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Optimization of a Novel Series of Ataxia-Telangiectasia Mutated (ATM) Kinase Inhibitors as Potential Radiosensitizing Agents Jaeki Min, Kexiao Guo, Praveen K. Suryadevara, Fangyi Zhu, Gloria Holbrook, Yizhe Chen, Clementine Feau, Brandon M Young, Andrew Lemoff, Michele C. Connelly, Michael B. Kastan, and R. Kiplin Guy J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01092 • Publication Date (Web): 03 Dec 2015 Downloaded from http://pubs.acs.org on December 6, 2015

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Journal of Medicinal Chemistry

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Optimization of a Novel Series of Ataxia-Telangiectasia Mutated (ATM) Kinase Inhibitors as Potential Radiosensitizing Agents

Jaeki Min†,#, Kexiao Guo%,§,#, Praveen K. Suryadevara†, Fangyi Zhu†, Gloria Holbrook†, Yizhe Chen†, Clementine Feau†, Brandon M. Young†, Andrew Lemoff†, Michele C. Connelly†, Michael B. Kastan§, and R. Kiplin Guy*†

†

Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, Tennessee 38105, United States %

Department of Oncology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, Tennessee 38105, United States

§

Department of Pharmacology and Cancer Biology, Duke Cancer Institute, Duke University School of Medicine, 422 Seeley Mudd Building, Durham, NC 27710, United States #

Contributed equally to this work

* Author to whom correspondence should be addressed; e-mail: [email protected]; Fax: 901-595-5715

ACS Paragon Plus Environment


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ABSTRACT We previously reported a novel inhibitor of the ataxia-telangiectasia mutated (ATM) kinase, which is a target for novel radiosensitizing drugs. While our initial lead, compound 4, was relatively potent and non-toxic, it exhibited poor stability to oxidative metabolism and relatively poor selectivity against other kinases.

The current study focused on balancing potency and selectivity with

metabolic stability through structural modification to the metabolized site on the quinazoline core. We performed extensive structure-activity and structure-property relationship studies on this quinazoline ATM kinase inhibitor in order to identify structural variants with enhanced selectivity and metabolic stability. We show that while the C-7-methoxy group is essential for potency, replacing the C-6-methoxy group considerably improves metabolic stability without affecting potency. Promising analogs 20, 27g and 27n were selected based on in vitro pharmacology and evaluated in murine pharmacokinetic and tolerability studies.

Compound 27g possessed

significantly improve pharmacokinetics relative to 4. Compound 27g was also significantly more selective against other kinases than 4. Therefore, 27g is a good candidate for further development as a potential radiosensitizer.


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INTRODUCTION ATM kinase, a member of the phosphatidylinositol 3-kinase (PI3K)-related kinase (PIKK) family,1 plays a central role in maintaining genome integrity by regulating the detection and repair of DNA double-strand breaks (DSBs).2-4 Unattended DSBs can lead to cell death, genomic instability, and tumorigenesis.5-7 The genetic disorder ataxia-telangiectasia (A-T), which results from mutations in the ATM gene, is characterized by genome instability, cerebral and thymic degeneration, immunodeficiency, radiosensitivity, and predisposition to cancer.8 Cells derived from A-T patients show abnormal cell cycle arrest in G1, S, and G2 phases; increased chromosomal breakage; and hypersensitivity to ionizing radiation and radiomimetic drugs. Loss of ATM kinase activity by gene mutation, small molecule inhibition, or gene expression down-regulation causes significant increases in the sensitivity of virtually every cell type to clinically relevant doses of irradiation, making the ATM kinase an attractive target for clinical radiosensitization.9, 10

ATM exists as an inactive dimer in unstressed cells. In response to ionizing radiation (IR) or other treatments that introduce DSBs, ATM kinase is rapidly activated by intermolecular autophosphorylation of ATM-serine 1981, leading to dimer dissociation.11 Activated ATM then phosphorylates a series of downstream substrates that regulate IR-induced cell cycle arrest in G1 phase (e.g., p53, Mdm2, and CHK2), S phase (e.g., NBS1, Smc1, BRCA1, and FancD2), and G2 phase (e.g., BRCA1 and Rad17).12 ATM phosphorylation of these and other targets enables optimal cellular response to DNA damage, while blockade of this signaling pathway by ATM inhibition results in significant radiosensitization.10, 13, 14



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4 Locally applied radiotherapy is a key component of treatment for a large percentage of cancers, but its efficacy can be limited by toxicity to normal tissues or resistance from tumor cells.10 Systemic administration of an effective sensitizer can complement radiotherapy by either lowering the dose of radiation needed to achieve a therapeutic effect, or enhancing efficacy toward resistant tumors. Since external beam radiation therapy is typically delivered locally to tumor sites in 1-2 Gy dose daily fractions over a period of weeks, the ideal radiosensitizer would possess a pharmacokinetic (PK) profile amenable to daily administration in conjunction with standard radiation dosing. Additionally, it must quickly yield efficacious drug concentrations in tissue, maintain exposure for several hours, and then rapidly clear from the body. To speed onset and reduce absorption-induced variability, parenteral dosing is preferred, but oral dosing could also be effective if absorption kinetics were predictable and relatively uniform. Only a handful of ATM inhibitors have been reported to date (Figure 1).15 The well-known nonspecific kinase inhibitors wortmannin and caffeine inhibit ATM, but are not particularly useful either as pharmacological probes or as leads for drug development due to poor selectivity.16 Compound 1 (KU55933)17, a relatively selective ATM inhibitor developed by Kudos Pharmaceuticals, was shown to sensitize human cancer cells to IR, but lacked oral bioavailability. Optimization of 1 led to the second generation compound 2 (KU60019)18 with a 10-fold better potency and oral availability. These compounds were effective in blocking the radiation-induced phosphorylation of key ATM targets in cells in vitro.18-20 However, these two inhibitors have not progressed into the clinic, likely due to challenges with their potency and pharmacological properties. Recently, compound 3 (NVPBEZ235)21, a PI3K/mTOR inhibitor currently in Phase I clinical trials, was reported to be an inhibitor of ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs), thus blocking both nonhomologous end joining and homologous recombination, the two major pathways of DSB


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5 repair. However, this compound is not progressing in clinical development because of toxicity and poor efficacy. The lack of a clinically viable ATM inhibitor at this time creates a need to develop alternate inhibitors with improved pharmacological and biological characteristics.

We recently reported a reasonably potent and moderately selective ATM inhibitor 4 (CP466722)14, 22

, which was identified through screening of a Pfizer kinase inhibitor collection. This novel

quinazoline inhibitor blocked ATM activity in cellular assays with an IC50 of 370 nM, equivalent to the first generation Kudos Pharmaceuticals compound, 1, and roughly 5-fold less potent against cells than the second generation compound, 2. We pursued a hit-to-lead process, including in vivo PK and pharmacodynamics (PD) studies to determine if the series could offer advantages over the other two existing ATM inhibitors.

To define the timescale on which 4 works in cells and thus define the desired exposure curve in vivo, cellular PD experiments were performed. HeLa cells were exposed to IR in the presence of varying concentrations of 4, and the phosphorylation of ATM substrates was monitored over time. We found that compound 4 is a reversible inhibitor of ATM that rapidly reaches maximal cellular effect, with 4 h or less of treatment duration being sufficient to enhance the IR-sensitivity of HeLa cells. Importantly, clonogenic survival assays with IR-treated cells from A-T patients do not show much difference in the presence or absence of 4, suggesting that the radiosensitization was in fact due to ATM inhibition and not an off-target effect.14

Below, we describe detailed structure-activity and

structure-property relationship studies (Figure 3) for this lead series. Overall, we maintained potency against ATM while significantly improving the metabolic stability of the series, making them suitable candidates for development into radiosensitizing drugs.



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RESULTS Chemistry. The general route used to synthesize most target compounds involved the condensation of 4-chloroquinazoline 11 with triazole 14 (Scheme 1). Compound 11 was synthesized from its 3, 4disubstituted benzoate ester precursor 8 in four steps. Nitration of 8 using HNO3 and AcOH at 50 oC afforded the nitrobenzoate in 92% average yield. The nitro group was then reduced to the corresponding amine 9 using one of two methods: FeCl3 and AcOH; or catalytic hydrogenation. Both methods gave an average 90% yield. The quinazoline ring was then installed by condensing amine 9 with formamide at 190 oC with 80% average yield. Heating of compound 10 in excess thionyl chloride afforded 4-chloro quinazoline 11 in 95% yield. The other intermediate 14 was synthesized in one pot by condensing 2-picolinic acid 12 and amino guanidine sulphate 13 at an elevated temperature with a quantitative yield.23-25 The nucleophilic displacement of chlorine in compound 11 by the most nucleophilic nitrogen on the triazole ring of 14 proceeded in the presence of Cs2CO3 in DMF at 100-130 oC, and produced the target compounds in variable yields (50-85%).

Modifications at the C-6 and C-7 positions to produce compounds 15-19 required the synthesis of alternate versions of intermediate 11 from commercially available precursors 8 as shown in Scheme 1. Introducing either electron withdrawing groups or an amine at C-6 or C-7 lowered the synthetic yields. The target intermediate for 19 was obtained from the etherification of 6,7-dihydroxy-4-chloro quinazoline with bromomethylcyclopropane in the presence of potassium carbonate. Compound 20 was synthesized from commercially available chloroquinazoline intermediate 11, as shown in Scheme 1. A set of varied lipophilic and hydrophobic groups 22a-j were introduced at the C-2 position of the quinazoline core from the commercially available quinazoline 21 and triazole 14 as


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7 shown in Scheme 2. Compounds 22a, g, h were synthesized from their commercially available precursors 21a, g, h respectively. Alkyl amines 22b-e were produced by heating compound 21a in NMP in the presence of the corresponding amine at 140 oC. Hydrolysis of 22e in the presence of lithium

peroxide afforded

acid

22f

in moderate

yields.

The

precursor 4-chloro-6,7-

dimethoxyquinazolin-2-amine21j was prepared from 2-amino-4,5-dimethoxy benzoate and cyanamide as described in literature.23-25

Compounds 23a-k (Table 3) were prepared by coupling of commercially available amines, anilines (23a-c) and several synthetic triazole fragments (23d-k) with 4-chloroquinazole intermediate 11g (Scheme 3). The synthesis of alternate triazole fragments was completed for each, using the commercially available carboxylic acids as shown in Scheme 1. Compounds 24a-f were synthesized from commercially available intermediates 11a, b, e, f following a published procedure (Scheme 3).13 Compounds 24b and 24d were prepared from 24a and 24c by catalytic hydrogenation.

Introducing hydrophobic side chains at C-6 was accomplished using the route shown in Scheme 4 to afford compounds 27a-s. Quinazoline 25 was synthesized from commercially available 6, 7dimethoxy-4-quinolone as described in the literature.19-20 In general, the reaction of 25 with the corresponding alcohols using Mitsunobu conditions provided intermediates 26d, 26e, 26g and with halogenated chains using inorganic base produced other intermediates 26a-c, 26f and 26h-o. Subsequent nucleophilic displacement of 14 afforded the target compounds in moderate to good yields.



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8 Compound 27i was synthesized from 25 as described in scheme 5. Compound 25 was treated with N-(3-chloropropyl)-2-((2S,6R)-2,6-dimethylmorpholino)acetamide21

34

(Scheme

S2)

using

potassium carbonate in DMF to prepare 26i with 58% yield, and further treated with 14 to afford compound 27i in 54% overall yield.

A series of carbamates27j-o were prepared from chlorinated propyl carbamate intermediates available either commercially or synthetically (30m-o, Scheme S1) coupled with 25 as shown in Scheme 4. Other C-6 linker analogs 27p-s were synthesized from key intermediate 26j by introducing new substituted chloroformates or trifluororacetyl group after deprotection of Boc group (Scheme 4).

All the compounds used in the studies described below were purified by reverse phase preparative HPLC to greater than 95% purity prior to biological studies. Identity was confirmed by both LC/MS and proton NMR analysis. Purity was confirmed by parallel LC/MS/ELSD/CLND with reported purities calculated as the average by all spectral methods.26 Compounds were formulated as stock solutions in DMSO with the concentrations being confirmed by CLND analysis.

In vitro ADME and Pharmacokinetic Study. Preliminary assessment of the physiochemical properties and membrane permeability of compound 4 revealed reasonable solubility in PBS (20 µM), reasonable permeability (100 x 10-6 cm/s), and good stability in aqueous buffers (t1/2 > 48 h). Intravenous administration of compound 4 to mice at 10 mg/kg and 20 mg/kg doses was well tolerated (Figure 4). Even though the peak plasma concentration was reached at 5 min with a maximal concentration of 4 µM, 1.4-fold higher than the cellular IC90, the total clearance was high


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9 (CL = 160 mL/h, t1/2 = 1 h at 10 mg/kg). In order to determine what drives the rapid clearance of 4, in vitro metabolic stability was measured using mouse, rat, dog, and human liver microsome models (Table 6). Compound 4 displayed rapid clearance in these models (mouse, t1/2 = 0.9 h; CLint = 61.7 mL/min/kg body weight), which correlated well with the PK data. Compound 4 was well distributed into the majority of mouse organs (Tables S2). Most organs had significantly higher peak levels and slower clearance than plasma.

Since the quinazoline methoxy group could be a susceptible metabolic site,27, 28 we evaluated the metabolic stability of 4 and identified the Phase I and II metabolites (Figure 2) through PK studies in mice using qualitative tandem mass spectrometry methods. The observed major metabolite 6 was generated by demethylation of one of the methoxy groups (C-6-methoxy). The minor metabolite 5 resulted from the loss of both methyl groups from the methoxys and a subsequent hydroxylation of the adjacent ring. The observed major Phase II metabolite 7 was the glucoronide of the phenol (6). To confirm their structures, the putative metabolites were synthesized (24b) and compared to the samples (6) obtained from the metabolism experiments. These experiments clearly supported the hypothesis that the initial demethylation step occurs at the C-6-methoxy and suggest this moiety should be modified to reduce demethylation.

Based on these observations, our primary goal became to decrease inhibitor clearance by roughly 4fold, while maintaining the volume of distribution. To identify a compound with these desired PK/PD properties, we sought replacements for the methyl of the C-6-methoxy group (Figure 2, R2) that could block the vulnerable metabolic site, but maintain or improve potency.



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10 Structure-Activity Relationship (SAR). To establish the SAR and optimize the potency and oral bioavailability, compound 4 was divided into four segments (A-D rings). We primarily focused our modifications on the B and D rings (Figure 2). Individual compounds were purified to better than 95% purity as confirmed by UPLC/MS and 1H-NMR.

Each compound was tested for ATM

inhibition using a cell-based assay that we developed.22 All inhibitory potencies are reported as IC50 values representing the mean of two independent assays, each run with internal triplicate for a total of six replicates (Tables 1-5). In order to provide a broader assessment of cytotoxicity, all the compounds were also tested for growth inhibitory activity against four mammalian cell lines (HEK293, HepG2, Raji, and BJ, Table S3) and solubility and permeability data were generated to aid in interpretation of SAR.

Given the in vivo metabolism of 4, we explored SAR for possible replacements of the methoxy groups at the C-6, 7 positions (Table 1). Complete deletion of methoxy groups (15a-d) or the introduction of a chloride (15e-h) at one of these methoxy sites strongly diminished activity. Replacement of both methoxy groups with fluorine (16) or a bridging ethylenedioxy group (17) also abrogated activity.

Due to the importance of the C-6 group in metabolism, a number of other substitutions were attempted. Translocation of the nitrogen in the pyridine ring from the 2-position to the 3- or 4positions (18a-b) caused strong or moderate decreases in potency, respectively. This suggests that the role of the nitrogen is not simply solvation; it may also contribute to an electrostatic interaction or to the conformation of the molecule by enforcing interactions between the C and D rings. Replacement of the pyridyl group with a phenyl ring (18c) showed a moderate decrease in potency,


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11 whereas further substituents at the para position (18d and 18e) or saturated ring systems (18f) diminished activity. Replacement of C-6-methoxy with a bulkier ether was also counterproductive (19). Surprisingly, the methyl group at the C-6 position could be replaced productively with a longer, linear, and more flexible side chain that incorporated a second oxygen atom (20). In summary, most changes to the major site of metabolism (C-6-methoxy) abolished or strongly attenuated activity. Likewise, most changes to the pyridyl ring strongly attenuated activity.

Based on precedent from other structurally similar kinase inhibitors, we attempted to introduce several additional substituents to the C-2 position of the quinazoline (Table 2). All the changes significantly reduced potency (22a-j), with most abolishing activity. Only small groups, such as methyl (22h) and amine (22j), were tolerated. Notably, replacement of the proton with an NH2 group (22j) was fairly well tolerated.

We further analyzed the role of the C and D rings in defining the core pharmacophore, as discussed in Table 3 (23a-k). Modification of a two-ring pyridyltriazole to single rings, such as pyridyl amines (23a-b), led to a complete loss of activity (Table 1). Replacement of the triazole C-ring with a phenyl (23c) led to a significant loss in potency (IC50 = 13 µM), as well as diminished solubility. Likewise, removal of the amino group from the triazole (23d-e) led to a loss in potency. These findings suggest that the nitrogens either form a hydrogen bond within the binding pocket, or intramolecularly enforce the inhibitor conformation. The replacement of the central triazole with pyrazole (23f) only caused a moderate decrease in potency but led to poor solubility. Replacement of the pyridyl ring in this context with a phenyl or substituted phenyl derivatives led to a dramatic or complete loss of activity (23g-i). However, movement of the pyridyl ring from the C-3 to C-4



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12 position on pyrazole was tolerated (23j). As expected, deletion of the pyridyl ring (23k) abolished activity.

Based on the preliminary SAR study, replacing the methoxy groups at C-6 and C-7 with other functional groups was expected to abrogate activity. To understand which methoxy group is more critical for potency, O-benzylated or demethylated derivatives 24a-f were synthesized and tested, with the expectation that either modification would be detrimental if the individual methoxy group was critical. Changes made to the C-7 methoxy resulted in much stronger effects on inhibitory potency than those made to C-6 (Table 4). For example, compound 24e is 20-fold more potent than 24f. This observation was consistent with prior SAR data (Tables 1-3) for this series and suggests that a compound with a C-7 methoxy and variant substitution at the C-6 position could maintain potency and exhibit better in vivo stability. Therefore, a systematic SAR study was undertaken to investigate the effect of C-6 substitutions on overall potency (Table 5).

Introducing a propyl-N, N-dimethylaminoether at the C-6 position (27a) reduced potency (IC50 = 7 µM). Other modifications of the amine, including replacement of the methyl groups with isopropyl groups (27b) or introduction of cyclic amines such as piperidine, pyrrolidine, and morpholine (27cf), maintained potency at roughly the same level with less than 3-fold variation. Interestingly, introducing a 2-methoxy ethoxy group at the C-6 position increased overall potency (27g) compared to other substituted alkyl amine linkers (27a-f). Subsequent studies focused on longer substituents (27i-s). Compound 27j, which has a 3-carbon chain capped with a bulky carbamate, displayed improved potency. However, it showed undesirable PK properties including poor solubility and a short half-life based on a microsomal stability study (data not shown). The majority of other linker


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13 analogs in the series maintained a potency of roughly 1 µM, regardless of substitution patterns. The only notable exceptions to this were the phenyl carbamate and n-pentylcarbamate, both of which were significantly less potent. Based on ATM inhibition activity and physicochemical property data on a total of 71 synthetic analogs, compounds 20, 27g, and 27n were selected as promising candidates for further evaluation.

We next tested the in vitro pharmacological parameters (Table 6), including permeability, solubility, and stability in biological media, for the top three candidates and the original hit compound 4 in order to identify the most suitable candidate for in vivo studies. All three new compounds had reasonable solubility (8 to 30 µM), although it was somewhat reduced for compounds 27g and 27n. All 4 compounds also had reasonable permeability (17 to 430 x 10-6 cm/s) although it was better for compounds 27g and 27n. Therefore no new issues with physiochemistry had arisen during lead optimization. All three new compounds were reasonably stable in buffer and plasma (> 24 h) so no intrinsic stability issues were expected. Likewise, all three new compounds were generally not tightly protein bound (with the exception of 20 by human plasma proteins).

The critical

discriminator between the compounds was predicted oxidative stability, which was measured using microsomal models for mouse, rat, dog, and human.

Compound 27g had significantly better

oxidative stability (t1/2, 2.0 h; CLint, 27.1 mL/min/kg for the mouse and > 4 h / < 12 mL/min/kg for all other species). Compound 20 also exhibited improved liver microsomal stability in rat, mouse, and dog models, but displayed reduced stability in human microsomes. All compound showed potential for saturation of liver metabolism as evidenced by increased stability at high concentrations (20 µM, Table S6) relative to low (0.8 µM, Table 6) in the microsomal models.



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14 In vivo Pharmacokinetics. Based on the result of the microsomal stability studies for top three new candidates, we selected compound 20 and 27g for in vivo pharmacokinetics using a single intravenous administration at 10 or 20 mg/kg to compare clearance directly with initial lead compound 4 (Figure 4). Both compound 20 and 27g were well tolerated in mice at these doses, with no significant evident toxicity based on a Functional Observational Battery after dosing and examining the gross pathology, histopathology, hematology, and clinical chemistries following sacrifice of the animals. The observed Cmax for compounds compound 20 and 27g at 20 mg/kg was 5.8 and 9.5 µM, respectively. These concentrations are 1.5 and 7-fold above the cellular IC50 of compound 20 and 27g, respectively (Table S2). The half-life of compound 20 was slightly lower than that of compound 4 in plasma. However, the AUC (area under the curve) of compound 20 is 1.5 fold higher than that of compound 4, showing a moderate increase in plasma exposure despite slightly faster clearance rate. Compound 27g showed both a prolonged half-life (19 h at 20 mg/kg) and enhanced plasma exposure (up to 30-fold of above that seen for compound 4), which substantiated the hypothesis that replacing the most metabolically labile methoxy groups at C-6 and C-7 significantly improved the in vivo metabolic stability.

To test whether these analogs inhibit ATM-dependent signaling pathways, we monitored the phosphorylation of ATM and its downstream targets, KAP1 and p53. In this cellular assay, MCF7 cells were pre-incubated for 30 min with 1, 4, 20, 27g, and 27n at three concentrations of 1, 5, and 10 µM. Next, the cells were exposed to 10 Gy of IR and phosphorylation of ATM, KAP1, and p53 were monitored using Western blot analysis. Control compounds, 1 and 4 inhibited the phosphorylation of ATM (Ser1981), KAP1 (Ser824) and p53 (Ser15) in a dose-dependent manner. Compounds 20 (Figure S3), 27g, and 27n (Figure 5) exhibited a similar dose-dependent inhibition;


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15 27g and 27n potencies of inhibition were comparable to compound 4, while compound 20 was the weakest inhibitor across all concentrations tested with respect to suppression of phospho p53 formation.

This experiment demonstrates that the optimized compound 27g remains on target for

ATM kinase with a similar affinity as the original lead.

We have shown in our previous publication that transient inhibition of ATM kinase by 4 is sufficient to sensitize cells to IR-induced DNA damage.14 Hence, a clonogenic assay was performed to evaluate the radiosensitization properties of analogs 20, 27g, and 27n (Figure 6). MCF7 cells were pre-treated with DMSO or the test compounds (10 µM), and irradiated with increasing doses of IR (0, 2, and 4 Gy). After 4h, the media was replaced with drug-free media and the cells were cultured for 10 days without selective pressure before the resulting colonies were counted. At a concentration of 10 µM, compounds 20 and 27n did not affect plating efficiency and cell viability compared to the negative control (DMSO), while cells treated with compounds 4 and 1 showed increased radiosensitization as previously published.14 Only 27g exhibited an enhanced radiosensitizing effect in comparison to the DMSO control, with a similar potency and efficacy to 4 and 1. This shows that the optimized compound 27g retains the efficacy of the original lead 4.

In order to determine if any drift in selectivity had occurred during the optimization process, compounds 4 and 27g were screened at fixed concentrations of 3 µM against a diverse panel of kinases and these results were compared with the previously reported kinase specificity of compound 218 as a reference (Table S4 and Figure S2, ScanMax by DiscoveRx Corp.). The original hit compound 4 inhibited both close family members (PI3K & PIKK) and also 106 (26%) kinases out of 451. However, analog 27g displayed significant improvements in overall selectivity by



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16 inhibiting only 41 (9%) kinases out of 451 in the panel. Interestingly, unlike compound 4, 27g did not show any significant inhibition of the MAPK kinases. This suggests that our modifications at the C-6 position not only maintained potency and improved stability, but also enhanced the overall selectivity to ATM. In order to quantitate the selectivity of 27g, we determined the Kd values of compound 27g for the 16 kinases from the panel that exhibited more than 95% inhibition at 3 µM (Table S5, Kd determination by DiscoveRx Corp.). Compound 27g inhibited substrate binding of 10 different kinases (Kd less than 1 µM) with relatively good potency. Two of these – TYK2 (Kd = 41 nM), which belongs to the JAK (Janus activated kinase) family of tyrosine kinases29, 30 and NEK6 (Kd = 130 nM), which belongs to the NIMA family kinases31, 32 – were inhibited with potency sufficient to expect efficacy in cells at the doses examined in this study. Neither is expected to have any effect on the DNA damage responses or the ATM pathway. Therefore, the data show that 27g is significantly more selective within the kinases than the original lead 4.

DISCUSSION AND CONCLUSIONS We previously reported the discovery of compound 4 as a potential inhibitor of ATM kinase. However, the compound showed an undesirable PK profile and was therefore not suitable for further development into a radiosensitizing agent.

We targeted the series to improve metabolic stability,

while maintaining or improving the cellular potency, with a secondary goal of improving selectivity. As shown above, we succeeded in improving the metabolic stability and selectivity of this quinazoline series by optimizing the C-6 and C-7 groups leading to compound 27g, which has significantly overall better exposure in rodents than 4. Western blot analysis of 27g in IR-treated MCF7 cells, demonstrated that it remains on target, reducing ATM autophosphorylation and subsequent phosphorylation of ATM substrates KAP1 and p53, both of which are critical to the


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17 DNA damage response pathway. Compound 27g exhibited significant stability and selectivity, while maintaining potency, and effectively sensitized cells to IR with low toxicity against human fibroblast cell lines. Given our pharmacokinetic data for the original lead that showed significant exposure in the lungs, these compounds could be beneficial for treating non-small-cell lung cancer, where radiosensitization by ATM inhibition may prove important.33

EXPERIMENTAL SECTION All materials were purchased from commercial suppliers and used without further purification. Analytical thin layer chromatography was performed using silica gel 60 F254 plate from EMD. Purification of compounds was done by either normal (Biotage Isolera) or reverse phase column chromatography (Waters prep-HPLC). Structures were determined by NMR spectroscopy and purity was determined by LC-MS/ELSD and HPLC. 1H NMR spectra were recorded on a Bruker 400 or 500 MHz instrument. Data were acquired using Masslynx v.4.1 and analyzed using the Openlynx software suite. The flow was then split to an evaporative light scattering detector (ELSD) and SQ mass spectrometer. The total flow rate was 1.0 mL/min and gradient program started at 90% A (0.1% formic acid in H2O), changed to 95 % B (0.1% formic acid in ACN), then to 90% A. The mass spectrometer was operated in positive-ion mode with electrospray ionization. The conditions were as follows: capillary voltage 3.4 kV, cone voltage 30 V, source temperature 130 °C, desolvation temperature 400 °C, desolvation gas 800 L/h, cone gas 100 L/h. A full scan range from m/z = 110-1000 in 0.2 s was used to acquire MS data. The ELSD-drift tube temperature was set at 52 °C. NMR spectra are recorded on a Bruker 400 or 500 MHz and referenced internally to the residual resonance in CDCl3 (δ 7.26 ppm) for hydrogen and (δ = 77 ppm) for carbon atoms. NMR peaks were assigned by MestRec (4.9.9.6) and MestReNova (5.2.2).



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18

Chemistry General procedure I for the synthesis of 3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (14). A finely grinded mixture of amino guanidine Sulfate (1.8 g, 10.46 mmol) and picolinic acid (1.3 g, 10.56 mmol) was taken into a 100 ml R.B flask. This mixture was heated to 210 °C for about 2 h on a metal bath (Wood's Metal Bath) and a reflux condenser. Reaction mixture first melts into a pale yellow liquid and then solidifies. The resulting product was used directly. Product is a pale yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.63 – 8.59 (m, 1H), 7.95 – 7.86 (m, 2H), 7.44 – 7.38 (m, 1H); 13C NMR (101 MHz, DMSO-d6) δ 158.73, 149.14, 148.18, 137.21, 123.93, 120.86. LCMS(ESI): m/z 162.2 [M+H]+.

1-(6,7-dimethoxyquinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (4). A mixture of 4chloro-6,7-dimethoxyquinazoline (0.733 g, 3.26 mmol) and 3-(pyridin-2-yl)-1H-1,2,4-triazol-5amine 14 (0.526 g, 3.26 mmol) in DMF ( 10 mL) was added cesium carbonate (1.27 g, 3.92 mmol). The resulting mixture was heated at 100 °C overnight. After completion, it was cooled, diluted with water and extracted with chloroform (Forms an emulsion). The organic layer is pooled, washed with brine and dried over MgSO4. The crude product was purified by HPLC. 1H NMR (400 MHz, CDCl3) δ 9.14 (s, 1H), 8.85 (s, 1H), 8.70 (ddd, J = 4.8, 1.9, 1.0 Hz, 1H), 8.08 (dt, J = 7.9, 1.1 Hz, 1H), 7.74 (td, J = 7.7, 1.8 Hz, 1H), 7.34 – 7.25 (m, 2H), 6.98 (s, 2H), 4.08 (s, 3H), 4.03 (s, 3H). . 13C NMR (101 MHz, CDCl3) δ 158.46 , 156.08 , 153.52, 151.34 , 150.74 , 150.24 , 150.15 , 149.32 , 136.59 , 124.26 , 121.35 , 111.65 , 107.06 , 106.14 , 77.21 , 56.42 , 56.26 . HRMS (ESI): [M+H] + calcd for C17H16N7O2+, 350.1365; found, 350.1368.


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19

General procedure II for the synthesis of quinazolines (15-27): To a mixture of substituted 4-Clquinazoline 11(1 mmol) and 3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine 14 (1.2 mmol) in DMF was added Cs2CO3 (1.5 mmol). The reaction mixture was heated at 90-130 oC for 6-18 h, with temperature and time varying depending on the reactivity. The reaction progress was monitored by UPLC and TLC. After the reaction, the mixture was allowed to cool and added to ice cold water and extracted into Chloroform (2X). The combined organic layers were dried over MgSO4 and purified by reverse-phase HPLC (XbridgeTM C18 5 µM OBD, 30 × 50 mm; mobile phase, water with 0.1% formic acid/acetonitrile with 0.1% formic acid; gradient (0−90% AcCN); flow rate, 4.0 mL/min) to afford the desired compound.

3-phenyl-1-(quinazolin-4-yl)-1H-1,2,4-triazol-5-amine (15a). Compounds 15a-h were synthesized from commercially available starting materials as described in the general procedure II. 1H NMR (400 MHz, DMSO-d6) δ 8.56 – 8.48 (m, 2H), 8.16 (d, J = 7.1, 2H), 7.88 – 7.81 (m, 1H), 7.72 (d, J = 8.0, 1H), 7.60 (t, J = 7.6, 1H), 7.53 – 7.46 (m, 2H), 7.46 – 7.40 (m, 1H). LCMS(ESI): m/z 288.3 [M+H]+.

3-(pyridin-2-yl)-1-(quinazolin-4-yl)-1H-1,2,4-triazol-5-amine (15b). 1H NMR (400 MHz, DMSOd6) δ 8.73 – 8.66 (m, 1H), 8.50 (d, J = 11.4, 2H), 8.25 (d, J = 7.4, 1H), 7.96 (td, J = 7.7, 1.8, 1H), 7.84 (dd, J = 11.1, 4.1, 1H), 7.72 (d, J = 8.1, 1H), 7.61 (t, J = 7.6, 1H), 7.47 (ddd, J = 7.5, 4.8, 1.1, 1H). LCMS(ESI): m/z 290.3 [M+H]+. 3-(4-methoxyphenyl)-1-(quinazolin-4-yl)-1H-1,2,4-triazol-5-amine (15c). 1H NMR (400 MHz, DMSO-d6) δ 8.57 – 8.47 (m, 2H), 8.39 (s, 0H), 8.09 (d, J = 8.7, 2H), 7.85 (dd, J = 11.1, 4.1, 1H),



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20 7.73 (d, J = 8.2, 1H), 7.61 (t, J = 7.6, 1H), 7.09 – 7.02 (m, 2H), 3.83 (s, 3H). LCMS (ESI): m/z 319.3 [M+H]+. 1

3-(4-chlorophenyl)-1-(quinazolin-4-yl)-1H-1,2,4-triazol-5-amine (15d).

H NMR (400 MHz,

DMSO-d6) δ 8.54 – 8.46 (m, 2H), 8.17 (d, J = 8.4, 2H), 7.85 (t, J = 7.0, 1H), 7.72 (d, J = 8.2, 1H), 7.64 – 7.52 (m, 3H). LCMS(ESI): m/z 232.7 [M+H]+. 1-(7-chloroquinazolin-4-yl)-3-phenyl-1H-1,2,4-triazol-5-amine (15e).

1

H NMR (400 MHz,

DMSO-d6) δ 8.54 (s, 1H), 8.48 (d, J = 8.0, 1H), 8.15 (d, J = 7.3, 2H), 7.77 (s, 1H), 7.65 (dd, J = 8.7, 2.1, 1H), 7.46 (dt, J = 25.2, 7.1, 3H). LCMS(ESI): m/z 323.7 [M+H]+. 1-(7-chloroquinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (15f). 1H NMR (400 MHz, DMSO-d6) δ 8.68 (d, J = 4.0, 1H), 8.51 – 8.43 (m, 2H), 8.35 (s, 1H), 8.23 (d, J = 7.8, 1H), 7.96 (td, J = 7.7, 1.8, 1H), 7.73 (s, 1H), 7.62 (dd, J = 8.7, 2.1, 1H), 7.46 (dd, J = 7.0, 5.4, 1H). LCMS(ESI): m/z 324.7 [M+H]+. 1-(7-chloroquinazolin-4-yl)-3-(4-methoxyphenyl)-1H-1,2,4-triazol-5-amine (15g). 1H NMR (400 MHz, DMSO-d6 ) δ 8.52 (s, 1H), 8.48 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 8.7, 2H), 7.76 (s, 1H), 7.63 (dd, J = 8.7, 2.1, 1H), 7.04 (d, J = 8.9, 2H), 3.82 (s, 3H). LCMS(ESI): m/z 353.7 [M+H]+. 3-(4-chlorophenyl)-1-(7-chloroquinazolin-4-yl)-1H-1,2,4-triazol-5-amine (15h). 1H NMR (400 MHz, DMSO-d6 ) δ 8.52 (s, 1H), 8.48 (d, J= 8 Hz, 1H), 8.17 (d, J = 8.4, 2H), 7.76 (s, 1H), 7.65 (dd, J = 8.7, 2.1, 1H), 7.59 – 7.53 (m, 2H). LCMS(ESI): m/z 358.2 [M+H]+. 1-(7-chloro-6-nitroquinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (15i).

1

H NMR

(400 MHz, CDCl3) δ 9.15 (d, J=2.8 Hz, 1H), 8.94 (s, 1H), 8.56 – 8.47 (m, 2H), 8.27 (s, 1H), 8.14 – 7.97 (m, 2H), 7.78 – 7.62 (m, 1H), 7.10 (s, 1H). LCMS(ESI): m/z 369.04 [M+H]+. 4-(5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-7-chloroquinazolin-6-amine (15j). A mixture of 15g (150 mg, 0.678 mmol) and SnCl2.2H2O in ethyl acetate (5 mL) was heated to reflux for 2 h.


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21 The mixture was cooled and diluted with EtOAc and washed with saturated NaHCO3 until a basic pH was acquired. The emulsion was filtered through Celite pad and washed with bicarbonate and extracted into EtOAc. The combined layers were evaporated to dryness, dissolved in DMSO, and purified by HPLC.1H NMR (400 MHz, DMSO-d6 ) δ 8.91 (d, J = 5.2 Hz, 3H), 8.62 (s, 2H), 8.47 (dd, J = 17.0, 7.9 Hz, 4H), 7.91 (t, J = 6.6 Hz, 2H), 7.81 (d, J = 6.3 Hz, 3H), 7.79 – 7.70 (m, 1H). LCMS(ESI): m/z339.7 [MH]+. 1-(7-methoxy-6-nitroquinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (15k). 1

H NMR (400 MHz, CDCl3) δ 9.19 (d, J=2.8 Hz, 1H), 9.01 (s, 1H) 8.92 (s, 1H), 8.54 – 8.37 (m, 1H),

8.27 (s, 2H), 8.16 – 7.94 (m, 2H), 7.65 – 7.60 (m, 1H), 6.98 (s, 1H), 3.98 (s, 3H). LCMS(ESI): m/z 365.2 [M+H]+. 4-(5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-7-methoxyquinazolin-6-amine (15l). 1

H NMR (400 MHz, CDCl3) δ δ 9.12 (d, J=2.8 Hz, 1H), 8.50 – 8.42 (m, 1H), 8.35 (s, 2H), 8.23 (s,

2H), 8.14 – 7.89 (m, 2H), 7.75 (s, 1H), 7.69 – 7.60 (m, 1H), 6.98 (s, 1H), 3.98 (s, 3H). LCMS(ESI): m/z 335.1 [M+H]+. 1-(6-nitro-7-(piperazin-1-yl)quinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine

(15m).

Compounds 15m-o were synthesized from corresponding starting materials as described below. Compound 15i (50 mg, 0.13 mmol) and piperazine (58 mg, 0.678 mmol) were mixed together in ethanol (3 mL) and the reaction mixture was heated at 75 oC for 4 h. The reaction mixture was cooled, evaporated, dissolved in DMSO, and purified by HPLC. 1H NMR (400 MHz, CDCl3) δ 8.74 (s, 1H), 8.56 – 8.43 (m, 1H), 8.21 (s, 2H), 8.14 – 7.95 (m, 2H), 7.78 – 7.62 (m, 1H), 7.10 (s, 1H), 3.13 (m, 4H), 3.01 (m, 4H). LCMS(ESI): m/z 419.4 [M+H]+. 1-(6-methoxyquinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (15n).

1

H NMR (400

MHz, CDCl3) δ 9.19 (d, J = 2.8 Hz, 1H), 8.99 (s, 1H), 8.78 (d, J = 4.8 Hz, 1H), 8.17 (d, J = 7.9 Hz,



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22 1H), 8.00 (d, J = 9.2 Hz, 1H), 7.83 (td, J = 7.7, 1.8 Hz, 1H), 7.63 (dd, J = 9.2, 2.8 Hz, 1H), 7.45 – 7.33 (m, 1H), 7.14 (s, 2H), 4.06 (s, 3H). LCMS(ESI): m/z 320.3 [M+H]+. 3-(pyridin-2-yl)-1-(7-(trifluoromethoxy)quinazolin-4-yl)-1H-1,2,4-triazol-5-amine (15o).

1

H

NMR (400 MHz, CDCl3) δ 9.93 (d, J = 9.6 Hz, 1H), 9.09 (s, 1H), 8.85 – 8.70 (m, 1H), 8.22 (dt, J = 7.9, 1.0 Hz, 1H), 7.92 – 7.76 (m, 2H), 7.69 – 7.55 (m, 1H), 7.40 (ddd, J = 7.5, 4.8, 1.2 Hz, 1H), 7.19 (s, 1H). LCMS(ESI): m/z 374.3 [M+H]+. 1-(6,7-difluoroquinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (16). To a mixture of 4chloro-6,7-difluoroquinazoline (100 mg, 0.499 mmol) and

3-(pyridin-2-yl)-1H-1,2,4-triazol-5-

amine (96 mg, 0.598 mmol) in the solvent mixture of CHCl3:DMF (3:1), added cesium carbonate (325 mg, 0.997 mmol). The reaction mixture was stirred at 75 oC overnight, then cooled and dissolved in DMSO and purified by HPLC. 1H NMR (400 MHz, DMSO-d6 ) δ 9.31 (s, 1H), 8.75 (d, J =4.9, 1H), 8.68 (d, J = 7.9, 1H), 8.25 (s, 2H), 8.15–7.99 (m, 2H), 7.84 (td, J = 7.9, 1.2 Hz, 1H), 7.42 (ddd, J = 7.9, 5.0, 1.0 Hz, 1H). LCMS(ESI): m/z 326.3 [M+H]+. 1-(7,8-dihydro-[1,4]dioxino[2,3-g]quinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (17). 1H NMR (400 MHz, CDCl3) δ 9.15 (s, 1H), 8.82 (d, J = 0.9 Hz, 1H), 8.70 (ddd, J = 4.8, 2.0, 1.1 Hz, 1H), 8.14 (dq, J = 8.0, 1.0 Hz, 1H), 7.76 (td, J = 7.8, 1.7 Hz, 1H), 7.42 (s, 1H), 7.33 – 7.25 (m, 1H), 7.16 (s, 2H), 4.46 – 4.32 (m, 5H). LCMS(ESI): m/z 348.3 [M+H]+. 1-(6,7-dimethoxyquinazolin-4-yl)-3-(pyridin-3-yl)-1H-1,2,4-triazol-5-amine (18a). Compounds 18a-f were synthesized from the corresponding starting materials as described in the general procedure II. 1H NMR (400 MHz, DMSO-d6) δ 9.29 (s, 1H), 8.63 (dd, J = 4.7, 1.6 Hz, 2H), 8.39 (s, 2H), 8.14 – 7.72 (m, 1H), 7.57 – 7.49 (m, 2H), 7.24 (s, 1H), 3.95 (s, 6H). LCMS(ESI): m/z 350.3 [M+H]+.


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23 1-(6,7-dimethoxyquinazolin-4-yl)-3-(pyridin-4-yl)-1H-1,2,4-triazol-5-amine (18b). 1H NMR (400 MHz, DMSO-d6) δ 8.95 (d, J = 13.5 Hz, 2H), 8.74 (d, J = 5.8 Hz, 2H), 8.19 (s, 1H), 7.97 (d, J = 6.0 Hz, 2H), 7.46 (s, 2H), 4.03 (d, J = 2.2 Hz, 6H). LCMS(ESI): m/z 350.3 [M+H]+. 1-(6,7-dimethoxyquinazolin-4-yl)-3-phenyl-1H-1,2,4-triazol-5-amine (18c). 1H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.94 (s, 1H), 8.17 – 8.07 (m, 4H), 7.56 – 7.42 (m, 4H), 4.03 (d, J = 2.8 Hz, 6H). LCMS(ESI): m/z 349.3 [M+H]+. 1-(6,7-dimethoxyquinazolin-4-yl)-3-(4-methoxyphenyl)-1H-1,2,4-triazol-5-amine

(18d).

1

H

NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.92 (s, 1H), 8.13 (s, 2H), 8.05 – 7.99 (m, 2H), 7.42 (s, 1H), 7.11 – 7.05 (m, 2H), 4.02 (d, J = 2.4 Hz, 6H), 3.83 (s, 3H). LCMS(ESI): m/z 379.3 [M+H]+. 3-(4-chlorophenyl)-1-(6,7-dimethoxyquinazolin-4-yl)-1H-pyrazol-5-amine (18e). 1H NMR (400 MHz, CDCl3) δ 9.06 (s, 1H), 8.81 (s, 1H), 7.77 – 7.69 (m, 2H), 7.37 – 7.29 (m, 2H), 7.22 – 7.16 (m, 1H), 7.27 (s, 1H), 5.99 (s, 2H), 5.87 (s, 1H), 4.01 (d, J = 2.7 Hz, 6H). LCMS(ESI): m/z 382.1 [M+H]+. 3-cyclohexyl-1-(6,7-dimethoxyquinazolin-4-yl)-1H-1,2,4-triazol-5-amine (18f). Procedure 1 was followed (33.5% yield).1H NMR (400 MHz, CDCl3) δ 8.36 (s, 1H), 8.21 (s, 1H), 7.54 (s, 1H), 6.98 (s, 1H), 3.83 (d, J = 4.1 Hz, 6H), 2.57 – 2.45 (m, 1H), 1.89 – 1.78 (m, 2H), 1.61 – 1.49 (m, 3H), 1.37 (m, 2H), 1.27 – 1.01 (m, 3H). LCMS(ESI): m/z 355.4 [M+H]+. 1-(6,7-bis(cyclopropylmethoxy)quinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (19). A mixture of 4-chloroquinazoline-6,7-diol (0.05 g, 0.254 mmol), (bromomethyl) cyclopropane (0.086 g, 0.636 mmol) and K2CO3 (0.105 g, 0.763 mmol) in DMF was heated to 80 oC for 6 h. It was cooled, diluted with water and extracted into ethyl acetate. The obtained product 4-chloro-6,7bis(cyclopropylmethoxy)quinazoline (0.05 g, 0.164 mmol) was treated with 3-(pyridin-2-yl)-1H1,2,4-triazol-5-amine (0.026 g, 0.164) according to the general procedure described above. The



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24 crude product was purified by column chromatography (97:3 CHCl3/MeOH) to obtain target compound. 1H NMR (400 MHz, CDCl3) δ 9.26 (s, 1H), 8.89 (s, 1H), 8.77 (td, J = 4.8, 1.8, 0.9 Hz, 1H), 8.16 (dt, J = 7.9, 1.1 Hz, 1H), 7.81 (td, J = 7.7, 1.8 Hz, 1H), 7.40 – 7.31 (m, 1H), 7.05 (s, 1H), 4.14 (d, J = 8.0 Hz, 2H), 4.11 (d, J = 8.0 Hz, 2H),1.61 (s, 3H), 0.75-0.70 (m, 4H),, 0.50 – 0.47 (m, 4H). LCMS(ESI): m/z 430.4 [M+H]+. 1-(6,7-bis(2-methoxyethoxy)quinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (20). To a solution of 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline (80 mg, 0.256 mmol) and 3-(pyridin-2yl)-1H-1,2,4-triazol-5-amine and in DMF (3 mL), was added cesium carbonate (167 mg, 0.512 mmol). The resulting mixture was heated at 70°C for 4 h. Next, the disappearance of starting material confirmed by TLC. The reaction mixture was poured into water and then extracted with DCM. The organic layer was dried over MgSO4, filtered and evaporated. The crude mixture was purified by flash column chromatography and re-purified by prep-HPLC. 1H NMR (500 MHz, CDCl3) δ 9.15 (s, 1H), 8.84 (s, 1H), 8.70 (d, J = 4.7 Hz, 1H), 8.10 (d, J = 7.9 Hz, 1H), 7.75 (t, J = 7.8 Hz, 1H), 7.32 – 7.27 (m, 2H), 6.98 (s, 2H), 4.37 (t, J = 5.1 Hz, 2H), 4.29 (t, J = 4.9 Hz, 2H), 3.84 (dq, J = 11.2, 6.3, 5.6 Hz, 4H), 3.43 (d, J = 3.3 Hz, 6H).

13

C NMR (125 MHz, CDCl3) δ 159.62 ,

158.48 , 155.67 , 153.51 , 151.22 , 150.71 , 150.09 , 149.70 , 149.31 , 136.59 , 124.25 , 121.48 , 111.61 , 107.93 , 107.44 , 70.57 , 70.40 , 68.54 , 68.51 , 59.41 , 59.40 . HRMS (ESI): [M+H]+ calcd for C21H24N7O4+, 438.1890; found, 438.1891. . 1-(2-chloro-6,7-dimethoxyquinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (22a). To a mixture of 2,4-Chloro-6,7-dimethoxy quinazoline 21a (0.1g, 0.386 mmol) and 3-(pyridin-2-yl)-1H1,2,4-triazol-5-amine 14 (0.075g, 0.463 mmol) in dry DMF (8 mL) was added Cs2CO3 (0.252g, 0.772 mmol). The resulting mixture was heated at 90 oC for 6-8 h. Then the reaction mixture was cooled to room temperature and poured onto ice cold water. The solid deposited was filtered off to get the pure compound in excellent yield. The remaining aqueous layer was extracted with chloroform (2x50 mL). The combined organic layers were dried over MgSO4, the solvent was evaporated and the residue was purified by reverse phase HPLC to afford compound 22a with 60.8%


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25 overall yield. 1H NMR (400 MHz, CDCl3) δ 9.26 (s, 1H), 8.81 – 8.74 (m, 1H), 8.14 (d, J = 8.0 Hz, 1H), 7.83 (td, J = 7.7, 1.7 Hz, 1H), 7.38 (m, 1H), 7.32 (s, 1H), 6.90 (s, 1H), 4.15 (s, 3H), 4.08 (s, 3H). LCMS(ESI): m/z 384.2 [MH]+. 1-(6,

7-dimethoxy-2-morpholinoquinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine

(22b). Compound 22a (0.05g, 0.13 mmol) and commercially available morpholine (0.057 g, 0.651 mmol) were mixed together in NMP (3 ml) and heated at 140 °C for 3-4 h under argon atmosphere. The reaction mixture was cooled to room temperature and poured into water. Then, NaCl was added. The resulting precipitate was collected by filtration, washed with water, dried to give 0.04 g (0.092 mmol, 70.7%) of the title compound. 1H NMR (400 MHz, CDCl3): δ 8.75-8.72 (m, 2H), 8.13 (d, J = 7.9 Hz, 1H), 7.79 (td, J = 7.7, 1.7 Hz, 1H), 7.36-7.33 (m, 1H), 7.00 (s, 1H), 6.61 (s, 2H), 4.03 (s, 3H), 4.02 (s, 3H) 3.87 – 3.81 (m, 8H). LCMS(ESI): m/z 435.0 [M+H]+. 1-(6,7-dimethoxy-2-(4-methylpiperazin-1-yl)quinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol5-amine (22c). Compound 22a (0.1g, 0.26 mmol) and commercially available N-methyl piperazine(0.13 g, 1.3 mmol) were mixed together in NMP (3 ml) and the reaction was carried out as described for the synthesis of compound 22b. The crude product was purified by HPLC to afford 22c (0.06 g, 51.5%) 1H NMR (400 MHz, CDCl3) CDCl3δ 8.76 (s, 1H), 8.75 (s, 1H), 8.14 (d, J = 7.9 Hz, 1H), 7.80 (td, J = 7.7, 1.7 Hz, 1H), 7.3-=7.34 (m, 1H), 7.00 (s, 1H), 6.71 (s, 2H), 4.04 (s, 3H), 4.03 (s, 3H), 3.04 – 3.92 (m, 4H), 2.65 (t, J = 5.1 Hz, 4H), 2.43 (s, 3H). LCMS(ESI): m/z 448.4 [M+H]+. 2-(4-(4-(5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)ethanol (22d). Compound 22a (0.1g, 0.26 mmol) and commercially available 2-(piperazin1-yl)ethanol (0.17 g, 1.3 mmol) were mixed together in NMP (5 ml) and the reaction was carried out as described for the synthesis of compound 22b. The crude product was purified by HPLC to afford



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26 22d (0.045 g, 36.2%). 1H NMR (400 MHz, CDCl3) δ 8.49 (s, 1H), 8.35 (s, 1H), 7.91 (s, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.22 (s, 1H), 6.78 (s, 1H), 3.81 (s, 3H), 3.79 (s, 3H) 3.42 (t, J = 11.1 Hz, 4H), 3.31 – 3.13 (m, 6H), 3.05 (d, J = 5.4 Hz, 2H). LCMS(ESI): m/z 478.5 [M+H]+. Ethyl 2-((4-(5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-6,7-dimethoxyquinazolin-2-yl)amino)acetate (22e). Compound 22a (0.038 g, 0.1 mmol) and commercially available ethyl 2aminoacetate hydrochloride (0.103 g, 1.0 mmol) were mixed together in NMP (5 ml) and the reaction was carried out as described for the synthesis of compound 22b. Next, ice cold water was added and extracted in ethyl acetate and the crude product was purified by HPLC to afford 22e (0.035 g, 78.0%). 1H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H), 8.10 (d, J = 7.9 Hz, 1H), 7.88 – 7.71 (m, 1H), 7.52 (s, 1H), 6.89 (s, 1H), 4.18 (q, 2H), 4.07 (s, 10H), 3.94 (s, 3H), 3.93 (s, 3H), 3.29-3.26 (m, 2H), 1.29 – 1.15 (m, 3H). LCMS(ESI): m/z 451.4 [M+H]+. 2-((4-(5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-6,7-dimethoxyquinazolin-2-yl)amino)acetic acid (22f). To a solution of compound 22e (0.02 g, 0.044 mmol) was diluted with a mixture of methanol (2 mL) and water (0.3 mL), and then treated with lithium peroxide (0.019 g, 0.022 mmol). The reaction mixture was stirred at room temperature for 0.45 h and acidified with 10% HCl and evaporated everything and re-dissolved in DMSO (1 mL) and purified by HPLC. 1H NMR (400 MHz, CD3OD) δ 13.93 (s, 1H), 8.65 (d, J = 4.2 Hz, 1H), 8.37 (s, 1H), 8.08 (d, J = 7.9 Hz, 1H), 8.00 – 7.90 (m, 1H), 7.84 (s, 1H), 7.44 (dd, J = 7.5, 4.8 Hz, 1H), 6.84 (s, 1H), 4.01 (s, 2H), 3.98 (s, 3H), 3.97 (s, 3H). LCMS(ESI): m/z 424.4 [M+H]+. 4-(5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-N,N-diethyl-6,7-dimethoxyquinazolin-2amine (22g). Compound 22a (0.05 g, 0.13 mmol) was dissolved in 5 mL of isopropanol. To this solution was added diethylamine (0.057 g, 0.78mmol) and HCl in dioxane (4.0 M, 0.1 mL). The resulting solution was stirred inside a microwave at 160 oC for 10 min. After cooling, TLC indicated


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27 the completion of the reaction. After removal of the solvent by rotary evaporation, the residue was re-dissolved in CH2Cl2 and washed with saturated NaHCO3 solution. The organic layer was dried, concentrated and purified by ISCO to give the desired compound (30 mg, 54.8%). 1H NMR (400 MHz, CDCl3) δ 8.81 (s, 1H), 8.77 (s, 1H), 8.15 (d, J = 7.9 Hz, 1H), 7.79 (td, J = 7.7, 1.7 Hz, 1H), 7.36-7.33 (m, 1H), 6.97 (s, 1H), 6.74 (s, 2H), 4.04 (s, 3H), 4.04 (s, 3H), 3.71 (q, J = 7.1 Hz, 4H), 1.29 (t, J = 7.1 Hz, 6H). LCMS(ESI): m/z 422.4 [M+H]+. 1-(6,7-dimethoxy-2-methylquinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (22h). To a solution of 4-Chloro-6,7-dimethoxy-2-methyl quinazoline 21g (0.1g, 0.419 mmol) and 3-(pyridin2-yl)-1H-1,2,4-triazol-5-amine 14 (0.075g, 0.503 mmol) in dry DMF (8 mL) was added Cs2CO3 (0.252g, 0.838 mmol). The mixture was heated at 90 oC for overnight. The mixture was cooled to room temperature and poured onto ice cold water and extracted with chloroform (2x50 mL). The combined organic layers were dried over MgSO4, evaporated and the residue was purified by reverse phase HPLC to afford compound 22h with 60.8% overall yield. 1H NMR (400 MHz, CD3OD): δ 8.91 (s, 1H), 8.52 (s, 1H), 7.98 (d, J = 7.9 Hz, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.32-7.27 (m, 1H), 7.14 (s, 1H), 4.21 (s, 13H), 3.92 (s, 3H), 3.89 (s, 3H) 2.64 (s, 3H). LCMS(ESI): m/z 364.1 [M+H]+. 1-(6,7-dimethoxy-2-phenylquinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (22i). 1

H NMR(400 MHz, CD3OD): δ 8.95 (s, 1H), 8.49 (s, 1H), 7.98 (d, J = 7.9 Hz, 1H), 7.76 (d, J = 7.7

Hz,1H), 7.68-7.59 (m, 3H), 7.44-7.37 (m, 2H), 7.30-7.27 (m, 1H), 7.16 (s, 1H), 3.93 (s, 6H) . LCMS(ESI): m/z 426.1 [M+H]+. 4-(5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-6,7-dimethoxyquinazolin-2-amine

(22j).

Compound synthesized using general procedure II for the synthesis of quinazolines as described above. A mixture of 4-chloro-6,7-dimethoxyquinazolin-2-amine 21j (0.1 g, 0.417 mmol), 3-(pyridin2-yl)-1H-1,2,4-triazol-5-amine 14 (0.134 g, 0.834 mmol) and Cs2CO3 ( 0.543, 1.66 mmol) in DMF



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28 (6 mL) was heated at 100 oC for 6 h. Next, the mixture was cooled, diluted with water (10 mL), extracted into CHCl3, and the organic layer was dried over sodium sulfate. After the removal of solvent, the crude extract was purified by flash column chromatography (chloroform/methanol 94:6) to give 0.056 g of desired compound. 1H NMR (400 MHz, CD3OD) δ 8.65 (s, 1H), 8.45 – 8.38 (m, 1H), 7.90 – 7.83 (m, 1H), 7.64-7.61 (m, 1H), 7.17-7.14 (m, 1H), 6.64 (s, 1H), 3.89 (s, 3H), 3.76 (s, 3H). LCMS(ESI): m/z 365.3 [M+H]+. 6,7-dimethoxy-N-(pyridin-2-ylmethyl)quinazolin-4-amine (23a). To a mixture of 4-chloro-6, 7dimethoxy quinazoline 11 (50 mg, 0.223 mmol) and pyridin-2-ylmethanamine (24.07 mg, 0.223 mmol) in DMF (0.8 mL) was added Cs2CO3 (0.145 g, 0.446 mmol). The reaction mixture was stirred at 130 oC for overnight. After completion, it was cooled, diluted with water and extracted in chloroform (2X30 mL). The combined organic layers were dried over magnesium sulphate and evaporated to dryness. The crude material was purified by HPLC to get the target compound with 45.5% overall yield. 1H NMR (400 MHz, DMSO-d6) δ 8.58 (t, J = 5.9 Hz, 1H), 8.55 – 8.45 (m, 1H), 8.29 (s, 1H), 7.78 – 7.65 (m, 2H), 7.32 (d, J = 7.9 Hz, 1H), 7.25 (ddd, J = 7.5, 4.9, 1.0 Hz, 1H), 7.11 (s, 1H), 4.85 (d, J = 5.7 Hz, 2H), 3.90 (s, 6H). LCMS(ESI): m/z 297.3 [M+H]+. 6, 7-dimethoxy-N-(pyridin-2-yl) quinazolin-4-amine (23b). Procedure followed as described for 15a using pyridine-2-amine as starting material. 1H NMR (400 MHz, DMSO-d6) δ 8.71 – 8.63 (m, 1H), 8.47 (s, 1H), 8.12 – 8.02 (m, 1H), 7.89 – 7.77 (m, 1H), 7.64 – 7.49 (m, 2H), 7.24 (s, 1H), 3.95 (s, 3H), 3.90 (s, 3H). LCMS(ESI): m/z 283.0 [M+H]+. 6,7-dimethoxy-N-(4-(pyridin-2-yl)phenyl)quinazolin-4-amine (23c). A mixture of 4-chloro-6,7dimethoxy quinazoline 11 (100 mg, 0.445 mmol) and 4-(pyridin-2-yl)aniline (83 mg, 0.490 mmol) in ethanol (3 mL) was heated to reflux for 6 hr. The reaction mixture was cooled evaporated, purified by column chromatography, and eluted at 95:5 (DCM/MeOH) to afford the target


Page 29 of 67

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29 compound in 68.9% yield. 1H NMR (400 MHz, CD3OD) δ 8.69 (s, 1H), 8.65 (m, 1H), 8.09 – 8.04 (m, 3H), 7.96 (ddd, J = 8.1, 7.2, 1.8 Hz, 1H), 7.93 – 7.88 (m, 3H), 7.77 (s, 1H), 7.42 (ddd, J = 7.3, 5.0, 1.4 Hz, 1H), 4.11 (s, 3H), 4.10 (s, 3H). LCMS(ESI): m/z 359.3 [M+H]+. 6,7-dimethoxy-4-(3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)quinazoline (23d). To a mixture of 4chloro-6,7-dimethoxy quinazoline 11 (102 mg, 0.456 mmol) and 2-(1H-1,2,4-triazol-3-yl)pyridine (80 mg, 0.547 mmol) in DMF (0.8 mL) was added Cs2CO3 (0.297 g, 0.912mmol). The reaction mixture was stirred at 130 oC for 4 h. After the completion, it was cooled, diluted with water and extracted in chloroform (2x 30 mL). The combined organic layers were dried over magnesium sulphate and evaporated to dryness. The crude material was purified by HPLC to get the target compound with 49.2 % overall yield. 1H NMR (400 MHz, DMSO-d6) δ 9.70 (s, 1H), 9.07 (s, 1H), 8.77 (d, J = 4.3 Hz, 1H), 8.67 (s, 1H), 8.25 (d, J = 7.8 Hz, 1H), 8.02 (t, J = 7.7 Hz, 1H), 7.56 – 7.51 (m, 2H), 4.05 (s, 3H), 4.00 (s, 3H). LCMS(ESI): m/z 335.3 [M+H]+. 6,7-dimethoxy-4-(4-(pyridin-2-yl)-2H-1,2,3-triazol-2-yl)quinazoline (23e). Procedure followed as described above using 2-(2H-1,2,3-triazol-4-yl) pyridine as key intermediate. 1H NMR (400 MHz, DMSO-d6) δ 9.15 (s, 1H), 8.86 (s, 1H), 8.76 (ddd, J = 4.8, 1.7, 0.9 Hz, 2H), 8.27 (s, 1H), 8.18 (d, J = 7.9 Hz, 1H), 8.02 (d, J = 1.8 Hz, 1H), 7.56 (s, 1H), 7.515-7.51 (m, 1H), 4.06 (s, 3H), 3.98 (s, 3H). LCMS(ESI): m/z 335.3 [M+H]+.

1-(6,7-dimethoxyquinazolin-4-yl)-3-(pyridin-2-yl)-1H-pyrazol-5-amine

(23f).

Procedure

followed as described above using 3-(pyridin-2-yl)-1H-pyrazol-5-amine as key intermediate. 1H NMR (400 MHz, CDCl3) δ 8.98 (s, 1H), 8.81 (s, 1H), 8.58 (dt, J = 4.8, 1.4 Hz, 1H), 7.99 (dt, J = 8.0, 1.1 Hz, 1H), 7.66 (td, J = 7.7, 1.8 Hz, 1H), 7.24 – 7.13 (m, 1H), 6.25 (s, 1H), 5.92 (s, 2H), 3.99 (d, J = 2.1 Hz, 6H). LCMS(ESI): m/z 349.3 [M+H]+.



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30 1-(6,7-dimethoxyquinazolin-4-yl)-3-phenyl-1H-pyrazol-5-amine (23g). Procedure followed as described above using 3-phenyl-1H-pyrazol-5-amine as key intermediate. 1H NMR (400 MHz, CDCl3) δ 9.15 (s, 1H), 8.80 (s, 1H), 6.00 (s, 2H), 7.85 – 7.78 (m, 2H), 7.36 (dd, J = 8.2, 6.4 Hz, 2H), 7.21 – 7.18 (m, 1H), 6.00 (s, 2H), 5.90 (s, 1H), 4.03 (s, 3H), 4.00 (s, 3H). LCMS(ESI): m/z 348.3 [M+H]+. 1-(6,7-dimethoxyquinazolin-4-yl)-3-(p-tolyl)-1H-pyrazol-5-amine (23h). Procedure followed as described above using 3-(p-toluyl)-1H-pyrazol-5-amine as key intermediate. 1H NMR (400 MHz, CDCl3) δ 9.17 (s, 1H), 8.80 (s, 1H), 7.75 – 7.66 (m, 2H), 7.23 – 7.14 (m, 3H), 6.00 (s, 2H), 5.87 (s, 1H), 4.03 (s, 3H), 4.01 (s, 3H), 2.34 (s, 3H). LCMS(ESI): m/z 362.4 [M+H]+. 3-(4-chlorophenyl)-1-(6,7-dimethoxyquinazolin-4-yl)-1H-pyrazol-5-amine

(23i).

Procedure

followed as described above using 3-(4-chlorophenyl)-1H-pyrazol-5-amine as key intermediate. 1H NMR (400 MHz, CDCl3) δ 9.06 (s, 1H), 8.81 (s, 1H), 7.77 – 7.69 (m, 2H), 7.37 – 7.29 (m, 2H), 7.22 – 7.16 (m, 1H), 7.27 (s, 1H), 5.99 (s, 2H), 5.87 (s, 1H), 4.01 (d, J = 2.7 Hz, 6H). LCMS(ESI): m/z 382.1 [M+H]+. 1-(6,7-dimethoxyquinazolin-4-yl)-4-(pyridin-2-yl)-1H-pyrazol-5-amine

(23j).

Procedure

followed as described above using 4-(pyridin-2-yl)-1H-pyrazol-5-amine as key intermediate. 1H NMR (400 MHz, CDCl3) δ 9.11 (d, J = 4.7 Hz, 2H), 8.84 (s, 1H), 8.58 (ddd, J = 5.0, 1.8, 1.0 Hz, 1H), 7.78 – 7.61 (m, 2H), 7.31 (s, 1H), 7.14 (ddd, J = 7.2, 5.0, 1.4 Hz, 1H), 5.81 (s, 2H), 5.30 (s, 1H), 4.10 (s, 3H), 4.07 (s, 3H). LCMS(ESI): m/z 349.3 [M+H]+. 1-(6,7-dimethoxyquinazolin-4-yl)-1H-1,2,4-triazole-3,5-diamine (23k). Procedure followed as described above using 1H-1,2,4-triazole-3,5-diamine as key intermediate. After the reaction, the crude material was purified on NH2 silica using a 25%-100% EtOAc/Hex gradient to give the desired product with 75 % overall yield. 1H NMR (400 MHz, CD3OD) δ 9.21 (s, 1H), 8.73 (s, 1H),


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31 9.27 – 9.17 (m, 1H), 7.81 (s, 1H), 7.26 (s, 1H), 4.05 (s, 3H), 4.03 (s, 3H). LCMS(ESI): m/z 288.2 [M+H]+. 1-(6-(benzyloxy)-7-methoxyquinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine

(24a).

Compound was synthesized from 6-(benzyloxy)-4-chloro-7-methoxyquinazoline, which was synthesized from literature available procedures1 coupled with 3-(pyridin-2-yl)-1H-1,2,4-triazol-5amine as described in general procedure II. 1H NMR (400 MHz, CDCl3) δ 9.21 (s, 1H), 8.84 (s, 1H), 8.72 (d, J = 4.7 Hz, 1H), 8.06 – 7.95 (m, 1H), 7.74 (td, J = 7.7, 1.8 Hz, 1H), 7.49 (dd, J = 6.6, 2.8 Hz, 2H), 7.35 – 7.15 (m, 2H), 7.26-7.22 (m, 3H) 6.90 (s, 2H), 5.38 (s, 2H), 4.03 (s, 3H). LCMS: 426.4 [M+H]+. 4-(5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-7-methoxyquinazolin-6-ol

(24b).

Prepared

from 24a upon debenzylation using 10% Pd-C in MeOH. 1H NMR (400 MHz, CD3OD) δ 9.28 (s, 1H), 8.87 (s, 1H), 8.70 (d, J = 4.8 Hz, 1H), 8.30 (d, J = 8.0 Hz, 1H), 7.97 (t, J = 7.7 Hz, 1H), 7.50 (t, J = 6.3 Hz, 1H), 7.37 (s, 1H), 4.12 (s, 3H). LCMS: 336.1 [M+H]+. 1-(7-(benzyloxy)-6-methoxyquinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine

(24c).

Synthesized from commercially available 7-(benzyloxy)-4-chloro-6-methoxyquinazoline and 3(pyridin-2-yl)-1H-1,2,4-triazol-5-amine as described in general procedure II. 1H NMR (400 MHz, CDCl3) δ 9.22 (s, 1H), 8.90 (s, 1H), 8.78 (ddd, J = 4.8, 1.9, 0.9 Hz, 1H), 8.15 (dt, J = 7.9, 1.1 Hz, 1H), 7.82 (td, J = 7.7, 1.8 Hz, 1H), 7.52 (dd, J = 8.2, 1.4 Hz, 2H), 7.45 – 7.34 (m, 5H), 7.00 (s, 2H), 5.37 (s, 2H), 4.15 (s, 3H). LCMS: 426.4 [M+H]+. 4-(5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-6-methoxyquinazolin-7-ol

(24d).

Prepared

from 24c upon debenzylation using 10% Pd-C in MeOH.1H NMR (400 MHz, CDCl3+ 1drop CD3OD) δ 9.13 (s, 1H), 8.84 (s, 1H),8.72 (s, 1H), 8.16 (d, J = 7.9 Hz, 1H), 7.90 (ddd, J = 7.2, 5.0, 1.4 Hz, 1H), 7.45-7.41 (m, 1H), 7.38 (s, 1H), 7.30 (s, 1H), 4.14 (s, 3H). LCMS: 336.1 [M+H]+.



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Page 32 of 67

32 1-(7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5amine (24e). Prepared from commercially available 4-(3-((4-chloro-7-methoxyquinazolin-6yl)oxy)propyl)morpholine and 3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine as described in general procedure II. 1H NMR (400 MHz, CD3OD) δ 9.07 (s, 1H), 8.83 (s, 1H), 8.65 (d, J = 4.7 Hz, 1H), 8.07 (d, J = 7.9 Hz, 1H), 7.80 (t, J = 7.7 Hz, 1H), 7.41 – 7.25 (m, 3H), 3.36 – 3.20 (m, 2H), 4.29 (t, J = 6.2 Hz, 2H), 4.01 (s, 3H), 3.69 (t, J = 4.7 Hz, 4H), 2.76 – 2.46 (m, 6H), 2.15 (p, J = 6.6 Hz, 2H). LCMS: 463.5 [M+H]+. 1-(6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5amine

(24f).

Prepared

from

morpholinopropoxy)quinazolin-4(3H)-one

commercially and

available

6-methoxy-7-(3-

3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine

as

described in general procedure II. 1H NMR (400 MHz, CDCl3) d 9.11 (s, 1H), 8.85 (s, 1H), 8.71 (d, J = 4.6, 1H), 8.07 (d, J = 7.9, 1H), 7.75 (t, J = 7.7, 1H), 7.39 - 7.26 (m, 2H), 4.25 (t, J = 6.5, 2H), 4.05 (s, 3H), 3.75 - 3.63 (m, 4H), 2.67 - 2.54 (m, 2H), 2.51 (s, 4H), 2.20 - 2.00 (m, 2H). LCMS: 463.5 [M+H]+. 1-(6-(3-(dimethylamino)propoxy)-7-methoxyquinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol5-amine (27a). The reactive key intermediate 3-((4-chloro-7-methoxyquinazolin-6-yl)oxy)-N,Ndimethylpropan-1-amine was prepared from a mixture of 4-chloro-7-methoxyquinazolin-6-ol, 3chloro-N,N-dimethylpropan-1-amine in DMF using potassium carbonate as discussed in scheme 4. 1

H NMR (400 MHz, CD3OD) δ 8.97 (s, 1H), 8.80 (s, 1H), 8.74 – 8.62 (m, 1H), 8.50 (s, 1H), 8.06 (d,

J = 7.9 Hz, 1H), 7.93 (td, J = 7.7, 1.7 Hz, 1H), 7.52-7.47 (m, 1H), 4.35 (t, J = 5.6 Hz, 2H), 4.08 (s, 3H), 3.44 (t, J = 7.2 Hz, 2H), 3.01 (s, 6H), 2.39 (p, J = 6.6 Hz, 2H). LCMS: 421.4 [M+H]+. 1-(6-(2-(diisopropylamino)ethoxy)-7-methoxyquinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol5-amine (27b). 1H NMR (400 MHz, CDCl3) δ 9.20 (s, 1H), 8.92 (s, 1H), 8.82 – 8.67 (m, 1H), 8.20


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33 (d, J = 7.9 Hz, 1H), 7.86 (td, J = 7.8, 1.7 Hz, 1H), 7.39 (d, J = 5.1 Hz, 2H), 7.29 (d, J = 16.8 Hz, 2H), 4.47 (t, J = 6.6 Hz, 2H), 4.07 (s, 3H), 3.49-3.45 (m, 2H), 3.31 (t, J = 6.6 Hz, 2H), 2.63 (d, J = 1.0 Hz, 1H), 1.25 (d, J = 6.6 Hz, 12H). LCMS: 463.2 [M+H]+. 1-(7-methoxy-6-(2-(pyrrolidin-1-yl)ethoxy)quinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5amine (27c). Procedure followed as described in 27a. 1H NMR 1H NMR (400 MHz, CD3OD) δ 9.10 (s, 2H), 8.85 (s, 1H), 8.76 – 8.64 (m, 1H), 8.38 (s, 2H), 8.11 (d, J = 7.9 Hz, 1H), 7.94 (td, J = 7.7, 1.7 Hz, 1H), 7.52 -7.50 (m, 1H), 4.65 – 4.52 (m, 2H), 4.10 (s, 3H), 3.87 – 3.75 (m, 2H), 3.70 – 3.52 (m, 4H), 2.68 (s, 1H), 2.17-2.14 (m, 4H). LCMS: 433.4 [M+H]+. 1-(7-methoxy-6-(2-(piperidin-1-yl)ethoxy)quinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5amine (27d). 1H NMR (400 MHz, CDCl3) δ 9.20 (s, 1H), 8.91 (s, 1H), 8.74 (d, J = 4.4 Hz, 1H), 8.11 (t, J = 14.0 Hz, 1H), 7.97 – 7.79 (m, 1H), 7.47 – 7.30 (m, 4H), 4.63 (t, J = 5.1 Hz, 2H), 4.03 (d, J = 18.5 Hz, 3H), 3.38 (dt, J = 22.6, 5.2 Hz, 2H), 3.02 (d, J = 37.3 Hz, 5H), 1.83 (dd, J = 11.3, 5.6 Hz, 5H), 1.63 (dd, J = 18.0, 12.2 Hz, 3H).LCMS: 447.5 [M+H]+. 1-(7-methoxy-6-(2-(4-methylpiperidin-1-yl)ethoxy)quinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4triazol-5-amine (27e). Procedure followed as described in 27a. 1H NMR (400 MHz, CDCl3) δ 9.22 (s, 1H), 8.92 (s, 1H), 8.77-8.76 (m, 1H), 8.17 (dt, J = 7.9, 1.1 Hz, 1H), 7.80 (td, J = 7.7, 1.8 Hz, 1H), 7.43 – 7.32 (m, 2H), 7.01 (s, 2H), 4.46 (t, J = 6.6 Hz, 2H), 4.07 (s, 3H), 3.08 – 2.87 (m, 3H), 2.21 – 2.06 (m, 2H), 1.77 – 1.54 (m, 4H), 1.45 – 1.16 (m, 3H), 0.93 (d, J = 6.3 Hz, 3H). LCMS: 461.5 [M+H]+. 1-(6-(3-(2,6-dimethylmorpholino)propoxy)-7-methoxyquinazolin-4-yl)-3-(pyridin-2-yl)-1H1,2,4-triazol-5-amine

(27f).

The

key

intermediate

4-(3-((4-chloro-7-methoxyquinazolin-6-

yl)oxy)propyl)-2,6-dimethylmorpholine prepared from 4-chloro-7-methoxyquinazolin-6-ol and 4-(3chloropropyl)-2,6-dimethylmorpholinein in DMF using potassium carbonate as described in scheme



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34 4. The reactive linker coupled to 4-chloro-7-methoxyquinazolin-6-ol to get the key intermediate as described above further coupled with triazole amine from the general procedure described above to obtain the target compound. 1H NMR (400 MHz, CDCl3) δ 9.18 (s, 3H), 8.93 (s, 3H), 8.79 (d, J = 4.7 Hz, 3H), 8.13 (d, J = 7.9 Hz, 3H), 7.85 (td, J = 7.7, 1.8 Hz, 3H), 7.39 (d, J = 6.5 Hz, 6H), 7.23 (s, 1H), 4.38 (t, J = 6.2 Hz, 6H), 1.14 – 1.12 (m, 0H), 4.07 (s, 3H), 3.87 (d, J = 9.0 Hz, 2H), 3.05 (d, J = 11.3 Hz, 2H), 2.84 (q, J = 10.9, 9.3 Hz, 2H), 2.63 (s, 2H), 1.99 (t, J = 10.9 Hz, 3H), 1.17 (d, J = 6.3 Hz, 6H). LCMS: 491.5 [M+H]+. 1-(7-methoxy-6-(2-methoxyethoxy)quinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (27g). A key intermediate 4-chloro-7-methoxy-6-(2-methoxyethoxy) quinazoline 26g was prepared from commercially available 4-chloro-7-methoxyquinazolin-6-ol 25 as described below. di-tertbutylazodicarboxylate (0.262 g, 1.13 mmol) was added portion wise at 0 oC to a stirred suspension of 4-chloro-7-methoxyquinazolin-6-ol (25, 0.22 g, 1.045 mmol), 2-methoxy ethanol (0.91 g, 6.27 mmol) and triphenylphosphine (0.329 g, 1.25 mmol) in dichloromethane (15 ml). The reaction mixture was stirred for 5 h and then the resulting orange solution was purified directly by silica gel chromatography eluting with a mixture of 10% ethyl acetate in DCM to afford the reactive key intermediate, which was directly coupled with triazole amine using general procedure described above. 1H NMR (400 MHz, CDCl3) δ 9.24 (s, 1H), 8.92 (s, 1H), 8.83 – 8.72 (m, 1H), 8.17 (d, J = 7.9 Hz, 1H), 7.82 (td, J = 7.7, 1.7 Hz, 1H), 7.41 – 7.30 (m, 2H), 7.05 (s, 2H), 4.52 – 4.41 (m, 2H), 4.07 (s, 3H), 3.98 – 3.83 (m, 2H), 3.49 (s, 3H). 13C NMR (125 MHz, DMSO) δ 159.71, 158.87, 156.26, 153.46, 151.57, 151.02, 150.37, 149.68, 149.31, 137.50, 124.94, 122.32, 111.24, 107.59, 107.08, 70.36, 68.25, 58.70, 56.66. HRMS (ESI): [M+H]+ calcd for C19H20N7O3+, 394.1627; found, 394.1620.


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35 2-((4-(5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-7-methoxyquinazolin-6-yl)oxy)-N,Ndiethylacetamide (27h). The reactive key intermediate 2-(4-chloro-7-methoxyquinazolin-6-yloxy)N,N-diethylacetamide was prepared from a mixture of

4-chloro-7-methoxyquinazolin-6-ol, 2-

chloro-N,N-diethylacetamide in DMF using potassium carbonate. It was further diluted with water and extracted in DCM and filtered to purify. The obtained intermediate was coupled with triazole amine using general procedures described above. 1H NMR (400 MHz, CDCl3) δ 9.06 (s, 1H), 8.91 (s, 1H), 8.81 – 8.67 (m, 1H), 8.20 (t, J = 14.1 Hz, 1H), 7.89 – 7.76 (m, 1H), 7.48 – 7.32 (m, 4H), 5.09 – 4.94 (m, 2H), 4.12 – 4.01 (m, 4H), 3.49 – 3.25 (m, 5H), 1.20 – 1.07 (m, 3H), 1.04 (t, J = 7.1 Hz, 3H). LCMS: 449.4 [M+H]+. N-(3-((4-(5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-7-methoxyquinazolin-6yl)oxy)propyl)-2-(2,6-dimethylmorpholino)acetamide (27i). The key synthetic intermediate N-(3((4-chloro-7-methoxyquinazolin-6-yl)oxy)propyl)-2-(2,6-dimethylmorpholino)acetamide

was

synthesized as described below. A solution of morpholine (10.91 mmol) in DCM (40 mL) was treated with 0.692 mL of benzyl 2-bromo acetate at 0 oC. The resulting solutions was stirred at ambient temperature for 16 h, filtered and concentrated. The residue was purified by silica gel chromatography using DCM/ethyl acetate to obtain benzyl 2-(2,6-dimethylmorpholino)acetate. Upon debenzylation in MeOH/Pd-C followed by treatment with 3-chloropropan-1-amine hydrochloride in the presence of DIEA in DCM, this resulted in N-(3-chloropropyl)-2-(2,6dimethylmorpholino)acetamide. The resulting linker was treated with 25 in the presence of K2CO3 in DMF, forming the target intermediate N-(3-((4-chloro-7-methoxyquinazolin-6-yl)oxy)propyl)-2-(2,6 dimethylmorpholino) acetamide with 62.3% overall yield. 1H NMR (400 MHz, CDCl3) δ 8.88 (s, 1H), 7.40 (s, 1H), 7.35 (s, 1H), 4.25 (t, J = 6.1 Hz, 2H), 4.06 (s, 3H), 3.61-3.59 (m, 4H), 2.99 (d, J =



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36 7.0 Hz, 2H), 2.66 (d, J = 10.4 Hz, 2H), 2.29 – 2.13 (m, 2H), 1.96-1.91 (t, J = 8.0 Hz, 2H), 1.52 (d, 8.0 Hz, 6H). LCMS: 423.1 [M+H]+. Tert-butyl

(3-((4-(5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-7-methoxyquinazolin-6-

yl)oxy)propyl)carbamate (27j).

The reactive key intermediate tert-butyl (3-((4-chloro-7-

methoxyquinazolin-6-yl)oxy)propyl) carbamate was prepared from a mixture of

4-chloro-7-

methoxyquinazolin-6-ol (0.95 mmol), commercially available tert-butyl (3-bromopropyl)carbamate (1.14 mmol) in DMF (5 mL) using potassium carbonate (1.42 mmol) as discussed above. The mixture was stirred at 65 oC for 4 hr, after the reaction mixture was cooled and diluted with water and extracted in chloroform. The organic layer was dried and evaporated. The crude extract was purified by flash chromatography on silica (20:1 DCM:MeOH) to give 0.2 g of (60.2 %). Thus obtained fragment coupled with 3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine as discussed above. 1H NMR (400 MHz, CDCl3) δ 9.19 (s, 1H), 8.93 (s, 1H), 8.77 (d, J = 4.8 Hz, 1H), 8.14 (d, J = 7.9 Hz, 1H), 7.84 (td, J = 7.7, 1.7 Hz, 1H), 7.42 – 7.28 (m, 2H), 6.99 (s, 2H), 5.52 (s, 1H), 4.39 (t, J = 5.9 Hz, 2H), 4.06 (d, J = s, 3H), 3.45 (d, J = 5.9 Hz, 2H), 2.25 – 2.06 (m, 2H), 1.46 (d, J = 6.4 Hz, 8H). LCMS: 493.5 [M+H]+. Tert-butyl


yl)oxy)butyl)carbamate (27k). The reactive key intermediate tert-butyl (4-((4-chloro-7methoxyquinazolin-6-yl)oxy)butyl)carbamate was prepared from 4-chloro-7-methoxyquinazolin-6ol and tert-butyl (4-bromobutyl)carbamate as discussed above. 1H NMR (400 MHz, CDCl3) δ 9.20 (s, 1H), 8.92 (s, 1H), 8.77 (m, 1H), 8.14 (dt, J = 7.9, 1.1 Hz, 1H), 7.83 (td, J = 7.7, 1.8 Hz, 1H), 7.42 – 7.30 (m, 2H), 6.99 (s, 2H), 1.66 – 1.52 (m, 4H), 4.33 (t, J = 6.4 Hz, 2H), 4.08 (s, 3H), 3.25 (d, J = 6.5 Hz, 2H), 2.08 – 1.96 (m, 2H), 1.82 – 1.70 (m, 2H), 1.43 (s, 9H). LCMS: 507.5 [M+H]+.


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37 Tert-butyl


yl)oxy)ethyl)carbamate (27l). 1H NMR (400 MHz, CDCl3) δ 9.13 (s, 1H), 8.90 (s, 1H), 8.73 (d, J = 4.8 Hz, 1H), 8.08 (d, J = 7.9 Hz, 1H), 7.88 (td, J = 7.7, 1.7 Hz, 1H), 7.35 – 7.26 (m, 2H), 6.99 (s, 2H), 5.54 (s, 1H), 4.28 (t, J = 5.9 Hz, 2H), 4.04 ( s, 3H), 3.35 (t, J = 5.9 Hz, 2H), 1.46 (s, 9H). LCMS: 479.0 [M+H]+. Ethyl


yl)oxy)propyl)carbamate

(27m).

Reactive

key

intermediate

ethyl

(3-((4-chloro-7-

methoxyquinazolin-6-yl)oxy)propyl)carbamate was prepared from commercially available 4-chloro7-methoxyquinazolin-6-ol and ethyl (3-chloropropyl)carbamate as described above 27a. 1H NMR (400 MHz, CDCl3) δ 9.18 (s, 1H), 8.93 (s, 1H), 8.78-8.76 (m, 1H), 8.15 – 8.13 (m, 1H), 7.84 (td, J = 7.7, 1.8 Hz, 1H), 7.43 – 7.30 (m, 2H), 7.02 (d, J = 13.4 Hz, 2H), 5.76 (s, 1H), 4.41 (t, J = 5.8 Hz, 2H), 4.17 – 4.03 (m, 4H), 3.50 (d, J = 5.7 Hz, 2H), 2.26 – 2.08 (m, 2H), 1.58 (s, 4H), 1.26 (q, J = 7.5 Hz, 3H). LCMS: 465.4 [M+H]+. Isopropyl


yl)oxy)propyl)carbamate

(27n).

Reactive

key

intermediate

ethyl

(3-((4-chloro-7-

methoxyquinazolin-6-yl)oxy)propyl)carbamate was prepared from commercially available 4-chloro7-methoxyquinazolin-6-ol and isopropyl (3-chloropropyl)carbamate as described above 27a. 1H NMR (400 MHz, CDCl3) δ 9.17 (s, 1H), 8.91 (s, 1H), 8.78-8.76 m, 1H), 8.13 (d, J = 7.9 Hz, 1H), 7.84 (td, J = 7.7, 1.7 Hz, 1H), 7.42 – 7.31 (m, 2H), 7.12 (d, J = 25.1 Hz, 2H), 5.73 (s, 1H), 4.92 (dt, J = 12.4, 6.2 Hz, 1H), 4.40 (t, J = 5.8 Hz, 2H), 4.10 (s, 3H), 3.50 (dd, J = 11.2, 5.9 Hz, 2H), 2.26 – 2.09 (m, 2H), 1.68 (s, 2H), 1.31 – 1.14 (m, 7H).

13

C NMR (125 MHz, DMSO) δ 159.68, 158.92,

156.42, 156.24, 153.38, 151.44, 150.94, 150.31, 149.64, 149.40, 137.44, 124.88, 122.31, 111.22,



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38 107.51, 106.98, 66.99, 66.74, 56.66, 37.78, 29.21, 22.52. HRMS (ESI): [M+H]+ calcd for C23H27N8O4+, 479.2155; found, 479.2158.

2,2,2-trifluoroethyl (3-((4-(5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-7-methoxy-quinazolin-6-yl)oxy)propyl)carbamate (27o). 1H NMR (400 MHz, CDCl3+ CD3OD) δ 9.13 (s, 1H), 8.95 (s, 1H), 8.74-8.72 (m, 1H), 8.16 – 8.13 (m,1H), 7.82 (td, J = 7.7, 1.8 Hz, 1H), 7.43 – 7.30 (m, 2H), 7.05 (d, J = 13.4 Hz, 2H), 5.76 (s, 1H),4.41 (t, J = 5.8 Hz, 2H), 4.19 – 4.05 (m, 4H), 3.50 (t, J = 5.7 Hz, 2H), 2.26 – 2.08 (m, 2H). LCMS: 519.2 [M+H]+.

Hexyl


yl)oxy)propyl)carbamate (27p). General procedure for the synthesis of carbamates:1-(6-(3aminopropoxy)-7-methoxyquinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine (1.0 mmol) was dissolved in DCM (3 mL), which was prepared from 27j by deprotection of the tertbutoxycarbonyl (Boc) group using HCl/THF. The mixture was cooled to 0 oC and treated with triethylamine (3.0 mmol) under nitrogen atmosphere. The resulting mixture was stirred for 15 min and then treated with the corresponding hexyl chloroformate (1.5), dissolved in DCM, added drop wise. The mixture was stirred at 0 oC for 1 h then stirred over night at room temperature. The contents were evaporated to dryness and re-dissolved in CHCl3 and washed with bicarbonate solution. The organic layer was evaporated to dryness and purified by flash chromatography on silica gel eluted at 20:1 DCM:MeOH. 1H NMR (400 MHz, CDCl3) δ 9.19 (s, 1H), 8.93 (s, 1H), 8.74 (t, J = 19.2 Hz, 1H), 8.14 (d, J = 7.9 Hz, 1H), 7.84 (td, J = 7.8, 1.8 Hz, 1H), 7.44 – 7.30 (m, 2H), 6.99 (d, J = 7.2 Hz, 2H), 5.71 (s, 1H), 4.41 (t, J = 5.8 Hz, 2H), 4.15 – 3.96 (m, 4H), 3.50 (d, J = 6.3


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39 Hz, 2H), 2.30 – 2.04 (m, 3H), 1.67 – 1.41 (m, 4H), 1.32 (dd, J = 17.8, 9.8 Hz, 6H), 0.88 (t, J = 6.8 Hz, 3H). LCMS: 479.5 [M+H]+. Phenyl


yl)oxy)propyl)carbamate (27q). 1H NMR (400 MHz, CDCl3) δ 9.22 (s, 1H), 8.94 (s, 1H), 8.75 (t, J = 18.4 Hz, 1H), 8.14 (d, J = 7.9 Hz, 1H), 7.83 (t, J = 6.9 Hz, 1H), 7.36 (dd, J = 15.7, 7.8 Hz, 3H), 7.15 (dd, J = 24.0, 7.7 Hz, 2H), 6.98 (s, 2H), 6.38 (s, 1H), 4.48 (t, J = 5.7 Hz, 2H), 4.12 – 4.02 (m, 3H), 3.65 – 3.49 (m, 2H), 2.35 – 2.17 (m, 2H). LCMS: 513.5 [M+H]+. neopentyl


yl)oxy)propyl)carbamate (27r). 1H NMR (400 MHz, CD3OD) δ 9.09 (s, 1H), 8.88 (s, 2H), 8.66 – 8.51 (m, 1H), 8.53 (d, J = 14.7 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.95 (t, J = 7.1 Hz, 1H), 7.48 – 7.41 (m, 1H), 4.32 (t, J = 6.0 Hz, 2H), 4.04 (s, 3H), 3.68 (d, J = 13.9 Hz, 3H), 2.08 (t, J = 6.4 Hz, 2H), 0.85 (s, 9H). LCMS: 507.5 [M+H]+. N-(3-((4-(5-amino-3-(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)-7-methoxyquinazolin-6yl)oxy)propyl)-2,2,2-trifluoroacetamide (27s). 1H NMR (400 MHz, CDCl3+ CD3OD) δ 9.03 (s, 1H), 8.96 (s, 1H), 8.72-8.68 (m, 1H), 8.20 – 8.15 (m,1H), 7.84 (td, J = 7.7, 1.8 Hz, 1H), 7.44 – 7.35 (m, 2H), 7.06 (d, J = 13.4 Hz, 2H), 5.76 (s, 1H),4.41 (t, J = 5.8 Hz, 2H), 4.19 – 4.05 (m, 2H), 4.03 (s, 3H), 2.26 – 2.08 (m, 2H). LCMS: 489.0 [M+H]+.

Biology. Cell Culture and Irradiation: MCF7 cells were obtained from ATCC (American Type Culture Collection, Manassas, VA), and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum at 37 °C in a humidified atmosphere (5% CO2). Cells were irradiated using a



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40 137Cs-source (Gamma cell 40-exactor, MDS Nordion, Ottawa, Ontario, Canada) at a dose of 2-10 Gy. In-Cell Western Assay: Quantitative immunofluorescence assay (In-Cell western assay) was used for testing ATM kinase inhibitors. 1x105 of MCF7 cells were plated in 384-well plates and incubated overnight in an incubator with 5% CO2 atmosphere at 37 °C. After incubation, using liquid handling equipment Biomek FX (Beckman Coulter) automation workstation, compounds were transferred into the wells of plates with DMSO included as a negative control and compound 4 as a positive control. Etoposide, A DNA damaging agent, was transferred to each well to a final concentration of 25 µM to activate ATM kinase. Next, cells were incubated at 37 °C for 1 h, then immediately fixed by adding formaldehyde to a final concentration of 4% (vol./vol.), and further incubated for 20 min at room temperature. After being washed 3 times with 1x PBS, cells were permeabilized by incubating with 1x PBS containing 0.1% Triton X-100 for 20 min. After permeabilization, cells were then incubated overnight at 4°C with 20 µL of p-KAP1 antibody (1:1000, Bethyl Laboratories). The next day, plates were washed three times with 1x PBS containing 0.1% Tween-20. Subsequently, 20 µL of IRDye 800CW Goat anti-Rabbit secondary antibody solution (1:5000, LI-COR life science) containing DNA stain DRAQ5 (1:5000) was added into each well. After 1 h incubation at room temperature, the plates were washed 3 times with 1x PBS containing 0.1% Tween-20, then turned upside down to remove washing buffer, and scanned on an Aerius automated Infrared Imaging system (Licor Biosciences) for quantitative analysis. Images for nuclear and p-KAP1 staining were obtained by using the 700 nm and 800 nm channels of the Aerius Infrared imaging system, respectively. Fluorescence intensities were measured using the software provided with the imaging system.


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41 Western Blotting: MCF7 cells were pretreated with ATM inhibitors or DMSO for 30 min and then irradiated at 10 Gy. 1 h after IR, cells were harvested and lysed in RIPA buffer (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.1% SDS, 1% NP-40) supplemented with protease and phosphatase inhibitor cocktails from Roche diagnostics. Lysates were centrifuged at 13000 x g for 20 min at 4˚C. After centrifugation, supernatants were transferred into new 1.5 mL tubes, protein concentrations were quantitated using DCTM Protein Assay from Bio-Rad laboratories (Hercules, CA). Equal amounts of total protein (100 µg) for each lysate was loaded on a 4-12% NuPAGEBis-Tris pre-cast gel (Life Technologies, Grand Island, NY). After running for 1.5 h under a constant 120 V, resolved proteins were electronically transferred onto 0.45 µM PVDF transfer membrane overnight with 800 mA in a TE 62 transfer unit (GE Healthcare Life Sciences, Pittsburgh, PA). After transfer, the membrane was cut into 4 strips for blotting of anti–phospho-ATM(S1981),

anti–phospho-KAP1(S824), anti–

phospho-p53(S15), and anti–β-actin respectively. Membranes were then blocked with 1xTrisbuffered saline with 0.1% Tween (TBST) buffer containing 5% non-fat milk for 1 h at RT. After blocking, membranes were rinsed with TBST buffer and incubated with different primary antibodies overnight at 4˚C with gentle agitation. Primary antibodies were prepared in 1:1000 dilutions in 1× Tris-buffered saline with Tween (TBST) buffer containing 5% bovine serum albumin (BSA). Antibodies used in this experiment include anti–phospho-ATM(S1981) (cat. 2152-1; Epitomics, Burlingame, CA), anti–phospho-p53(S15) (cat. 9284L; Cell Signaling, Danvers, MA), anti– phospho-KAP1(S824) (cat. A300-767A; Bethyl Laboratories, Montgomery, TX), anti-total-ATM (cat. ab59541; Abcam, Cambridge, MA), and anti–β-actin (cat.A5316; Sigma, St. Louis, MO). The next day, after removing primary antibodies, membranes were washed 4 times for 5 min/wash with TBST. Next, membranes were incubated in the dark for 1 h at RT with gentle agitation with IRDye 800CW goat anti-rabbit or anti-mouse secondary bodies (LI-COR Biosciences, Lincoln, NE) in



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42 1:5000 dilution in TBST containing 5% non-fat milk. After incubation, membranes were washed 4 times for 5 min/wash with TBST and then visualized using an Odyssey CLx infrared imaging system (LI-COR Biosciences, Lincoln, NE). Clonogenic assay: 0.5x106 of MCF7 cells were plated in each well in a 6-well plate and incubated overnight at 37˚C. The next day, cells were pre-treated with DMSO, or various ATM kinase inhibitors at a final concentration of 10 µM. After 1 h of pretreatment with inhibitors, cells were irradiated at 2 or 4 Gy and incubated for another 4 h with DMSO or ATM inhibitors. Cells were then harvested, re-suspended in media, and counted using a cell counter. A total of 2,000 cells for each treatment were placed in a 10-cm plate in the absence of inhibitors, and were cultured for 10 days. After a 10-day incubation, the culture media was removed. Cells were washed with PBS once. Then 5 ml of a mixture of 6.0% glutaraldehyde and 0.5% crystal violet was added into each plate and incubated for 30 min. Next, the glutaraldehyde crystal violet mixture was decanted. The plates were rinsed with tap water, and air dried at room temperature. Colonies containing over 50 cells were counted for each plate. This experiment was run in triplicate. The average numbers of colonies were calculated and normalized to that of the DMSO-treated plates. Surviving fractions for each treatment were plotted using GraphPad Prism software. In vivo Pharmacokinetic Study: Female C57BL/6 mice of 22-27 grams were purchased from Charles River Laboratories (Wilmington, MA). Food and water were provided ad libitum. For each compound, 18 mice were divided into 3 dosage groups of 0, 10, and 20 mg/kg. Solubilized compound in formulation (5/45/50, EtOH/PPG/PBS (7.4), HβCD 10 mM, with 5% DMSO) was given to each animal by tail vein injection at a volume of 4 ml/kg. 0.1 ml blood was collected retroorbitally from a different mouse within each dosage group at 5, 15 min, 30 min, 1 h, 4 h, and 24 h post administration (n= 6 for 6 time points). A terminal blood collection via cardiac puncture was


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43 conducted on each animal to get 0.5-0.7 mL blood for 48 h time point. Blood samples were treated with 10% EDTA disodium at collection. Each blood sample was centrifuged for 3 min at 13,200 rpm in a desktop centrifuge to get plasma samples. Each sample was combined with three volumes of internal standard (2 uM warfarin in acetonitrile) and centrifuged at 3 min at 13,200 rpm twice. The supernatant was transferred to a 96-well assay plate and concentration was determined with UPLC/MS (Waters Corp., Milford, MA). Quantification was based on the peak area ratio (compound of interest / internal standard).

ANCILLARY INFORMATION Supporting Information Experimental procedures for early ADMET and MTD study; Permeability data for all compounds; in vivo PK and metabolic identification data for compound 4; synthetic scheme for 19, 20, and linker intermediates for 27m-o and 27i; Cytotoxicity data against HepG2, HEK293, and Raji cell lines for all compounds; kinome scan analysis for compound 4 and 27g; Kds for selected kinases of compound 27g; Western blot analysis for the effect of compounds 1, 4, and 20; Liver microsome stability data for compound 4, 20 and 27g; Complete blood count data, blood chemistry panel and pathological review of tissues for compound 20 and 27g.

Corresponding Author *E-mail: [email protected]. Phone: 901-595-5714. Fax: 901-595-5715

ABBREVIATIONS USED



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44

ATM, ataxia-telangiectasia mutated; PI3K, phosphatidylinositol 3-kinase; PIKK, phosphatidylinositol 3kinase-related kinase; DSB, DNA double-strand break; IR, ionizing radiation; NBS1, Nijmegen Breakage Syndrome Gene; Smc, structural maintenance of chromosome; FancD2, Fanconi anemia group D2; Rad17, Cell cycle checkpoint protein RAD17; mTOR, Mammalian Target of Rapamycin; DNA-PKcs,

DNA-

dependent protein kinase catalytic subunit; HNO3, Nitric acid; AcOH, acetic acid; FeCl3, Iron(III) chloride; Cs2CO3, cesium carbonate; ELSD, evaporative light scattering detector; CLND, Chemiluminescent nitrogen detection; CLint, intrinsic clearance; Cmax, Maximum Concentration observed; KAP1, KRAB domainassociated protein 1

Reference

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Abraham, R. T. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes

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Shiloh, Y. ATM and related protein kinases: safeguarding genome integrity. Nat. Rev.

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Bassing, C. H.; Alt, F. W. The cellular response to general and programmed DNA double

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Khanna, K. K.; Jackson, S. P. DNA double-strand breaks: signaling, repair and the cancer

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van, G. D. C.; Hoeijmakers, J. H.; Kanaar, R. Chromosomal stability and the DNA double-

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Shiloh, Y. Ataxia-telangiectasia and the Nijmegen breakage syndrome: related disorders but

genes apart. Annu. Rev. Genet. 1997, 31, 635-662. 9.

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and normal tissues to radiation. Nat. Rev. Drug Discov. 2013, 12, 526-42. 11.

Bakkenist, C. J.; Kastan, M. B. DNA damage activates ATM through intermolecular

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Kastan, M. B.; Bartek, J. Cell-cycle checkpoints and cancer. Nature 2004, 432, 316-23.

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Khalil, H. S.; Tummala, H.; Hupp, T. R.; Zhelev, N. Pharmacological inhibition of ATM by

KU55933 stimulates ATM transcription. Exp. Biol. Med. 2012, 237, 622-34. 14.

Rainey, M. D.; Charlton, M. E.; Stanton, R. V.; Kastan, M. B. Transient Inhibition of ATM

kinase Is sufficient to enhance cellular sensitivity to ionizing radiation. Cancer Res. 2008, 68, 74667474. 15.

Andrs, M.; Korabecny, J.; Nepovimova, E.; Jun, D.; Hodny, Z.; Moravcova, S.; Hanzlikova,

H.; Kuca, K. The development of ataxia telangiectasia mutated kinase inhibitors. Mini-Rev. Med. Chem. 2014, 14, 805-811.



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46 16.

Sarkaria, J. N.; Tibbetts, R. S.; Busby, E. C.; Kennedy, A. P.; Hill, D. E.; Abraham, R. T.

Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizing agent wortmannin. Cancer Res. 1998, 58, 4375-82. 17.

Hickson, I.; Zhao, Y.; Richardson, C. J.; Green, S. J.; Martin, N. M. B.; Orr, A. I.; Reaper, P.

M.; Jackson, S. P.; Curtin, N. J.; Smith, G. C. M. Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res. 2004, 64, 91529159. 18.

Golding, S. E.; Rosenberg, E.; Valerie, N.; Hussaini, I.; Frigerio, M.; Cockcroft, X. F.;

Chong, W. Y.; Hummersone, M.; Rigoreau, L.; Menear, K. A.; O'Connor, M. J.; Povirk, L. F.; van, M. T.; Valerie, K. Improved ATM kinase inhibitor KU-60019 radiosensitizes glioma cells, compromises insulin, AKT and ERK prosurvival signaling, and inhibits migration and invasion. Mol. Cancer Ther. 2009, 8, 2894-2902. 19.

Hollick, J. J.; Rigoreau, L. J. M.; Cano-Soumillac, C.; Cockcroft, X.; Curtin, N. J.; Frigerio,

M.; Golding, B. T.; Guiard, S.; Hardcastle, I. R.; Hickson, I.; Hummersone, M. G.; Menear, K. A.; Martin, N. M. B.; Matthews, I.; Newell, D. R.; Ord, R.; Richardson, C. J.; Smith, G. C. M.; Griffin, R. J. Pyranone, thiopyranone, and pyridone inhibitors of phosphatidylinositol 3-kinase related kinases. Structure-activity relationships for DNA-dependent protein kinase inhibition, and identification of the first potent and selective inhibitor of the ataxia telangiectasia mutated kinase. J. Med. Chem. 2007, 50, 1958-1972. 20.

Li, Y.; Yang, D.-Q. The ATM inhibitor KU-55933 suppresses cell proliferation and induces

apoptosis by blocking AKT in cancer cells with overactivated AKT. Mol. Cancer Ther. 2010, 9, 113-125.


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47 21.

Mukherjee, B.; Tomimatsu, N.; Amancherla, K.; Camacho, C. V.; Pichamoorthy, N.; Burma,

S. The dual PI3K/mTOR inhibitor NVP-BEZ235 is a potent inhibitor of ATM- and DNA-PKCsmediated DNA damage responses. Neoplasia 2012, 14, 34-43. 22.

Guo, K. X.; Shelat, A. A.; Guy, R. K.; Kastan, M. B. Development of a cell-based, high-

throughput screening assay for ATM kinase inhibitors. J. Biomol. Screen. 2014, 19, 538-546. 23.

Bridges, A. J.; Zhou, H.; Cody, D. R.; Rewcastle, G. W.; McMichael, A.; Showalter, H. D.

H.; Fry, D. W.; Kraker, A. J.; Denny, W. A. Tyrosine kinase inhibitors: unusually steep structureactivity relationship for analogs of 4-(3-bromoanilino)-6,7-dimethoxyquinazoline (PD 153035), a potent inhibitor of the epidermal growth factor receptor. J. Med. Chem. 1996, 39, 267-76. 24.

Hennequin, L. F.; Thomas, A. P.; Johnstone, C.; Stokes, E. S. E.; Ple, P. A.; Lohmann, J.-J.

M.; Ogilvie, D. J.; Dukes, M.; Wedge, S. R.; Curwen, J. O.; Kendrew, J.; Lambert-van, d. B. C. Design and structure-activity relationship of a new class of potent VEGF receptor tyrosine kinase inhibitors. J. Med. Chem. 1999, 42, 5369-5389. 25.

Matsuno, K.; Ushiki, J.; Seishi, T.; Ichimura, M.; Giese, N. A.; Yu, J.-C.; Takahashi, S.; Oda,

S.; Nomoto, Y. Potent and selective inhibitors of platelet-derived growth factor receptor phosphorylation. 3. Replacement of quinazoline moiety and improvement of metabolic polymorphism of 4-[4-(N-Substituted (thio)carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives. J. Med. Chem. 2003, 46, 4910-4925. 26.

Lemoff, A.; Yan, B. Dual detection approach to a more accurate measure of relative purity in

high-throughput characterization of compound collections. J. Comb. Chem. 2008, 10, 746-751. 27.

McKillop, D.; Hutchison, M.; Partridge, E. A.; Bushby, N.; Cooper, C. M. F.; Clarkson-

Jones, J. A.; Herron, W.; Swaisland, H. C. Metabolic disposition of gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, in rat, dog and man. Xenobiotica 2004, 34, 917-934.



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48 28.

McKillop, D.; Partridge, E. A.; Hutchison, M.; Rhead, S. A.; Parry, A. C.; Bardsley, J.;

Woodman, H. M.; Swaisland, H. C. Pharmacokinetics of gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, in rat and dog. Xenobiotica 2004, 34, 901-915. 29.

Leonard, W. J.; O'Shea, J. J. JAKS and STATS: biological implications. Annu. Rev.

Immunol. 1998, 16, 293-322. 30.

Ghoreschi, K.; Laurence, A.; O'Shea, J. J. Janus kinases in immune cell signaling. Immunol.

Rev. 2009, 228, 273-287. 31.

Li, M. Z.; Yu, L.; Liu, Q.; Chu, J. Y.; Zhao, S. Y. Assignment of NEK6, a NIMA-related

gene, to human chromosome 9q33.3 -> q34.11 by radiation hybrid mapping. Cytogenet. Cell Genet. 1999, 87, 271-272. 32.

Kandli, M.; Feige, E.; Chen, A.; Kilfin, G.; Motro, B. Isolation and characterization of two

evolutionarily conserved murine kinases (NEK6 and NEK7) related to the fungal mitotic regulator, NIMA. Genomics 2000, 68, 187-196. 33.

Toulany, M.; Mihatsch, J.; Holler, M.; Chaachouay, H.; Rodemann, H. P. Cisplatin-mediated

radiosensitization of non-small cell lung cancer cells is stimulated by ATM inhibition. Radiother. Oncol. 2014, 111, 228-36.

Figure, Scheme, and Table Legends Figure 1. Structures of representative ATM inhibitors. The median potency (IC50) of the inhibitors is shown, measured using either the in vitro kinase assay (biochemical) or the in-cell Western assay (cellular).


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49 Figure 2. Observed in vivo metabolism of compound 4. Three metabolites were identified: phase I metabolites 5 and 6 and phase II metabolite 7.

Figure 3. Outline of lead optimization studies of compound 4 presented herein, indicating positions varied in the studies.

Figure 4. Pharmacokinetics of compounds 4, 20, and 27g observed during in vivo murine dose ranging studies by the intravenous route. Graph shows plasma concentrations of parent molecule versus time after dosing. Each compound (20 mg/kg) was dissolved in 5% DMSO with a final formulation of 5/45/50 (v/v/v), EtOH/PPG/PBS (7.4), HβCD 10 mM) and dosed via tail vein injection. At 5 min, 15 min,30 min, 1 h, , 4 h, 24 and 48 h, blood was taken and plasma was analyzed by LC/MS. Concentrations of all three compounds vs time were plotted.

Figure 5. Inhibition of ATM driven phosphorylation events by selected analogs after IR-induced DNA damage. MCF7 cells were treated with DMSO or compounds 1, 4, 27g, or 27n at concentrations of 1, 5, or 10 µM for 30 min before mock-IR (control) or IR (10 Gy). Cells were lysed 1 h after IR and probed by Western blot analysis using anti-phospho-ATM, anti-phosphoKAP1, or anti-phospho-p53 antibodies. Total ATM and beta-actin protein levels were used as loading controls.

Figure 6. Sensitization of MCF7 cells to IR-induced DNA damage by compounds 1, 4, 20, 27g, or 27n. MCF7 cells were plated in triplicate and incubated for 16 h. Cells were pre-incubated with DMSO or 10 µM of compound 4, 1, 27g, 27n, or 20 for 10 min before being exposed to escalating



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Page 50 of 67

50 doses of IR (0-4 Gy). Media was removed 4 h after IR and replaced with fresh media without drugs. After 10 days of incubation, the cells were fixed and stained with crystal violet (0.5%). Surviving colonies on each plate were counted.

Scheme 1. General route for the synthesis of substituted triazol 14 and quinazoline derivatives 15-20 Scheme 2. Synthesis of C-2-substituted quinazolines 22a-j Scheme 3. Synthesis of compounds 23a-k and 24a-f Scheme 4. General synthesis of compounds 27a-s containing linkers at the C-6 position Scheme 5. Synthetic route for the preparation of compound 27i

Table 1. SAR exploration of the B ring modified at C-6,7 positions for compounds 15a-20 Table 2. SAR exploration of the A ring modified at the C-2 position for compounds 22a-j Table 3. SAR analysis of modification to the C and D rings for compounds 23a-k Table 4. Selected modifications of the B ring for compounds 24a-f Table 5. Further SAR studies of substituents at C-6 for compounds 27a-s Table 6. In vitro pharmacology of selected ATM inhibitors

.


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51

Figure 1.



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Page 52 of 67

52

Ph

e as

II

or uc l g

Figure 2.

Figure 3.


at id n o

n io

Page 53 of 67

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53

Figure 4.



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Page 54 of 67

54

Figure 5.


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55

Figure 6.



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Page 56 of 67

56

Scheme 1. General route for the synthesis of substituted triazol 14 and quinazoline derivatives 15-20a

a

Reagents and conditions: (a) 70% HNO3, AcOH, 50 oC, 3 h, 91-94%; (b) Fe/FeCl3, AcOH, EtOH, H2O, or H2Pd-C, MeOH, 80 oC, 90%; (c) NH2CHO, 190 oC, 75-86%; (d) SOCl2, catalytic DMF, 85 oC, 95%; (e) substituted 1,2,4-triazol-5-amines, Cs2CO3, DMF, 90 oC, 5085%; (f) Reflux/Wood’s mal bath, 210 oC, 2 h, quant.


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57 Scheme 2. Synthesis of C-2-substituted quinazolines 22a-ja

a

Reagents and conditions: (a) Cs2CO3, DMF, 90 oC, 6 h, 61%; (b) morpholine (for 22b), NMethyl piperizine (for 22c), 2-(piperazine-1-yl) ethanol (for 22d), or Ethyl-2-amino acetate. HCl (for 22e), NMP, 140 oC, 4 h, 52-70%, diethylamine, i-PrOH, 4 M HCl/dioxane, Microwave, 160 oC, 10 min, 55% (for 22i); (c) Li2O2, MeOH:H2O (3:1), 40 min, rt, 45% (for 22f). Scheme 3. Synthesis of compounds 23a-k and 24a-fa

a

Reagents and conditions: (a) amines or anilines or substituted triazoles, Cs2CO3, DMF, 90 ~130 oC, 6~16 h, 46-75%; (b) 14, Cs2CO3, DMF, 90 oC, 6 h, 65-75%; (c) 10% Pd-C/ MeOH, 80-85%



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Page 58 of 67

58

Scheme 4. General synthesis of compounds 27a-s containing linkers at the C-6 positiona

a

Reagents and conditions: (a) substituted alcohols (correspond to 27d, 27e, and 27g), DTAD, TPP, DCM, 0 °C-rt, 55-70%; (b) substituted alkyl halides (correspond to 27a-c, 27f, and 27h-o), K2CO3, DMF, 90 °C, 70-75%; (c) 14, Cs2CO3, DMF, 90 °C, 6-8 h, 50-65%; (d) HCl/THF, rt, 1 h, 95%; (e) substituted chloroformates (for 27p-r) or CF3C(O)Cl (for 27s), TEA/DCM, 0 °C-rt, overnight, 70%85% Scheme 5. Synthetic route for the preparation of compound 27ia

a

Reagents and conditions: (a) 34, K2CO3, DMF, 90 °C, 4 h, 62%; (b) 14, Cs2CO3, DMF, 90 °C, 4 h, 54%


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59 Table 1.

Compd

R1

1

R2

R3

KU55933

ATM Inhibition IC50 (µM)a

Solubility pH=7.4 (µM)b

EC50 BJ (µM)

0.3 ± 0.1

NDc

ND

4

-OMe

-OMe

2-pyridyl

0.4 ± 0.2

28 ± 2

>50

15a

-H

-H

phenyl

>40

1±1

>50

15b

-H

-H

2-pyridyl

>40

9±1

>50

15c

-H

-H

p-methoxy phenyl

>40

5±1

>50

15d

-H

-H

p-chloro phenyl

>40

1±1

>50

15e

-H

-Cl

phenyl

>40

0.3 ± 1

>50

15f

-H

-Cl

2-pyridyl

>40

9±1

>50

15g

-H

-Cl

p-methoxy phenyl

>40

2±1

>50

15h

-H

-Cl

p-chloro phenyl

>40

5±1

>47

15i

-NO2

-Cl

2-pyridyl

>40

3±1

>13

15j

-NH2

-Cl

2-pyridyl

>40

3±1

27

15k

-NO2

-OMe

2-pyridyl

31.1 ± 8.7

2±1

>13

15l

-NH2

-OMe

2-pyridyl

>40

3±1

ND

15m

-NO2

2-pyridyl

>40

4±1

ND

15n

-OMe

-H

2-pyridyl

16.9 ± 7.1

7±1

>27

15o

-H

-OCF3

2-pyridyl

>40

3±1

>13

16

-F

-F

2-pyridyl

>40

ND

ND

2-pyridyl

>40

60 ± 4

>55

3-pyridyl

15.0 ± 0.1

27 ± 6

>71

17 18a

-OMe

-OMe



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Page 60 of 67

60 18b

-OMe

-OMe

4-pyridyl

5.0 ± 3.5

ND

ND

18c

-OMe

-OMe

phenyl

2.0 ± 1.1

1 ±1

>50

18d

-OMe

-OMe

p-methoxy phenyl

>40

2±1

>50

18e

-OMe

-OMe

p-chloro phenyl

>40

1±1

>50

18f

-OMe

-OMe

cyclohexyl

>40

35 ± 1

>27

2-pyridyl

>40

1±1

>27

2-pyridyl

2.3 ± 0.7

30 ± 1

>27

19 O

20

O

a

Values are means of three independent experiments, each run in triplicate. bValues are means of two independent experiments, each run in triplicate. cND, not determined

Table 2.

Compd

R4



EC50 BJ (µM)

4

-H

0.4 ± 0.2

28 ± 2

>50

22a

>40

13

22b

>40

5±1

>27

22c

>40

30 ± 1

>13

22d

>40

58 ± 1

>27

22e

>40

48 ± 2

>27

22f

>40

3±1

>25


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61

22g

>40

6±1

>9

22h

10.1 ± 1.3

16 ± 1

>13

22i

>40

2±1

>27

22j

2.1 ± 0.6

27 ± 1

>27

a

Values are means of three independent experiments, each run in triplicate. bValues are means of two independent experiments, each run in triplicate. cDL, detection limit

Table 3.



EC50 BJ (µM)

4

0.4 ± 0.2

28 ± 2

>50

23a

>40

160 ± 2

>27

23b

>40

NDc

>27

23c

12.5 ± 2.1

3±1

>27

23d

5.3 ± 1.2

30 ± 2

>27

23e

13.3 ± 4.0

3±1

>27

Compd

R



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Page 62 of 67

62

23f

1.6 ± 0.7

1±1

>27

23g

20.6 ± 5.1

ND

ND

23h

>40

ND

ND

23i

>40

ND

ND

23j

2.4 ± 0.1

1±1

>27

23k

>40

83 ± 3

>33

a



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63 Table 4. N N H2N

N

R1O

N

R2O

Compd

R1

4

-Me

24a 24b

-H

24c

-Me

24d

-Me

24e

24f

-Me

N

N



EC50 BJ (µM)

-Me

0.4 ± 0.2

28 ± 2

>50

-Me

8.6 ± 2.1

58 ± 2

>27

-Me

12.6 ± 1.5

77 ± 2

>27

>40

68 ± 1

>27

-H

>40

44 ± 4

>27

-Me

2.2 ± 0.3

38 ± 1

>27

>40

66 ± 3

>54

R2

a

Values are means of three independent experiments, each run in triplicate. bValues are means of two independent experiments, each run in triplicate.



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Page 64 of 67

64

Table 5.

Compd

R1



EC50 BJ (µM)

4

-Me

0.4 ± 0.2

28 ± 2

>50

27a

6.6 ± 1.4

53 ± 2

>25

27b

6.1 ± 0.8

33 ± 1

>25

27c

2.7 ± 0.7

NDc

>25

27d

5.6 ± 0.1

36 ± 1

>12

27e

1.7 ± 0.1

24 ± 1

>8

27f

1.6 ± 0.4

8±1

>25

27g

1.2 ± 0.3

8±1

>13

27h

5.3 ± 0.9

42 ± 2

>25


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65

27i

1.7 ± 0.3

34 ± 1

>25

27j

0.9 ± 0.1

2±1

>12

27k

0.8 ± 0.1

0.5 ± 0.1

>7

27l

0.8 ± 0.1

2±1

>15

27m

0.8 ± 0.3

16 ± 1

>11

27n

1.0 ± 0.2

18 ± 1

>13

27o

1.2 ± 0.5

0.1 ± 1

>11

27p

28.6 ± 8.3

1±1

ND

27q

23.5 ± 3.7

6±1

ND

27r

1.9 ± 0.6

0.2 ± 1

ND

27s

2.9 ± 0.6

1±1

>7

a




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Page 66 of 67

66 Table 6. In vitro properties

4

20

27g

27n

Solubility (µM)

pH = 7.4

28.8

30.1

7.8

2.2

Permeability (10-6 cm/s)

pH = 7.4

95.9

16.6

196.3

432.5

Mouse

0.9/61.7

2.4/22.9

2.0/27.1

0.2/329.4

Rat

0.8/57.8

>4.0/4.0/4.0/48 >48 19 ± 1 ND >48 >48 77.9 ± 2.2 78.7 73.1 ± 4.2 ND 86.7 ± 1.4 98.3 ND ND >48 >48 >48 >48 >48 >48 ND = not determined

>4.0/4.0/48 >24 >48 94.1 90.1 79.6 97.7 >24 >24 >24

1.1/18.8 >4.0/24 >24 >24 ND ND ND ND >24 >24 >24

Liver microsome stability t1/2 (h) /CLint (mL/min/kg) Plasma stability (h)

Plasma protein binding (%)

PBS stability (h)

Human Dog Mouse Rat Human Mouse Rat Human Dog pH = 7.4 pH = 5.0 pH = 3.0


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67 Table of Contents graphic


Optimization of a Novel Series of Ataxia ... - ACS Publications

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