Identification of a Potent, Selective, and Efficacious Phosphatidylinositol 3-Kinase í (PI3Kí) Inhibitor for the Treatment of Immunological Disorders

Article abstract of DOI:10.1021/acs.jmedchem.7b00618

PI3Kí plays an important role controlling immune cell function and has therefore been identified as a potential target for the treatment of immunological disorders. This article highlights our work toward the identification of a potent, selective, and efficacious PI3Kí inhibitor. Through careful SAR, the successful replacement of a polar pyrazole group by a simple chloro or trifluoromethyl group led to improved Caco-2 permeability, reduced Caco-2 efflux, reduced hERG PC activity, and increased selectivity profile while maintaining potency in the CD69 hWB assay. The optimization of the aryl substitution then identified a 4′-CN group that improved the human/rodent correlation in microsomal metabolic stability. Our lead molecule is very potent in PK/PD assays and highly efficacious in a mouse collagen-induced arthritis model.

Full text of DOI:10.1021/acs.jmedchem.7b00618

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
Identification of a potent, selective, and efficacious phosphatidylinositol
3-kinase # (PI3K#) inhibitor for the treatment of immunological disorders
Qingjie Liu, Qing Shi, David Marcoux, Douglas G. Batt, Lyndon Cornelius, Lan-Ying Qin, Zheming
Ruan, James Neels, Myra Beaudoin Bertrand, Anurag S. Srivastava, Ling Li, Robert Joseph Cherney,
Hua Gong, Scott H. Watterson, Carolyn Weigelt, Kathleen M. Gillooly, Kim W. McIntyre, Jenny
H. Xie, Mary T. Obermeier, Aberra Fura, bogdan sleczka, Kevin Stefanski, R. Marcus Fancher,
Shweta Padmanabhan, Thatipamula RP, Ipsit Kundu, Kallem Rajareddy, Rodney Smith, James K.
Hennan, Dezhi Xing, Jingsong Fan, Paul C. Levesque, Qian Ruan, Sidney Pitt, Rosemary Zhang,
Donna Pedicord, Jie Pan, Melissa Yarde, Hao Lu, Jonathan Lippy, christine goldstine, Stacey Skala,
Richard A. Rampulla, Arvind Mathur, Anuradha Gupta, Pirama Nayagam Arunachalam, John S.
Sack, Jodi K. Muckelbauer, Mary Ellen Cvijic, Luisa M. Salter-Cid, Rajeev S Bhide, Michael A Poss,
John Hynes, Percy H Carter, John E. Macor, stefan ruepp, Gary L. Schieven, and Joseph A Tino
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 25 May 2017
Downloaded from http://pubs.acs.org on May 25, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Hennan, James; Bristol-Myers Squibb Co, Discovery
Toxicology/Cardiovascular Safety
Xing, Dezhi; Bristol-Myers Squibb
Fan, Jingsong; Bristol-Myers Squibb
Levesque, Paul; Bristol-Myers Squibb,
Ruan, Qian; Bristol-Myers Squibb Com., Biotransformation
Pitt, Sidney; Bristol-Myers Squibb Company
Zhang, Rosemary; Bristol-Myers Squibb Company
Pedicord, Donna; Bristol-Myers Squibb Company
Pan, Jie; Bristol-Myers Squibb Company
Yarde, Melissa; Bristol-Myers Squibb Pharmaceutical Research and
Development, LEAD EVALUATION
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
Lu, Hao; Bristol-Myers Squibb Company
Lippy, Jonathan; Bristol-Myers Squibb Company
goldstine, christine; Bristol-Myers Squibb Company
Skala, Stacey; Bristol-Myers Squibb Pharmaceutical Research and
Development, Discovery Biology
Rampulla, Richard; Bristol-Myers Squibb Pharmaceutical Research and
Development, Discovery Chemistry
Mathur, Arvind; Bristol-Myers Squibb Company
Gupta, Anuradha; Syngene International Pvt Ltd, Biocon BMS Research
Center
Arunachalam, Pirama Nayagam; Syngene, Synthetic Chemistry
Sack, John; Bristol-Myers Squibb, Dept. of Macromolecular Crystallography
Muckelbauer, Jodi; Bristol-Myers Squibb Pharmaceutical Research and
Development, Molecular Structure and Design, Molecular Discovery
Technologies
Cvijic, Mary; Bristol-Myers Squibb Pharmaceutical Research and
Development, Leads Discovery & Optimization
Salter-Cid, Luisa; Bristol-Myers Squibb Pharmaceutical Research and
Development, Discovery Biology
Bhide, Rajeev; Bristol-Myers Squibb Co., Chemistry
Poss, Michael; Bristol-Myers Squibb, Early Discovery Chemistry
Hynes, John; Bristol-Myers Squibb, Discovery Chemistry
Carter, Percy; BMS, R&D
Macor, John; Sanofi,
ruepp, stefan; Bristol-Myers Squibb Company
Schieven, Gary; Bristol-Myers Squibb,, Immunology biology
Tino, Joseph; Bristol-Myers Squibb Pharmaceutical Research and
Development, Immunoscience Chemistry
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 61
1
2
3
4
5
6
7
8
9
Identification of a potent, selective, and efficacious
phosphatidylinositol 3ꢀkinase δ (PI3Kδ) inhibitor for
the treatment of immunological disorders
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
Qingjie Liu,a Qing Shi,a David Marcoux*,a Douglas G. Batt,a Lyndon Cornelius,a Lan-Ying Qin,a
Zheming Ruan,a James Neels,a Myra Beaudoin-Bertrand,a Anurag S. Srivastava,a Ling Li,a
Robert J. Cherney,a Hua Gong,a Scott H. Watterson,a Carolyn Weigelt,a Kathleen M. Gillooly,a
Kim W. McIntyre,a Jenny H. Xie,a Mary T. Obermeier,a Aberra Fura,a Bogdan Sleczka,a Kevin
Stefanski,a R. M. Fancher,a Shweta Padmanabhan,b Thatipamula RP,b Ipsit Kundu,b Kallem
Rajareddy,c Rodney Smith,a James K. Hennan,a Dezhi Xing,a Jingsong Fan,a Paul C. Levesque,a
Qian Ruan,a Sidney Pitt,a Rosemary Zhang,a Donna Pedicord,a Jie Pan,a Melissa Yarde,a Hao
Lu,a Jonathan Lippy,a Christine Goldstine,a Stacey Skala,a Richard A. Rampulla,a Arvind
Mathur,a Anuradha Gupta,b Pirama Nayagam Arunachalam,b John S. Sack,a Jodi K.
Muckelbauer,a Mary Ellen Cvijic,a Luisa M. Salter-Cid,a Rajeev S. Bhide,b Michael A. Poss,a
John Hynes,a Percy H. Carter,a John E. Macor,d Stefan Ruepp,a Gary L. Schieven,a Joseph A.
Tinoa
aResearch & Development, BristolꢀMyers Squibb Company, Route 206 & Province Line Road,
Princeton, NJ 08543
ACS Paragon Plus Environment
1

Page 3 of 61
Journal of Medicinal Chemistry
1
2
bDepartment of Discovery Synthesis, Biocon BristolꢀMyers Squibb Research Centre, Biocon
Park, Bommasandra IV phase, Jigani Link Road, Bengaluru – 560099, India
3
4
5
6
7
8
9
cCurrent address: School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo,
Texaꢀ79106
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
dCurrent address: Integrated Drug Discovery, Sanofi, 153 2nd Ave, Waltham, MA 02451
PI3Kδ, PI3Kγ, KLH, hERG, CIA, pyrrolotriazine
Abstract
PI3Kδ plays an important role controlling immune cell function and has therefore been
identified as a potential target for the treatment of immunological disorders. This article
highlights our work towards the identification of a potent, selective, and efficacious PI3Kδ
inhibitor. Through careful SAR, the successful replacement of a polar pyrazole group by a
simple chloro or trifluoromethyl group led to improved Cacoꢀ2 permeability, reduced Cacoꢀ2
efflux, reduced hERG PC activity, and increased selectivity profile while maintaining potency in
the CD69 hWB assay. The optimization of the aryl substitution then identified a 4’ꢀCN group
that improved the human/rodent correlation in microsomal metabolic stability. Our lead molecule
is very potent in PK/PD assays and highly efficacious in a mouse collagenꢀinduced arthritis
model.
Introduction
ACS Paragon Plus Environment
2

Journal of Medicinal Chemistry
Page 4 of 61
1
2
3
4
5
6
7
8
9
Over the last few decades, phosphatidylinositol 3ꢀkinases (PI3Ks) have been identified as
important targets for the potential treatment of oncologic and immunologic disorders.1 Class I
PI3Ks, comprising PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ, have been targeted the most. The primary
cellular function of class I PI3Ks is to phosphorylate phosphatidylinositol bisphosphate (PIP2),
converting it to phosphatidylinositol triphosphate (PIP3).2 PIP3 then interacts with a variety of
protein kinases that possess a pleckstrin homology (PH) domain, such as AKT, PDK1, and BTK.
For example, the activation of AKT contributes to the control of growth, survival, and
proliferation of hematopoietic cells through the stimulation of the mTOR pathway.3
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
PI3Kγ and PI3Kδ have been shown to be more suitable targets for the treatment of
immunological disorders because they are predominantly expressed in hematopoietic cells.
PI3Kα and β, on the other hand, are ubiquitously expressed and are reported to play a role in
sugar metabolism.4 PI3Kα is also reported to affect platelet function.5 Selectivity over PI3Kγ
may also be desirable considering its expression level in cardiomyocytes6 and the reduced ability
7
of PI3Kγ KO mice to cope with cardiac distress. In the last decade, PI3Kδ has been revealed to
play an important role controlling immune cell function.8 Guided by these reports and as part of
an effort to identify new potential treatments for immunological disorders, this article will
,
9
10
discuss work towards the identification of a potent, selective, and efficacious PI3Kδ inhibitor.
11
Several PI3Kδ inhibitors with varying degrees of selectivity over PI3Kγ have been reported.
Two of them, highlighted in Figure 1, have been studied in late stage clinical trials for oncologic
disorders. Idelalisib (1a) was approved by the FDA for the treatment of blood cancer while
Duvelisib (1b) was recently terminated in a phase III clinical trial. Both PI3Kδ inhibitors showed
a ~20ꢀ30ꢀfold selectivity over PI3Kγ. In this article, PI3Kδ inhibitors with high selectivity over
ACS Paragon Plus Environment
3

Page 5 of 61
Journal of Medicinal Chemistry
1
2
3
4
PI3Kα, β, and γ as well as the efficacy of a lead molecule in a mouse preclinical model of
5
arthritis are disclosed.
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 1. Clinical PI3Kδ inhibitors
Chemistry.
12
Pyrrolotriazine 2 was recently reported as a very potent PI3Kδ inhibitor (Figure 2). Despite
excellent potency in a CD69 human whole blood (hWB) assay, this molecule lacked selectivity
over PI3Kα and PI3Kγ. Furthermore, its high polar surface area (PSA = 124 Å2) resulted in poor
in vitro ADME properties.
Figure 2. Previously reported PI3Kδ inhibitor 2
ACS Paragon Plus Environment
4

Journal of Medicinal Chemistry
Page 6 of 61
1
2
3
4
5
6
7
8
9
The first area of the molecule that was explored was the heterocyclic Cꢀ5 moiety. In order to
do so, the synthetic plan described in Schemes 1 and 2 was followed. The piperazinone ring was
prepared by reacting 3ꢀbromoaniline (3) with bromoacetyl bromide 4 yielding amide 5 in high
yield. Silver mediated displacement of the tertiary bromide 5 with ethanol amine (6) afforded
alcohol 7. Mitsunobu reaction on alcohol 7 followed by acetylation afforded piperazinone 9. Pdꢀ
catalyzed cross coupling of arylbromide 9 with bispinacol diboronate gave rise to the desired
boronic ester 10. With respect to the synthesis of the core, two procedures were utilized
depending on the heterocyclic moiety (Scheme 2). 1,5ꢀPyrazoles were prepared from the
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
13
previously disclosed pyrrolotriazine 11. Hydrolysis of ester 11 followed by Weinreb’s amide
formation afforded amide 12. The addition of an alkynyl lithium reagent to amide 12 followed by
the addition of dimethylamine afforded ketone 13. Condensation with various hydrazines 14
gave rise to 1,5ꢀpyrazoles 15 as the major regioisomer. Finally, Suzuki crossꢀcoupling with
boronic ester 10 led to the desired analogues. Alternatively, previously disclosed bromo
compound 17 was allowed to react with boronate ester 10 under Suzuki crossꢀcoupling reaction
condition. The resulting pyrrolotriazine 18 was then iodinated at the Cꢀ5 position using NIS
which facilitated SAR studies using palladiumꢀcatalyzed cross coupling reactions.
o
o
o
a NꢀethylꢀNꢀisopropylpropanꢀ2ꢀamine, DCM, 0 C, 98%; b silver oxide, MeCN, 23 C, 75%; c PPh3, DIAD, THF, 23 C, 90%; d AcCl, Et3N,
DCM, 0 oC, 93%; e bispinacol diboronate, PdCl2(dppf), KOAc, dioxane, 90 oC, 68%.
Scheme 1. Synthesis of piperazinone intermediate 10
ACS Paragon Plus Environment
5

Page 7 of 61
Journal of Medicinal Chemistry
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
o
o
a NaOH, water/MeOH 40 C; b Weinreb’s salt, T3P, NꢀethylꢀNꢀisopropylpropanꢀ2ꢀamine, THF, 23 C, 81% (2 steps); c ethynyltrimethylsilane,
BuLi, THF, ꢀ78 oC; d Me2NH, EtOH/Water, 90 oC, 89% (2 steps); e TEA, EtOH, 80 oC, 85ꢀ94%; f 10, PdCl2(dppf), K3PO4, dioxane, 100 oC, 55ꢀ
86%; g NIS, THF, 23 oC, 88%
Scheme 2. Synthetic schemes for Cꢀ5 heterocyclic SAR
Results and discussion.
Table 1 summarizes different Cꢀ5 heterocyclic moieties that were initially studied. Reduction
of PSA using hydrophobic side chains (see 21 and 22) provided improved Cacoꢀ2 (AꢀB)
permeability. 2,2,2ꢀTrifluoroethyl derivative 22 showed a similar selectivity profile to 2,
although it is about eightꢀfold less potent in the CD69 hWB assay. 1,4ꢀPyrazole analogues 23
and 24, on the other hand, exhibited improved PI3Kδ selectivity while being about twoꢀfold
more potent in the hWB assay compared to 22. However, their high efflux ratios resulted in low
mouse in vivo exposures (exposure for 24 in BALB/c mice @ 10 mg/kg P. O.: 470 ± 120 nM
Cmax, 1100 ± 320 nM•h AUC24h). A variety of heteroaromatic moieties were then studied (25 ꢀ
30). Although the Cacoꢀ2 efflux ratios and selectivity profiles were much improved in most
cases, the low hWB potency precluded further investigation. Considering its combination of
good potency and lower Cacoꢀ2 efflux, trifluoroethylpyrazole 22 was selected for further
profiling.
Table 1. SAR of the Cꢀ5 heterocyclic group
ACS Paragon Plus Environment
6

Journal of Medicinal Chemistry
Page 8 of 61
1
2
3
4
5
6
7
8
9
R2
21
23
N
R1
=
R2
=
N
N
N
Me
NH2
R1
22
24
CF3
Cl
N
Me
N
N
N
N
S
N
O
N
Me
Me
S
27
O
26
N
Me
O
N
25
N
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
i-Pr
i-Pr
N
Me
N
O
30
N
N
O
29
28
PI3K
PI3K
PI3K
Caco-2 A-B
(nm/s)b
δ
γ
α
hWB CD69
IC50 (nM)
IC50 (nM)a selectivitya selectivitya
21
2.0
3 ± 1
4.1
11x
37x
25x
110x
110x
170x
33x
38
36 (15)
54 (6)
22
23
58 ± 39
24 ± 7
210x
360x
43x
35 (18)
23 (28)
<36 (>13)
24 1.9 ± 0.5
31 ± 11
100 ± 20
25
26
3.3
4.2
79x
110x
410
240 (1)
27
28
3.3
5.5
60x
100x
96x
160
170 (2)
120 (4)
210x
500 ± 280
29 1.4 ± 0.6
30 1.4 ± 0.5
280x
140x
160x
260x
140 ± 110
280 ± 130
120 (4)
ND
a ADP Glo functional assay b Efflux ratio are found in parenthesis
Figure 3. Xꢀray coꢀcrystal structure of pyrazole 22 bound to PI3Kδ (PBD ID: 5EM)
Figure 3 represents an Xꢀray coꢀcrystal structure of pyrazole 22 in PI3Kδ. Key interactions
between 22 and the hinge included V828 and E826, and a close interaction between the carbonyl
ACS Paragon Plus Environment
7

Page 9 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
of the acetamide group and T750. The pyrazole group of 22 filled a hydrophobic pocket formed
by I825, and participated in an edgeꢀtoꢀface interaction with Y813. Pyrazole 22 was further
profiled, and the results are provided in Table 2. This molecule was potent in an IFNγ hWB
assay, was moderately stable in microsomal stability assays, and displayed a 30% free fraction in
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
14
human serum. Despite an efflux ratio of 6 , moderate exposures were observed in BALB/c mice
at a dose of 10 mg/kg P. O. (1800 ± 300 nM Cmax, 4100 ± 1200 nM•h AUC24h). The in vitro
15
cardiac channel safety profile (hERG patch clamp (PC) IC50 ~17 ꢀM) and high free fraction of
22 suggested a potentially narrow safety window with respect to cardiovascular liabilities. This
was confirmed upon evaluation of 22 in a rabbit electrophysiology (EP) study where QTc
prolongation exceeded 10 ms at free concentration levels that were deemed unacceptable for
progression. Additional undesirable effects included concentrationꢀdependent increases in heart
rate and decreases in blood pressure.
Table 2. Profiling of pyrazole 22
Parameters
Results
58 ± 39
71 ± 40
hWB CD69 (IC50; nM)
hWB IFNγ (IC50; nM)
CYP inhibition
> 20 ꢀM (1A2, 2B6, 2C8,
2C9, 2C19, 2D6, 3A4)
Met. Stab. (h, r, m; % rem)
80, 70, 62
t1/2 (h, r, m; min)
57, 31, 26
Prot. Binding (h, r, m, rb; %
free)
30, 31, 32, 26
Cacoꢀ2 (nm/s)
54 (AꢀB) (efflux ratio = 6)
23 ± 2, 42 ± 6, 59 ± 3
hERG PC (% inh. @ 3, 10, 30
ꢀM)
Cmax = 1800 ± 300 nM
AUC = 4100 ± 1200 nM•h
In vivo PK (BALB/c; 10 mg/kg)
ACS Paragon Plus Environment
8

Journal of Medicinal Chemistry
Page 10 of 61
1
2
3
4
5
6
7
8
In order to reduce cardiovascular liabilities, SAR efforts were focused on reducing hERG PC
activity (QTc prolongation), increasing PI3Kγ selectivity (potential hemodynamic effect),16
reducing the free Cmax by improving hWB potency, increasing protein binding, and improving
ADME properties to achieve a lower peak to trough ratio. In order to accomplish this,
replacement of the relatively large and polar Cꢀ5 heterocyclic moiety was pursued. Smaller
groups at Cꢀ5 were investigated, and the results are summarized in Table 3. As seen with
analogue 18, which showed low affinity for PI3Kδ, a steep SAR was observed when the Cꢀ5
position was unsubstituted. However, simple methyl or nitrile substitution improved hWB
potencies comparable to 22 while also improving PI3Kγ selectivity and permeability (see 31 and
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
17
32). Unfortunately, the hERG PC activity of nitrile 32 was comparable to that of pyrazole 22.
The hERG activity could be lowered by introducing a trifluoromethyl group or a halogen at Cꢀ5.
Indeed, the hERG inhibition at 3 ꢀM was reduced to 12ꢀ14% with analogues 33, 35, and 36.
More importantly, their improved protein binding profiles (7ꢀ12% free in human serum), their
increased PI3Kγ selectivities (>100x), and their reduced Cacoꢀ2 efflux ratios (1ꢀ2) indicated that
these molecules were worthy of further investigation. It is believed that the reduction of PSA (97
Å2 for 33 and 34) is responsible for these improvements. Although iodo analogue 19 and bromo
analogue 36 are more potent in the hWB assay, these Cꢀ5 substituents were deprioritized due to
their light sensitivity which was identified in a reactive oxygen generation assay (ROS, data not
shown).
Table 3. Replacement of Cꢀ5 heterocyclic moiety
ACS Paragon Plus Environment
9

Page 11 of 61
Journal of Medicinal Chemistry
1
2
3
NH2
R
4
5
N
N
N
6
7
8
O
Me
Me
O
N
N
Me
9
PI3Kδ
IC50 (nM) selectivity
Caco-2 A-B
(nm/s)a
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
PI3K hWB CD69
Prot Bind
(h; % free)
hERG PC
(% inh @ 3 ꢀM)
γ
R
IC50 (nM)
18
H
>150
9
ND
ND
ND
ND
17
30
12
23
9
ND
ND
31 Me
240x
110x
270x
120x
180x
74 ± 34
36 ± 17
72 ± 46
570
110 (5)
84 (7)
32 CN 5 ± 2
28 ± 4
12 ± 1
ND
CF
33
34
2.4 ± 0.8
20
290 (1)
190 (2)
200 (2)
3
F
35 Cl
3 ± 1
95 ± 21
12 ± 2
36 Br 2.1 ± 0.9
19 2.0 ± 0.9
75x
97x
39 ± 14
35
290 (1)
250 (1)
7
5
14 ± 2
ND
I
a Efflux ratio are found in parenthesis
This effective replacement of the relatively large pyrazole at Cꢀ5 in 22 by a simple chloro or
trifluoromethyl group was surprising. To understand this further, a computational model of
analog 35 based on the Xꢀray coꢀcrystal structure of pyrazole 22 in PI3Kδ, showed that the fairly
large chloro group filled the hydrophobic pocket nicely as well as interacted with Y813 in a
similar fashion to pyrazole 22, making it an adequate replacement (Figure 4). In contrast,
reduced affinity was observed when a smaller fluoro group was introduced at Cꢀ5 which from
the modeling did not appear to fill the pocket as well (see 34, Table 3).
ACS Paragon Plus Environment
10

Journal of Medicinal Chemistry
Page 12 of 61
1
2
3
4
Figure 4. Computational model of chloro compound 35, based on the Xꢀray coꢀcrystal
5
6
7
18
structure of pyrazole 22 bound to the kinase domain of PI3Kδ (PDB ID: 5EM).
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
Unfortunately, trifluoromethyl analogue 33 and chloro analogue 35 suffered from a
human/mouse disconnect in an in vitro metabolic stability assay (Figure 5). In vitro liver
microsome metabolite identification revealed that both 33 and 35 underwent extensive oxidation
on the piperazinone ring especially in rodents (Figure 5). This posed a problem since the in vivo
models for program progression were in rodents making it difficult to assess proof of concept in
an efficacy study. In an effort to saturate clearance mechanisms, high dose mouse PK studies
with chloro compound 35 were conducted. Unfortunately, even at doses of 30 mg/kg and 100
mg/kg, the exposures were low with AUC24h of 2.9 ± 0.3 ꢀM•h and 17 ± 6 ꢀM•h, respectively.
These exposures were much lower than those observed with pyrazole 22 at a 10 mg/kg dose
(vide supra).
Figure 5. Microsomal stability and metabolite identification of trifluoromethyl analogue 33 and
chloro analogue 35
As part of the effort to improve the microsomal stability of these molecules, new piperazinone
motifs and phenyl substitutions were pursued to sterically block cytochrome P450s from
accessing the metabolic soft spots. Of the two approaches, modification of the phenyl
ACS Paragon Plus Environment
11

Page 13 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
substitution was found to be more fruitful. New analogs were prepared using the synthetic route
shown in Scheme 1, which was adapted by varying the substitution on the 3ꢀbromoaniline
starting material. The corresponding boronic ester was prepared and coupled under Suzuki crossꢀ
coupling reaction conditions with bromo compound 37 (eq 1, Table 4) to provide the analogues
presented in Table 4. It was anticipated that substitution at Cꢀ2 and/or Cꢀ4 would be of interest
considering their proximity to the heterocyclic ring. Unfortunately, the introduction of a methyl
group at the Cꢀ2 position did not improve microsomal stability and showed weak activity at
PI3Kδ (see 39, Table 4). On the other hand, a much better correlation between human and mouse
microsomal stability was observed when a methyl group was introduced at the Cꢀ4 position (see
40, Table 4). Importantly, hWB potency was also improved by about twoꢀfold. The improvement
in potency was surprising since this substitution was originally predicted to extend towards a
solvent exposed area of the PI3Kδ binding pocket. It was hypothesized that Cꢀ4 substitution
locks rotation of the piperazine ring in its preferred bioactive conformation. Although,
substitution at Cꢀ5 significantly improved rat microsomal stability, hWB potency was greatly
diminished (see 41, Table 4). Cꢀ4 substitution, therefore, appeared to be a viable strategy for
improving in vitro mouse metabolic stability.
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
Table 4. The effect of substitution on the phenyl ring
ACS Paragon Plus Environment
12

Journal of Medicinal Chemistry
Page 14 of 61
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
Considering poor microsomal stability can result from aromatic methyl substitution, a variety
of groups were investigated at the Cꢀ4 position (Table 5). Overall, several groups were tolerated.
In all cases, metabolic stability was improved compared to 35. However, when the Cꢀ5 position
of the pyrrolotriazine ring was substituted by a chloro group (see 42 – 45, Table 5), potencies in
both the IFNγ and CD69 hWB assays were reduced. The racemic secondary alcohol 46 showed
good potency in the hWB CD69 assay but had low Cacoꢀ2 AꢀB permeability and a high efflux
ratio. Nonetheless, this compound provided a potential prodrug handle if needed. Nitrile 47
showed good potency in the hWB IFNγ but lesser potency in the hWB CD69. Interestingly,
when the Cꢀ5 position of the pyrrolotriazine ring was substituted with a trifluoromethyl group
(see 48 – 52, Table 5), hWB potencies were generally improved. This was especially true for
racemic secondary alcohol 51 and nitrile 52 which exhibited hWB potencies <36 nM. Other
groups at Cꢀ4 such as trifluoromethyl, methoxy, and sulfone were less potent in these assays (see
48 – 50, Table 5). From Table 5, nitrile 52 was preferred as it had the best balance of potency,
selectivity, free fraction and permeability.
Table 5. Optimization of the Cꢀ4 phenyl substitution
ACS Paragon Plus Environment
13

Page 15 of 61
Journal of Medicinal Chemistry
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
While maintaining the nitrile group at the Cꢀ4 position, the addition of a methyl group was
19
studied on the piperazinone ring (Table 6). In all cases, the potencies of these molecules in the
biochemical assay were found to be similar to the trifluoromethyl derivative 52. However, the
hWB potency of analogue 54b provided an improvement over 52 in the CD69 hWB assay.
Interestingly, its enantiomer 54a is less potent by about fiveꢀfold in the same assay. Although
trifluoromethyl analogue 54b may offer improved potency over trifluoromethyl analogue 52, the
latter was selected for additional studies due to its improved PI3Kγ selectivity profile.
Table 6. Study of the addition of methyl groups to the piperazinone ring
ACS Paragon Plus Environment
14

Journal of Medicinal Chemistry
Page 16 of 61
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
Trifluoromethyl analogue 52 was profiled further in vitro (Table 7). This molecule showed
>100ꢀfold selectivity over the other PI3K isoforms. Kinome selectivity was also excellent with
>660ꢀfold selectivity over MNK1 and others in HTRF assays (235 kinases tested including
phosphatidylinositol 3ꢀkinaseꢀrelated kinases). Importantly, 52 had an improved human/rodent in
vitro stability correlation and a good permeability profile. This translated to much improved
mouse PK (BALB/c, 10 mg/kg; P. O.) compared to pyrazole 22, with a Cmax of 8.2 ± 1.2 ꢀM and
an AUC24h of 17 ± 3 ꢀM•h. PK studies in higher species were also performed revealing excellent
oral bioavailability across species (Table 8). Clearance was found to be higher in mouse but low
in dog resulting in a longer IV t1/2 in dog of 29 ± 8 h. However, the incorporation of the nitrile
group reintroduced hERG PC activity. Although this level of activity was similar to pyrazole 22,
it was hoped that the improved protein binding profile, PK, potency and PI3Kγ selectivity of 52
would help mitigate cardiovascular risk. Compound 52 was therefore evaluated in a rabbit EP
study (Table 9). Some degree of QTc prolongation was observed at doses of 10 and 20 mg/kg,
while the 3 mg/kg dose did not produce significant changes. Although hemodynamic effects
were profound at the 20 mg/kg dose, it is important to note that observed concentrations at 3
mg/kg represent approximately a 10ꢀfold safety margin over the predicted free Cmax (vide infra).
ACS Paragon Plus Environment
15

Page 17 of 61
Journal of Medicinal Chemistry
1
2
3
4
Table 7. Profiling of trifluoromethyl 52
5
6
7
Parameters
Results
8
9
1.7 ± 0.9
ADPꢀGlo PI3Kδ (IC50; nM)
HTRF PI3Kδ (IC50; nM)
ADPꢀGlo PI3Kγ (IC50; nM)
ADPꢀGlo PI3Kα (IC50; nM)
ADPꢀGlo PI3Kβ (IC50; nM)
hWB CD69 (IC50; nM)
hWB IFNγ (IC50; nM)
CYP inhibition
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
<0.1 (n = 7)
170 ± 70 (100x)
1200 ± 500 (700x)
> 5000
35 ± 20
35 ± 15
> 20 ꢀM (1A2, 2B6, 2C8,
2C6, 2C19, 2D6, 3A4)
Met. Stab. (h, r ,m; % rem)
94,91,81
In vitro t1/2 (h, r ,m; min)
>120,67,72
16, 11, 14, 9
Prot. Binding (h, r ,m,rb; %
free)
PAMPA pH7.4 (nm/s)
Cacoꢀ2 (nm/s)
590
110 (AꢀB) (efflux ratio = 4)
25 ± 5, 40 ± 10
hERG PC (% inh. @ 3, 10 ꢀM)
Kinase selectivity (HTRF
assay) (IC50; nM)
PI3Kγ = 41 ± 11
MNK1 = 66 ± 24
PI3Kα = 370 ± 65
MLCK = 510 ± 110
CLK4 = 700 ± 500
Others > 2000
Table 8. Pharmacokinetic profile of trifluoromethyl 52 in preꢀclinical species
ACS Paragon Plus Environment
16

Journal of Medicinal Chemistry
Page 18 of 61
1
2
3
4
5
6
7
8
9
Vss
(L/kg)
t1/2
(h)
Cmax
(ꢀM)
Dose
Route
Dose
(mg/kg)
AUC
(ꢀM h)
CL
(mL/min/kg)
Species
F%
mouse
IV
PO
IV
3
6
2
5
1
2
1
17 ± 4
2.3 ± 0.3
0.24 ± 0.04
4.6 ± 1.3
2.7 ± 0.3
0.5 ± 0.1
4.9 ± 1.2
3.5 ± 0.2
5.9 ± 0.8
14 ± 2
12 ± 6
54 ± 25
90 ± 37
94 ± 21
71 ± 34
75 ± 28
rat
PO
IV
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
dog
0.61 ± 0.06 29 ± 8
PO
IV
6.3 ± 3.4
monkey
3.6 ± 0.8
10 ± 2
Table 9. In vivo rabbit EP study with trifluoromethyl 52
Compound Free Compound QTcf Heart Rate Blood Pressure
Dose
(ms)
(%)
(%)
(mg/kg) Conc. (ꢀM) Conc. (ꢀM)
3
10 ± 2
27 ± 3
54 ± 9
0.9 ± 0.2 +2.2 ± 0.5 +3.3 ± 0.5
ꢀ4.1 ± 1.0
ꢀ22 ± 4
10
20
2.4 ± 0.5
4.8 ± 1.0
+13 ± 2 +27 ± 3
+25 ± 4 +100 ± 10
ꢀ58 ± 10
Trifluoromethyl analogue 52 was further evaluated in a basophil ex vivo pharmacodynamic
study. This assay consisted of stimulating blood taken from mice 1 hour after oral administration
of 52 and antiꢀmIgE. The levels of CD63+ cells were determined, and the results are shown in
Figure 6. Analogue 52 demonstrated a dose dependent inhibition of CD63+ cells that was
maximal with a dose of 10 mg/kg. This data indicated a mouse ex vivo EC50 of 2.0 ± 0.8 nM and
an EC90 of 9 ± 5 nM. These values appear to be consistent with the mWB assay (IC50 = 5.1 ± 1.8
nM).
Figure 6. AntiꢀmIgE basophile mouse ex vivo assay with trifluoromethyl 52
ACS Paragon Plus Environment
17

Page 19 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Analogue 52 was further evaluated in a keyhole limpet hemocyanin (KLH) mouse humoral
model. The compound was administered orally BID at five different doses to mice that had been
immunized intraperitoneally with KLH. On day 13, blood levels of CD69 and IgG titers at
trough (24h) were recorded following ex vivo KLH treatment (Table 10). Importantly, the IgG
response was suppressed in a doseꢀproportional fashion and this correlated with the inhibition of
CD69 measured ex vivo. The data indicated that greater inhibition of CD69 was required (based
on calculated % inhibition) for robust inhibition of IgG, although this could be due to the fact
that IgG levels were recorded at trough. Overall, based on CD69 inhibition, the results from the
study suggested three different doses (0.5, 2 and 5 mg/kg BID) for an in vivo efficacy study.
Table 10. Results of the mouse KLH study for compound 52
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
Arthritis was induced in mice by injecting collagen in the tail of arthritis prone DBAꢀ1 mice.
Analogue 52 was dosed in a preventive mode orally BID for 42 days, starting on day 1 (Figure
7). Methotrexate was used as a positive control. Although lower than expected exposures were
observed for 52 compared to healthy DBAꢀ1 mice (data not shown), a doseꢀdependent reduction
of the clinical score was observed. Doses of 2 and 5 mg/kg showed greater than 50% suppression
of paw swelling. Taking the exposure into consideration, an EC50 = 10 nM @ 24 h (ED50 of
~1.25 mg/kg) was derived. This level of efficacy was similar to other compounds reported in the
20
literature, and corresponded to about a twoꢀfold multiple of the IC50 at trough.
ACS Paragon Plus Environment
18

Journal of Medicinal Chemistry
Page 20 of 61
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 7. Mouse CIA efficacy model with trifluoromethyl compound 52
Conclusion.
In conclusion, several highly potent and selective PI3Kδ inhibitors were identified. The initial
analogue, pyrazole 2, suffered from poor ADME properties and kinase selectivity. Two
significant improvements to 2 were made. First, replacement of the polar pyrazole group by a
simple chloro or trifluoromethyl group led to improved Cacoꢀ2 permeability, reduced Cacoꢀ2
efflux, and an improved selectivity profile while maintaining potency in a CD69 hWB assay.
Furthermore, analogues 33 and 35 demonstrated reduced activity in a hERG PC assay and a
better protein binding profile which together may mitigate cardiovascular risk. However, these
molecules suffered from an undesired human/rodent disconnect in metabolic stability. Second,
the identification of nitrile 52 improved mouse metabolic stability while maintaining an excellent
profile. This molecule was shown to be very potent ex vivo in a mouse basophil assay as well as
in vivo in a mouse KLH study. Furthermore, 52 was shown to be efficacious in a mouse arthritis
model. Finally, from this effort, trifluoromethyl analogue 52 was selected for additional
preclinical toxicity studies, including evaluation for cardiovascular risk in higher species.
ACS Paragon Plus Environment
19

Page 21 of 61
Journal of Medicinal Chemistry
1
2
3
4
Experimental data
5
6
7
8
9
All commercially available chemicals and solvents were used without further purification.
Reactions were performed under an atmosphere of nitrogen. All new compounds gave
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
1
1
satisfactory H NMR and/or LC/MS results. H NMR spectra were obtained on a Bruker 400
MHz or a JEOL 500 MHz NMR spectrometer using TMS as internal reference. Electrospray
ionization (ESI) mass spectra were obtained on a Waters ZQ single quadrupole mass
spectrometer. We do not believe that this series of compounds has a PAINS liability. All
compounds were tested in four different PI3K isoforms ADPꢀGlo assays as well as in >200
kinases HTRF assays. Clear isoform selectivity as well as general kinase selectivity for the
compounds discussed in the paper support that this is not an issue. Additionally, in vivo efficacy
studies have confirmed activity for compound 52. The purity of tested compounds determined by
analytical HPLC was >95%. HPLC methods are listed below:
Method A. Waters Acquity UPLC, BEH C18 2.1 x 50 mm, 1.7 ꢀm particles; Mobile Phase A:
98:2 water:acetonitrile with 0.05% TFA; Mobile Phase B: acetonitrile with 0.05% TFA,
o
temperature 50 C; Gradient 2ꢀ98% B over 1 min then 0.5 min hold at 100% B. Flow 0.8
mL/min; Detection: UV at 200 nm.
Method B. Waters Acquity UPLC, BEH C18 2.1 x 50 mm, 1.7 ꢁm particles; Mobile Phase A:
5:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water
with 10 mM ammonium acetate; Temperature: 50 °C; Gradient: 0ꢀ100% B over 2 min, then a
0.5ꢀmin hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm.
Method C. Sunfire C18 3.0 x 150 mm, 3.5 ꢁm particles; Mobile Phase A: 5:95
acetonitrile:water with 0.05% TFA; Mobile Phase B: 95:5 acetonitrile:water with 0.05 TFA;
ACS Paragon Plus Environment
20

Journal of Medicinal Chemistry
Page 22 of 61
1
2
3
4
5
6
Temperature: 25 °C; Gradient: 0ꢀ100% B over 3 minutes, then a 1ꢀmin hold at 100% B; Flow:
1.0 mL/min; Detection: UV at 254 nm.
7
8
9
Method D (Preparative). Luna C18 30 x 100 mm, 5 ꢁm particles; 2 mL injection; Mobile
Phase: 0.1% TFA in Water; Mobile Phase B: 0.1% TFA in acetonitrile; temperature: 50 °C;
Gradient: 20ꢀ100% B over 5 min, then a 10 min hold at 100% B, Flow: 30 mL/min; Detection:
UV at 220 nm.
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
4-Acetyl-1-(3-(4-amino-5-(1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-5-yl)pyrrolo[2,1-
f][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (2). A pressure vial was charged
with boronic ester 10 (52 mg, 0.18 mmol), water (0.3 mL), 7ꢀbromoꢀ5ꢀ(1ꢀ(tetrahydroꢀ2Hꢀpyranꢀ
4ꢀyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)pyrrolo[2,1ꢀf][1,2,4]triazinꢀ4ꢀamine (15a) (33 mg, 0.15 mmol), Na2CO3
(49 mg, 0.46 mmol), and PdCl2(dppf)ꢀDCM adduct (25 mg, 0.03 mmol) in dioxane (1.2 mL).
The mixture was purged with nitrogen and then the vessel was sealed and stirred at 110 ºC for 5
h. The reaction mixture was cooled, diluted with 15 mL of water and extracted with ethyl
acetate. The combined organic layers were washed with brine, dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified by preparative HPLC (Method
D)
to
obtain
4ꢀacetylꢀ1ꢀ(3ꢀ(4ꢀaminoꢀ5ꢀ(1ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ꢀ1Hꢀpyrazolꢀ5ꢀ
yl)pyrrolo[2,1ꢀf][1,2,4]triazinꢀ7ꢀyl)phenyl)ꢀ3,3ꢀdimethylpiperazinꢀ2ꢀone (22 mg, 65% yield).
HPLC tr = 2.90 min (Method C). LCMS (ESI) m/z calcd for C28H32N8O3 [
M+H]+ 529.3, found
1
529.3. H NMR (500 MHz, DMSOꢀd6) δ 8.11ꢀ8.04 (m, 2H), 8.04 (s, 1H), 7.68 (s, 1H), 7.54 (t,
J=7.9 Hz, 1H), 7.35 (d, J=8.4 Hz, 1H), 7.27 (s, 1H), 6.47 (s, 1H), 4.35 (m., 1H), 3.92ꢀ3.85 (m,
2H), 3.83 (m., 2H), 3.77 (m, 2H), 3.43ꢀ3.40 (m, 2H), 2.20ꢀ2.00 (m, 2H), 2.10 (s, 3H), 1.78 (m,
2H), 1.70 (s, 6H).
ACS Paragon Plus Environment
21

Page 23 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2-Bromo-N-(3-bromophenyl)-2-methylpropanamide (5). To a mixture of 3ꢀbromoaniline (3)
(2.0 g, 12 mmol) and NꢀethylꢀNꢀisopropylpropanꢀ2ꢀamine (4.0 mL, 23 mmol) in DCM (100 mL)
was cooled to 0 ºC and 2ꢀbromoꢀ2ꢀmethylpropanoyl bromide (4) (2.2 mL, 17 mmol) was added
dropwise. The mixture was allowed to warm up to room temperature over 2 h and was diluted
with DCM (100 mL). The organic phase was washed with water, 1 N HCl, saturated NaHCO3
and brine. The resulting solution was dried over Na2SO4 and concentrated to give the titled
compound in 98% yield which was used without further purification. HPLC tr = 1.01 min
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
(Method A). LCMS (ESI) m/z calcd for C10H11Br2NO [
M+H]+ 321.6, found 321.7.
N-(3-Bromophenyl)-2-((2-hydroxyethyl)amino)-2-methylpropanamide (7). Crude bromide
5 and 2ꢀaminoethanol (6) (1.4 g, 23 mmol) were dissolved in acetonitrile (95 mL) followed by
the addition of silver oxide (5.4 g, 23 mmol). The mixture was stirred at room temperature for 1
h, and then filtered through Celite. The filtrate was diluted with ethyl acetate (250 mL), and
washed with saturated NaHCO3 and brine. The resulting solution was dried over Na2SO4 and
concentrated to give crude solid which was used without further purification. (75% yield).
HPLC tr = 1.95 min (Method C). LCMS (ESI) m/z calcd for C12H17BrN2O2 [
M+H]+ 302.6,
found 302.7. 1H NMR (400 MHz, chloroformꢀd) δ 9.79 (br. s., 1H), 7.90 (t, J=2.0 Hz, 1H), 7.54
(dt, J=7.8, 1.7 Hz, 1H), 7.27 ꢀ 7.11 (m, 2H), 3.86 ꢀ 3.75 (m, 2H), 2.83 ꢀ 2.69 (m, 2H), 1.44 (s,
6H).
1-(3-Bromophenyl)-3,3-dimethylpiperazin-2-one (8). To a solution of diisopropyl
azocarboxylate (2.4 g, 12 mmol) in THF (50 mL) at room temperature was added
triphenylphosphine (3.1 g, 12 mmol). After 10 min, a yellow solid formed. A solution of Nꢀ(3ꢀ
bromophenyl)ꢀ2ꢀ((2ꢀhydroxyethyl)amino)ꢀ2ꢀmethylpropanamide (7) (1.2 g, 4 mmol) in of THF
(50 mL) was added. After stirring overnight at room temperature, the mixture was diluted with
ACS Paragon Plus Environment
22

Journal of Medicinal Chemistry
Page 24 of 61
1
2
3
4
5
6
7
8
9
100 mL of ethyl acetate and was washed with saturated NaHCO3 and brine. The resulting
solution was dried over Na2SO4 and concentrated under reduced pressure. The residue was
purified by acid/base extraction to give 1ꢀ(3ꢀbromophenyl)ꢀ3,3ꢀdimethylpiperazinꢀ2ꢀone in 90%
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
yield. HPLC tr = 1.71 min (Method C). LCMS (ESI) m/z calcd for C12H15BrN2O [
M+H]+ 284.8,
1
found 284.8. H NMR (400 MHz, chloroformꢀd) δ 7.50 ꢀ 7.45 (m, 1H), 7.44 ꢀ 7.38 (m, 1H),
7.29 ꢀ 7.22 (m, 2H), 3.75 ꢀ 3.67 (m, 2H), 3.31 ꢀ 3.22 (m, 2H), 1.50 (s, 6H).
4-Acetyl-1-(3-bromophenyl)-3,3-dimethylpiperazin-2-one (9). To a solution of 1ꢀ(3ꢀ
bromophenyl)ꢀ3,3ꢀdimethylpiperazinꢀ2ꢀone (8) (0.82 g, 2.7 mmol) in DCM (10 mL) was added
triethylamine (1 mL) and acetic anhydride (0.33 g, 3.2 mmol) and the resulting mixture was
stirred for 2 h at room temperature. The reaction was diluted with DCM (20 mL), washed with
water, saturated NaHCO3, and brine. The resulting solution was dried over Na2SO4 and
concentrated under reduced pressure. The crude mixture was purified by silica gel
chromatography eluting with a gradient of 0% to 100% ethyl acetate in hexanes to give 4ꢀacetylꢀ
1ꢀ(3ꢀbromophenyl)ꢀ3,3ꢀdimethylpiperazinꢀ2ꢀone (9) (0.9 g, 93%). HPLC tr = 0.79 min (Method
1
A). LCMS (ESI) m/z calcd for C14H17BrN2O2
[
M+H]+ 326.7, found 326.8. H NMR (400MHz,
chloroformꢀd) δ 7.51 (dt, J=1.9, 1.0 Hz, 1H), 7.44 (dt, J=7.3, 1.9 Hz, 1H), 7.33 ꢀ 7.22 (m, 2H),
3.83 ꢀ 3.73 (m, 4H), 2.20 (s, 3H), 1.86 (s, 6H).
4-Acetyl-3,3-dimethyl-1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazin-
2-one (10). A mixture of 4ꢀacetylꢀ1ꢀ(3ꢀbromophenyl)ꢀ3,3ꢀdimethylpiperazinꢀ2ꢀone (9) (1.95 g,
6.00 mmol), 4,4,4ʹ,4ʹ,5,5,5ʹ,5ʹꢀoctamethylꢀ2,2ʹꢀbi(1,3,2ꢀdioxaborolane) (1.7 g, 6.60 mmol),
PdCl2(dppf)ꢀDCM adduct (0.150 g, 0.180 mmol) and potassium acetate (1.2 g, 12 mmol) in
dioxane (15 mL) in a capped pressure reaction vial was degassed by vacuum then filled with
nitrogen. This process was repeated twice. The reaction mixture was placed in a 90 ºC heating
ACS Paragon Plus Environment
23

Page 25 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
block for 2.5 h. The reaction mixture was filtered through Celite and the filtrate was purified by
silica gel chromatography eluting with a gradient of 0% to 100% ethyl acetate in hexanes
7
8
9
affording
4ꢀacetylꢀ3,3ꢀdimethylꢀ1ꢀ(3ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀ
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
yl)phenyl)piperazinꢀ2ꢀone (1.75 g, 4.7 mmol, 68% yield) as a white solid. HPLC tr = 0.87 min
1
(Method A). LCMS (ESI) m/z calcd for C19H27BN2O4
[
M+H]+ 359.1, found 359.0. H NMR
(400 MHz, chloroformꢀd) δ 7.77ꢀ7.73 (m, 1H), 7.68 (d, J=1.5 Hz, 1H), 7.45ꢀ7.42 (m, 2H), 3.84ꢀ
3.74 (m, 4H), 2.20 (s, 3H), 1.86 (s, 6H), 1.37 (s, 12H).
(E)-1-(4-Amino-7-bromopyrrolo[2,1-f][1,2,4]triazin-5-yl)-3-(dimethylamino)prop-2-en-1-
one (13). To a solution of ethynyltrimethylsilane (1.7 mL, 12 mmol) in THF (30 mL) at ꢀ78 ºC
was added nꢀbutyllithium (4.6 mL, 11 mmol) dropwise. After stirring for 5 min at ꢀ78 ºC, 4ꢀ
21
aminoꢀ7ꢀbromoꢀNꢀmethoxyꢀNꢀmethylpyrrolo[2,1ꢀf][1,2,4]triazineꢀ5ꢀcarboxamide (12) (1.2 g, 4
mmol) was added as a solution in THF (6 mL). After 30 min, acetic acid (0.6 mL) was added to
give a bright yellow solution. The mixture was partitioned between ethyl acetate (40 mL) and
water (20 mL). The organic layer was washed with brine, dried over Na2SO4 and concentrated to
afford alkyne 13 (1.3 g) as a yellow solid used without further purification. The solid (1.1 g, 3.3
mmol) was suspended in ethanol (16 mL) and dimethylamine (0.620 mL, 4.9 mmol, 40%
solution in water) was added. The mixture was heated at 80 ºC for 0.5 h and then cooled to room
temperature. The solids were removed through filtration and were washed with ethanol, ethyl
acetate and hexanes and volatiles were removed under vacuum providing (E)ꢀ1ꢀ(4ꢀaminoꢀ7ꢀ
bromopyrrolo[2,1ꢀf][1,2,4]triazinꢀ5ꢀyl)ꢀ3ꢀ(dimethylamino)propꢀ2ꢀenꢀ1ꢀone (904 mg, 2.91 mmol,
89% yield) as a yellow/green solid. HPLC tr = 0.86 min (Method C). LCMS (ESI) m/z calcd for
1
C11H12BrN5O
[
M+H]+ 310.1, found 310.2. H NMR (400 MHz, DMSOꢀd6) δ 10.86 (br. s., 1H),
ACS Paragon Plus Environment
24

Journal of Medicinal Chemistry
Page 26 of 61
1
2
3
4
8.17 (br. s., 1H), 8.03ꢀ7.93 (m, 1H), 7.78 (d, J=12.1 Hz, 1H), 7.64ꢀ7.56 (m, 1H), 5.92 (d, J=12.1
5
6
Hz, 1H), 3.17 (s, 3H), 2.97 (s, 3H).
7
8
9
7-Bromo-5-(1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-
amine (15a). (E)ꢀ1ꢀ(4ꢀaminoꢀ7ꢀbromopyrrolo[2,1ꢀf][1,2,4]triazinꢀ5ꢀyl)ꢀ3ꢀ(dimethylamino)propꢀ
2ꢀenꢀ1ꢀone (13) (1.0 g, 3.2 mmol) was suspended in ethanol (40 mL) and (tetrahydroꢀ2Hꢀpyranꢀ
4ꢀyl)hydrazine, HCl salt (0.92 g, 6.3 mmol) was added. The suspension was stirred vigorously
for 2 min and triethylamine (2.2 mL, 16 mmol) was added. The mixture was refluxed for 18 h
then cooled to room temperature followed by the addition of water (50 mL). The mixture was
concentrated to 15 mL. The solid formed was filtered and dried under vacuum providing 7ꢀ
bromoꢀ5ꢀ(1ꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)ꢀ1Hꢀpyrazolꢀ5ꢀyl)pyrrolo[2,1ꢀf][1,2,4]triazinꢀ4ꢀamine
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
(3.24 g, 94% yield) as a white solid. HPLC tr = 0.69 min (Method A). LCMS (ESI) m/z calcd for
1
C14H15BrN6O
[
M+H]+ 363.1, found 363.2. H NMR (400 MHz, DMSOꢀd6) δ 8.06 (s, 1H), 7.65
(d, J=1.5 Hz, 1H), 7.04 (s, 1H), 6.42 (d, J=1.8 Hz, 1H), 4.33ꢀ4.23 (m, 1H), 3.87 (dd, J=11.2, 3.5
Hz, 2H), 3.37ꢀ3.31 (m, 2H), 2.06 (qd, J=12.2, 4.7 Hz, 2H), 1.84ꢀ1.68 (m, 2H).
4-Acetyl-1-(3-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-
one (18). A pressure vial was charged with boronic ester 10 (500 mg, 1.5 mmol), water (3 mL),
7ꢀbromopyrrolo[2,1ꢀf][1,2,4]triazinꢀ4ꢀamine (17) (330 mg, 1.5 mmol), Na2CO3 (489 mg, 4.61),
and PdCl2(dppf)ꢀDCM adduct (251 mg, 0.308 mmol) in dioxane (12 mL). The mixture was
purged with nitrogen, the vessel was sealed and stirred at 100 ºC for 5 h. The reaction mixture
was cooled, diluted with 15 mL of water and extracted with ethyl acetate. The combined organic
layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The
residue was purified by silica gel chromatography eluting with a gradient of 0% to 100% ethyl
acetate in hexanes to obtain 4ꢀacetylꢀ1ꢀ(3ꢀ(4ꢀaminopyrrolo[2,1ꢀf][1,2,4]triazinꢀ7ꢀyl)phenyl)ꢀ3,3ꢀ
ACS Paragon Plus Environment
25

Page 27 of 61
Journal of Medicinal Chemistry
1
2
3
4
dimethylpiperazinꢀ2ꢀone (320 mg, 60% yield). HPLC tr = 1.90 min (Method C). LCMS (ESI)
5
6
1
m/z calcd for C20H22N6O2
[
M+H]+ 379.3, found 379.4. H NMR (400 MHz, chloroformꢀd) δ
7
8
9
8.02ꢀ7.99 (m, 2H), 7.94ꢀ7.88 (m, 1H), 7.54ꢀ7.48 (m, 1H), 7.31ꢀ7.27 (m, 1H), 6.92 (d, J=4.6 Hz,
1H), 6.73 (d, J=4.6 Hz, 1H), 3.92ꢀ3.87 (m, 2H), 3.85ꢀ3.79 (m, 2H), 2.21 (s, 3H), 1.90 (s, 6H).
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
4-Acetyl-1-(3-(4-amino-5-iodopyrrolo[1,2-f][1,2,4]triazin-7-yl)phenyl)-3,3-
dimethylpiperazin-2-one (19). A solution of 4ꢀacetylꢀ1ꢀ(3ꢀ(4ꢀaminopyrrolo[2,1ꢀf][1,2,4]triazinꢀ
7ꢀyl)phenyl)ꢀ3,3ꢀdimethylpiperazinꢀ2ꢀone (18) (300 mg, 0.8 mmol) and Nꢀiodosuccinimide (180
mg, 0.8 mmol) in DMF (10 mL) was stirred under nitrogen at room temperature for 15 h. More
NIS (45 mg, 0.2 mmol) and TFA (1 drop) were added to the reaction mixture and stirring was
continued for 2 h allowing full conversion. The reaction mixture was diluted with saturated
NaHCO3 (50 mL) and stirred vigorously for 15 min. The precipitated solid was collected by
filtration, washed with water, and then diethyl ether. The solid was dried under vacuum
affording
4ꢀacetylꢀ1ꢀ(3ꢀ(4ꢀaminoꢀ5ꢀiodopyrrolo[2,1ꢀf][1,2,4]triazinꢀ7ꢀyl)phenyl)ꢀ3,3ꢀ
dimethylpiperazinꢀ2ꢀone (350 mg, 0.7 mmol, 88% yield) as a light brown solid. HPLC tr = 0.7
M+H]+ 505.1, found 505.0. 1H NMR
min (Method A). LCMS (ESI) m/z calcd for C20H21I1N6O2
[
(500 MHz, DMSOꢀd6) δ 8.01ꢀ7.96 (m, 2H), 7.93 (s, 1H), 7.50 (t, J=7.9 Hz, 1H), 7.37ꢀ7.31 (m,
2H), 3.84ꢀ3.73 (m, 4H), 2.10 (s, 3H), 1.70 (s, 6H).
4-Acetyl-1-(3-(4-amino-5-(1-cyclopropyl-1H-pyrazol-5-yl)pyrrolo[2,1-f][1,2,4]triazin-7-
yl)phenyl)-3,3-dimethylpiperazin-2-one (21). Compound synthesized according to the
procedure outlined for the synthesis of pyrazole 2. HPLC tr = 2.99 min (Method C). LCMS (ESI)
m/z calcd for C26H28N8O2 [
M+H]+ 485.2, found 485.4. 1H NMR (500 MHz, DMSOꢀd6) δ 8.12 (s,
1H), 8.07 ꢀ 7.99 (m, 2H), 7.60 ꢀ 7.50 (m, 2H), 7.45 ꢀ 7.31 (m, 2H), 6.45 (s, 1H), 3.86 ꢀ 3.73 (m,
4H), 2.08 (s, 3H), 1.79 ꢀ 1.64 (m, 7H), 1.09 ꢀ 1.00 (m, 2H), 0.92 ꢀ 0.80 (m, 2H).
ACS Paragon Plus Environment
26

Journal of Medicinal Chemistry
Page 28 of 61
1
2
3
4
4-Acetyl-1-(3-(4-amino-5-(1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl)pyrrolo[2,1-
5
6
f][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (22). Compound synthesized
7
8
9
according to the procedure outlined for the synthesis of pyrazole 2. HPLC tr = 2.61 min (Method
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
1
C). LCMS (ESI) m/z calcd for C25H25F3N8O2
[
M+H]+ 527.2, found 527.4. H NMR (400 MHz,
methanolꢀd4) δ 8.92 ꢀ 8.80 (m, 3H), 8.59 (s, 1H), 8.40 ꢀ 8.30 (m, 1H), 8.20 ꢀ 8.13 (m, 1H), 8.05
(s, 1H), 7.43 (s, 1H), 5.91 ꢀ 5.78 (m, 2H), 4.70 ꢀ 4.52 (m, 4H), 2.91 (s, 3H), 1.63 (s, 6H).
4-Acetyl-1-(3-(4-amino-5-(1-methyl-1H-pyrazol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-7-
yl)phenyl)-3,3-dimethylpiperazin-2-one (23).
A
mixture of 4ꢀacetylꢀ1ꢀ(3ꢀ(4ꢀaminoꢀ5ꢀ
iodopyrrolo[2,1ꢀf][1,2,4]triazinꢀ7ꢀyl)phenyl)ꢀ3,3ꢀdimethylpiperazinꢀ2ꢀone (19) (100 mg, 0.2
mmol), 1ꢀmethylꢀ4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ1Hꢀpyrazole (50 mg, 0.24
mmol), PdCl2(dppf)ꢀDCM adduct (8 mg, 10 µmol) and K3PO4 (2N, 0.2 mL, 0.4 mmol) in
dioxane (3 mL) in a capped pressure reaction vial was degassed by vacuum and back filled with
o
nitrogen twice. The vial was placed in a 90 C heating block and stirred for 3 h. The crude
material was purified via preparative HPLC (Method D) yielding 40 mg of the desired product.
1
HPLC tr = 0.61 min. LCMS (ESI) m/z calcd for C24H26N8O2
[
M+H]+ 459.2, found 459.2. H
NMR (500 MHz, DMSOꢀd6) δ 8.08 ꢀ 8.01 (m, 1H), 8.01 ꢀ 7.88 (m, 3H), 7.69 ꢀ 7.59 (m, 1H),
7.57 ꢀ 7.45 (m, 1H), 7.36 ꢀ 7.24 (m, 1H), 7.06 (s, 1H), 3.91 (s, 3H), 3.85 ꢀ 3.68 (m, 4H), 2.09 (s,
3H), 1.69 (s, 6H).
4-Acetyl-1-(3-(4-amino-5-(1-cyclopropyl-1H-pyrazol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-7-
yl)phenyl)-3,3-dimethylpiperazin-2-one (24). Compound synthesized according to the
procedure outlined for the synthesis of pyrazole 23. HPLC tr = 1.90 min (Method C). LCMS
1
(ESI) m/z calcd for C26H28N8O2
[
M+H]+ 485.2, found 485.4. H NMR (500 MHz, DMSOꢀd6) δ
8.08 ꢀ 8.01 (m, 1H), 8.01 ꢀ 7.88 (m, 3H), 7.69 ꢀ 7.59 (m, 1H), 7.57 ꢀ 7.45 (m, 1H), 7.36 ꢀ 7.24 (m,
ACS Paragon Plus Environment
27

Page 29 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
1H), 7.06 (s, 1H), 3.85 ꢀ 3.68 (m, 5H), 2.09 (s, 3H), 1.69 (s, 6H), 1.20 ꢀ 1.08 (m, 2H), 1.08 ꢀ 0.90
(m, 2H).
7
8
9
4-Acetyl-1-(3-(4-amino-5-(2-chloro-1-methyl-1H-imidazol-5-yl)pyrrolo[2,1-
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
f][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (25). Compound synthesized
according to the procedure outlined for the synthesis of pyrazole 23. HPLC tr = 2.64 min
1
(Method C). LCMS (ESI) m/z calcd for C24H25Cl1N8O2
[
M+H]+ 493.2, found 493.4. H NMR
(500 MHz, DMSOꢀd6) δ 8.13 ꢀ 8.06 (m, 1H), 8.06 ꢀ 7.93 (m, 2H), 7.57 ꢀ 7.47 (m, 1H), 7.37 ꢀ
7.27 (m, 1H), 7.20 (s, 1H), 6.85 ꢀ 6.72 (m, 2H), 6.68 ꢀ 6.55 (m, 1H), 3.87 ꢀ 3.67 (m, 4H), 2.74 (s,
1H), 2.10 (s, 3H), 1.71 (s, 6H).
4-Acetyl-1-(3-(4-amino-5-(oxazol-2-yl)pyrrolo[2,1-f][1,2,4]triazin-7-yl)phenyl)-3,3-
dimethylpiperazin-2-one (26). A mixture of 4ꢀacetylꢀ1ꢀ(3ꢀ(4ꢀaminoꢀ5ꢀbromopyrrolo[2,1ꢀ
f][1,2,4]triazinꢀ7ꢀyl)phenyl)ꢀ3,3ꢀdimethylpiperazinꢀ2ꢀone (37) (30 mg, 0.07 mmol), 1ꢀ
tributyltinoxazole (27 mg, 0.07 mmol), Pd2(dba)3 (6 mg, 7 µmol) and tri(2ꢀfuryl)phosphine (3
o
mg, 0.01 mmol) in DMF (1 mL) was heated at 120 C for 1 h. The crude material was purified
via preparative HPLC (Method D) yielding 7 mg of the desired product. HPLC tr = 0.69 min
(Method A). LCMS (ESI) m/z calcd for C23H23N7O3 [
M+H]+ 446.2, found 446.1. 1H NMR (500
MHz, DMSOꢀd6) δ 10.31 (s, 1H), 8.41 (s, 1H), 8.26 (s, 1H), 8.14 ꢀ 7.93 (m, 3H), 7.62 (s, 1H),
7.58 ꢀ 7.49 (m, 1H), 7.49 ꢀ 7.41 (m, 1H), 7.41 ꢀ 7.31 (m, 1H), 3.89 ꢀ 3.66 (m, 4H), 2.11 (s, 3H),
1.71 (s, 6H).
4-Acetyl-1-(3-(4-amino-5-(5-methyl-1,3,4-thiadiazol-2-yl)pyrrolo[2,1-f][1,2,4]triazin-7-
yl)phenyl)-3,3-dimethylpiperazin-2-one (27). Compound synthesized according to to the
procedure outlined for the synthesis of pyrazole 23. HPLC tr = 0.67 min (Method A). LCMS
ACS Paragon Plus Environment
28