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Discovery of Novel 3-Quinoline Carboxamides as Potent, Selective and Orally Bioavailable Inhibitors of Ataxia Telangiectasia Mutated (ATM) Kinase Sébastien L. Degorce, Bernard Barlaam, Elaine Cadogan, Allan Paul Dishington, Richard Ducray, Steven C. Glossop, Lorraine A. Hassall, Franck Lach, Alan Lau, Thomas M. McGuire, Thorsten Nowak, Gilles Ouvry, Kurt G. Pike, and Andrew G. Thomason J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00519 • Publication Date (Web): 03 Jun 2016 Downloaded from http://pubs.acs.org on June 4, 2016

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Discovery of Novel 3-Quinoline Carboxamides as Potent, Selective and Orally Bioavailable Inhibitors of Ataxia Telangiectasia Mutated (ATM) Kinase Sébastien L. Degorce,*,†,‡ Bernard Barlaam,† Elaine Cadogan,† Allan Dishington,† Richard Ducray,‡ Steven C. Glossop,† Lorraine A. Hassall,† Franck Lach,‡ Alan Lau,† Thomas M. McGuire,† Thorsten Nowak,† Gilles Ouvry,‡ Kurt G. Pike,† and Andrew G. Thomason.† †

Oncology Innovative Medicines Unit, AstraZeneca, Mereside, Alderley Park, Macclesfield,

Cheshire SK10 4TG, United Kingdom;



Oncology Innovative Medicines Unit, AstraZeneca,

Centre de Recherches, Z.I. la Pompelle, BP1050, 51689 Reims Cedex 2, France.

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ABSTRACT

A novel series of 3-quinoline carboxamides has been discovered and optimized as selective inhibitors of the Ataxia Telangiectasia Mutated (ATM) Kinase. From a modestly potent HTS hit (4), we identified molecules such as 6-[6-(methoxymethyl)-3-pyridinyl]-4-{[(1R)-1-(tetrahydro2H-pyran-4-yl)ethyl]amino}-3-quinolinecarboxamide

(72)

and

7-fluoro-6-[6-

(methoxymethyl)pyridin-3-yl]-4-{[(1S)-1-(1-methyl-1H-pyrazol-3-yl)ethyl]amino}quinoline-3carboxamide (74) as potent and highly selective ATM inhibitors with overall ADME properties suitable for oral administration. 72 and 74 constitute excellent oral tools to probe ATM inhibition in vivo. Efficacy in combination with the DSB-inducing agent irinotecan was observed in a disease relevant model.

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INTRODUCTION The Ataxia Telangiectasia Mutated (ATM) kinase1 is an atypical serine/threonine protein kinase involved in DNA damage responses (DDR).2 It belongs to the so-called PIKK (PhosphatidylInositol 3’-Kinase [PI3K]-related kinase) family, together with DNA-dependent Protein Kinase (DNAPK), Ataxia Telangiectasia mutated and RAD3-related (ATR) kinase, themselves also key DDR targets, and mammalian Target Of Rapamycin (mTOR). ATM detects DNA double strand breaks (DSBs),3 and signals activation of downstream DNA repair and cell cycle checkpoint. As DSBs can be caused by ionizing radiation (IR) or chemically induced DNA damage (e.g. the DNA topoisomerase I inhibitor irinotecan4), the inhibition of ATM in combination with clinically induced DNA damage in tumour cells could bring important benefits to cancer patients by potentiating the effects of such agents or techniques.5, 6 Few inhibitors of ATM have been previously reported, and their use has been limited by their weak potency and/or lack of PIKK-selectivity, sometimes combined with unsuitable pharmacokinetic properties. Amongst those, 1 (KU-55933)7 and its improved version 2 (KU60019)8 from Kudos Pharmaceuticals (now part of AstraZeneca) and the structurally distinct quinazoline 3 (CP-466722)9 from Pfizer are probably the most studied. Pyran-4-one 1 is a potent and reasonably selective inhibitor of ATM with demonstrated activity in cells, but suffers from poor solubility, high turnover in hepatocytes, and therefore could not be used as an in vivo tool orally. It was later optimized to 2, a roughly 10-fold more potent ATM inhibitor with higher solubility and reduced hERG activity, but in our hands remained a short half-life molecule despite improved metabolic stability (Table 1). After oral dosing, only low exposure in mice could be achieved, insufficient to achieve durable ATM inhibition. Quinazoline 3 was, in our hands, less potent both in enzyme and cell assays than 2, and with similar selectivity against

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PI3K and PIKK kinases; however, despite lower lipophilicity 3 also suffered from moderate to high turnover in rodent hepatocytes, which made it unsuitable as an in vivo tool. Other pan-PI3K and/or pan-PIKK inhibitors showing good pharmacokinetic properties have also been reported to inhibit ATM.10 Unfortunately, these could not be used as ATM in vivo probes because of their lack of specificity. Our ambition was therefore to identify a novel, potent and selective ATM inhibitor with good oral bioavailability to evaluate the potential of more specific ATM inhibition in vivo. Figure 1. Selected examples of reported ATM inhibitors.

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SYNTHESIS Compounds reported herein were synthesized as shown in Schemes 1–4. 3-Quinoline carboxamides substituted with an aryl group at C6 and an amine at C4 were generally made using the versatile 6-bromo-4-chloroquinoline-3-carboxamide 11 via Suzuki cross-coupling with aryl boronic acids/esters followed by nucleophilic aromatic substitution (SNAr) with primary amines, or by reversing these steps. In some cases where the aryl boronic acids/esters were either unavailable or unreactive, the Suzuki cross-coupling was reversed and 6-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)-3-quinolinecarboxamides (e.g. 30) and aryl halides were used instead, as described in Scheme 1. 6-(3-Pyridinyl)-3-quinolinecarboxamides 13-23 were obtained via Suzuki cross-coupling first to give key intermediate 4-chloro-6-(3-pyridinyl)-quinoline-3-carboxamide 12, followed by SNAr with the appropriate primary amine or thiol. Synthesis of the 6-pyrazole analogue 25 was achieved in a similar manner, whilst 27-29 were obtained via SNAr first to give key intermediate 6-bromo-4-[(2-ethylbutyl)amino]-3-quinolinecarboxamide 26, followed by Suzuki crosscoupling. Compounds 31-37 were made from 4-[(2-ethylbutyl)amino]-6-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)-3-quinolinecarboxamide

30

and

the

corresponding

aryl

bromides/chlorides. 4-[(2-Ethylbutyl)amino]-6-(5-pyrimidinyl)-3-quinolinecarboxamide 33 was also obtained in this way using (2-chloro-5-pyrimidinyl)boronic acid in the cross-coupling step, with subsequent de-halogenation by hydrogenation. Likewise, cross-coupling with (5-bromo-2pyridinyl)methanol

provided

intermediate

6-[6-(hydroxymethyl)-3-pyridinyl]-3-

quinolinecarboxamide 35. After bromination of the alcohol, the methoxy analogue 36 and the methylsulfonyl analogue 37 were obtained by nucleophilic displacement with sodium methoxide or sodium methanesulfinate.

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O-linked 3-quinolinecarboxamide 40 was obtained using ethyl 6-bromo-4-chloroquinoline-3carboxylate 38 via displacement of the 4-chloro with 2-ethylbutyl alcohol with NaH in THF followed by treatment of the ester with aqueous ammonia (Scheme 2). 8-Methyl quinoline 43 was also obtained using the corresponding commercially available 3-carboxylate ester 41 which was saponified and reacted with thionylchloride and then aqueous ammonia to give the corresponding 4-chloro-6-bromo-3-carboxamide intermediate 42. Similar chemistry as shown in Scheme 1 was subsequently applied to achieve the desired substitution at C4 and C6. Further substitution on the quinoline core was completed by constructing the core using the appropriate anilines and acrylates to afford the desired key 4-chloro-6-bromo intermediates (Scheme 3). In the case of analogues 49-51, the 3-cyano quinolones 46-48 were formed by condensation of the appropriate aniline and either ethyl-2-cyano-3-ethoxybut-2-enoate (46) or ethyl-2-cyano-3-ethoxyacrylate (47-48). Conversion of the 3-cyano into the desired 3carboxamide was performed using acetaldoxime and palladium acetate. Analogues 58-60 were obtained in a similar way via condensation of the appropriate aniline and diethyl (ethoxymethylene)malonate, followed by cyclisation in diphenylether at high temperature. The 3-esters were then treated as described above to afford the 4-chloro-6-bromo-3-quinoline carboxamides 55-57 which were substituted at C4 and C6 in the usual way. Combinations 72-76 shown in Scheme 4 were performed in a similar manner as described above, using the handcrafted 2-(methoxymethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)pyridine 63 (obtained in two steps from (5-bromo-2-pyridinyl)methanol 61) or 2-fluoro-6(methoxymethyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine 64 (also obtained in two steps from 3-bromo-6-(bromomethyl)-2-fluoro-pyridine 62). Likewise, 1,3,4-trisubstituted pyrazoles 77-78 were synthesized in a similar way using the corresponding amines:

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fluoropyrazole 67 was obtained via fluorination of tert-butyl [(1S)-1-(1-methyl-1H-pyrazol-3yl)ethyl]carbamate 65 with Selectfluor; methylpyrazole 68 was obtained in a three step procedure involving reductive amination of 1-(1,4-dimethyl-1H-pyrazol-3-yl)ethanone 66 with the Ellman chiral auxiliary (R)-2-methylpropane-2-sulfinimide.11

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Scheme 1.a Synthesis of 3-quinoline carboxamides 13-37 from 6-bromo-4-chloroquinoline-3carboxamide 11.

a

Reagents and conditions: (a) 3-BPin-pyridine, Cs2CO3, Pd(PPh3)4, dioxane: water, 100 °C, 5 h (72%); (b) primary amine or cyclopentanethiol (15), DIPEA, DMF (13, 17-19, 23), NMP (1416, 20-21) or DMA (22-23), 80-100 °C; 10-16 h (19-85%); (c) 1-methyl-4-BPin-1H-pyrazole, Cs2CO3, Pd(PPh3)4, dioxane:water, 100 °C, 2 h (96%); (d) 2-ethylbutan-1-amine, DIPEA, DMF, 80 °C, 18 h (61%); (e) aryl boronic acid, Cs2CO3, Pd(PPh3)4, dioxane:water, 150 °C, 30 min (µwave) or 80 °C, 24 h (23-43%); (f) B2Pin2, (dppf)PdCl2, KOAc, THF, reflux, 3 d (50%); (g) aryl chloride/bromide, Na2CO3, (dppf)PdCl2, dioxane, 90 °C, 1 h (µwave, 30-60%); (h) step g,

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then NaHCO3, Pd/C, MeOH, 25 °C, 16 h (33, 51%); (i) i) PBr3, DCM, reflux, 16 h; ii) NaOMe, MeOH, 50 °C, 1 h (34%); (j) sodium methanesulfinate, DMA, 40 °C, 30 min (55%). Scheme 2.a Synthesis of 3-quinoline carboxamides 40 and 43 from ethyl 6-bromo-4chloroquinoline-3-carboxylates.

a

Reagents and conditions: (a) ethylbutyl alcohol, NaH, THF, RT, 2 h (quantitative); (b) i) 3pyridinylboronic acid, Na2CO3, Pd(PPh3)4, dioxane:water, 90 °C, 14 h (54%); ii) NH4OH, H2O, RT, 14 h (34%); (c) i) NaOH, H2O, 90-100 °C, 2 h; ii) SOCl2, DMF, 75-80 °C, 1 h; iii) NH4OH, H2O/acetone, 90 °C, 10-15 min (57-81%); (d) 3-pyridinylboronic acid, Cs2CO3, Pd(PPh3)4, dioxane:water, 100 °C, 3 h (32%); (e) 2-ethylbutan-1-amine, DIPEA, DMA, 100 °C, 2 h (72%).

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Scheme 3.a Synthesis of 5-, 7- or 8- substituted 3-quinoline carboxamides through construction of the quinoline core.

a

Reagents and conditions: (a) ethyl-2-cyano-3-ethoxybut-2-enoate (46) or ethyl 2-cyano-3ethoxyacrylate (47-48), EtOH, 80 °C, 5 h (46, 61%; 47-48, 72%); (b) i) POCl3, 110 °C, 8 h; ii) 2ethylbutan-1-amine or tetrahydro-2H-pyran-4-amine, K2CO3, DMF, RT; 14 h (25-43%); (c) 3pyridinylboronic acid, Na2CO3, Pd(PPh3)4, dioxane:water, 90 °C, 3 h (42-66%); (d) acetaldoxime, Pd(OAc)2, EtOH, 80 °C, 6 h (9-14%); (e) aniline, diethyl (ethoxymethylene)malonate, EtOH, 80 °C, 5 h, then Ph2O, 240 °C, 3 h; (f) i) LiOH, THF:EtOH:H2O, 55 °C, 1 h; ii) SOCl2, DMF, 70 °C, 3 h; iii) NH4OH, H2O, RT, 10-15 min (4151% over 4 steps); (g) tetrahydro-2H-pyran-4-amine, DIPEA, DMF, 100 °C; 14 h (25-86%).

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Scheme 4.a Final combinations

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a

Reagents and conditions: (a) iodomethane, NaH, THF, −20 °C, 1 h (92%); (b) B2Pin2, (dppf)PdCl2, KOAc, THF, reflux, 16 h (quant.); (c) NaOMe, MeOH, −30 °C, 1 h (74%); (d) (1S)1-(1-methyl-1H-pyrazol-3-yl)ethanamine, Selectfluor, MeCN, 20-80 °C, 60 h (25%); (e) (R)-2methylpropane-2-sulfinimide, TiOPr4, 2-methyltetrahydrofuran, 70 °C, 18 h, then s-BuLi, −78 °C, 1 h (89%); (f) HCl, dioxane, RT, 1 h (quant.); (g) (1S)-1-(1-methyl-1H-pyrazol-3yl)ethanamine or 67 or 68, DIPEA, DMA, 100 °C, 10-18 h (13-100%); (h) 63 or 64, Cs2CO3, Pd(PPh3)4, dioxane:water, 80 °C, 2.5 h (51-72%).

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RESULTS AND DISCUSSION A directed subset screen focused on likely kinase inhibitors from the AstraZeneca compound collection was conducted in a HT29 cell-based radiation-activated ATM assay and trisubstituted quinolines 4-6 caught our attention amongst an interesting cluster, with either a 2ethylbutylamino or benzylamino side chains at C4 and a primary carboxamide at C3 (Table 1). Selectivity was assessed using enzyme and cell assays previously reported by our group against a selection of PI3K and PIKK enzymes.12-17 Quinoline 4 was found to be a sub-micromolar inhibitor of ATM phosphorylation in cells, and its selectivity profile against members of the PIKK family and PI3K isoforms was encouraging, with >10-fold enzyme selectivity against DNAPK and PI3Kα, and >400-fold against mTOR and PI3Kβ, although selectivity against ATR in cells needed improving (10-fold) against ATR, suggesting that the 6-cyano group present in both 4 and 5 was sub-

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optimal. However, 6 was found to be roughly equipotent against DNAPK and PI3Kα, and generally even more promiscuous than both 4 and 5, suggesting that the combination of two pendant aryl groups was undesirable for selectivity. Table 1.a Profile of ATM hit 4-6 vs reported ATM inhibitors 1-3.

Entry

1

2

3

4

5

6

ATM enzyme IC50b 1 >1 >1 >1 0.204 0.045 PI3Kγ enzyme IC50 ATM cell IC50b 1.22 0.149 3.42 0.820 14.4 0.109 d ATR cell IC50 >30 8.50 >30 4.40 4.70 1.90 logD7.4e 4.0 3.6 2.6 3.5 3.5 >4.3 LLEf 1.9 3.2 2.9 2.6 1.3 30

ATR cell IC50a >30

8

H

9

>30

>30

10

>30

>30

All three compounds were tested inactive up to 30 µM in three independent measurements.

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Further follow-up testing around 4 led us to make the following observations: a) favored C4 analogues were all bearing an NH linker (in line with our internal H-bond hypothesis) and an extra methylene linker was preferred; b) a potency gain was observed with α-substituted groups (e.g. benzylamino vs. α-methyl benzylamino), although the preferred stereochemistry was unclear, as shown in Figure 2; c) evidence showed that 6-aryl groups brought a lot of potency to the molecules, in line with the 5/6 matched pair. Figure 2. Follow-up testing around hit 4. Every marker represents a pre-existing collection molecule tested against a cell-based ATM assay, and molecules are grouped on the x axis by type of group linker at the C4 position. Matched molecular pairs are connected. R4 R6

O NH2

N

With these results in hand, we synthesized and evaluated 3-pyridyl quinoline 13, which showed a 10-fold improvement in cell potency over our initial hit 4, combined with increased selectivity over ATR in cells (>150-fold vs 11

43/140

0.67

4.0

3.1

40

N/A

>30

>30

42/260

2.1

>4.3

-

14

N/A

0.137

1.2

23/65

0.57

3.6

3.3

15

N/A

9.7

>23

18/120

5.1

3.2

1.9

16

N/A

0.132

3.2

34/70

3.8

3.1

17

N/A

0.954

3.1

4.3

14

14/38

3.6

2.5

4.3

22

(R)

1.13

>18

3.4/120

2.8

2.3

3.6

23

(S)

0.124

0.64

10/31

11

2.5

4.4

0.730

3.5

14/46

4.3

2.3

3.8

a

Experimental assays as per Table 1 unless otherwise specified. b All IC50 data are expressed in micromolar (µM) and are the means of at least n=3 independent measurements. Each has a SEM ± 0.2 log units. c Hepatocyte intrinsic clearance expressed in µL/min/106 cells. d Inhibition of hERG channel in a electrophysiology (IonWorks™) assay.20

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Investigation at C6 A large investigation at C6 was carried out in parallel to that at C4 with the 2-ethylbutylamino side chain in place at C4, and allowed us to identify interesting aryl groups that are summarized in Table 4. There seemed to be some degree of tolerance for a range of 6-aryl groups, although close analogues of the 3-pyridyl such as the 4-pyridyl quinoline 27 or diazines 31-33 were all less active than parent 13 and displayed equally poor intrinsic clearances. The 3-pyridyl / 4pyridyl matched pair seemed to indicate a key role for the pyridine nitrogen when in the 3position, whilst any additional nitrogen in the ring was disfavored. Interestingly, the ATM potency of 6-(1-methylpyrazol-4-yl)quinoline 25 was within 10-fold of 13, and showed some improvements of hepatocyte turnover. The real breakthrough however was observed with psubstituted analogues: 6-(6-methyl-3-pyridyl)quinoline 34 showed a slight increase in cell potency compared to the unsubstituted 3-pyridyl, and 6-[4-(methoxymethyl)phenyl]quinoline 28 or 6-[4-(methylsulfonyl)phenyl]quinoline 29 had remarkably good potencies considering they lacked the key pyridyl nitrogen. This led us to make the 3-pyridyl analogues 36-37 in the hope of seeing the combined beneficial effects of the p-substitution and the 3-pyridyl. This was observed with quinoline 36 where the introduction of a nitrogen led to an 8-fold cell potency improvement relative to 28. We were also delighted to observe no activity against ATR in cells up to 30 µM and much improved kinase selectivity as exemplified by weak or no inhibition of enzymatic activity of KDR and GSK3β (IC50 = 82 µM and >100 µM respectively). In addition, despite a high logD7.4, 36 showed a low turnover in human hepatocytes, although it remained high in rat hepatocytes. Interestingly, the same was not observed with 37, with which only a modest improvement in potency was achieved. It is also noteworthy that despite a lower logD7.4, the sulphone showed no improvement in metabolic stability in rat hepatocytes.

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Table 4.a Selection of C6 substituents.

Entry

R6

ATM cell IC50b

ATR cell IC50b

Hu/Rat heps CLintc

hERG IC50

logD7.4

LLE

13

0.073

>11

43/140

0.67

4.0

3.1

25

0.525

7.1

13/74

1.2

3.4

2.7

27

0.418

0.73

37/76

0.62

3.3

3.1

31

0.571

>15

30/93

2.3

3.4

2.8

32

0.908

2.6

18/95

1.6

3.1

2.9

33

1.840

>18

46/140

0.76

3.3

2.4

34

0.050

5.5

7.3/150

3.3

>4.3

30

21/76

18

4.1

2.3

29

0.070

>30

23/210

2.1

3.5

3.7

35

0.087

>13

150/>300

3.1

3.7

3.4

36

0.045

>30

4.3

30

15/200

1.9

3.1

4.3

a

Experimental assays as per Table 3 unless otherwise specified. b All IC50 data are expressed in micromolar (µM) and are the means of at least n=3 (ATM) or n=2 (ATR) independent measurements. Each has a SEM ± 0.2 log units. c Hepatocyte intrinsic clearance expressed in µL/min/106 cells.

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Investigation of the core Further investigation of the core was performed using 13 and 17 as reference compounds. Simple tetra-substituted quinolines were synthesized in an attempt to assess the potential for further substitution on the core and corroborate the postulated binding mode, and the results are summarized in Table 5. Methyl substitution at C2 exemplified with 2-methylquinoline 49 resulted in an inactive compound (>30 µM), which could be interpreted either by lack of necessary space to accommodate the substituent close to the hinge region of the enzyme or deleterious effect on the conformation of the 3-carboxamide, in turn affecting that of the 4substitutent. Similarly, 5-fluoro derivative 50 was equally inactive, presumably due to a detrimental conformational effect on the C4 amine. Interestingly, 50 showed reduced lipophilicity compared to parent 17 (∆logD7.4=−0.4), which led to decreased activity against hERG (13 µM). 8-Substitution by either a fluoro (59) or a chloro (60), also led to inactive molecules, which was consistent with the postulated binding mode, although the 8-methyl quinoline 43 had some residual activity, but was significantly less potent than the parent 13 (6.0 µM, >80-fold). 7-Substitution proved to be the most interesting, with 7-fluoro analogue 51 being equipotent to the parent 17, with the advantage of improved selectivity over ATR (>30 µM, >50fold selectivity vs.~ 3-fold selectivity). 7-Chloro 58, however, showed a loss of potency (c.a. 3fold) accompanied with an increase in lipophilicity resulting in decreased LLE (2.8 vs 4.1).

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Table 5.a Core substitution.

Entry

R4

R2

R5

R7

R8

ATR cell IC50b >11

Hu/Rat heps CLintc 43/140

hERG IC50

logD7.4

LLE

H

ATM cell IC50b 0.073

13

H

H

H

0.67

4.0

3.1

49

CH3

H

H

H

>30

>30

6/43

0.68

2.7

-

43

H

H

H

CH3

6.0

>30

52/89

0.36

17 50 51 58 59 60

H H H H H H

H F H H H H

H H F Cl H H

H H H H F Cl

0.954 >30 0.606 3.25 >30 >30

3.1 >30 >30 >30 >30 >30

30

3.2/6.9

17

3.0

5.0

a

Experimental assays as per Table 3 unless otherwise specified. b All IC50 data are expressed in micromolar (µM) and are the means of at least n=3 independent measurements. Each has a SEM ± 0.2 log units. c Hepatocyte intrinsic clearance expressed in µL/min/106 cells.

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Journal of Medicinal Chemistry

Figure 3. Evolution of the selectivity of ATM inhibitors from lead 4. Every marker represents an IC50 ratio of selected targets over ATM. Markers are connected and colored by target: enzymatic ratios for DNAPK, mTOR, PI3Kα, PI3Kβ, PI3Kγ, GSK3β and KDR (left); cellular ratios for ATR, DNAPK, PI3Kα and PI3Kβ (right).

Table 7.a Selectivity data for 72 and 74. Assay type Enzyme inhibition

Kinase 72 74 ATM 27 12.8 PI3Kβ β >1 7.1 PI3Kγγ >100 >100 GSK3β β >100 79 KDR 0.046 0.033 Cellular inhibition ATM >30 >19 ATR >30 18.8 PI3Kα α >30 >17.5 PI3Kβ β >30 >30 DNAPK a All IC50 data are expressed in micromolar (µM).

On the basis of their high solubility, high permeability and low turnover in hepatocytes across four species, pharmacokinetic parameters were measured for quinolines 72 and 74 (Table 8). Low to moderate in vivo clearances in rat and dog were observed, with moderate to good bioavailability in both species. 74 had lower clearance (13 mL/min/kg) and higher bioavailability (71%) than 72 (50 mL/min/kg and 31%) in dog as predicted from observed differences in vitro

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in hepatic metabolism (CLint = 2 vs 11 µL/min/106 cells, respectively) and improved permeability (Papp = 14 and 5.2 x10-6 cm.s-1 respectively). Furthermore, good exposure was seen with both 72 and 74, achieving free plasma concentration over the ATM cell IC50 for over 10 hours in mice following doses of 50 mg/kg, where AUC’s of 40 and 33 µM.h and Cmax‘s of 6 and 17 µM were measured respectively. The encouraging pharmacokinetic parameters of 72 and 74 meant that both compounds are suitable for use to evaluate the specific inhibition of ATM in vivo. Efficacy for 72 will be reported in due course, whilst we report herein efficacy following treatment with 74 as a representative compound of this series. Table 8. Physical, pharmacokinetic and other properties of 72 and 74. Parameter 72 74 a Solubility (µM) 590 69 -6 -1 b Caco2 A–B Papp (10 cm.s ) 5.2 14 Hu/Mu/Rat/Dog PPB (%free)c 29/24/16/49 18/17/12/18 Hu/Mu/Rat/Dog heps Clint