Discovery of Mammalian Target of Rapamycin (mTOR) Kinase Inhibitor CC-223

Article abstract of DOI:10.1021/acs.jmedchem.5b00626

We report here the synthesis and structure-activity relationship (SAR) of a novel series of mammalian target of rapamycin (mTOR) kinase inhibitors. A series of 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were optimized for in v

Full text of DOI:10.1021/acs.jmedchem.5b00626

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Article
Discovery of Mammalian Target of Rapamycin (mTOR) Kinase Inhibitor CC-223
Deborah S. Mortensen, Sophie M. Perrin-Ninkovic, Graziella Shevlin, Jingjing Zhao, Garrick
Packard, Sogole Bahmanyar, Matthew Correa, Jan Elsner, Roy Harris, Branden G.S. Lee, Patrick
Papa, Jason S. Parnes, Jennifer R. Riggs, John Sapienza, Lida Tehrani, Brandon Whitefield,
Julius Apuy, René R. Bisonette, James C. Gamez, Matt Hickman, Godrej Khambatta, Jim Leisten,
Sophie X. Peng, Samantha J. Richardson, 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 • Publication Date (Web): 17 Jun 2015
Downloaded from http://pubs.acs.org on June 17, 2015
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Discovery of Mammalian Target of Rapamycin
(mTOR) Kinase Inhibitor CCꢀ223
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Deborah S. Mortensen*, Sophie M. Perrin-Ninkovic, Graziella Shevlin, Jingjing Zhao, Garrick
Packard, Sogole Bahmanyar, Matthew Correa, Jan Elsner, Roy Harris, Branden G.S. Lee,
Patrick Papa, Jason S. Parnes, Jennifer R. Riggs, John Sapienza, Lida Tehrani, Brandon
Whitefield, Julius Apuy, René R. Bisonette, James C. Gamez, Matt Hickman, Godrej Khambatta,
Jim Leisten, Sophie X. Peng, Samantha J. Richardson, 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.
Running Title: Discovery of CCꢀ223
Keywords: Mammalian target of rapamycin, mTOR kinase, kinase inhibitors,
PI3K/AKT/mTOR pathway, oncology
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Abstract.
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We report here the synthesis and structureꢀactivity relationship (SAR) of a novel series of
mammalian target of rapamycin (mTOR) kinase inhibitors. A series of 4,6ꢀ or 1,7ꢀdisubstitutedꢀ
3,4ꢀdihydropyrazino[2,3ꢀb]pyrazineꢀ2(1H)ꢀones were optimized for in vivo efficacy. These
efforts resulted in the identification of compounds with excellent mTOR kinase inhibitory
potency, with exquisite kinase selectivity over the related lipid kinase PI3K. The improved PK
properties of this series allowed for exploration of in vivo efficacy and ultimately the selection of
CCꢀ223 for clinical development.
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Introduction.
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The phosphoinositide 3ꢀkinase (PI3K)/protein kinase B (AKT) pathway is frequently
disgregulated in human cancers through multiple mechanisms, including loss of function
mutations or promoter hypermethylation of the tumor suppressor PTEN or activating mutations
of the PIK3CA oncogene.1 The mammalian target of rapamycin (mTOR) kinase is found in two
distinct multiꢀprotein signaling complexes, mTOR complexꢀ1 (mTORC1) and mTOR complexꢀ2
(mTORC2), which function as critical mediators of the PI3K/AKT pathway.2 While mTORC1
is responsible for regulating protein synthesis and growth,3 mTORC2 has been shown to
phosphorylate and activate AKT,4 a key kinase in the control of cell growth, metabolism and
survival. Rapamycin analogs such as temsirolimus and everolimus, allosteric inhibitors that
generally do not inhibit mTORC2 and have been shown to only partially inhibit the mTORC1
complex have achieved some clinical success, though it is hypothesized that mTOR kinase
(TORK) inhibitors, blocking both mTORC1 and mTORC2 signaling, will have expanded
therapeutic potential.5 An attractive drug target, mTOR kinase has been the focus of much
investigation in the pharmaceutical industry and ATPꢀcompetitive inhibitors selective for mTOR
kinase relative to the related PI3Kꢀα, such as OSIꢀ0276, AZD80557, AZD20147, MLN01288, CCꢀ
1159 and CCꢀ22310,11, have entered clinical trials.
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We have previously reported the identification of a series of 1,6ꢀsubstituted imidazo[4,5ꢀ
b]pyrazinꢀ2ꢀone based mTOR kinase inhibitors following an HTS campaign and initial
optimizations.12 Optimization in this series provided analogs with excellent potency and
selectivity over PI3Kα, such as 1 (CC214ꢀ1) and 2 (Figure 1A). Further optimization to achieve
good oral PK properties however, proved to be a challenge. We therefore evaluated core
modifications to expand the chemical matter available to the program. Insertion of a methylene
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unit in the imidazo ring led to the identification of two new series of potent and selective mTOR
kinase inhibitors (Figure 1B). We have previously disclosed the initial results of our first ring
expansion effort resulting in the identification of an in vivo tool compound CC214ꢀ2 (26)13, here
we describe the SAR exploration in both ring expansion series and the selection of CCꢀ223 for
clinical development.
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Figure 1. (A) Representative analogs from initial compound series. (B) Core modification of the
imidazo[4,5ꢀb]pyrazinꢀ2ꢀones to give 3,4ꢀdihydropyrazino[2,3ꢀb]pyrazinꢀ2(1H)ꢀones.
Chemistry
Compounds from the 4,6ꢀdisubstitutedꢀ3,4ꢀdihydropyrazino[2,3ꢀb]pyrazineꢀ2(1H)ꢀone
(RE1) series were synthesized by the methods in Scheme 1. Amine addition to either 2ꢀbromoꢀ
(3a) or 2ꢀiodoꢀNꢀ(3,5ꢀdibromopyrazinꢀ2ꢀyl)acetamide (3b) afforded the desired addition and ring
closure products 4aꢀi. Three methods were employed to achieve the aryl substitution at C6 of
the 3,4ꢀdihydropyrazino[2,3ꢀb]pyrazineꢀ2(1H)ꢀone core. In one method, Suzuki coupling of
intermediate 4a with aryl boronic acid pinacol esters, followed by acid catalyzed deprotection,
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when relevant, afforded the desired compounds 9, 13 and 15. Alternatively, 4aꢀi were subjected
to Stille coupling with 2ꢀ(5ꢀ(trimethylꢀstannyl)pyridinꢀ2ꢀyl)propanꢀ2ꢀol to afford analogs 11, 22,
24, 26, 28, 29, 31, 35, and 36. Finally, bromide 4a was converted to stannane 5a. Stille
coupling of 5a with aryl bromides, followed by acid catalyzed deprotection, when relevant, gave
compounds 17, 18 and 20.
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Compounds from the 1,7ꢀdisubstitutedꢀ3,4ꢀdihydropyrazino[2,3ꢀb]pyrazineꢀ2(1H)ꢀone
(RE2) series were synthesized by the methods in Scheme 2. Addition of amines to ethyl (3,5ꢀ
dibromopyrazinꢀ2ꢀyl)glycinate (6), followed by acid catalyzed ring closure gave the desired
intermediates (7aꢀi). As with the RE1 analogs, three methods were employed in completing the
aryl substitution, now at the C7 of the 3,4ꢀdihydropyrazino[2,3ꢀb]pyrazineꢀ2(1H)ꢀone core.
Suzuki coupling of intermediate 7a with aryl boronic acid pinacol esters, followed by acid
catalyzed deprotection, when needed, afforded the desired compounds 10, 14, and 16. Stille
coupling of 7aꢀi with 2ꢀ(5ꢀ(trimethylꢀstannyl)pyridinꢀ2ꢀyl)propanꢀ2ꢀol afforded analogs 12, 23,
25, 27, 30, 32, 33, 34, and 37. Alternatively the reactivity of the Stille coupling partners was
reversed with the conversion of bromide 7a to stannane 8a. Aryl bromides were reacted with 8a
under Stille coupling conditions, followed by acid catalyzed deprotection, when needed, to
provide compounds 19 and 21.
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Scheme 1.
Synthesis of 4,6ꢀdisubstitutedꢀ3,4ꢀdihydropyrazino[2,3ꢀb]pyrazineꢀ2(1H)ꢀones
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(RE1).
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Reagents and conditions: (a) R1ꢀNH2, acetonitrile, DIPEA, rt or 40ꢀ70 °C, 0.5ꢀ16 h, yield 24ꢀ
79%; (b) R2ꢀboronate ester, sodium carbonate in water, PdCl2(dppf)ꢀCH2Cl2, dioxane or DMF,
microwave 120 °C, 15ꢀ30 min, yield 25ꢀ74%; (c) HCl (4N in dioxane or 6N aqueous), ethanol or
dioxane, rt or 45ꢀ90 °C, 1ꢀ12 h, yield 22ꢀ39% (d) 2ꢀ(5ꢀ(trimethylꢀstannyl)pyridinꢀ2ꢀyl)propanꢀ2ꢀ
ol, PdCl2(dppf)ꢀCH2Cl2 or Pd(PPh3)2Cl2, DMF, 110ꢀ140 °C, 1ꢀ2 h, yield 9ꢀ66%; (e)
hexamethylditin, Pd(PPh3)4, dioxane, 100 °C, 5 h, yield 66%; (f) R2ꢀbromide, PdCl2(dppf)ꢀ
CH2Cl2or Pd2(dba)3 and P(oꢀtol)3 and triethylamine, DMF, 100ꢀ120 °C, 1ꢀ4 h, yield 15ꢀ74%.
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Scheme 2.
Synthesis of 1,7ꢀdisubstitutedꢀ3,4ꢀdihydropyrazino[2,3ꢀb]pyrazineꢀ2(1H)ꢀones
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(RE2).
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Reagents and conditions: (a) R1ꢀNH2, DMSO and/or DIPEA, 100ꢀ150 °C, 1ꢀ48 h, yield 43ꢀ
74%; (b) acetic acid or TFA in methanol, 90ꢀ120 °C, 2ꢀ24 h, 33ꢀ76%; (c) R2ꢀboronate ester,
sodium carbonate in water, PdCl2(dppf)ꢀCH2Cl2 or Pd(PPh3)2Cl2, dioxane, 130ꢀ150 °C, 1ꢀ4 h,
yield 8ꢀ44%; (d) HCl (4N in dioxane or 6N aqueous), ethanol, 50ꢀ100 °C, 0.2ꢀ1 h, yield 39ꢀ86%
(e) 2ꢀ(5ꢀ(trimethylꢀstannyl)pyridinꢀ2ꢀyl)propanꢀ2ꢀol, PdCl2(dppf)ꢀCH2Cl2 or Pd(dtbpf)Cl2 or
Pd(PPh3)2Cl2, DMF, 110ꢀ140 °C, 0.3ꢀ2 h, yield 14ꢀ68%; (f) hexamethylditin, Pd(PPh3)4,
dioxane, 110 °C, 1 h, yield 55%; (g) R2ꢀbromide, Pd(dtbpf)Cl2 or Pd2(dba)3 and P(oꢀtol)3 and
triethylamine, DMF, 120 °C, 1 h, yield 33ꢀ55%.
Results and Discussion
We first examined whether the SAR requirements of the C6/C7 position for the ring
expansion series would translate as compared to the original imidazoꢀpyrazinone series. With
the N1/N4 ethyleneꢀtetrahydropyran constant, we found the SAR for mTOR kinase potency
tracked from the original series, with benzylic alcohols, aryl/heteroaryl triazoles and 6,5ꢀfused
heterocycles each maintaining potency in both isomers of the ring expansion series (Table 1).
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Table 1. SAR of mTOR Kinase and PI3Kα for compounds 9ꢀ21.
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mTOR
PI3K
Kinase
IC50 (µM)b
Analog
C6/C7
Core
IC50 (µM)a
RE1 0.066 ± 0.032
RE2 0.025 (n=1)
RE1 0.103 ± 0.031
RE2 0.033 ± 0.008
RE1 0.008 ± 0.007
NTc
NT
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NT
NT
0.53
RE2 0.004 ± 0.001
RE1 0.004 ± 0.001
RE2 0.005 (n=1)
0.10
0.20
0.12
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RE1 0.018 ± 0.005
1.6
17
RE1 0.090 ± 0.015
RE2 0.014 ± 0.002
2.3
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19
0.51
RE1 0.122 ± 0.030
RE2 0.072 ± 0.028
amean± SEM, n=2 or more, cNT = not tested
NT
NT
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21
b
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As observed in the imidazoꢀpyrazinone series, the triazole phenyl substituted analogs
proved to be among the most potent, with single digit nanomolar IC50 values against mTOR
kinase. As we evaluated the PK properties of the ring expansion series, we first looked at the
more potent triazoleꢀphenyl compounds. While the RE2 analog of the triazoleꢀphenyl (14) was
not tested in PK studies due to poor in vitro metabolic stability (RatS9 48% remaining at 60
min), the RE1 triazoleꢀphenyl analog (13) showed an improvement in IV clearance properties as
compared to CC214ꢀ1, but unfortunately, showed no exposure upon oral dosing (Table 2).
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A.
RatPlasma Concentration of Compounds 1,2, 11 & 12
2.5
2.0
1.5
1.0
0.5
0.0
Dosed 5 or 10 mg/kg p.o. in 0.5% CMC/0.25% Tween 80 (N=3)
CC214-1 (1)
Compound 2
Compound 11
Compound 12
0
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12
Time (h)
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Rat Plasma Concentration of Compounds 2, 11 & 12
Normalized to pAkt-Cellular IC50 Values
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Compound 2
Compound 11
Compound 12
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Time (h)
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Figure 2. (A) Rat PK profiles of 1, 13, 2, 11 and 12 show the ringꢀexpanded analogs of 2
provided improved oral exposure. (B) Potency normalized exposures further illustrates benefit of
new series.
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Table 2. Rat PK parameters for compounds CC214ꢀ1 (1), 2, 11, 12 and 13.
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Mean (± SD) Value
Rat PK Parameter
CC214ꢀ1
10 mg/kg
0.024±0.019
1.0 ± 0.7
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po Dose a
po Cmax (µM)
10 mg/kg
5 mg/kg
1.8 ± 0. 5
0.5 ± 0
4.8 ± 0.8
43 ± 9
5 mg/kg
1.5 ± 0.7
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11 ± 6
~100
5 mg/kg
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0.6 ± 0.3
14 ± 2.1
58 ± 18
9.0 ± 2.1
73 ± 3
po tmax (hr)
BLQb
po AUC(0ꢀ∞) (µMꢁhr)
po F(%)
0.11 ±0.08
0.85 ± 0.66
32 ± 9
iv CL (mL/min/kg)c
13.8 ± 5.7
61 ± 8
15 ± 2
23 ± 2
Rat S9 Stabilityd
93 ± 12
91± 2
95 ± 4
aformulated as a suspension in aqueous 0.5% CMC/0.25% Tweenꢀ80, BLQ=below limit of
b
quantitation, limit of quantitation 0.00213 µM, dose 2 mg/kg, d(% remaining at 60 min)
c
We next evaluated the direct comparisons of the matched ring expansion analogs to the
compound which gave the best PK profile in the original imidazo[4,5ꢀb]pyrazinꢀ2ꢀone series, 2.
Both analogs in the ring expansion series, 11 and 12, provided equivalent or improved PK
profiles as compared to the imidazo[4,5ꢀb]pyrazinꢀ2ꢀone counterpart, 2 (Figure 2A and Table 2).
In addition to the favorable PK profiles, these ring expansion analogs also showed improved
potency and cellular efficacy as compared to the previous series. The potency normalized
exposures, shown in Figure 2B, further illustrate the improved potential of the new cores,
suggesting the ring expansion series could be optimized for in vivo testing.
Given the improved PK profiles of the ring expansion series substituted with the
pyridinylꢀisopropyl alcohol aryl group, we sought to explore the SAR requirements of the N4
and N1 position in the RE1 and RE2 series, respectively. The SAR on mTOR kinase in the
previous series suggested favorable interactions for an Hꢀbond or polar substituent in the binding
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pocket of the N1/N4 substituent. In addition to potential potency benefits, the improvement in
drugꢀlike properties, such as LogD, led us to primarily focus on ether or alcohol substituents
(Table 3).
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Comparison of the directly connected THP (25) with 27 and 12 shows that extension of
the THP into the binding pocket results in an improvement in potency. The shorter but slightly
more flexible methoxyethyl analog 23 has similar potencies as compared to the directly
connected THP analog, 25. Comparison of analog 25 with the substituted cyclohexyl analogs
34 and 37, or 24 with 36, also suggests extension of the polar group further into the binding
pocket generally affords improved potencies. The comparison of cis/trans pairs 30/32, 33/34, or
35/36 suggests that proper positioning of the polar group in the binding pocket plays a role in
potency. Intriguingly in the directly connected hydroxyꢀ or methoxyꢀcyclohexyl analogs, the
transꢀisomer demonstrated a potency benefit relative to the cis, while the cis-isomer proved more
potent when the group was extended by a methylene further into the binding pocket (30 vs 32).
This cis/trans preferences shown here held across multiple other subseries in the course of the
program, although in the absence of a protein coꢀcrystal structure we can only speculate about
proper alignment for favorable interactions.
These SAR explorations also showed the RE2 series to be generally more potent than the
corresponding RE1 analogs. Both ring expansion series maintained selectivity over PI3Kꢀα,
particularly in the pyridylꢀisopropanol aryl subseries. Compounds from the C6/C7 arylꢀtriazole
subseries picked up some potency against PI3K (Table 1), but maintained >20ꢀ88 fold mTOR
kinase selectivity. In the pyridylꢀisopropanol aryl subseries analogs maintained >20ꢀ400 fold
selectivity over PI3K, and no analog demonstrated a PI3K IC50 value below 1 ꢂM (Table 3).
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Table 3. SAR of mTOR Kinase and PI3Kα for compounds 11, 12 and 22ꢀ37.
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
mTOR
PI3K
Kinase
IC50 (µM) b
Analog
N4/N1
Core
IC50 (µM)a
0.436 ± 0.111
0.175 ± 0.013
0.185 ± 0.025
0.168 ± 0.122
0.106 ± 0.026
0.046 ± 0.019
RE1
RE2
RE1
RE2
RE1
RE2
10.5
6.2
22
23
4.7
24
3.2
25
26 (CC214ꢀ2)13
>30
3.8
27
11
12
RE1 0.103 ± 0.031
RE2 0.033 ± 0.008
NTc
NT
RE1 0.108 ± 0.035
17.7
28
RE1 0.149 ± 0.039
RE2 0.012 ± 0.003
RE1 0.170 ± 0.009
RE2 0.097 ± 0.043
7.6
2.5
29
30
31
32
20.2
4.7
RE2 0.203 ± 0.093
RE2 0.038 ± 0.023
RE1 0.300 ± 0.104
2.8
1.9
33
34
11.1
35
36
RE1 0.103 ± 0.017
RE2 0.010 ± 0.001
16.4
4.0
37
b
amean ± SEM, n=2 or more, cNT = not tested
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With potent and selective inhibitors of mTOR kinase identified, we evaluated the
inhibitors’ effect on the mTOR pathway in PCꢀ3 prostate cancer cells. Inhibition of pS6
(TORC1) and pAkt(S473) (TORC2) were assessed following 1 hour of compound treatment.
We also assessed proliferation as a functional effect resulting from mTOR pathway inhibition in
these cells. As expected from targeting the mTOR kinase domain, inhibition of both
TORꢀcomplexes was observed and this pathway inhibition lead to antiꢀproliferative effects. In
PCꢀ3 prostate cancer cells a good correlation was demonstrated between the enzymatic mTOR
kinase potency and cellular biomarker potency. Potency of both TORC1 and TORC2 inhibition
correlated with inhibition of cellular growth in the proliferation assays, suggesting that in this
cell line the PI3K/mTOR pathway is driving cell survival (Figure 3 and Sup. Table 1ꢀ2).
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. Correlation of mTOR kinase enzyme and cellular biomarker (pS6 and pAkt), colored
by proliferation potency (green 0.034 to >5 µM red). Supplemental tables 1 and 2 provide
individual compound data (mean ± SEM).
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Having identified a number of compounds in the C6/7ꢀpyridylꢀisopropanol aryl ring
expansion series with mTOR kinase potency, selectivity and cellular efficacy, we next examined
the rodent PK profiles of the analogs to support in vivo efficacy studies. In vitro metabolic
stability studies were used to select analogs for in vivo evaluation. Compounds such as 28, 29,
30 and 31 did not meet the stability criteria to support in vivo evaluation (Table 4). The
remaining analogs exhibited in vitro stability values of ≥73% remaining after 60 minute
incubation with rat liver S9 enzyme fractions. Results of rat oral PK studies demonstrated that
most of the analogs in this subseries provided equivalent or improved PK profiles as compared to
our initial analogs 11 and 12. Compounds generally showed good absorption properties,
reaching Cmax values as high as 6.3 ꢂM while providing high AUCs with good duration of
exposure, corresponding to oral bioavailability ranging from 43ꢀ100% (Table 4).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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37
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52
53
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Based on these results, compounds were selected for single dose PK/PD studies assessing
mTOR pathway biomarker inhibition in tumor bearing mice. PCꢀ3 tumorꢀbearing mice were
administered with a single dose of test compound, dosed orally at 100 mg/kg, and plasma and
tumor samples were collected at various time points for analysis. Significant inhibition of mTOR
pathway markers pS6 and pAktS473 were observed, indicating inhibition of both mTORC1
(pS6) and mTORC2 (pAktS473). Most analogs tested demonstrated full biomarker inhibition in
the PCꢀ3 tumor model through 24 hours (Table 5). Over the course of our in vivo studies on, we
observed reasonable PK/PD correlations for compounds in this series, including those discussed
here. When a compound maintained exposure at sufficient levels to inhibit the target, as
estimated from the cellular IC50 values, biomarker suppression was observed. Lesser inhibition
of both pS6 and pAkt were observed as compound levels decreased in plasma and tumor. We
typically observed suppression of ≥80% and ≥60% for pS6 and pAkt, respectively, when tumor
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concentrations were >5 fold over the respective cellular IC50 values (Sup. Tables 2ꢀ4). Because
of the intricacies of the PK/PD studies, and to better correlate effects at low compound levels, we
conducted single dose biomarker studies using lower doses of 37 (1, 10 or 25 mg/kg). We
constructed a refined PK/PD model with the findings and the results have been published
elsewhere.10
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
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43
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45
46
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50
51
52
53
54
55
56
57
58
59
60
Table 4. In Vitro and In Vivo PK Properties.
Rat Oral PK Parametera
Rat S9
Met.
Rat IV
CL
AUC(0ꢀ∞)
(µMꢁhr)
Stabilityb (mL/min/kg)c
Analog
Cmax (µM)
F(%)
89 ± 2
~100
NTd
8.1 ± 0.9
NT
NCe
~100
NC
23
26
27
11
12
28
29
30
31
32
34
35
36
37
5.6 ± 2.4
3.3 ± 0.7
1.6 ± 0.53
1.5 ± 0.7
1.7 ± 0.4
29 ± 4.5
28 ± 3
9. 9 ± 1.8
11 ± 6
14 ± 2.1
NT
83 ± 3
95 ± 4
73 ± 3
16 ± 0.5
39 ± 1
14 ± 1
77 ± 2
55 ± 6
97 ± 6
87 ± 2
94 ± 5
81 ± 9
23.3 ± 1.9
9.0 ± 2.1
NT
~100
58 ± 18
NT
NT
NT
NT
NT
NC
0.70 ± 0.23 7.25 ± 2.25
NT
NT
NT
NC
NC
2.20 ± 1.12 9.23 ± 2.45
NT
2.77 ± 0.77
6.3 ± 0.9
2.9 ± 0.9
26.6 ± 7.5
90 ± 22
1.7 ± 0.5
70 ± 28
59 ± 8.4
4.9 ± 0.7
26 ± 0.7
adose 5 mg/kg in aqueous 0.5% CMC/0.25% Tweenꢀ80, mean , b(% remaining
at 60 min), cdose 2 mg/kg, dNT: not tested, eNC: IV study not available.
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Table 5. In Vitro Cellular Potency and In Vivo Single Dose Biomarker Inhibition.
5
6
7
Analog 23
26
27
11
12
31
35
36
37
8
9
a
0.65 0.39 0.2 0.24 0.24 0.23 0.53 0.34 0.03
Cellular pS6 IC50
2 h
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
NTc
82
88
86
88
83
NT
93
94
91
87
63
64
0
71
80
88
59
NT
80
64
0
80
54
52
70
87
90
85
85
NT
91
4 h
In Vivo pS6b
8 h
91
92
91
91
24 h
NT 13.2 NT
37.6
18.2 5.2
10 7.6 11.3 0.3
6
30.3 NT
6.4 12.9
NT
2 h
Fold Tumor
Conc./pS6
5.1 27.4 2.7
9.4 3.5
4
6.8
4.7
1
2.4 12.1 140.3
4 h
Cellular
5.5
4.5
2.6
3.7
7.7
9.3
31
8 h
IC50
39.3
24 h
a
0.61 0.32 0.13 0.11 0.09 0.16 0.33 0.35 0.01
Cellular pAkt IC50
NT
87
84
77
85
73
71
65
NT
86
81
80
74
63
67
28
82
63
78
69
NT
55
55
0
72
34
59
45
75
62
69
61
NT
80
2 h
4 h
In Vivo
pAktb
82
8 h
86
24 h
NT 16.0 NT 13.2 80.7 NT 10.2 12.6
NT
2 h
Fold Tumor
Conc./pAkt
40.0 6.3 42.2 5.9 10.7 9.8
19.4 6.4 14.5 7.6 14.8 6.8
10.6 9.3 17.3 0.6 12.1 1.4
3.9 11.7 421.0
4 h
Cellular
4.2
5.9
7.5
93.0
8 h
IC50
9.0 118.0
24 h
a µM, SEM for cellular data available in supplemental material, b% inhibition, cNT=not tested.
Based on both rat PK profiles and the singleꢀdose PK/PD studies, compounds 11, 12, 26,
36 and 37 were selected for evaluation in PCꢀ3 tumor bearing efficacy models. Tumor volumes
were determined prior to the initiation of treatment and were considered as the starting volumes.
When tumors reached approximately 125 mm3, mice were randomized and treated once daily
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(qd) or twice daily (bid) orally with vehicle or various doses of test compound, at a dose volume
of 5 mL/kg. The bid doses were administered with a 10 h separation between the morning and
evening doses. Animals were treated for 21 days and the final tumor volume reductions reported
here were measured following the final day of dosing. All analogs assessed showed doseꢀ and
scheduleꢀdependent inhibition of tumor growth in the PCꢀ3 model. The maximum observed
efficacy for each compound, at a tolerated dose, is shown in Table 6. Compound 11 required
twice daily dosing to achieve efficacy, a finding that is consistent with the minimal inhibition
observed at 24 hours with 11 in the biomarker study. The remaining compounds achieved
significant efficacy with once daily dosing. When dosed once daily at 50 mg/kg, analogs 12, 26
and 36 showed 59%, 84% and 90% tumor volume reductions (TVR= (vehicle ꢀ treated /
vehicle)x100%), respectively. Compound 37 achieved similar efficacy (87% TVR) at a lower
dose of 25 mg/kg. While the PK properties of 26 and 36 are similar to or better than those of 37,
the superior cellular potency of 37 translated to efficacy at lower doses.
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
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43
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48
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50
51
52
53
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55
56
57
58
59
60
Table 6. In Vivo Efficacy Studies in PCꢀ3 Xenograft.a
Maximum Efficacy with
Tolerated Dose
Dose (mg/kg)
100 bid
50 qdb
TVR (%)
72
59
84
90
87
11
12
26
36
37
50 qd
50 qd
25 qd
aAssay details published previously.10 bHighest dose tested.
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The data described herein, along with assessment in additional in vivo models, safety
studies and higher species PK informed the selection of 37, CCꢀ223, for clinical development.
CCꢀ223 demonstrates the excellent kinase selectivity achieved in this series. When screened
against a commercial panel of >240 kinases, other than mTOR kinase, only 3 were targets were
inhibited >80% at 10 µM. Followꢀup studies showed only two of these were inhibited with IC50
values below 1 ꢂM; Flt4 (0.651 ꢂM) and cFMS (0.028 ꢂM). The kinsase selectivity was
confirmed in a cellular assessment, where no kinase other than mTOR was identified when cells
were treated with 1 ꢂM of 37 and assayed for kinase activity in ActivX KiNavtiv™ profiling.10
When tested at concentrations up to 10 ꢂM, CCꢀ223 does not inhibit CYP enzymes and when
screened in a single point assay at 10 ꢂM against a Cerep receptor and enzyme panel no target
was inhibited >60%. CCꢀ223 was negative in AMES mutagenicity and in vitro micronucleus
tests and has a hERG (human etherꢀaꢀgoꢀgoꢀrelated gene) ion channel IC50 value of 33 ꢂM. The
expanded in vitro and in vivo characterization of the mTOR kinase activity of CCꢀ223 has been
reported.10
10
11
12
13
14
15
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17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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36
37
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Conclusion
In summary we have described the SAR and optimization of a series of 4,6ꢀ or 1,7ꢀ
disubstitutedꢀ3,4ꢀdihydropyrazino[2,3ꢀb]pyrazineꢀ2(1H)ꢀone based mTOR kinase inhibitors.
The series maintains mTOR kinase potency with exquisite kinase selectivity including the related
PI3Kα. Focused investigation in the C6/7ꢀ(6ꢀ(2ꢀhydroxypropanꢀ2ꢀyl)pyridinꢀ3ꢀyl)ꢀsubstituted
analogs allowed for the identification of a number of compounds with oral PK properties suitable
for in vivo studies. This work ultimately led to the identification of CCꢀ223, with superior
physicochemical and pharmacokinetic properties, excellent kinase selectivity, demonstrated
efficacy across multiple in vivo solid tumor models with oral dosing, in addition to favorable in
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vivo and in vitro safety profiles, suitable for clinical development. The results of the Phase I
clinical investigations with CCꢀ223 have been reported.11
7
8
9
Materials and Methods
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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35
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Chemistry 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 airꢀsensitive reactions
were carried out under a positive pressure of an inert nitrogen atmosphere. Reported yields are
1
unoptimized. H 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). Semiꢀ
preparative 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 (compounds 20, 21 and 32 were tested at 94%
purity). Compounds were analyzed for purity by one of two methods: (A) Gradient (0ꢀ75%
acetonitrile + 0.1% formic acid in water + 0.1% formic, over 7 min, followed by 75%
acetonitrile + 0.1% formic acid for 2 min); Flow Rate 1 mL/min, Column Phenomenex Geminiꢀ
NX 5u C18 110A (50x4.60mm); (B) Gradient (0ꢀ75% 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 GeminiꢀNX 5u C18 110A (250x4.60mm). Melting points
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were determined on either a manual Electrothermal MelꢀTemp® or Stanford Research Systems’
OptiMelt System and are uncorrected. Elemental analysis was performed at Robertson Microlit
Laboratories, Ledgewood, New Jersey.
10
11
12
13
14
15
16
17
18
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21
22
23
24
25
26
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56
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58
59
60
Synthetic procedures for all intermediates 4aꢀi, 5a, 7aꢀi and 8a are included in the
supplemental material. All assay procedures have been published previously.10
General Procedure A (9, 10, 13ꢀTHP, 14ꢀTHP, 15ꢀTHP, 16ꢀTHP). Substrate, boronate ester
and [1,1'ꢀbis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with 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 15ꢀ30 min or on an oil bath at 130ꢀ150 °C for 1ꢀ4 h. The cooled reaction solutions
were filtered through Celite and the filter cake was washed with ethyl acetate. The filtrate and
ethyl acetate wash were combined and solvent was removed under reduced pressure. The
resulting products were purified using silica gel chromatography or reverse phase HPLC.
General Procedure B (13ꢀ19). 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 45ꢀ110 °C for
0.2ꢀ12 h. The solutions were concentrated under reduced pressure and products were purified
using reverse phase HPLC.
General Procedure C (11, 12, 17ꢀTHP, 18ꢀTHP, 19ꢀTHP, 20ꢀ37). Aryl bromide, stannane and
palladium catalyst were combined in triethylamine and/or DMF. The solution was then heated at
100ꢀ140 °C for 0.3ꢀ4 h. The reaction mixtures were partitioned between organic solvent (ethyl
acetate or methylene chloride) and water. The organic solvents were dried and concentrated
under reduced pressure. Products were purified using silica gel chromatography or reverse phase
HPLC.
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6ꢀ(4ꢀ(2ꢀHydroxypropanꢀ2ꢀyl)phenyl)ꢀ4ꢀ(2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ethyl)ꢀ3,4ꢀ
5
6
7
8
dihydropyrazino[2,3ꢀb]pyrazinꢀ2(1H)ꢀone (9). 4a (0.250 g, 0.733 mmol) and 2ꢀ(4ꢀ(4,4,5,5ꢀ
tetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenyl)propanꢀ2ꢀol (0.192 g, 0.733 mmol) and [1,1'ꢀ
bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0.030g,
0.037 mmol) were reacted according to Procedure A to give 9 (0.074 g, 0.19 mmol, 25% yield,
HPLC purity (A) >99%). 1H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 7.98 (s, 1H), 7.89 (d,
J=8.39 Hz, 2H), 7.53 (d, J=8.39 Hz, 1H), 5.04 (s, 1H), 4.16 (s,1H), 3.82 (dd, J=11.1, 2.39 Hz,
2H), 3.61 (t, J=7.59, Hz 2H), 3.25 (t, J=9.59 Hz, 3H), 1.70 (s, 1H), 1.66 (s, 1H), 1.58 (m, 3H),
1.44 (s, 6H), 1.25 (m, 2H); MS (ESI) m/z 397.2 [M+1]+; mp 210ꢀ212 °C.
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
7ꢀ(4ꢀ(2ꢀHydroxypropanꢀ2ꢀyl)phenyl)ꢀ1ꢀ(2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ethyl)ꢀ3,4ꢀ
dihydropyrazino[2,3ꢀb]pyrazinꢀ2(1H)ꢀone (10). 7a (500.0 mg, 1.465 mmol), 2ꢀ(4ꢀ(4,4,5,5ꢀ
tetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenyl)propanꢀ2ꢀol (461 mg, 1.758 mmol) and [1,1'ꢀ
bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (53.6 mg,
0.073 mmol) were reacted according to Procedure A to provide the title compound (94.7 mg,
1
0.239 mmol, 16.3% yield, HPLC purity (A) >99%). H NMR (400 MHz, DMSOꢀd6) δ 8.19 (s, 1
H), 7.83 ꢀ 7.87 (m, 2 H), 7.50 ꢀ 7.54 (m, 3 H), 5.03 (s, 1 H), 4.18 (d, J=1.6 Hz, 2 H), 4.07 ꢀ 4.13
(m, 3 H), 3.83 (dd, J=10.3, 3.3 Hz, 3 H), 3.22 ꢀ 3.31 (m, 2 H), 1.69 (d, J=12.1 Hz, 3 H), 1.53 ꢀ
1.62 (m, 4 H), 1.44 (s, 6 H), 1.16 ꢀ 1.29 (m, 2 H); MS (ESI) m/z 397.2 [M+1]+; mp 198ꢀ199 °C.
6ꢀ(6ꢀ(2ꢀHydroxypropanꢀ2ꢀyl)pyridinꢀ3ꢀyl)ꢀ4ꢀ(2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ethyl)ꢀ3,4ꢀ
dihydropyrazino[2,3ꢀb]pyrazinꢀ2(1H)ꢀone (11).
(trimethylstannyl)pyridinꢀ2ꢀyl)propanꢀ2ꢀol (9.67
4a (10.0 g, 29.3 mmol), 2ꢀ(5ꢀ
g, 32.2 mmol) and [1,1'ꢀ
bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (4.79 g,
5.86 mmol) were reacted according to Procedure C to give the title compound (3.95 g, 9.94
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mmol, 34 % yield, HPLC purity (A) >98%). 1H NMR (400 MHz, DMSOꢀd6) δ 11.32 (s, 1 H),
9.06 (d, J=1.95 Hz, 1 H), 8.27 (dd, J=8.59, 2.34 Hz, 1 H), 8.06 (s, 1 H), 7.72 (d, J=8.20 Hz, 1 H),
5.27 (s, 1 H), 4.19 (s, 2 H), 3.81 (dd, J=11.52, 2.54 Hz, 2 H), 3.62 (t, J=7.03 Hz, 2 H), 3.25 (td,
J=11.62, 1.76 Hz, 2 H), 1.68 (d, J=12.49 Hz, 2 H), 1.51 ꢀ 1.63 (m, 3 H), 1.46 (s, 6 H), 1.16 ꢀ 1.29
(m, 2 H); MS (ESI) m/z 398.1 [M+1]+; mp 239ꢀ241 °C; Anal. (C21H27N5O3ꢀ0.4H2O) Calc. C:
62.34, H: 6.92, N: 17.31; Found C: 62.47, H: 7.07, N: 17.45; KF = 1.80%.
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7ꢀ(6ꢀ(2ꢀHydroxypropanꢀ2ꢀyl)pyridinꢀ3ꢀyl)ꢀ1ꢀ(2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ethyl)ꢀ3,4ꢀ
dihydropyrazino[2,3ꢀb]pyrazinꢀ2(1H)ꢀone (12). 2ꢀ(5ꢀ(Trimethylstannyl)pyridinꢀ2ꢀyl)propanꢀ
2ꢀol
(2.80
g,
9.35
mmol),
7a
(2.90
g,
8.50
mmol)
and
[1,1'ꢀ
bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0.69 g,
0.85 mmol) were reacted according to Procedure C to give the title compound (2.15 g, 2.60
mmol, 68% yield, HPLC purity (A) >98%). 1H NMR (400 MHz, DMSOꢀd6) δ 9.03 (d, J=2.3
Hz, 1 H), 8.27 (s, 1 H), 8.23 (dd, J=8.2, 2.3 Hz, 1 H), 7.71 (d, J=8.2 Hz, 1 H), 7.68 (s, 1 H), 5.25
(s, 1 H), 4.20 (s, 2 H), 4.10 (t, J=6.8 Hz, 2 H), 3.82 (dd, J=11.3, 2.7 Hz, 2 H), 3.26 (t, J=10.7 Hz,
2 H), 1.69 (d, J=12.5 Hz, 2 H), 1.57 (t, J=5.3 Hz, 3 H), 1.46 (s, 6 H), 1.09 ꢀ 1.31 (m, 2 H); MS
(ESI) m/z 398.4 [M+1]+; mp 158ꢀ160 °C; Anal. (C21H27N5O3ꢀ0.1H2O) Calc. C: 62.46, H: 6.69,
N: 17.43; Found C: 62.81, H: 6.69, N: 17.43; KF = 0.48%.
6ꢀ(4ꢀ(4Hꢀ1,2,4ꢀTriazolꢀ3ꢀyl)phenyl)ꢀ4ꢀ(2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ethyl)ꢀ3,4ꢀ
dihydropyrazino[2,3ꢀb]pyrazinꢀ2(1H)ꢀone (13). 4a (0.500 g, 1.46 mmol), 1ꢀ(tetrahydroꢀ2Hꢀ
pyranꢀ2ꢀyl)ꢀ3ꢀ(4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenyl)ꢀ1Hꢀ1,2,4ꢀtriazole (0.677
g, 1.905 mmol) and [1,1'ꢀbis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with
dichloromethane (0.060 g, 0.073 mmol) were reacted according to Procedure A to give 13ꢀTHP
(0.347 g, 0.710 mmol, 48 % yield). MS (ESI) m/z 406.2 [M+1]+. Procedure B gave 13 (0.135 g,
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0.330 mmol, 22 % yield, HPLC purity (A) >97%). H NMR (400 MHz, DMSO-d6) δ 11.31 (s,
5
1H), 8.64 (s, 1H), 8.08 (s, 5H), 4.19 (s, 2H), 3.82 (dd, J= 2.39, 11.19 Hz, 2H), 3.64 (t, J=7.19,
2H), 3.26 (t, J=9.99 Hz, 2H), 1.70 (d, J=13.99 Hz, 2H), 1.59 (m, 3H), 1.25 (m, 2H); MS (ESI)
m/z 406.2 [M+1]+; mp 301ꢀ303 °C.
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7ꢀ(4ꢀ(1Hꢀ1,2,4ꢀTriazolꢀ5ꢀyl)phenyl)ꢀ1ꢀ(2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ethyl)ꢀ3,4ꢀ
dihydropyrazino[2,3ꢀb]pyrazinꢀ2(1H)ꢀone (14). 7a (0.5 g, 1.465 mmol), 1ꢀ(tetrahydroꢀ2Hꢀ
pyranꢀ2ꢀyl)ꢀ3ꢀ(4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenyl)ꢀ1Hꢀ1,2,4ꢀtriazole (0.573
g, 1.612 mmol) and [1,1'ꢀbis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with
dichloromethane (0.060 g, 0.073 mmol) were reacted according to Procedure A followed by
Procedure B to give the title compound (0.050 g, 0.123 mmol, 8.4 % yield over two steps, HPLC
1
purity (A) >99%). H NMR (400 MHz, DMSOꢀd6) δ 8.64 (br. s., 1 H), 8.29 (br. s., 4 H), 8.07
(br. s., 4 H), 7.65 (br. s., 1 H), 4.21 (s, 2 H), 4.12 (t, J=7.0 Hz, 2 H), 3.84 (dd, J=11.3, 2.7 Hz, 2
H), 3.28 (td, J=11.7, 2.0 Hz, 2 H), 1.71 (d, J=12.9 Hz, 2 H), 1.59 (t, J=5.7 Hz, 3 H), 1.13 ꢀ 1.32
(m, 2 H); MS (ESI) m/z 406.0 [M+1]+; mp 237ꢀ239 °C.
6ꢀ(2ꢀMethylꢀ1ꢀ(1Hꢀ1,2,4ꢀtriazolꢀ3ꢀyl)phenyl)ꢀ4ꢀ(2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ethyl)ꢀ3,4ꢀ
dihydropyrazino[2,3ꢀb]pyrazinꢀ2(1H)ꢀone (15). 4a (0.212 g, 0.621 mmol), 3ꢀ(3ꢀmethylꢀ4ꢀ
(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenyl)ꢀ1ꢀ(tetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)ꢀ1Hꢀ1,2,4ꢀ
triazole (0.250 g, 0.677 mmol) and [1,1'ꢀbis(diphenylphosphino)ferrocene]dichloropalladium(II)
complex with dichloromethane (0.023 g, 0.028 mmol) were reacted according to Procedure A to
give (15ꢀTHP) (0.21 g, 0.42 mmol, 74 % yield). MS (ESI) m/z 504.5 [M+1]+. Procedure B gave
15 (0.066 g, 0.16 mmol, 35 % yield, HPLC purity (A) >97%). 1H NMR (400 MHz, DMSOꢀd6)
δ 11.28 (s, 4 H), 8.64 (s, 2 H), 7.85 ꢀ 7.97 (m, 7 H), 7.57 ꢀ 7.64 (m, 3 H), 7.49 (d, J=7.42 Hz, 2
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H), 4.18 (s, 8 H), 3.77 (dd, J=12.49, 5.08 Hz, 8 H), 3.54 (t, J=7.22 Hz, 7 H), 3.22 (s, 8 H), 1.46 ꢀ
1.67 (m, 18 H), 1.09 ꢀ 1.25 (m, 7 H); MS (ESI) m/z 420.3 [M+1]+; mp 262ꢀ265 °C.
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8
9
7ꢀ(2ꢀMethylꢀ4ꢀ(1Hꢀ1,2,4ꢀtriazolꢀ3ꢀyl)phenyl)ꢀ1ꢀ(2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ethyl)ꢀ3,4ꢀ
dihydropyrazino[2,3ꢀb]pyrazinꢀ2(1H)ꢀone hydrochloride (16). 3ꢀ(3ꢀMethylꢀ4ꢀ(4,4,5,5ꢀ
tetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenyl)ꢀ4ꢀ(tetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)ꢀ4Hꢀ1,2,4ꢀtriazole
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(200
mg,
0.542
mmol),
7a
(185
mg,
0.542
mmol)
and
[1,1'ꢀ
bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (22.12
mg, 0.027 mmol) were reacted according to Procedure A followed by Procedure B to give the
title compound (0.11 g, 0.26 mmol, 44 % yield over two steps, HPLC purity (A) >96%). 1H
NMR (400 MHz, DMSOꢀd6) δ 8.56 (br. s., 1 H), 7.97 (s, 1 H), 7.91 (d, J=7.8 Hz, 1 H), 7.84 (s, 1
H), 7.55 (d, J=7.8 Hz, 1 H), 4.21 (s, 2 H), 3.95 ꢀ 4.12 (m, 2 H), 3.78 (dd, J=10.9, 2.7 Hz, 2 H),
3.22 (t, J=10.7 Hz, 2 H), 2.49 (br. s., 3 H), 1.62 (d, J=12.5 Hz, 2 H), 1.44 ꢀ 1.58 (m, 3 H), 1.05 ꢀ
1.24 (m, 2 H); MS (ESI) m/z 420.4 [M+1]+; mp 150 °C (dec).
6ꢀ(6ꢀ(4Hꢀ1,2,4ꢀTriazolꢀ3ꢀyl)pyridinꢀ3ꢀyl)ꢀ4ꢀ(2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ethyl)ꢀ3,4ꢀ
dihydropyrazino[2,3ꢀb]pyrazinꢀ2(1H)ꢀone (17). 5ꢀBromoꢀ2ꢀ(1ꢀ(tetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)ꢀ
1Hꢀ1,2,4ꢀtriazolꢀ3ꢀyl)pyridine (0.305 g, 0.987 mmol), 5a (0.419 g, 0.987 mmol) and
tris(dibenzylideneacetone)dipalladium(0) (0.090 g, 0.099 mmol) and triꢀoꢀtolylphosphine (0.060
g, 0.197 mmol) were reacted according to Procedure C followed by Procedure B to give the title
1
compound (0.112 g, 0.276 mmol, 28 % yield over two steps, HPLC purity (A) >99%). H NMR
(400 MHz, DMSOꢀd6) δ 11.41 (br. s., 1H), 9.28 (d, J = 1.56 Hz, 1H), 8.49 (dd, J = 1.95, 8.20
Hz, 1H), 8.29 (br. s., 1H), 8.20 (s, 1H), 8.16 (d, J = 8.59 Hz, 1H), 4.22 (s, 2H), 3.83 (dd, J =
2.93, 11.13 Hz, 2H), 3.64 (t, J = 7.03 Hz, 2H), 3.21 ꢀ 3.31 (m, 2H), 1.69 (d, J = 12.49 Hz, 2H),
1.49 ꢀ 1.65 (m, 3H), 1.16 ꢀ 1.31 (m, 2H); MS (ESI) m/z 407.3 [M+1]+.
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6ꢀ(2ꢀMethylꢀ6ꢀ(4Hꢀ1,2,4ꢀtriazolꢀ3ꢀyl)pyridinꢀ3ꢀyl)ꢀ4ꢀ(2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ethyl)ꢀ
3,4ꢀdihydropyrazino[2,3ꢀb]pyrazinꢀ2(1H)ꢀone (18). 3ꢀBromoꢀ2ꢀmethylꢀ6ꢀ(1ꢀ(tetrahydroꢀ2Hꢀ
pyranꢀ2ꢀyl)ꢀ1Hꢀ1,2,4ꢀtriazolꢀ3ꢀyl)pyridine (0.87 g, 2.69 mmol), 5a (1.144 g, 2.69 mmol),
tris(dibenzylideneacetone)dipalladium(0) (0.271 g, 0.296 mmol) and triꢀoꢀtolylphosphine (0.180
g, 0.592 mmol) were reacted according to Procedure C to give 18ꢀTHP (1.0 g, 1.98 mmol, 73.6
% yield). MS (ESI) m/z 505.5 [M+1]+. Procedure B gave 18 (0.326 g, 0.78 mmol, 39 % yield,
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60
1
HPLC purity (A) >95%). H NMR (400 MHz, DMSOꢀd6) δ 11.36 (s, 1H), 7.98 (s, 2H), 7.69 (s,
1H), 4.21 (s, 2H), 3.78 (dd, J = 2.93, 11.13 Hz, 2H), 3.49 ꢀ 3.61 (m, 2H), 3.22 (td, J = 1.76,
11.62 Hz, 2H), 2.69 (s, 3H), 1.44 ꢀ 1.71 (m, 5H), 1.08 ꢀ 1.27 (m, 2H); MS (ESI) m/z 421.1
[M+1]+; mp 200ꢀ201 °C.
7ꢀ(2ꢀMethylꢀ6ꢀ(1Hꢀ1,2,4ꢀtriazolꢀ3ꢀyl)pyridinꢀ3ꢀyl)ꢀ1ꢀ(2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ethyl)ꢀ
3,4ꢀdihydropyrazino[2,3ꢀb]pyrazinꢀ2(1H)ꢀone (19). 3ꢀBromoꢀ2ꢀmethylꢀ6ꢀ(1ꢀ(tetrahydroꢀ2Hꢀ
pyranꢀ2ꢀyl)ꢀ1Hꢀ1,2,4ꢀtriazolꢀ3ꢀyl)pyridine (0.38 g, 1.18 mmol), 8a (0.5 g, 1.18 mmol) and [1,1'ꢀ
bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0.015 g,
0.024 mmol) were reacted according to Procedure C to give 19ꢀTHP (0.33 g, 0.65 mmol, 55 %
yield). MS (ESI) m/z 505.5 [M+1]+. Procedure B gave 19 (0.51 g, 1.19 mmol, 86 % yield,
1
HPLC purity (B) >99%). H NMR (400 MHz, DMSOꢀd6) δ 8.09 (s, 1H), 7.96 ꢀ 8.04 (m, 2H),
7.89 ꢀ 7.94 (m, 1H), 7.66 ꢀ 7.77 (m, 1H), 4.22 (s, 2H), 4.04 (t, J = 7.61 Hz, 2H), 3.78 (dd, J =
2.93, 10.35 Hz, 2H), 3.17 ꢀ 3.28 (m, 2H), 2.62 ꢀ 2.77 (m, 3H), 1.63 (d, J = 14.84 Hz, 2H), 1.54
(t, J = 5.86 Hz, 3H), 1.10 ꢀ 1.24 (m, 2H); MS (ESI) m/z 421.1 [M+1]+; mp 168ꢀ170 °C; Anal.
(C21H24N8O2ꢀ0.4 H2O) Calc. C: 58.98, H: 5.84, N: 26.20; Found C: 58.88, H: 5.83, N: 25.98, KF
= 1.72%.
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6ꢀ(1HꢀIndolꢀ5ꢀyl)ꢀ4ꢀ(2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ethyl)ꢀ3,4ꢀdihydropyrazino[2,3ꢀ
b]pyrazinꢀ2(1H)ꢀone (20). 5a (0.700 g, 1.647 mmol), 5ꢀbromoꢀ1Hꢀindole (0.339 g, 1.729
mmol) and [1,1'ꢀbis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with
dichloromethane (0.120 g, 0.165 mmol) were reacted according to Procedure C to give the title
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11
12
13
14
15
16
17
18
19
20
21
22
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60
1
compound (0.092 g, 0.244 mmol, 14.80 % yield, HPLC purity (A) 94%). H NMR (400 MHz,
DMSOꢀd6) δ 11.17 (s, 2H), 8.18 (s, 1H), 8.00 (s, 1H), 7.74 (dd, J = 1.95, 8.59 Hz, 1H), 7.44 (d, J
= 8.59 Hz, 1H), 7.37 (t, J = 2.54 Hz, 1H), 6.47 (t, J = 1.95 Hz, 1H), 4.16 (s, 2H), 3.83 (dd, J =
2.73, 11.32 Hz, 2H), 3.65 (t, J = 7.22 Hz, 2H), 3.27 (td, J = 1.76, 11.62 Hz, 2H), 1.72 (d, J =
12.89 Hz, 2H), 1.54 ꢀ 1.65 (m, 3H), 1.20 ꢀ 1.32 (m, 2H); MS (ESI) m/z 378.1 [M+1]+; mp 272ꢀ
274 °C.
7ꢀ(1HꢀIndolꢀ5ꢀyl)ꢀ1ꢀ(2ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ethyl)ꢀ3,4ꢀdihydropyrazino[2,3ꢀ
b]pyrazinꢀ2(1H)ꢀone (21). 8a (0.750 g, 1.764 mmol), 5ꢀbromoꢀ1Hꢀindole (0.363 g, 1.852
mmol), tris(dibenzylideneacetone)dipalladium(0)
(0.162 g, 0.176 mmol) and triꢀoꢀ
tolylphosphine (0.107 g, 0.353 mmol) were reacted according to Procedure C to give the title
compound (0.222 g, 0.588 mmol, 33.3 % yield, HPLC purity (A) 94%). 1H NMR (400 MHz,
DMSOꢀd6) δ 11.15 (br. s., 1H), 8.19 (s, 1H), 8.13 (s, 1H), 7.70 (dd, J = 1.56, 8.59 Hz, 1H), 7.44
(d, J = 8.59 Hz, 1H), 7.36 (t, J = 2.73 Hz, 1H), 7.34 (s, 1H), 6.45 (br. s., 1H), 4.10 ꢀ 4.18 (m,
4H), 3.83 (dd, J = 2.54, 11.52 Hz, 2H), 3.28 (td, J = 1.56, 11.52 Hz, 2H), 1.72 (d, J = 12.89 Hz,
2H), 1.53 ꢀ 1.64 (m, 3H), 1.20 ꢀ 1.31 (m, 2H); MS (ESI) m/z 378.2 [M+1]+; mp = 268ꢀ270 °C.
6ꢀ(6ꢀ(2ꢀHydroxypropanꢀ2ꢀyl)pyridinꢀ3ꢀyl)ꢀ4ꢀ(2ꢀmethoxyethyl)ꢀ3,4ꢀdihydropyrazino[2,3ꢀ
b]pyrazinꢀ2(1H)ꢀone (22). 4b (0.251, 0.836 mmol), 2ꢀ(5ꢀ(trimethylstannyl)pyridinꢀ2ꢀyl)propanꢀ
2ꢀol (0.251 g, 0.836 mmol) and [1,1'ꢀbis(diphenylphosphino)ferrocene]dichloropalladium(II)
complex with dichloromethane (0.114 g, 0.139 mmol) were reacted according to Procedure C to
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give the title compound (0.055 g, 0.16 mmol, 23 % yield, HPLC purity (A) 99%). H NMR (400
MHz, DMSOꢀd6) δ 11.34 (s, 1 H), 9.06 (d, J=1.56 Hz, 1 H), 8.28 (dd, J=8.39, 2.15 Hz, 1 H),
8.07 (s, 1 H), 7.71 (d, J=8.98 Hz, 1 H), 5.27 (s, 1 H), 4.26 (s, 2 H), 3.77 (m, J=5.08 Hz, 2 H),
3.66 (t, J=5.86 Hz, 2 H), 3.29 (s, 3 H), 1.46 (s, 6 H); MS (ESI) m/z 344.1 [M+1]+; mp 170ꢀ172
°C.
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19
20
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7ꢀ(6ꢀ(2ꢀHydroxypropanꢀ2ꢀyl)pyridinꢀ3ꢀyl)ꢀ1ꢀ(2ꢀmethoxyethyl)ꢀ3,4ꢀdihydropyrazino[2,3ꢀ
b]pyrazinꢀ2(1H)ꢀone (23). 7b (230.0 mg, 0.801 mmol), 2ꢀ(5ꢀ(trimethylstannyl)pyridinꢀ2ꢀ
yl)propanꢀ2ꢀol (240.0 mg, 0.801 mmol) and dichlorobis(triphenylphosphine)palladium(II) (117.0
mg, 0.160 mmol) were reacted according to Procedure C to give the title compound (104.0 mg,
1
0.303 mmol, 37.8 % yield, HPLC purity (A) 96%). H NMR (400 MHz, DMSOꢀd6) δ 9.02 (d,
J=1.6 Hz, 1 H), 8.27 (s, 1 H), 8.24 (dd, J=8.6, 2.3 Hz, 1 H), 7.71 (d, J=0.8 Hz, 1 H), 7.69 (s, 1
H), 5.25 (s, 1 H), 4.28 (t, J=6.2 Hz, 2 H), 4.20 (d, 2 H), 3.60 (t, J=6.2 Hz, 2 H), 3.26 (s, 3 H),
1.46 (s, 6 H); MS (ESI) m/z 344.3 [M+1]+; mp 160ꢀ161 °C.
6ꢀ(6ꢀ(2ꢀHydroxypropanꢀ2ꢀyl)pyridinꢀ3ꢀyl)ꢀ4ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ꢀ3,4ꢀ
dihydropyrazino[2,3ꢀb]pyrazinꢀ2(1H)ꢀone (24).
(trimethylstannyl)pyridinꢀ2ꢀyl)propanꢀ2ꢀol (0.389
4c (0.338 g, 1.079 mmol), 2ꢀ(5ꢀ
g, 1.295 mmol) and [1,1'ꢀ
bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0.176 g,
0.216 mmol) were reacted according to Procedure C to give the title compound (0.085 g, 0.230
1
mmol, 21.3 % yield, HPLC purity (A) 99%). H NMR (400 MHz, DMSOꢀd6) δ 11.34 (s, 1H),
9.08 (dd, J = 0.73, 2.29 Hz, 1H), 8.30 (dd, J = 2.34, 8.30 Hz, 1H), 8.09 (s, 1H), 7.72 (dd, J =
0.76, 8.32 Hz, 1H), 5.27 (s, 1H), 4.64 ꢀ 4.76 (m, 1H), 4.12 (s, 2H), 3.97 (dd, 2H), 3.47 ꢀ 3.55 (m,
1H), 1.79 ꢀ 1.94 (m, 2H), 1.59 ꢀ 1.71 (m, 2H), 1.46 (s, 6H); MS (ESI) m/z 370.3 [M+1]+; mp
150ꢀ151 °C.
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Journal of Medicinal Chemistry
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7ꢀ(6ꢀ(2ꢀHydroxypropanꢀ2ꢀyl)pyridinꢀ3ꢀyl)ꢀ1ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ꢀ3,4ꢀ
5
6
dihydropyrazino[2,3ꢀb]pyrazinꢀ2(1H)ꢀone (25). 7c (250.0 mg, 0.798 mmol), 2ꢀ(5ꢀ
7
8
9
(trimethylstannyl)pyridinꢀ2ꢀyl)propanꢀ2ꢀol
(239
mg,
0.798
mmol)
and
[1,1'ꢀ
10
11
12
13
14
15
16
17
18
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59
60
bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (58.4
mg, 0.080 mmol) were reacted according to Procedure C to give the title compound (42.3 mg,
1
0.115 mmol, 14.34 % yield, HPLC purity (A) >99%). H NMR (400 MHz, DMSOꢀd6) δ 9.05 (d,
J=2.3 Hz, 1 H), 8.29 (s, 1 H), 8.26 (dd, J=8.2, 2.3 Hz, 1 H), 7.71 (d, J=8.6 Hz, 1 H), 7.63 (s, 1
H), 5.26 (br. s., 1 H), 5.10 ꢀ 5.20 (m, 1 H), 4.15 (d, J=1.6 Hz, 2 H), 4.00 (dd, J=10.9, 4.3 Hz, 2
H), 3.40 ꢀ 3.49 (m, 2 H), 2.80 (qd, J=12.4, 4.3 Hz, 2 H), 1.57 (dd, J=12.1, 1.6 Hz, 2 H), 1.47 (s, 6
H); MS (ESI) m/z 370.2 [M+1]+.
6ꢀ(6ꢀ(2ꢀHydroxypropanꢀ2ꢀyl)pyridinꢀ3ꢀyl)ꢀ4ꢀ((tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)methyl)ꢀ3,4ꢀ
dihydropyrazino[2,3ꢀb]pyrazinꢀ2(1H)ꢀone (CC214ꢀ2, 26). 4d (35.98 g, 110 mmol), 2ꢀ(5ꢀ
(trimethylstannyl)pyridinꢀ2ꢀyl)propanꢀ2ꢀol
(33.0
g,
110
mmol)
and
[1,1'ꢀ
bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (8.05 g,
11.00 mmol) were reacted according to Procedure C to give the title compound (15.08 g, 39.3
mmol, 35.8% yield, HPLC purity (A) >99%). Combined batches were recrystallized from
1
ethanol. H NMR (400 MHz, DMSOꢀd6) δ 11.32 (s, 1 H), 9.07 (d, J=1.56 Hz, 1 H), 8.29 (dd,
J=8.59, 2.34 Hz, 1 H), 8.05 (s, 1 H), 7.72 (d, J=8.20 Hz, 1 H), 5.26 (s, 1 H), 4.21 (s, 2 H), 3.83
(d, J=2.73 Hz, 2 H), 3.51 (d, J=7.42 Hz, 2 H), 3.27 (t, J=11.32 Hz, 2 H), 2.09 (br. s., 1 H), 1.61
(d, J=11.3 Hz, 2 H), 1.46 (s, 6 H), 1.24 ꢀ 1.38 (m, 2 H); MS (ESI) m/z 384.2 [M+1]+; mp 268 ꢀ
269 °C; Anal. (C20H25N5O3) Calc. C: 62.65, H: 6.57, N: 18.26; Found C: 62.25, H: 6.55, N:
18.19.
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