (25 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM TCEP, 10% glycerol, 250 mM imidazole). The eluted protein was treated with TEV protease during overnight dialysis against lysis buffer. The cleaved protein was passed through the Ni-NTA resin again, and the flow-through fraction was collected. ACK1 protein was further purified on a Superdex200 size exclusion column equilibrated with 25 mM HEPES, pH 7.7, 300 mM NaCl, 20

mM MgCl2, 0.5 mM TCEP, and 10% glycerol. Fraction containing ACK1 protein was concentrated to 5−6 mg/mL for crystallization. Crystallization, 10d Soaking, and Data Collection. Crystallization was accomplished by the hanging-drop vapor diffusion method at 18 °C. Apo crystals of human ACK1 were obtained from a 4 μL drop consisting of 2 μL of well solution [24−30% (w/v) PEG 3350, 100 mM Bis-tris propane, pH 6.1−6.7, 200 mM ammonium sulfate, and 10 mM DTT] and 2 μL of protein. The crystals grew within 2−3 days. A cocrystal in complex with 10d was obtained by soaking the apo crystal in 125 μM 10d for 24 h in the presence of 28% PEG 3350, 100 mM Bis-tris propane, pH 6.1, 200 mM ammonium sulfate, and 10 mM DTT. Crystals were cryoprotected by dipping them into a solution of mother liquid and 20% glycerol (v/v). Diffraction data was collected on the 5C beamline at the Pohang Accelerator Laboratory (Pohang, South Korea). Date processing and scaling were performed using HKL2000 (HKL Research, Inc., Charlottesville, VA).30 Structure Determination and Refinement. Molecular replacement was used to determine the structure of ACK1 in complex with 10d using a previously solved structure (PDB 4EWH) as a search model with water and ligand removed. Rigid body refinement, followed by simulated annealing at 5000 K, was conducted using PHENIX (Python-Based Hierachical Environment for Integrated Xtallography).31 Subsequently, refinement was conducted in alternating cycles of manual model building in COOT (Crystallographic Object Oriented Toolkit),32 followed by refinement in PHENIX until the R factors converged. Data collection and refinement statistics are shown in Table 7. The coordinate and structural factors of the complex structure have been deposited in the Protein Data Bank (PDB 5ZXB). Molecular structure images were generated using the PyMOL Molecular Graphics System. Western Blot. Cells (2 × 106 cells) were treated with indicated compounds for 2 h and briefly washed with ice-cold PBS once. Thereafter, cells were subjected to lysis in a NP40 buffer (50 mM Tris-HCl pH7.5, 1% NP40, 1 mM EDTA, 150 mM NaCl, 5 mM Na3VO4 and 2.5 mM NaF) containing 1× protease inhibitor cocktail (Roche). The equal amount of lysate was separated by SDS-PAGE gel and transferred to PVDF membrane. All primary antibodies were uniformly prediluted in TBS-T at 1:1000 (v/v) except for actin (1:5000), while all secondary antibodies were prediluted in 5% skim milk (in TBS-T) at 1:5000. All primary antibody used for phosphoform of p70S6K1 (Thr421/Ser424, no. 9204), AKT (S473, no. 9171), pJNK (T183/Y185 no. 9251) p-p38 (Thr180/Tyr182 no. 9216), and cleaved caspase-3 (no. 9661) were purchased from Cell Signaling Technologies (Massachusetts, USA). Antibodies for β-actin (SC47778), cleaved PARP (SC-7150), and HRP-conjugated secondary antibodies for rabbit and mouse (SC-2305, SC-2318) were products of Santa Cruz Biotech (California, USA). Flow Cytometric Analysis. Cells (2 × 106 cells per vial) were prepared and incubated for 24 h with test compounds. For harvesting, cells were centrifuged at 1000 rpm for 2 min. After brief ice-cold PBS washing, the cells were stained with Alexafluor488 conjugated annexin V (A13201) and propidium iodide (P3560) according to the manufacturer’s instruction (Thermo Fisher Scientific, USA). Thereafter, apoptotic cells were determined by FACS Canto II (BD Biosciences, USA). Apoptosis and Cell-Cycle Analysis. For apoptosis assay, cells (2 × 106 cells per vial) were prepared and incubated for 24 h with test compounds. Cells were centrifuged at 1000 rpm for 2 min. After icecold DPBS washing, the cells were stained with Alexafluor488 conjugated annexin V (A13201) and propidium iodide (P3560) according to the manufacturer’s instruction (Thermo Fisher Scientific, USA). Thereafter, apoptotic cells were determined by FACS Canto II (BD Biosciences, USA). For cell-cycle analysis, cells were treated with compounds for 24 h and then fixed in 70% ethanol at −20 °C overnight. Cells were harvested by centrifugation at 500g, washed with ice-cold DPBS, and then suspended in propidium iodide/RNase solution (Cell Signaling Technology, no. 4087) and incubated for 30 min in a dark condition before flow cytometer analysis (Accuri C6, BD Biosciences). 8371



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Anchorage Independent Assay. On the 1.5% bottom agar, cells in the complete media containing 0.7% agar was plated at a density of 5000 cells in 6-well plates. The cells were incubated with indicated compounds for 2 w at 37 °C and 5% CO2. Spheroids were stained using iodonitrotetrazolium chloride (Sigma-Aldrich) for 24 h. The entire area of each well was photographed without magnification, and the average number and size of colonies in each well were counted using ImageJ software. In Vivo Efficacy Study. All animal procedures were approved by the KIST Laboratory Animal Facilities and Use Committee and carried out in AAALAC-accredited facilities. Female immunodeficient mice (n = 4 per cohort) aged 6 weeks (Orientbio Inc., Korea) received 3 × 106 Ba/F3-NRAS-G12D cells by lateral tail vein injection. Following treatments were administered for each cohort: vehicle alone, 15 or 25 mg/kg/day 10k, and 8 mg/kg/day GNF-7. Oral administration was initiated on day 6 for Ba/F3-NRAS-G12D cells. 10k formulated in 5% NMP, 30% PEG E400, 15% solutol, and 50% 0.05 M citric acid was administered for 8 weeks (po, qd). Actuarial survival was analyzed by the Kaplan−Meier methodology. For xenograft mouse model, OCI-AML3 cell line (1 × 106 cells) was subcutaneously injected into right flanks of mice. At day 21, OCIAML-3 (n = 5 per cohort) implanted mice received 15 or 25 mg/kg/ day 10k, GNF7 (8 mg/kg/day), or vehicle alone for 3 weeks (po, qd). Mouse weight and tumor size were measured once every 2 days. To determine tumor volume by external caliper, the greatest longitudinal diameter (length) and the greatest transverse diameter (width) were determined. Tumor volumes were calculated by the modified ellipsoidal formula. Significant differences between values were determined using the Log-rank (Mantel-Cox) test for trend. P values 0.05 were assigned to be significant. tumor volume =

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Author Contributions

Hanna Cho and Injae Shin contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Korea Institute of Science and Technology (KIST), the KU-KIST Graduate School of Converging Science and Technology Program, a grant (D33400) from the Korea Basic Science Institute, Support for Candidate Development Program (NRF2016M3A9B5940991), and Pioneer Research Center Program (NRF-2014M3C1A3051476) of the National Research Foundation of Korea funded by the Ministry of Science and ICT.

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ABBREVIATIONS USED ACK1, activated Cdc42-associated tyrosine kinase 1; GCK, germinal center kinase; AML, acute myeloid leukemia; PARP, poly(ADP-ribose) polymerase; mTOR, mammalian target of rapamycin complex; Xphos, 2-dicyclohexylphosphino-2′,4′,6′triisopropylbiphenyl; Pd2(dba)3, tris(dibenzylideneacetone)dipalladium(0); HATU, 1-[Bis(dimethylamino)methylene]1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; PTPN1, tyrosine-protein phosphatase nonreceptor type 1; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; TEV, tabacco etch virus; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid

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ASSOCIATED CONTENT

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00882.

(1) Wilson, C. Y.; Tolias, P. Recent advances in cancer drug discovery targeting RAS. Drug Discovery Today 2016, 21, 1915−1919. (2) (a) Johnson, D. B.; Smalley, K. S.; Sosman, J. A. Molecular pathways: targeting NRAS in melanoma and acute myelogenous leukemia. Clin. Cancer Res. 2014, 20, 4186−4192. (b) Weisberg, E.; Nonami, A.; Chen, Z.; Liu, F.; Zhang, J.; Sattler, M.; Nelson, E.; Cowens, K.; Christie, A. L.; Mitsiades, C.; Wong, K.-K.; Liu, Q.; Gray, N.; Griffin, J. D. Identification of Wee1 as a novel therapeutic target for mutant RAS-driven acute leukemia and other malignancies. Leukemia 2015, 29, 27−37. (c) Sachs, Z.; LaRue, R. S.; Nguyen, H. T.; Sachs, K.; Noble, K. E.; Hassan, N. A. M.; Diaz-Flores, E.; Rathe, S. K.; Sarver, A. L.; Bendall, S. C.; Ha, N. GA.; Diers, M. D.; NOlan, G. D.; Shannon, K. M.; Largaespada, D. A. NRASG12V oncogene facilitates self-renewal in a murine model of acute myelogenous leukemia. Blood 2014, 124, 3274−3283. (d) Li, Q.; Haigis, K. M.; McDaniel, A.; Harding-Theobald, E.; Kogan, S. C.; Akagi, K.; Wong, J. C.; Braun, B. S.; Wolff, L.; Jacks, T.; Shannon, K. Hematopoiesis and leukemogenesis in mice expressing oncogenic NrasG12D from the endogenous locus. Blood 2011, 117, 2022−2032. (e) Li, Q.; Bohin, N.; Wen, T.; Ng, V.; Magee, J.; Chen, S. C.; Shannon, K.; Morrison, S. J. Oncogenic Nras has bimodal effects on stem cells that sustainably increase competitiveness. Nature 2013, 504, 143−147. (3) Nonami, A.; Sattler, M.; Weisberg, E.; Liu, Q.; Zhang, J.; Patricelli, M. P.; Christie, A. L.; Saur, A. M.; Kohl, N. E.; Kung, A. L.; Yoon, H.; Sim, T.; Gray, N. S.; Griffin, J. D. Identification of novel therapeutic targets in acute leukemias with NRAS mutations using a pharmacologic approach. Blood 2015, 125, 3133−3143. (4) Shah, A.; Andersson, T. M. L.; Rachet, B.; Björkholm, M.; Lambert, P. C. Survival and cure of acute myeloid leukaemia in England, 1971−2006: a population−based study. Br. J. Haematol. 2013, 162, 509−516.

In vitro selectivity profiling of 10k at 1 μΜ against 351 human kinases, antiproliferative activity of selected compounds in ACK1/GCK double knockdown OCIAML3 cells, dose-dependent induction of apoptosis by compound 10k, FACS analysis for the selected compounds in Ba/F3-NRAS-G12D and OCI-AML3 cell lines (PDF) PDB-formatted coordinates for computational models (ZIP) Molecular formula strings (XLSX) Accession Codes

PDB ID code for ACK1-10d complex is 5ZXB. Authors will release the atomic coordinates and experimental data upon article publication.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +82-2-958-6437. E-mail: [email protected], tbsim@ korea.ac.kr. ORCID

Wooyoung Hur: 0000-0002-8034-0501 Nathanael S. Gray: 0000-0001-5354-7403 Taebo Sim: 0000-0003-3015-2059 8372



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