Optimization of a Series of Triazole Containing Mammalian Target of

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Optimization of a Series of Triazole Containing Mammalian Target of Rapamycin (mTOR) Kinase Inhibitors and the Discovery of CC-115 Deborah S. Mortensen, Sophie M. Perrin-Ninkovic, Graziella Shevlin, Jan Elsner, Jingjing Zhao, Brandon Whitefield, Lida Tehrani, John Sapienza, Jennifer R. Riggs, Jason S. Parnes, Patrick Papa, Garrick Packard, Branden G.S. Lee, Roy Harris, Matthew Correa, Sogole Bahmanyar, Samantha J. Richardson, Sophie X. Peng, Jim Leisten, Godrej Khambatta, Matt Hickman, James C. Gamez, René R. Bisonette, Julius Apuy, Brian E. Cathers, Stacie Canan, Mehran F. Moghaddam, Heather K. Raymon, Peter Worland, Rama Krishna Narla, Kimberly E. Fultz, and Sabita Sankar J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00627 • Publication Date (Web): 23 Jun 2015 Downloaded from http://pubs.acs.org on June 25, 2015

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Journal of Medicinal Chemistry

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Optimization of a Series of Triazole Containing Mammalian Target of Rapamycin (mTOR) Kinase Inhibitors and the Discovery of CC-115 Deborah S. Mortensen*, Sophie M. Perrin-Ninkovic, Graziella Shevlin, Jan Elsner, Jingjing Zhao, Brandon Whitefield, Lida Tehrani, John Sapienza, Jennifer R. Riggs, Jason S. Parnes, Patrick Papa, Garrick Packard, Branden G.S. Lee, Roy Harris, Matthew Correa, Sogole Bahmanyar, Samantha J. Richardson, Sophie X. Peng, Jim Leisten, Godrej Khambatta, Matt Hickman, James C. Gamez, René R. Bisonette, Julius Apuy, Brian E. Cathers, Stacie S. Canan, Mehran F. Moghaddam, Heather K. Raymon, Peter Worland, Rama Krishna Narla, Kimberly E. Fultz and Sabita Sankar Celgene Corporation, 10300 Campus Pointe Drive, Suite 100, San Diego, California 92121. Keywords:

Mammalian target of rapamycin, mTOR kinase, kinase inhibitors,

PI3K/AKT/mTOR pathway, oncology

ACS Paragon Plus Environment

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Abstract.

We report here the synthesis and structure-activity relationship (SAR) of a novel series of triazole containing mammalian target of rapamycin (mTOR) kinase inhibitors. SAR studies examining the potency, selectivity and PK parameters for a series of triazole containing 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones resulted in the identification of triazole containing mTOR kinase inhibitors with improved PK properties. Potent compounds from this series were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC-3 cancer cells, in vitro and in vivo. When assessed in efficacy models, analogs exhibited dose-dependent efficacy in tumor xenograft models. This work resulted in the selection of CC-115 for clinical development.


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Introduction. The mammalian target of rapamycin (mTOR) kinase is a critical mediator of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT pathway).1 Multiple mechanisms, including loss of function mutations or promoter hypermethylation of the tumor suppressor PTEN or activating mutations of the PIK3CA oncogene contribute to the frequent dysregulation of this pathway in human cancers.2 signaling complexes.

mTOR kinase functions in two distinct multi-protein

mTOR complex-1 (mTORC1) is responsible for regulating protein

synthesis and growth,3 and mTOR complex-2 (mTORC2) has been shown to phosphorylate and activate AKT,4 a key kinase in the control of cell growth, metabolism and survival. Rapamycin analogs have been shown to stimulate the upstream kinase AKT by releasing the feedback inhibition of PI3K via p70S6 kinase and Insulin Receptor Substrate 1. This may explain at least in part, the resistance to rapamycin analogs exhibited by the majority of cancer cell lines and tumors.5

It is hypothesized that ATP-competitive mTOR kinase inhibitors, blocking both

mTORC1 and mTORC2 signaling, will have expanded therapeutic potential.6 The first generation of reported ATP-competitive mTOR kinase inhibitors to enter clinical trials presented as dual inhibitors of mTOR kinase and the related lipid kinase, PI3K-alpha.7-9 Agents selectively targeting mTOR kinase, have since entered clinical investigation.10-12 We have previously described13 the results of our efforts on the optimization of a 1,6substituted-imidazo[4,5-b]pyrazin-2-one series from an HTS hit. While compounds in the initial 1,6-substituted-imidazo[4,5-b]pyrazin-2-one series were optimized to afford compounds with excellent potency, PK remained an issue, with potent compounds such as CC214-1(Figure 1), showing negligible oral bioavailability. We have also investigated core modifications14-15, through insertion of a methylene into the imidazo-ring, resulting in two ring-expansion series


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which maintained or improved mTOR kinase potencies, such as 2, 3 and 4. Following the initial analog comparisons, we focused our efforts on the C6/7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)substitution because of the PK advantage of these analogs. Exploration and optimization of the C6/7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-substituted

4,6-

or

1,7-disubstituted-3,4-

dihydropyrazino[2,3-b]pyrazine-2(1H)-one ATP-competitive mTOR kinase inhibitors for in vitro potency and in vivo efficacy, lead to the identification of clinical candidate CC-223.15 However, we remained interested in the more potent, triazole containing subseries and herein we describe our efforts to explore the SAR and optimize the PK properties of the triazole containing analogs resulting in the selection of CC-115 for clinical development.

Figure 1. CC-223, representative analogs from imidazo[4,5-b]pyrazin-2-one compound series CC214-1 and 1 and initial analogs 2-4 in the ring expansion 3,4-dihydropyrazino[2,3-b]pyrazin2(1H)-one series.


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Chemistry Compounds in both ring-expansion series were synthesized utilizing synthetic routes as described for our previous efforts.15 Specifically the 4,6-disubstituted-3,4-dihydropyrazino[2,3b]pyrazine-2(1H)-one (RE1) series discussed herein were synthesized by the methods in Scheme 1.

The 6-bromo-4-substituted-3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-ones 5a-d were

prepared through amine addition to 2-halo-N-(3,5-dibromopyrazin-2-yl)acetamide as described previously.15

Bromides 5a-d were converted to stannanes 6a-d.

Stille coupling with aryl

bromides, followed by acid catalyzed deprotection, when relevant, gave compounds 16, 23 and 25. In one instance the triazole ring was constructed after the Stille coupling of 6b with 5bromo-6-methylpicolinonitrile, followed by hydrolysis of the nitrile to the primary amide, reaction with N,N-dimethylformamide dineopentyl acetal, followed by treatment with hydrazine to give 17. Scheme 1.

Synthesis of 4,6-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones

(RE1).

Reagents and conditions: (a) hexamethylditin, Pd(PPh3)4, dioxane, 90-100 °C, 1-5 h, yield 54-69%; (b) R2-bromide, Pd2(dba)3, P(o-tol)3, triethylamine, DMF, 95-100 °C, 1-2 h, yield 7380%; (c) HCl (4N in dioxane), ethanol, 50-80 °C, 1-3 h, yield 17-86%; (d) TFA, sulfuric acid, rt, 19 h, yield 69 %; (e) DMF:dineopenyl acetal, hydrazine, acetic acid, 85 °C, 10 min, yield 49% . Compounds from the 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones (RE2) series were synthesized by the methods in Scheme 2.

The 7-bromo-1-substituted-3,4-

dihydropyrazino[2,3-b]pyrazin-2(1H)-ones 7a-h were prepared through amine addition to ethyl


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(3,5-dibromopyrazin-2-yl)glycinate, followed by acid catalyzed ring closure, as described previously.15 Suzuki coupling of intermediate 7c, e, i, j and k with aryl boronic acid pinacol esters, followed by acid catalyzed deprotection, when relevant, afforded the desired compounds 14, 18, 22, 24 and 26. Alternately, stannane intermediates 8a-h were prepared and reacted with aryl bromides under Stille coupling conditions, followed by acid catalyzed deprotection, when relevant, to provide compounds 12,13, 15 and 19-21. Scheme 2.

Synthesis of 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones

(RE2).

Reagents and conditions: (a) R2-boronate ester, sodium or potassium carbonate in water, PdCl2(dppf)-CH2Cl2, dioxane or DMF, 65-120 °C, 0.5-3 h, yield 49-85%; (b) HCl (4N in dioxane or 6N aqueous), ethanol or methanol, rt-70 °C, 0.6-5 h, yield 28-86% ; (c) hexamethylditin, Pd(PPh3)4, dioxane, 100-150 °C, 0.5-4 h, yield 21-77%; (d) R2-bromide, Pd(dtbpf)Cl2 or Pd(PPh3)2Cl2 or Pd2(dba)3 and P(o-tol)3 and triethylamine, DMF, 110-130 °C, 14 h, yield 8-82%. Results and Discussion Of the seven triazole substituted analogs initially prepared in the ring expansion series, four demonstrated sufficient in vitro metabolic stability to advance into in vivo PK assessment.


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While these analogs demonstrated good cellular potency, they generally suffered from poor oral exposures (Table 1). Within this series, we found the replacement of the phenyl substituent (3) with pyridyl (9) maintained cellular inhibition of Akt phosphorylation and resulted in improvement of the rat oral PK. Addition of a 2-methyl group was also acceptable, showing a slight loss of potency in the RE1 series (10), while conferring excellent cellular potency for the RE2 compound (11). These analogs also showed an improvement in oral exposure profiles with 11 displaying an AUC nearly 300 fold over the cellular biomarker IC50 value. While the potency and rat PK profile of 11 was attractive, the duration of exposure was short, due to moderately high clearance (38 mL/min/kg) and short MRT (0.69 h), with a corresponding oral bioavailability of 27%. Table 1. pAkt(S473) cellular potency and Rat PK parameters for compounds 3, 9, 10 and 11.

Rat PO PK Value (5 mg/kg)a Core

pAkt(S473) IC50 (µM)

3

RE1

0.034

9

RE1

0.024

0.02 ± 0.01

0.9 ± 0.8

0.18 ± 0.07

10

RE1

0.106

0.09 ± 0.05

4.8 ± 3.8

0.64 ± 0.48

11

RE2

0.005

0.59 ± 0.11

0.5 ± 0

1.47 ± 0.23

Analog

a

C6/C7

Cmax (µM)

Tmax (h)

AUC (µM-h)

BLQb

mean±standard deviation, bBLQ=below limit of quantitation, limit of quantitation 0.0021 µM.


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We undertook a SAR exploration of the 2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl substituted analogs to determine the minimal requirements of the N1/N4 substituent for mTOR kinase potency and to explore the effects of this substitution on the PK profile. Beginning from the original 4-tetahydropyranyl-ethyl analogs, 6 and 7, we found the introduction of an amine was tolerated with a very small loss in potency for the morpholino-ethyl 12 (Table 2). Compared to the findings in the C6/7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-substitution previously described15, the position or extension of the polar moiety on the N1/N4 group was not as critical to potency in the triazole subseries as was evident from the equi-potent analogs such as 7, 13 and 21.

Smaller groups, such methoxy-ethyl, did not improve potency in the C6/7-(6-(2-

hydroxypropan-2-yl)pyridin-3-yl)-series.

However, within the triazole subseries, significant

potency was achieved with small groups such as iso-propyl (22), methoxy-ethyl (24) or ethyl (26). We found that the 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-one (RE2) consistently provided improved potency as compared to their 4,6-disubstituted-3,4dihydropyrazino[2,3-b]pyrazine-2(1H)-one (RE1) comparator (6 vs. 7, 17 vs. 18, 23 vs. 24 and 25 vs. 26). At the time of this work, no protein crystal structures of the mTOR kinase binding domain had been reported.

Based on the results of SAR from the initial 1,6-substituted-imidazo[4,5-

b]pyrazin-2-one series, compounds were originally docked into a homology model of the mTOR ATP binding site. The core 3-NH and 4-N were proposed as the hinge-binding donor-acceptor motif, with the C6-Aryl group extending back into the catalytic pocket and the N1 substituent either reaching into the ribose binding pocket or extending towards the solvent exposed region as reported previously.13 Similar binding modes for the ring expansion analogs were proposed


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based on comparative SAR studies, with the 1-NH/4-NH and 8-N/5-N of the cores serving as the hinge-binding motifs. A structure of the mTOR kinase domain has since published16 and we reevaluated our homology model docking results in comparison to docking results using the published mTOR structure. We found that the overall homology model, and more specifically the binding site residue positions, were in good agreement with the published structure. The original proposed binding modes were supported and the docking result for compound 26 in the mTOR kinase binding site is shown in Figure 2. The core presents a dual hinge binding motif with the 4-NH providing a hydrogen bond donor and the 6-N an acceptor. The N1-ethyl group binds in the hydrophobic g-loop. The triazole moiety is extended deep into the catalytic pocket and makes multiple potential hydrogen bonding interactions, likely explaining the improved potency of the triazoles as compared to the C6/7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)substituted analogs (Figure 2 and Supplemental Figure 1).

Figure 2. 26 docked into the mTOR kinase binding pocket.


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Table 2. SAR of mTOR kinase and PC-3 cellular potency.a

PC-3 Cellular Potency IC50 (µM) Core

mTOR Kinase IC50(µM)

10

RE1

0.099

0.108b

0.106

0.573

11

RE2

0.014

0.008

0.005

0.034

12

RE2

0.030

0.054

0.112

0.413

13

RE2

0.012

0.015 b

0.004

0.082

14

RE2

0.001

0.004

0.009

0.024

15

RE2

0.031

0.027

0.098

0.142

16

RE1

0.039

0.085 b

0.131

0.423

17

RE1

0.041

0.203 b

0.120

0.478

18

RE2

0.015

0.020 b

0.013

0.038

19

RE2

0.005

0.006

0.006

0.037

20

RE2

0.011

0.012

0.027

0.119

21

RE2

0.028

0.010

0.035

0.102

22

RE2

0.030

0.044 b

0.049

0.149

23

RE1

0.057

0.066

0.230

0.363

24

RE2

0.020

0.010

0.017

0.137

25

RE1

0.090

0.053

0.369

0.889

Analog

N4/N1

p-p70S6K pAkt(S473)

Prolif.

RE2 0.021 0.023b 0.022 0.138 26 a b SEM for data available in supplemental material, pS6 IC50 (not p-p70S6)


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Compounds’ effects in PC-3 cancer cell lines were evaluated.

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Inhibition of the mTOR

pathway was assessed through evaluation of the phosphorylation of p70S6K or S6 (TORC1) and Akt(S473) (TORC2).

We also assessed proliferation as a functional effect of mTOR pathway

inhibition in these cells. As expected for mTOR kinase domain inhibitors, our compounds showed potent inhibition of both TORC1 and TORC2, as well as inhibition of cell proliferation (Table 2). In vitro ADME properties for analogs from this series were assessed. The estimated metabolic stability was evaluated using both rat and human liver S9 fractions. In vitro permeability data in Caco-2 cells was collected for all compounds which advanced into in vivo PK studies.

Analogs

from this series typically showed good metabolic stability, with ≥70% remaining in both rat and human, with a few exceptions (Table 3).

Analogs 14 and 16, both containing readily

metabolized methoxy-cyclohexyl moiety, suffered from poor stability in at least one species, though not all analogs with methyl-ethers were unstable (for example 15, 17, 18, 23 and 24). Comparison of cis/trans pairs 14/15 and 16/17 indicated the trans isomer to be the more stable of the pair. The small methoxy-ethyl analogs 23 and 24, proved to be highly stable with ≥90% remaining in both rat and human.

Permeability assessment showed the analogs to have

reasonable to excellent permeability (Papp = 1.6 to 30.4 x10-6 cm/s). Most compounds showed low efflux potential, with 11, 19 and 24 showing the highest efflux potential with B-A/A-B ratios of 12.9, 18.1 and 7.7, respectively.


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Table 3. In Vitro ADME Properties

S9 Met. Stabilitya Analog

a

N4/N1

Caco-2 Permeability Papp A-Bb Papp B-Ab B-A/A-B

Core

Rat

Human

10

RE1

80 ± 2

90 ± 5

8.0

21.8

2.7

11

RE2

76 ± 6

100

4.2

54.7

12.9

12

RE2 96 ± 14

13

RE2

100

100

14

RE2

39 ± 2

10 ± 6

NT

15

RE2

100

90 ± 1

NT

16

RE1

90 ± 3

36 ± 3

NT

17

RE1

89 ± 4

87 ± 4

14.8

20.2

1.4

18

RE2

72 ± 2

82 ± 13

9.7

59.5

6.2

19

RE2

100

100

1.6

29.2

18.1

20

RE2

91 ± 5

97 ± 5

22

RE2

100

99 ± 6

37.6

45.2

1.2

23

RE1

97 ± 2

100

16. 2

19.1

1.2

24

RE2

91 ± 9

100

5.0

38.8

7.7

25

RE1

89 ± 7

100

NTc

86 ± 3 9.7

14.5

1.5

NT

NT

100 100 30.4 38.1 1.3 26 RE2 b -6 c mean±standard deviation (% remaining at 60 min), (x 10 cm/s), NT: not tested.


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The compounds that advanced into in vivo PK assessment, and the resulting rat PK parameters, are shown in Table 4.

Compounds were formulated as suspensions in aqueous 0.5%

carboxymethyl cellulose and 0.25% Tween-80 and were administered by oral gavage at 5 mg/kg. Our goal in this exploration was to determine if we could improve the duration of oral exposure in the trizaole series compared to our starting analogs 10 and 11. Most analogs tested showed exposure improvements over these analogs with higher Cmax and/or AUC values (Table 4). The notable exception, with very poor exposure, was compound 19.

This poor oral exposure

correlates with the poor permeability and high efflux potential predicted by Caco-2 (Table 3). In addition to the potency benefit of the RE2 over RE1 analogs discussed above, the 1,7disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-one (RE2) generally provided improved exposure as compared to their 4,6-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-one (RE1) comparator (10 vs. 11, 17 vs. 18 and 25 vs. 26). Potency normalized parameters further highlight the combined potency and exposure benefit with the RE2 series with AUC values 300 to 1200 fold above the pAkt cellular IC50 values as compared to only 6 to 80 fold for the corresponding RE1 compounds (10 vs. 11, 17 vs. 18, 23 vs. 24 and 25 vs. 26). The best exposure improvements were found in the analogs with smaller N1/4 substitution, analogs 22-26, and these compounds showed oral bioavailability ranging from 50-100%. Though some potency loss was seen in going to the smaller N1 in compounds such as 22, 24 and 26 as compared to 10, the improved exposures provided compounds with improved potency normalized exposures (Table 4), even with the loss in potency.


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Table 4. In Vivo PK Properties.a

Rat IV PK Parametersb

Cmax AUC(0-∞) F(%) (µM) (µM·hr) 0.09 ± 0.64 ± NTe NCf 10 0.05 0.48 0.69 ± 0.59 ± 1.47 ± 37.9 ± 5.5 28 ± 6 11 0.11 0.23 0.08 0.23 ± 2.65 ± NT NC 13 0.07 0.94 0.53 ± 4.03 ± NT NC 17 0.17 0.83 0.84 ± 6.96 ± 16.0 ± 7.5 6.5 ± 4.6 49 ± 21 18 0.27 1.17 0.03 ± 0.07 ± NT NC 19 0.01 0.01 6.97 ± 2.08 ± 22.02 ± 64 ± 24 3.9 ± 0.2 22 0.47 9.48 0.45 2.43 ± 18.91 ± 16.9 ± 1.3 1.8 ± 0.3 ~100 23 0.57 3.56 1.96 ± 14.03 ± 34.7 ± 4.8 2.2 ± 0.2 ~100 24 0.42 1.99 0.55 ± 5.27 ± 23.8 ± 4.2 1.2 ± 0.3 50 ± 24 25 0.05 2.33 1.67 ± 27.46 ± 7.35 ± 4.3 ± 0.4 78 ± 20 26 0.42 1.44 2.13 a Reported values are mean±standard deviation, bdose 2 mg/kg, CMC/Tween suspension, dfold over pAkt cellular IC50 value, eNT: calculated, IV study not available. Analog

CL

(mL/min/kg)

Potency Normalized PK Parametersd

Rat PO PK Parametersc

MRT (h)

Cmax

AUC(0-∞)

0.8

6.0

118.0

294.0

57.5

662.5

4.4

33.6

64.6

535.2

5.0

11.7

42.4

449.4

10.6

82.2

115.3

825.3

1.5

14.3

75.9

1248.2

c

dose 5 mg/kg as not tested, fNC: not

Encouraged by the observed exposures, compounds 18, 22, 24 and 26 were advanced into single dose PK/PD studies assessing mTOR pathway biomarker inhibition in tumor bearing mice. PC-3 tumor-bearing mice were administered with a single dose of test compound, dosed orally at either 1 or 10 mg/kg, and plasma and tumor samples were collected at various time points for analysis. Significant inhibition of both mTORC1 (pS6) and mTORC2 (pAktS473) was observed for all compounds and the level of biomarker inhibition correlated to plasma compound levels (Table 5). While compounds 18 and 22 maintained biomarker inhibition through 10 hours


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when dosed at 10 mg/kg, 24 and 26 sustained inhibition though 24 hours. At the 1 mg/kg dose all four compounds showed significant inhibition at 1 and 3 hours, with 24 and 26 demonstrating inhibition through 10 hours.

Based on these results and the desire to maintain pathway

suppression throughout the dosing period in efficacy studies, we chose to explore 18 and 22 using a twice daily (bid) dosing schedule. Compounds 24 and 26 were evaluated using both once (qd) and twice (bid) daily dosing schedules. Table 5. In Vivo Single Dose Biomarker Inhibition and Plasma Levels. Compound

18

24

26

10 1 10 1 10 1 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg 1h 81 75 91 93 85 85 3h 89 61 96 94 91 89 a p-S6 6h 87 25 96 90 94 83 10 h 86 0 96 74 94 79 24 h 0 0 89 0 93 0 1h 82 73 89 89 88 80 3h 76 67 88 69 88 65 a p-Akt 6h 80 44 82 46 87 70 10 h 76 0 85 62 86 65 24 h 0 0 82 37 78 19 3.05 ± 0.36 ± 5.37 ± 0.38 ± 6.78 ± 0.53 ± 1h 1.27 0.06 1.47 0.15 2.32 0.03 1.16 ± 0.18 ± 2.35 ± 0.25 ± 5.69 ± 0.34 ± 3h 0.31 0.07 0.90 0.23 1.92 0.02 Plasma 0.97 ± 0.11 ± 1.00 ± 0.10 ± 2.10 ± 0.13 ± Conc. 6h 0.26 0.03 0.71 0.03 0.43 0.02 (µM) 0.88 ± 0.02 ± 0.46 ± 0.07 ± 1.18 ± 0.12 ± 10 h 0.12 0.01 0.26 0.02 0.40 0.01 0.06 ± 0.001 ± 0.16 ± 0.80 ± 0.005 ± 24 h BLQb,c BLQc BLQd 0.02 0.001 0.07 0.18 0.003 a b % inhibition compared to vehicle control, BLQ: below limit of quantification, cLimit of quantification (LOQ)=0.0062 µM, dLOQ=0.0009 µM. Dose

10 mg/kg 90 89 88 86 60 84 68 34 41 0 1.32 ± 0.24 0.50 ± 0.17 0.321± 0.02 0.23 ± 0.10

22

1 mg/kg 78 64 0 0 0 66 31 0 0 0 0.098 ± 0.044 0.085 ± 0.006 0.043 ± 0.023 0.022 ± 0.016

PC-3 tumor bearing mice were treated once daily (qd) or twice daily (bid), with bid doses administered with a 10 h separation between the morning and evening doses. Tumor volumes,


15

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determined prior to the initiation of treatment, were considered as the starting volumes (average starting volumes were ~125 mm3). Compounds were dosed for 21 days and the final tumor volume reductions reported here were measured following the final day of dosing. All compounds demonstrated dose dependant tumor volume reductions (TVR = (vehicle - treated / vehicle)x100%) and all doses used were well tolerated with minimal body weight loss. When dosed at 2 mg/kg bid, compounds 18, 22 and 24 demonstrated 45%, 56% and 62% tumor volume reductions, respectively (Figure 3A, Figure 3B and Figure 3C). At the highest dose level assessed, 5 mg/kg bid, 58%, 67% and 74% tumor volume reductions were achieved, respectively. The 5 mg/kg bid dose for all three compounds was well tolerated, suggesting increased efficacy may be reached at higher doses. Compound 24 showed similar efficacy when tested at 10 mg/kg qd or 5 mg/kg bid (68% vs. 74% TVR). Similarly 24 at 5 mg/kg qd or 2 mg/kg bid gave efficacy of 57% vs. 62% TVR, respectively, suggesting there was in this case at best only a small benefit of the twice daily dosing schedule (Figure 3C). Compound 26 was tested at lower doses of 0.25, 0.5 and 1 mg/kg bid or 1 mg/kg qd, with observed corresponding tumor volume reductions of 46%, 57%, 66% and 57% respectively (Figure 3D). Here we also found no apparent benefit to twice daily dosing. When mice received 1 mg/kg/day of 26, whether as a single 1 mg/kg dose or when split as a 0.5 mg/kg bid dose, 57% tumor volume was observed. These experiments were designed to identify the minimum dose required to achieve ≈60% tumor volume reductions, a defined minimal efficacious dose. The minimal efficacious dose was determined to be 5 mg/kg bid for 18, 2 mg/kg bid for 22, 2 mg/kg bid for 24 and 1 mg/kg bid for 26.


16


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A.

B.

C.

D.

Page 18 of 41

Figure 3. PC-3 tumor xenograft studies with (A) 18, (B) 22, (C) 24 and (D) 26 CC-115. The data described here along with assessment in additional in vivo models, safety studies and higher species PK informed the selection of 26 (CC-115) for clinical development. In a kinase selectivity assessment against a panel of 250 protein kinases at 3 µM, only one kinase other than mTOR kinase was identified with more than 50% inhibition (cFMS 57%, IC50 = 2.0 µM).17 Of the PI3K related kinases (PIKKs) tested, CC-115 proved to be equipotent against DNA PK (IC50


17

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= 0.015 µM) and demonstrated 40 to >1000 fold selectivity against the remaining PIKKs tested; PI3K-alpha (IC50 = 0.85 µM), ATR (50% inhibition at 30 µM) and ATM (IC50 > 30 µM) (Supplemental Table 3). CC-115 showed good in vivo PK profiles across multiple species with 53%, 76% and ~100% oral bioavailability in mouse, rat and dog, respectively (Supplemental Table 4). The IC50 values for CC-115 are >10 µM against a panel of CYP enzymes and >33 µM for the hERG (human ether-a-go-go-related gene) ion channel. When screened in a single point assay at 10 µM against a Cerep receptor and enzyme panel only one target was inhibited >50% (PDE3, IC50 = 0.63µM). CC-115 was negative in the AMES mutagenicity test, both with and without S9 fractions. The on-target mTOR kinase effects of CC-115 are expected to be similar to those shown by other mTOR kinase selective clinical candidates such as OSI02718, AZD805519, CC-22320, MLN012821 or AZD2014.19 Off-target activities for these compounds may contribute to distinct activity and/or toxicity profiles, and PK properties may also impact the clinical success among these compounds. Clinical trials for both OSI027 and AZD8055 have been halted, with poor human PK being cited for one.19 We have completed studies showing a potential advantage of CC-115 as compared to CC-223, due in part to the added DNA-PK inhibition of the former. This comparison and the expanded in vitro and in vivo characterization of CC-115 will be reported elsewhere. Conclusion In summary we have described the SAR and optimization of a series of triazole containing 4,6or

1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-one

inhibitors.

based

mTOR

kinase

The series maintains mTOR kinase potency with exquisite kinase selectivity

including the related lipid kinase PI3Kα.

A focused investigation of 2-methyl-6-(1H-1,2,4-


18


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Page 20 of 41

triazol-3-yl)pyridin-3-yl substituted analogs, identified smaller substitutions in the N1/N4 position which maintained potency and improved oral PK properties. This work ultimately led to the identification of CC-115, with favorable physicochemical and pharmacokinetic properties, demonstrated in vivo mTOR pathway inhibition and tumor growth inhibition, as well as a good in vitro and in vivo safety profile, suitable for clinical development. CC-115 is currently under Phase I clinical investigation. Materials and Methods Compounds were named using ChemDraw Ultra.

All materials were obtained from

commercial sources and used without further purification, unless otherwise noted. Chromatography solvents were HPLC grade and used as purchased. All air-sensitive reactions were carried out under a positive pressure of an inert nitrogen atmosphere. Reported yields are unoptimized. 1H NMR spectra were obtained on a Varian 400 MHz spectrometer with tetramethylsilane (TMS) as the internal standard. Chemical shifts (δ) are reported in ppm downfield of TMS and coupling constants (J) are given in Hz. Thin Layer Chromatography (TLC) analysis was performed on Whatman thin layer plates. LCMS analysis was performed on a PE Sciex ESI MS or Agilent 1100 MS. Preparative reverse phase HPLC was performed on a Shimadzu system equipped with a Phenomenex 15 micron C18 column (250 x 50 mm). Semipreparative reverse phase HPLC was performed on a Shimadzu system equipped with a Phenomenex 15 micron C18 column (250 x 10 mm). The purity of final tested compounds was typically determined to be > 95% by HPLC, conducted on an Agilent 1100 system using a reverse phase C18 column and diode array detector (compound 14 was tested at 94% purity). Compounds were analyzed by one of four methods: (A) Gradient (0-75% acetonitrile + 0.1% formic acid in water + 0.1% formic, over 7 min, followed by 75% acetonitrile + 0.1% formic


19

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acid for 2 min); Flow Rate 1 mL/min, Column Phenomenex Gemini-NX 5u C18 110A (50x4.60mm); (B) Gradient (0-75% acetonitrile + 0.1% formic acid in water + 0.1% formic, over 20 min, followed by 75% acetonitrile + 0.1% formic acid for 5 min); Flow Rate 1 mL/min, Column Phenomenex Gemini-NX 5u C18 110A (250x4.60mm); (C) Gradient (15-100% methanol + 0.05% formic acid in water + 0.05% formic, over 5.5 min, followed by 100% methanol + 0.05% formic acid for 4 min); Flow Rate 1 mL/min, Column Phenomenex GeminiNX 5u C18 110A (50x4.60mm); (D) Gradient (15-100% methanol + 0.05% formic acid in water + 0.05% formic, over 20 min, followed by 100% methanol + 0.05% formic acid for 5 min); Flow Rate 1 mL/min, Column Phenomenex Gemini-NX 5u C18 110A (250x4.60mm). Melting points were determined on either a manual Electrothermal Mel-Temp® or Stanford Research Systems’ OptiMelt System and are uncorrected. Elemental analysis was performed at Robertson Microlit Laboratories, Ledgewood, New Jersey. Synthetic procedures for intermediates 5a-c, 7b-h, 7j have been published previously.15 Procedures for intermediates 5d, 6a-d, 7a, 7i, 7k and 8a-h are included in the supplemental material. General Procedure A (12, 13, 15-THP, 16-THP, 17-nitrile, 19-THP, 20-THP, 21-THP, 23THP, 25-THP) Aryl bromide, stannane and palladium catalyst were combined in triethylamine and/or DMF. The solution was then heated at 100-140 °C for 0.3-4 h. The reaction mixtures were partitioned between organic (ethyl acetate or methylene chloride) and water. The organics were dried and concentrated under reduced pressure. Resulting products were purified using silica gel chromatography or reverse phase HPLC. General Procedure B (14-THP, 18-THP, 22-THP, 24-THP, 26-THP) Substrate, boronate ester

and

[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II)

complex

with


20


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Page 22 of 41

dichloromethane or dichlorobis(triphenylphosphine)palladium(II) were combined in dioxane or DMF. Sodium carbonate in water was then added. The solution was then heated at 120 °C in a microwave reactor for 15-30 min or on an oil bath at 130-150 °C for 1-4 h. The cooled reaction solutions were filtered through Celite and the filter cake was washed with ethyl acetate. Filtrate and ethyl acetate wash were combined and solvent removed under reduced pressure. Products were purified using silica gel chromatography or reverse phase HPLC. General Procedure C (14-16, 18-26) Substrate was dissolved in ethanol or dioxane and treated with HCl (4N in dioxane or 6N aqueous). The reaction mixtures were stirred at rt or 45110 °C for 0.2-12 h. The solutions were concentrated under reduced pressure and products were purified using reverse phase HPLC. 7-(2-Methyl-6-(4H-1,2,4-triazol-3-yl)pyridin-3-yl)-1-(2-morpholinoethyl)-3,4dihydropyrazino[2,3-b]pyrazin-2(1H)-one (12).

8a (275.0 mg, 0.645 mmol), 3-bromo-2-

methyl-6-(4-(tetrahydro-2H-pyran-2-yl)-4H-1,2,4-triazol-3-yl)pyridine (209 mg, 0.645 mmol) and dichlorobis(triphenylphosphine)palladium(II) (47.2 mg, 0.065 mmol) were reacted according to Procedure A to give the title compound (23.0 mg, 0.055 mmol, 8.46 % yield, HPLC purity (A) 96%). 1H NMR (400 MHz, DMSO-d6) δ 7.99 (br. s., 2 H), 7.93 (s, 1 H), 7.72 (br. s., 1 H), 4.22 (d, J=1.6 Hz, 2 H), 4.14 (t, J=7.2 Hz, 2 H), 3.49 (t, J=4.5 Hz, 4 H), 2.71 (br. s., 3 H), 2.52 - 2.56 (m, 2 H), 2.41 (br. s., 4 H); MS (ESI) m/z 422.2 [M+1]+; mp 166-168 °C. 7-(2-Methyl-6-(4H-1,2,4-triazol-3-yl)pyridin-3-yl)-1-((tetrahydro-2H-pyran-4-yl)methyl)3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-one (13). 8b (0.344 g, 0.837 mmol), 3-bromo-2methyl-6-(4H-1,2,4-triazol-3-yl)pyridine

(0.200

g,

0.837

mmol),

tris(dibenzylideneacetone)dipalladium(0) (0.153 g, 0.167 mmol) and tri-o-tolylphosphine (0.025 g, 0.084 mmol) were reacted according to Procedure A to give the title compound (0.12 g, 0.373,


21

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mmol, 14.2 % yield, HPLC purity (A) >99%). 1H NMR (400 MHz, DMSO-d6) δ 8.28 (br. s., 1 H), 7.99 (s, 2 H), 7.93 (s, 2 H), 7.73 (s, 1 H), 4.23 (s, 2 H), 3.94 (d, J=7.03 Hz, 2 H), 3.80 (d, 2 H), 3.20 (d, 2 H), 2.71 (s, 2 H), 2.00 - 2.13 (m, 1 H), 1.53 (d, J=12.89 Hz, 2 H), 1.18 - 1.34 (m, 2 H); MS (ESI) m/z 407.3 [M+1]+. 1-(((cis)-4-Methoxycyclohexyl)methyl)-7-(2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl)3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-one (14) 7c (493 mg, 1.39 mmol), 2-methyl-6-(1(tetrahydro-2H-pyran-2-yl)-1H-1,2,4-triazol-3-yl)pyridin-3-yl boronic acid (400 mg, 1.39 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (102 mg, 0.10 mmol) were reacted according to Procedure B to give 14-THP (350 mg, 0.68 mmol, 49 % yield). MS (ESI) m/z 519.7 [M+1]+. Procedure C gave 14 (65 mg, 0.15 mmol, 31 % yield, HPLC purity (A) 94%). 1H NMR (400 MHz, DMSO-d6) δ 7.97 (br. s., 2 H), 7.92 (s, 1 H), 7.72 (br. s., 1 H), 4.23 (s, 2 H), 3.90 (d, J=7.03 Hz, 2 H), 3.18 (s, 3 H), 2.70 (s, 3 H), 1.83 - 1.93 (m, 1 H), 1.77 (br. s., 2 H), 1.24 - 1.43 (m, 6 H); MS (ESI) m/z 435.5 [M+1]+; mp 205 °C (dec). 1-(((trans)-4-Methoxycyclohexyl)methyl)-7-(2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-one (15) 8d (1.50 g, 3.42 mmol), 3-bromo-2methyl-6-(1-(tetrahydro-2H-pyran-2-yl)-1H-1,2,4-triazol-3-yl)pyridine (1.10 g, 3.42 mmol) and dichloro[1,1'-bis(ditert-butylphosphino)ferrocene]palladium (44 mg, 0.068 mmol) were reacted according to Procedure A to give 15-THP (1.22 g, 2.35 mmol, 69 % yield). MS (ESI) m/z 519.6 [M+1]+ Procedure C gave 15 (350 mg, 0.81 mmol, 34 % yield, HPLC purity (A) >99%).

1

H

NMR (400 MHz, DMSO-d6) δ 7.98 (s, 2 H), 7.92 (s, 1 H), 7.73 (s, 1 H), 4.23 (s, 2 H), 3.88 (d, J=6.64 Hz, 2 H), 3.20 (s, 3 H), 3.00 - 3.10 (m, 1 H), 2.70 (s, 3 H), 1.97 (d, J=9.76 Hz, 2 H), 1.64 - 1.82 (m, 3 H), 0.92 - 1.13 (m, 4 H); MS (ESI) m/z 435.4 [M+1]+. ; mp 155 °C (dec).


22


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Page 24 of 41

4-((cis)-4-Methoxycyclohexyl)-6-(2-methyl-6-(4H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4dihydropyrazino[2,3-b]pyrazin-2(1H)-one (16). 6a (0.292 g, 0.687 mmol), 3-bromo-2-methyl6-(1-(tetrahydro-2H-pyran-2-yl)-1H-1,2,4-triazol-3-yl)pyridine

(0.244

g,

0.756

mmol),

tris(dibenzylideneacetone)dipalladium(0) (0.063 g, 0.069 mmol) and tri-o-tolylphosphine (0.042 g, 0.137 mmol) were reacted according to Procedure A to give 16-THP (0.279 g, 0.687 mmol, 80 % yield). MS (ESI) m/z 505.6 [M+1]+. Procedure C gave 16 (0.040 g, 0.095 mmol, 17 % yield, HPLC purity (A) 99%).

1

H NMR (400 MHz, METHANOL-d4) δ 7.88 - 8.13 (m, 2 H),

7.65 (s, 1 H), 4.58 (s, 1 H), 4.16 (s, 2 H), 3.47 (br. s., 1 H), 3.22 - 3.32 (m, 66 H), 2.73 (s, 3 H), 2.08 (br. s., 2 H), 1.91 (br. s., 2 H), 1.56 (br. s., 4 H); MS (ESI) m/z 421.2 [M+1]+; mp 192195 °C. 4-((trans)-4-Methoxycyclohexyl)-6-(2-methyl-6-(4H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4dihydropyrazino[2,3-b]pyrazin-2(1H)-one (17).

6b (0.315g, 0.741 mmol), 5-bromo-6-

methylpicolinonitrile (0.161 g, 0.815 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.068 g, 0.074 mmol) and tri-o-tolylphosphine (0.045 g, 0.148 mmol) were reacted according to Procedure A to give coupled product 17-nitrile (0.225 g, 0.595 mmol, 80 % yield). MS (ESI) m/z 379.8 [M+1]+. 5-(8-((trans)-4-Methoxycyclohexyl)-6-oxo-5,6,7,8-tetrahydropyrazino[2,3b]pyrazin-2-yl)-6-methylpicolinonitrile (0.225 g, 0.595 mmol) was diluted with trifluoroacetic acid (4 mL) and sulfuric acid (1 mL) and stirred at rt for 19 h. The solution was poured into ice (100 mL) and neutralized to pH 10 with solid potassium carbonate. The resulting precipitate was filtered and washed with additional water followed by hexanes to afford the desired amide (0.163 g, 0.411 mmol, 69 % yield). MS (ESI) m/z 397.6 [M+1]+. 5-(8-((trans)-4-Methoxy-cyclohexyl)6-oxo-5,6,7,8-tetrahydropyrazino[2,3-b]pyrazin-2-yl)-6-methylpicolinamide (0.163 g, 0.411 mmol) was diluted with N,N-dimethylformamide dineopentyl acetal (1.5 mL, 0.411 mmol) and


23

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tetrahydrofuran (2 mL). The solution was heated in a screw capped flask at 85 °C for 1 h. The solution

was

condensed

to

afford

(E)-N-((dimethylamino)methylene)-5-(8-((trans)-4-

methoxycyclohexyl)-6-oxo-5,6,7,8-tetrahydropyrazino[2,3-b]pyrazin-2-yl)-6methylpicolinamide and was used without purification. MS (ESI) m/z 452.4 [M+1]+. (E)-N((Dimethylamino)methylene)-5-(8-((trans)-4-methoxycyclohexyl)-6-oxo-5,6,7,8tetrahydropyrazino[2,3-b]pyrazin-2-yl)-6-methylpicolin-amide (0.185 g, 0.410 mmol), hydrazine (0.386 mL, 12.29 mmol) and glacial acetic acid (5 mL, 87 mmol) were combined in a sealed tube and heated to 85 °C for 10 min and cooled to ambient temperature. The solution was purged with nitrogen to remove excess acetic acid. The resulting tan solid slurry was diluted with water, subjected to sonicaiton and filtered. The solid was washed with additional water, minimal acetonitrile, then with hexanes to afford 17 (0.084 g, 0.200 mmol, 49 % yield, HPLC purity (A) 95% ). 1H NMR (400 MHz, METHANOL-d4) δ 7.86 - 8.10 (m, 2 H), 7.66 (s, 1 H), 4.43 - 4.65 (m, 1 H), 4.16 (s, 2 H), 3.26 - 3.32 (m, 28 H), 3.15 - 3.26 (m, 1 H), 2.74 (s, 3 H), 2.20 (d, J=12.10 Hz, 2 H), 1.83 (br. s., 2 H), 1.72 (qd, J=12.76, 3.12 Hz, 2 H), 1.25 - 1.40 (m, 2 H); MS (ESI) m/z 421.0 [M+1]+; mp 165-167 °C. 1-((trans)-4-methoxycyclohexyl)-7-(2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4dihydropyrazino[2,3-b]pyrazin-2(1H)-one (18).

2-Methyl-6-(1-(tetrahydro-2H-pyran-2-yl)-

1H-1,2,4-triazol-3-yl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (91.65 g, 248 mmol), 7e (84.8 g, 248 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (10.0 g, 5 mol%) were reacted according to Procedure B to give 18-THP (107 g, 212 mmol, 85% yield). MS (ESI) m/z 505.6 [M+1]+. Procedure C gave 18 (80.4 g, 120 mmol, 57% yield, HPLC purity (D) >99%). 1H NMR (400 MHz, METHANOL-d4) δ 8.59 (br. s., 1H), 8.11 (br. s., 1H), 7.87 - 8.03 (m, 3H), 7.83 (s, 1H), 4.92 - 5.05 (m, 1H), 4.16


24


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Page 26 of 41

(s, 2H), 3.32 (s, 3H), 3.16 - 3.25 (m, 1H), 2.72 (s, 3H), 2.59 (qd, J = 3.12, 12.89 Hz, 2H), 2.15 (d, J = 11.32 Hz, 2H), 1.74 (d, J = 10.54 Hz, 2H), 1.18 - 1.35 (m, 2H); MS (ESI) m/z 421.0 [M+1]+; mp 145-147 °C; Anal. (C21H24N8O2-0.37H2O) Calc. C: 59.05, H: 5.84, N: 26.23; Found C: 59.40, H: 5.90, N: 25.83; KF = 1.54%. 1-((cis)-4-Hydroxycyclohexyl)-7-(2-methyl-6-(4H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4dihydropyrazino[2,3-b]pyrazin-2(1H)-one

(19).

3-Bromo-2-methyl-6-(4-(tetrahydro-2H-

pyran-2-yl)-4H-1,2,4-triazol-3-yl)pyridine (0.211 g, 0.653 mmol), 8f (0.276 g, 0.544 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.050 g, 0.054 mmol) and tri-o-tolylphosphine (0.033 g, 0.109 mmol) were reacted according to Procedure A to give 19-THP (0.270 g, 0.447 mmol, 82 % yield). MS (ESI) m/z 491.2 [M+1]+. Procedure C gave 19 (0.090 g, 0.221 mmol, 50 % yield, HPLC purity (B) >99%). 1H NMR (400 MHz, DMSO-d6) δ 8.14 (br. s., 2H), 7.92 - 8.01 (m, 2H), 7.64 (br. s., 1H), 4.84 (tt, J = 3.27, 12.35 Hz, 1H), 4.37 (d, J = 1.95 Hz, 1H), 4.14 (s, 2H), 3.86 (br. s., 1H), 2.82 - 2.97 (m, 2H), 2.75 (s, 3H), 1.77 (d, J = 12.49 Hz, 2H), 1.46 (t, J = 13.47 Hz, 2H), 1.34 (d, J = 10.15 Hz, 2H); MS (ESI) m/z 407.3 [M+1]+. 1-(trans-4-Hydroxycyclohexyl)-7-(2-methyl-6-(4H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4dihydropyrazino[2,3-b]pyrazin-2(1H)-one (20).

3-Bromo-2-methyl-6-(4-(tetrahydro-2H-

pyran-2-yl)-4H-1,2,4-triazol-3-yl)pyridine (0.275 g, 0.849 mmol), 8g 0.291 g, 0.708 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.065 g, 0.071 mmol) and tri-o-tolylphosphine (0.043 g, 0.142 mmol) were reacted according to Procedure A to give 20-THP (0.336 g, 0.537 mmol, 76 % yield). MS (ESI) m/z 491.2 [M+1]+. Procedure C gave 20 (0.130 g, 0.320 mmol, 58 % yield, HPLC purity (B) >99%). 1H NMR (400 MHz, DMSO-d6) δ 8.27 (br. s., 1H), 8.00 (s, 2H), 7.95 (s, 1H), 7.65 (s, 1H), 4.85 (tt, J = 3.71, 12.10 Hz, 1H), 4.59 (d, J = 3.51 Hz, 1H), 4.13 (d, J


25

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= 1.56 Hz, 2H), 3.35 - 3.47 (m, 1H), 2.72 (s, 3H), 2.41 - 2.58 (m, 2H), 1.91 (d, J = 9.76 Hz, 2H), 1.61 (d, J = 10.93 Hz, 2H), 1.15 - 1.32 (m, 2H); MS (ESI) m/z 407.3 [M+1]+. 7-(2-Methyl-6-(4H-1,2,4-triazol-3-yl)pyridin-3-yl)-1-(tetrahydro-2H-pyran-4-yl)-3,4dihydropyrazino[2,3-b]pyrazin-2(1H)-one hydrochloride (21). 8h (530.0 mg, 1.335 mmol), 3-bromo-2-methyl-6-(1-(tetrahydro-2H-pyran-2-yl)-1H-1,2,4-triazol-3-yl)pyridine

(431

mg,

1.335 mmol) and dichlorobis(triphenylphosphine)palladium(II) (98 mg, 0.133 mmol) were reacted according to Procedure A to give 21-THP, followed by Procedure C to give 21 (32.6 mg, 0.076 mmol, 5.69 % yield, HPLC purity (B) 98%).

1

H NMR (400 MHz, DMSO-d6) δ 7.91 -

8.05 (m, 3 H), 7.68 (br. s., 1 H), 5.07 - 5.17 (m, 1 H), 4.15 (d, J=1.6 Hz, 2 H), 3.95 (dd, J=11.1, 3.7 Hz, 2 H), 3.35 - 3.40 (m, 2 H), 2.63 - 2.77 (m, 5 H), 1.52 - 1.60 (m, 2 H); MS (ESI) m/z 393.2 [M+1]+. 1-Isopropyl-7-(2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3b]pyrazin-2(1H)-one (22)

2-Methyl-6-(1-(tetrahydro-2H-pyran-2-yl)-1H-1,2,4-triazol-3-yl)-3-

(4,4,5,5-tetramethyl-1,3,2-dioxa-borolan-2-yl)pyridine (3.7 g, 9.99 mmol), 7i (2.46 g, 9.08 mmol)

and

[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II)

complex

with

dichloromethane (0.332 g, 0.454 mmol) were reacted according to Procedure B to give 22-THP (3.3 g, 7.60 mmol, 84 % yield). MS (ESI) m/z 435.5 [M+1]+. Procedure C gave 22 (1.02 g, 2.91 mmol, 27.7 % yield, HPLC purity (D) >99%). 1H NMR (400 MHz, METHANOL-d4) δ 7.92 8.04 (m, 2 H), 7.82 (s, 1 H), 5.36 (quin, J=6.93 Hz, 1 H), 4.17 (s, 2 H), 2.70 (s, 3 H), 1.51 (d, J=7.03 Hz, 6 H); MS (ESI) m/z 351.0 [M+1]+; mp 186-188 °C; Anal. (C17H18N8O-0.46 H2O) Calc. C: 56.93, H: 5.32, N: 31.24; Found C: 57.34, H: 5.19, N: 31.21; KF = 2.32%. 4-(2-Methoxyethyl)-6-(2-methyl-6-(4H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-one (23).

3-Bromo-2-methyl-6-(1-(tetrahydro-2H-pyran-2-yl)-


26


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1H-1,2,4-triazol-3-yl)pyridine

(2.56

g,

7.92

mmol),

6c

Page 28 of 41

(2.94

g,

7.92

mmol),

tris(dibenzylideneacetone)dipalladium(0) (0.80 g, 0.87 mmol) and tri-o-tolylphosphine (0.53 g, 1.74 mmol) were reacted according to Procedure A to give 23-THP (2.80 g, 6.22 mmol, 78 % yield). MS (ESI) m/z 451.5 [M+1]+. Combined batches of 23-THP (5.0 g, 11.1 mmol) were reacted according to Procedure C to give 23 (3.50 g, 9.55 mmol, 86% yield). Combined batches were recrystallized from ethanol (HPLC purity (B) >99%).

1

H NMR (400 MHz, DMSO-d6) δ

8.29 (br. s., 1H), 7.98 (s, 2H), 7.72 (s, 1H), 4.28 (s, 2H), 3.67 - 3.73 (m, 2H), 3.58 - 3.64 (m, 2H), 3.27 (s, 3H), 2.69 (s, 3H); MS (ESI) m/z 367.3 [M+1]+; mp 262 - 264 °C; Anal. (C17H18N8O2) Calc. C: 55.73, H: 4.95, N: 30.58; Found C: 55.45, H: 4.67, N: 30.52. 1-(2-Methoxyethyl)-7-(2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4dihydropyrazino[2,3-b]pyrazin-2(1H)-one (24).

7j (1.20 g, 4.18 mmol), 2-methyl-6-(4-

(tetrahydro-2H-pyran-2-yl)-4H-1,2,4-triazol-3-yl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)pyridine

(1.702

g,

4.60

mmol)

and

[1,1'-bis(diphenylphosphino)ferrocene]di-

chloropalladium(II) complex with dichloromethane (0.341 g, 0.418 mmol) were reacted according to Procedure B to give 24-THP (1.44 g, 3.20 mmol, 76 % yield).

MS (ESI) m/z

451.5 [M+1]+. Combined batches of 24-THP (2.20 g, 4.88 mmol) were reacted according to Procedure C followed by recyrstallization from ethanol gave 24 (1.17 g, 3.19 mmol, 65 % yield, HPLC purity (C) >99%). 1H NMR (400 MHz, DMSO-d6) δ 8.10 (br. s., 1 H), 7.98 (br. s., 1 H), 7.94 (s, 1 H), 7.73 (br. s., 1 H), 4.13 - 4.28 (m, 4 H), 3.55 (t, J=6.25 Hz, 2 H), 3.24 (s, 3 H), 2.70 (br. s., 3 H); MS (ESI) m/z 367.0 [M+1]+; mp 244-246 °C; Anal. (C17H18N8O2) Calc. C: 55.73, H: 4.95, N: 30.58; Found C: 55.52, H: 4.91, N: 30.38. 4-Ethyl-6-(2-methyl-6-(4H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3b]pyrazin-2(1H)-one

(25).

3-Bromo-2-methyl-6-(1-(tetrahydro-2H-pyran-2-yl)-1H-1,2,4-


27

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


triazol-3-yl)pyridine

(0.474

g,

1.466

mmol),

6d

(0.5

g,

1.466

mmol),

tris(dibenzylideneacetone)dipalladium(0) (0.148 g, 0.161 mmol) and tri-o-tolylphosphine (0.098 g, 0.323 mmol) were reacted according to Procedure A to give 25-THP (0.450 g, 1.070 mmol, 73.0 % yield). MS (ESI) m/z 421.5 [M+1]+ Procedure C gave 25 (0.200 g, 0.595 mmol, 57.7 % yield, HPLC purity (B) 99%). 1H NMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H), 7.99 (br. s., 2H), 7.70 (s, 1H), 4.20 (s, 2H), 3.56 (q, J = 6.90 Hz, 2H), 2.70 (br. s., 3H), 1.15 (t, J = 7.03 Hz, 3H); MS (ESI) m/z 337.7 [M+1]+. 1-Ethyl-7-(2-methyl-6-(1H-1,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3b]pyrazin-2(1H)-one (26).

7k (29.49 g, 114.7 mmol), 2-methyl-6-(1-(tetrahydro-2H-pyran-2-

yl)-1H-1,2,4-triazol-3-yl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine

(42.50

g,

114.7 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (4.65 g, 5 mol%) were reacted according to Procedure B to give 26-THP (27.3 g, 65.37 mmol, 57% yield).

MS (ESI) m/z 421.6 [M+1]+.

Procedure C followed by

recrystallization from ethanol gave 26 (17.5 g, 52.0 mmol, 81% yield, HPLC purity (D) >99%). 1

H NMR (400 MHz, DMSO-d6) δ 7.99 (s, 2H), 7.93 (s, 1H), 7.72 (s, 1H), 4.22 (s, 2H), 4.05 (q, J

= 6.77 Hz, 2H), 2.71 (s, 3H), 1.18 (t, J = 7.03 Hz, 3H); MS (ESI) m/z 337.0 [M+1]+; mp 263265 °C; Anal. (C16H16N8O) Calc. C: 57.13, H: 4.79, N: 33.31; Found C: 59.95, H: 5.07, N: 33.23. Molecular Modeling

All structure preparation, docking, and protein-ligand complex

minimizations were carried out using methodology implemented in the Schrodinger SmallMolecule Drug Discovery Suite 2015-1: Protein Preparation Wizard; Epik version 3.1, Schrödinger, LLC, New York, NY, 2015; Impact version 6.6, Schrödinger, LLC, New York, NY, 2015; Prime version 3.9, Schrödinger, LLC, New York, NY, 2015. Maestro, version 10.1,


28


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Page 30 of 41

Schrödinger, LLC, New York, NY, 2015. LigPrep, version 3.3, Schrödinger, LLC, New York, NY, 2015. Glide, version 6.6, Schrödinger, LLC, New York, NY, 2015. MacroModel, version 10.7, Schrödinger, LLC, New York, NY, 2015. mTOR and PI3K Kinase Enzyme Assays An HTR-FRET substrate phosphorylation assay was employed for mTOR kinase, as described previously.13

PI3Kα IC50 determinations were

outsourced to Carna Biosciences (Japan) using the mobility shift assay format. Compounds were assessed against concentrations of ATP at approximately the Km for the assay, with average ATP Km of 15 µM and 50 µM for the mTOR and PI3K assays, respectively. PC-3 Cellular Assays. PC-3 cells were purchased from and verified by American Tissue Culture Collection and were cultured in growth media as recommended by the vendor. For biomarker studies cells were treated for 1 h and then assayed for pS6 and pAkt levels using MesoScale technology. For proliferation experiments, cells were treated with compound and then allowed to grow for 72 h. All data were normalized and represented as a percentage of the DMSO-treated cells. Results were then expressed as IC50 values. Full experimental details have been previously published.13 In Vivo Studies. All animal studies were performed under protocols approved by Institutional Animal Care and Use Committees. Single dose biomarker and multi-day efficacy studies were performed as previously published.20 Supporting Information Supporting information includes full experimental details for preparation of intermediates, further details on CC-115 docking results (Supplemental Figure 1), a table of enzyme and cellular data that includes SEM (Supplemental Table 1), single point kinase panel results for CC115 (Supplemental Table 2), CC-115 PIKK data with SEM (Supplemental Table 3) and CC-115


29

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cross species PK data (Supplemental Table 4) . This material is available free of charge via the Internet at http://pubs.acs.org.

Author Information *Corresponding Author: Tel: 858-795-4951, [email protected] Present Addresses: Current address information for R. Bisonette, B.Lee, S. Sankar, G. Shevlin and G.Packard may be available from corresponding author upon request. Notes: The authors declare no competing financial interest. All authors are currently employees of Celgene, except S.Sankar, B.Lee, G.Shevlin, R. Bisonette, and G.Packard, who were employees of Celgene at the time of their contribution to this work. Acknowledgements The authors thank the Celgene San Diego DMPK department for plasma and tumor compound level analysis, the Celgene San Diego CLMD group for their excellent support throughout the project, and Kirsten Blumeyer for coordination of outsourced compound testing. Abbreviations AKT, Protein Kinase B; ATM, ataxia telangiectasia mutated; ATR, atazia telangiectasis and Rad3-related protein kinase; AUC, area under the curve; cFMS (CSF1R), colony stimulating factor 1 receptor tyrosine kinase; Cmax, maximum concentration; mTOR, Mammalian Target of Rapamycin; mTORC1, mTOR Complex 1; mTORC2, mTOR Complex 2; pAKT, phosphorylated

AKT;

pAKT(S473),

phosphorylated

AKT

at

Serine

473;

PI3K,

phosphatidylinositol 3-Kinase; PIK3CA, Gene coding 110 kDa catalytic subunit of PI3K alpha; PIKK,

Phosphatidylinositol

3-Kinase-related

Kinase;

PK/PD,

Pharmacokinetic/


30


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Page 32 of 41

Pharmacodynamic; pS6RP or pS6, Phosphorylated Ribosomal protein S6; S6RP or S6, Ribosomal protein S6; SEM, Standard error of the mean; TVR, tumor volume reduction. References 1.

Laplante M.; Sabatini D.M. mTOR Signaling at a glance. J. Cell Sci. 2009; 122: 35893594.

2.

Fruman D.A.; Rommel C. PI3K and cancer: lessons, challenges and opportunities. Nat. Rev. Drug Discov. 2014; 13: 140-156.

3.

Kim D.H.; Sarbassov D.D. Ali S.M,.; King J.E.;, Latek R.R.; Erdjument-Bromage H.; Tempst T.; Sabatini D.M. mTOR Interacts with Raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell. 2002; 110: 163-175.

4.

Sarbassov D.D.; Guertin D.A.; Ali S.M.; Sabatini D.M. Phosphorylation and regulation of AKT/PKB by the Rictor-mTOR complex. Science. 2005; 307: 1098-1101.

5.

Gibbons J.J.; Abraham R.T.; Yu K. Mammalian target of rapamycin: discovery of rapamycin reveals a signaling pathway important for normal and cancer cell growth. Semin. Oncol. 2009; 36 Suppl 3(S3-S17).

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Rodon J.; Dienstmann R.; Serra V.; Tabernero J. Development of PI3K inhibitors: lessons learned from early clinical trials. Nat. Rev. Clin. Oncol. 2013; 10: 143-153

10. Bendell J.C.; Kelley R.K.; Shih K.C.; Grabowsky J.A.; Bergsland E.; Jones S.; Martin T.; Infante J.R.; Mischel P.S.; Matsutani, T.; Xu S.; Wong L.; Liu Y.; Wu X.; Mortensen D.S.; Chopra R.; Hege K.; Munster P.N. A phase I dose-escalation study to assess safety, tolerability, pharmacokinetics and preliminary efficacy of the dual mTORC1/mTORC2 kinase inhibitor CC-223 in patients with advanced solid tumors or multiple myeloma. Cancer. 2015, In Press. 11. Pike K.G.; Malagu K.; Hummerstone M.G.; Menear K.A.; Duggan H.M.E.; Gomez S.; Martin N.M.B.; Ruston L.; Pass S.L.; Pass M. Optimization of potent and selective dual mTORC1 and mTORC2 inhibitors: the discovery of AZD8055 and AZD2014. Bioorg. Med. Chem. Lett. 2013; 23: 1212-1216. 12. Janes M.R.; Vu C.; Mallaya S.; Shieh M.P.; Limon J.J.; Liu L-S.; Jessen K.A.; Martin M.B.; Ren P.; Lilly M.B.; Sender L.S.; Liu Y.; Rommel C.; Fruman D.A. Efficacy of the investigations mTOR kinase inhibitor MLN0128/INK128 in models of B-cell acute lymphoblastic leukemia. Leukemia. 2013; 27: 586-594 13. Mortensen D.S.; Perrin-Ninkovic S.M.; Harris R.; Lee B.G.S.; Shevlin G.; Hickman M.; Khambatta G.; Bisonette R.R.; Fultz K.E.; Sankar S. Discovery and SAR exploration of a


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novel series of imidazo[4,5-b]pyrazin-2-ones as potent and selective mTOR kinase inhibitors. Bioorg. Med. Chem. Lett. 2011; 21: 6793-6799. 14. Mortensen D.S.; Sapienza J.; Lee B.G.S; Perrin-Ninkovic S.M.; Harris R.; Shevlin G.; Parnes J.S.; Whitefield B.; Hickman M.; Khambatta G.; Bisonette R.R.; Peng S.; Gamez J.C.; Leisten J.; Narla R.K.; Fultz K.E.; Sankar S. Use of core modification in the discovery of CC214-2, an orally available, selective inhibitor of mTOR kinase. Bioorg. Med. Chem. Lett. 2013; 23: 1588-1591. 15. Mortensen D.S.; Perrin-Ninkovic S.M.; Shevlin G.; Zhao J.; Packard G.; Bahmanyar S.; Correa M.; Elsner J.; Harris R.; Lee B.G.S.; Papa P.; Parnes J.S.; Riggs J.R.; Sapienza J.; Tehrani L.; Whitefield B.; Apuy J.; Bisonette R.R.; Gamez J.C.; Hickman M.; Khambatta G.; Leisten J.; Peng S.X.; Richardson S.J.; Cathers B.E.; Canan S.S.; Moghaddam M.F.; Raymon H.K.; Worland P.; Narla R.K.; Fultz K.E.; Sankar S. Discovery of mammalian target of rapamycin (mTOR) kinase inhibitor CC-223. J. Med. Chem. 2015 Submitted. 16. Yang H.; Rudge D.G.; Koos J.D.; Vaidialingam B.; Yang H.J., Pavletich N.P. mTOR kinase structure, mechanism and regulation. Nature 2013; 497: 217-223. 17. SelectScreen® Kinase Profiling Services: Life Technologies, Inc., Madison WI, USA. 18. Bhagwat S.V.; Gokhale P.C.; Crew A.P.; Cooke A.; Yao Y.; Mantis C.; Kahler J.; Workman J.; Bittner M.; Dudkin L.; Epstein D.M.; Gibson N.W.; Wild R.; Arnold L.D.; Houghton P.J.; Pachter J.A. Preclinical characterization of OSI-027, a potent and selective inhibitor of mTORC1 and mTORC2: distinct from rapamycin. Mol. Cancer Ther. 2011; 10: 1394-1401.


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19. Pike K.G.; Malagu K.; Hummerstone M.G.; Menear K.A.; Duggan H.M.E.; Gomez S.; Martin N.M.B.; Ruston L.; Pass S.L.; Pass M. Optimization of potent and selective dual mTORC1 and mTORC2 inhibitors: the discovery of AZD8055 and AZD2014. Bioorg. Med. Chem. Lett. 2013; 23: 1212-1216. 20. Mortensen D.S.; Fultz K.E.; Xu S.; Xu W.; Packard G.; Khambatta G.; Gamez J.C.; Leisten J.; Zhao J.; Apuy J.; Ghoreishi K.; Hickman M.; Narla R.K.; Bissonette R.; Richardson S.; Peng S.X.; Perrin-Ninkovic S.; Tran T.; Shi T.; Yang W.Q.; Tong Z.; Cathers B.E.; Moghaddam M.F.; Canan S.S.; Worland P.J.; Sankar S.; Raymon H.K. CC-223, a potent and selective inhibitor of mTOR kinase: in vitro and in vivo characterization. Mol. Cancer Therp. 2015; 14: 1295-1305. 21. Janes M.R.; Vu C.; Mallaya S.; Shieh M.P.; Limon J.J.; Liu L-S.; Jessen K.A.; Martin M.B.; Ren P.; Lilly M.B.; Sender L.S.; Liu Y.; Rommel C.; Fruman D.A. Efficacy of the investigations mTOR kinase inhibitor MLN0128/INK128 in models of B-cell acute lymphoblastic leukemia. Leukemia. 2013; 27: 586-594.


34


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TOC GRAPHIC


35

Page 37 of 41

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Figure 2. 26 docked into the mTOR kinase binding pocket. 100x68mm (110 x 110 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. PC-3 tumor xenograft studies with (A) 18, (B) 22, (C) 24 and (D) 26 CC-115. 157x150mm (300 x 300 DPI)


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Optimization of a Series of Triazole Containing Mammalian Target of

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