Discovery and Optimization of Quinazolinone-pyrrolopyrrolones as Potent and Orally Bioavailable Pan-Pim Kinase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.6b00610

The high expression of proviral insertion site of Moloney murine leukemia virus kinases (Pim-1, -2, and -3) in cancers, particularly the hematopoietic malignancies, is believed to play a role in promoting cell survival and proliferation while suppressing apoptosis. The three isoforms of Pim protein appear largely redundant in their oncogenic functions. Thus, a pan-Pim kinase inhibitor is highly desirable. However, cell active pan-Pim inhibitors have proven difficult to develop because Pim-2 has a low K_m for ATP and therefore requires a very potent inhibitor to effectively block the kinase activity at cellular ATP concentrations. Herein, we report a series of quinazolinone-pyrrolopyrrolones as potent and selective pan-Pim inhibitors. In particular, compound 17 is orally efficacious in a mouse xenograft model (KMS-12 BM) of multiple myeloma, with 93% tumor growth inhibition at 50 mg/kg QD upon oral dosing.

Full text of DOI:10.1021/acs.jmedchem.6b00610

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Article
Discovery and Optimization of Quinazolinone-pyrrolopyrrolones
as Potent and Orally Bioavailable pan-Pim Kinase Inhibitors
Liping H. Pettus, Kristin L. Andrews, Shon K. Booker, Jie Chen, Victor J. Cee, Frank Chavez Jr., Yuping Chen,
Heather Eastwood, Nadia Guerrero, Bradley James Herberich, Dean Hickman, Brian A Lanman, Jimmy Laszlo
III, Matthew Randolph Lee, J. Russell Lipford, Bethany Mattson, Christopher Mohr, Yen Nguyen, Mark H
Norman, David Powers, Anthony B. Reed, Karen Rex, Christine Sastri, Nuria A. Tamayo, Paul Wang, Jeffrey
T. Winston, Bin Wu, Tian Wu, Ryan P. Wurz, Yang Xu, Yihong Zhou, Andrew S Tasker, and Hui-Ling Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00610 • Publication Date (Web): 10 Jun 2016
Downloaded from http://pubs.acs.org on June 11, 2016
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Discovery and Optimization of Quinazolinoneꢀ
pyrrolopyrrolones as Potent and Orally Bioavailable
panꢀPim Kinase Inhibitors
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Liping H. Pettus,* Kristin L. Andrews, Shon K. Booker, Jie Chen, Victor J. Cee, Frank
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Chavez Jr., Yuping Chen, Heather Eastwood, Nadia Guerrero, Bradley Herberich, Dean
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Wu, Tian Wu, Ryan P. Wurz, Yang Xu, Yihong Zhou, Andrew S. Tasker , and HuiꢀLing
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Wang
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Department of Therapeutic Discovery ꢀ Medicinal Chemistry; Molecular Structure;
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Pharmacokinetics and Drug Metabolism; Oncology Research; Pharmaceutics; Discovery
Technologies; Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
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ABSTRACT
The high expression of proviral insertion site of Moloney murine leukemia virus kinases
(Pimꢀ1, ꢀ2, and ꢀ3) in cancers, particularly the hematopoietic malignancies, is believed to play a
role in promoting cell survival and proliferation while suppressing apoptosis. The three isoforms
of Pim protein appear largely redundant in their oncogenic functions. Thus, a panꢀPim kinase
inhibitor is highly desirable. However, cell active panꢀPim inhibitors have proven difficult to
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develop because Pimꢀ2 has a low K for ATP and therefore, requires a very potent inhibitor to
m
effectively block the kinase activity at cellular ATP concentrations. Herein, we report a series of
quinazolinoneꢀpyrrolopyrrolones as potent and selective panꢀPim inhibitors. In particular,
compound 17 is orally efficacious in a mouse xenograft model (KMSꢀ12 BM) of multiple
myeloma, with 93% tumor growth inhibition at 50 mg/kg QD upon oral dosing.
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INTRODUCTION
The proviral integration site of Moloney murine leukemia virus proteins 1, 2, and 3 (Pimꢀ
1, Pimꢀ2, and Pimꢀ3) are a family of constitutively active serine/threonine kinases that are
involved in the survival and proliferation of hematopoietic cells in response to cytokines, stress,
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and growth factors signaling downstream of JAK/STAT and NFꢀκB activation. The signaling
pathway of the Pim kinases parallels that of the PI3K/Akt/mTOR axis and both pathways share
overlapping targets, such as Bclꢀ2ꢀassociated death promoter (BAD) and the protein synthesis
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,4
regulator eIF4EꢀBP1. Phosphorylation of BAD increases Bclꢀ2 activity and thus promotes cell
survival, whereas phosphorylation of eIF4EꢀBP1 releases eIF4E to promote mRNA translation
and cellular growth. The Pimꢀ1, ꢀ2, and ꢀ3 proteins are expressed at low levels in normal tissues
but overexpressed in hematologic and solid tumor malignancies including multiple myeloma,
1
acute myeloid leukemia, prostate cancer, and gastric and liver carcinomas. Pim triple knockout
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,6
mice are small but viable, fertile, and show subtle hematologic changes. Therefore, the
inhibition of the Pim kinases presents a promising clinical pathway to treat Pim overexpressing
forms of cancers.
The kinase domains of the Pim family are structurally unique in the hinge region of the
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ATPꢀbinding pocket, which contains a tertiary amide (proline) not found in other kinases. This
unique hinge region feature can be exploited to achieve selectivity against other protein kinases.
The three isoforms of Pim kinases share a high level of sequence homology within the family
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(>61%) and appear largely redundant in their oncogenic functions. Thus, a panꢀPim kinase
inhibitor is highly desirable. Among the three isoforms, Pimꢀ2 has the lowest K (~ 4 ꢀM) for
m
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ATP, about 100ꢀ and 10ꢀfold lower than those of Pimꢀ1 and Pimꢀ3, respectively. Hence, cell
active panꢀPim inhibitors have been more challenging to identify than Pimꢀ1 or Pimꢀ3 inhibitors.
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Many research groups have disclosed ATPꢀcompetitive Pim kinase inhibitors of diverse
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chemical scaffolds with a wide range of cell potency.
In particular, shown in Figure 1
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are the structures of three clinical molecules 27 (PIM447), 28 (SGIꢀ1776), and 29 (AZDꢀ
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16,17
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1208),
as well as 30, the macrocyclic inhibitor from Amgen. While basic Pim inhibitors 27
(Pimꢀ2 K = 18 pM) and 29 (Pimꢀ2 K = 160 pM) were very potent in enzymatic assays, they
i
i
displayed >1000ꢀfold enzyme to cell shift. On the other hand, neutral Pim inhibitor 30 had only
about 100ꢀfold enzyme to cell shift, but the flat molecule had limited solubility in aqueous
medium (40 and 295 ꢀM in PBS (pH 7.4) and FaSIF (pH 6.8), respectively. Herein, we describe
the development of a novel series of quinazolinoneꢀpyrrolopyrrolones as potent and selective
panꢀPim inhibitors that led to the identification of compound 17, which is orally efficacious in a
mouse xenograft model (KMSꢀ12 BM) of multiple myeloma.
Figure 1. Structures and potency of publicly disclosed Pim kinases inhibitors.
cpd
Pimꢀ1
Pimꢀ2
Pimꢀ3
KMSꢀ11 luc KMSꢀ12 BM
IC (ꢁM)
K (nM) K (nM) K (nM) EC (ꢁM)
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7 (PIM447)*
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8 (SGIꢀ1776)* 16
9 (AZD1208)* 0.017
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* Data from Reference 14; Data from Reference 18.
We recently disclosed a highly potent and selective series of 2ꢀarylꢀ6,7ꢀdihydroꢀ1Hꢀ
pyrrolo[3,2ꢀc]pyridinꢀ4(5H)ꢀones, exemplified by 1 (Scheme 1), as panꢀPim kinase
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inhibitors.
Compound 1 was potent in both biochemical (IC = 0.3 nM for both Pimꢀ1 and
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Pimꢀ2) and cellular (KMSꢀ12 BM, IC = 23 nM) assays. In addition, 1 was efficacious in a
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KMSꢀ12 BM multiple myeloma xenograft model in mice (78% TGI @ 50 mg/kg QD oral
dosing). Although 1 possessed desirable pharmacokinetic (PK) properties in rat (CL = 0.4
L/h/kg, V = 1.0 L/kg, T1/2 = 3.5 h, %F = 37), it had rapid intravenous (IV) clearance (CL = 3.4
SS
L/h/kg, over liver blood flow) and a short halfꢀlife (T1/2 = 0.6 h) in dog. Therefore, this
compound was not suitable for further advancement as safety studies in a nonꢀrodent species
(
such as dog) would have been difficult. We sought to improve the PK profile of this series while
maintaining the good potency in vitro and in vivo. The rat and dog plasma protein binding
unbound fractions (fu) of 1 were measured (using rapid equilibrium dialysis) to be 1.7% and
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.0%, respectively, which calculated the unbound clearance CL of 1 in rat and dog to be 23.5
u
L/h/kg and 56.6 L/h/kg, respectively. The intrinsic clearance (CL ) of 1 in rat liver microsomes
int
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(
RLM) and dog liver microsomes (DLM) was 189 and 203 ꢁL/min/mg, respectively. The CLint
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of 1 in rat hepatocytes and dog hepatocytes was 39 and 32 ꢀL/min/10 cells, respectively. The in
vitro and in vivo pharmacokinetics data of 1 in rat and dog appeared to be poorly correlated.
Although metabolic pathways besides hepatic oxidations might be involved, we decided to look
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for clues in the microsomal stability first. Metabolite identification studies showed that when 1
was incubated with RLM, compound 2 (derived from hydroxylation of the t-butyl group) was the
major metabolite. When 1 was incubated with DLM, compound 3 (formed from dehydrogenation
of the 6,7ꢀdihydroꢀ1Hꢀpyrrolo[3,2ꢀc]pyridinꢀ4(5H)ꢀone) was the major metabolite. With this
information in hand, we prepared a series of analogs designed to block these oxidations, focusing
initially on replacement of the 6,7ꢀdihydroꢀ1Hꢀpyrrolo[3,2ꢀc]pyridinꢀ4(5H)ꢀone with a
metabolically more stable bicycle and, subsequently, on the leftꢀhand portion of the molecule,
modifying the aminoꢀquinoxaline.
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Scheme 1. In vitro metabolism of compound 1.
F
F
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O
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N
N
RLM
DLM
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OH
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Pim-1 IC50
Pim-2 IC50
0.9 nM
0.3 nM
Pim-1 IC50
Pim-2 IC50
0.3 nM
0.3 nM
Pim-1 IC50
Pim-2 IC50
0.2 nM
0.1 nM
KMS-12 BM IC50 143 nM
KMS-12 BM IC50 23 nM
KMS-12 BM IC50 148 nM
RESULTS AND DISCUSSION
In Vitro SAR. In biochemical assays, the compounds were evaluated for their ability to
inhibit the phosphorylation of a BAD peptide at Serine 112 (S112) by fullꢀlength recombinant
human Pimꢀ1 and Pimꢀ2 proteins. Our experience across multiple scaffolds in the Pim program
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2,23
had been that Pimꢀ3 activity was in general comparable to that of Pimꢀ1.
Therefore, the Pimꢀ
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enzyme assay was not routinely performed. In cellꢀbased assays, the compounds were
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measured for their ability to inhibit the phosphorylation of BAD at S112 in KMSꢀ12 BM cells in
the presence of 20% fetal bovine serum (FBS) using flow cytometry. KMSꢀ12 BM is a multiple
myeloma cell line with endogenous Pimꢀ1, ꢀ2, and ꢀ3 proteins, and a relatively high level of Pimꢀ
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expression. For an initial assessment of metabolism, the compounds were evaluated in RLM
and HLM, and the data, reported as intrinsic clearance (CL ), were used to prioritize potent
int
compounds for additional PK studies.
In the initial phase of this investigation, we focused our SAR work on the rightꢀhand
portion of compound 1, searching for a more metabolically stable replacement for the 6,7ꢀ
dihydroꢀ1Hꢀpyrrolo[3,2ꢀc]pyridinꢀ4(5H)ꢀone motif. As observed previously in our SAR
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development for compound 1, the C7ꢀF in 1 offered about 3ꢀfold improvement in enzyme and
cellular potency over a C7ꢀH in 4 (structure and data are shown in Table 1), whereas in vitro and
in vivo metabolic stability in rats was comparable. For this part of SAR investigation, to
facilitate syntheses, we elected to keep the leftꢀhand portion of the analogs constant as 3ꢀ(tertꢀ
butylamino)ꢀ2ꢀmethylquinoxalinꢀ5ꢀyl and compare their data to that of compound 4, then make a
C7ꢀF analog on an optimized right hand piece. Moving the pyrroleꢀnitrogen in 4 to the
bridgehead position in 5 (KMSꢀ12 BM, IC50 = 4.56 ꢀM, RLM CLint = 205 ꢀL/min/mg), or
homologating the [5,6]ꢀfused bicycle in 4 to the [5,7]ꢀfused bicycle in 6 (KMSꢀ12 BM, IC =
5
0
1
.63 ꢀM, RLM CL = 169 ꢀL/min/mg) resulted in much weaker cellular potency and higher rat
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microsomal turnover when compared to 4 (KMSꢀ12 BM, IC50 = 110 nM, RLM CLint = 86
L/min/mg). Contraction of the [5,6]ꢀfused bicycle in 4 to the [5,5]ꢀfused bicycle gave rise to
ꢀ
1
2
compound 7, and through substitutions at C6’ (R and R ), a set of 5,6ꢀdihydropyrrolo[3,4ꢀ
b]pyrrolꢀ4(1H)ꢀones, 8, 9, 10, 11, and 12. Compared to 4, the 5,6ꢀdihydropyrrolo[3,4ꢀb]pyrrolꢀ
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(1H)ꢀones 7, 8, 9, 10, and 11 have improved cellular potency, with pBAD IC values of 44, 65,
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0, 22 and 78 nM, respectively. Interestingly, between the set of enantiomers 8 and 9, the (R)ꢀ
’ꢀMe enantiomer (9) was not only more potent in both enzymatic and cellꢀbased assays, but also
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had lower microsomal turnover. The [5,5]ꢀfused bicycles, 7, 9, 10, and 11, were further profiled
in rat IV/PO PK. Among these four compounds, the C6’ꢀunsubstituted analog 7 had the highest
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RLM turnover (CL = 96 ꢀL/min/mg) and the highest IV clearance (1.6 L/h/kg). The (R)ꢀ6’ꢀ(2ꢀ
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aminoethyl) analog 10 had poor rat PK profile, with moderate clearance (CL = 1.4 L/h/kg), high
volume of distribution (Vss = 21.2 L/kg) and low bioavailability (%F <1), a feature common to
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other basic amineꢀderived Pim inhibitors from our Pim program. In rat PK, both compound 9
CL = 0.5 L/h/kg, %F = 62) and 6ꢀspirocyclopropane 11 (CL = 0.6 L/h/kg, %F = 100) had
(
similar IV clearance to that of 4 (CL = 0.7 L/h/kg, %F = 6), but with much higher oral
bioavailability. Expansion of the 6ꢀspirocyclopropane in 11 (Pimꢀ2 IC = 0.5 nM, KMSꢀ12 BM
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IC = 78 nM) to a 6ꢀspiropyran in 12 (Pimꢀ2 IC = 117 nM, KMSꢀ12 BM IC = 5.76 ꢀM)
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resulted in a significant loss of potency. Chlorination at pyrroleꢀC3’ in 9 (KMSꢀ12 BM, IC =
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0 nM, RLM CL = 50 ꢀL/min/mg) provided 13 (KMSꢀ12 BM, IC = 407 nM, RLM CL =
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143 ꢀL/min/mg) that had weaker potency and higher microsomal turnover. Lastly, the
replacement of pyrrole in 7 with a thiophene gave rise to 5,6ꢀdihydroꢀ4Hꢀthieno[2,3ꢀc]pyrrolꢀ4ꢀ
one 14 and 4Hꢀthieno[2,3ꢀc]pyrrolꢀ6(5H)ꢀone 15, which displayed reduced potency and
microsomal stability when compared to pyrrole 7.
Table 1. Potency, microsomal stability, and rat PK data of compounds 1, 4ꢀ15, and 26.
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c
d
Pimꢀ1 Pimꢀ2 KMSꢀ12
rat IV / PO PK
Vss T1/2
(L/h/kg) (L/kg) (h)
R / HLM CLint
2
cpd
R
IC50
IC50
BM IC50
CL
b
(µL/min/mg)
%F
a
a
a
(nM) (nM)
(nM)
e1
1
4
0.3
0.8
0.3
0.6
23
189 / 66
86 / 70
0.4
0.7
1.0
4.2
3.5
4.7
37
e2
110
6
5
6
7
6.6
2.6*
1.2
6.1
8.1*
0.9
4560
1635
44
205 / 85
169 / 67
96 / 45
h
NT
NT
f
1.6
2.3
1.7
36
8
9
1.6
0.7
1.8
1.0
65
30
130 / 89
50 / 37
NT
f
0.5
1.3
3.0
62
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g
1
0
1
0.6
0.4
0.3
0.5
22
<14 / <14
1.4
0.6
21.2
1.3
17.5
4.5
<1
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
f
1
78
52 / 35
100
*
*
12
44
117
5760
45 / <14
NT
1
3
0.5
6.4
1.0
6.8
407
143 / 111
NT
14
2870
326 / 102
373 / 104
NT
NT
1
2
5
6
2.5
0.1
2.5
0.1
1380
29
f
52 / 35
0.5
1.6
2.9
54
a
*
Average of at least 2 determinations unless indicated by (n = 1), for full statistical information,
b
see the Supporting Information; In vitro microsomal stability (n = 1) measurements were
conducted in the presence of NADPH at 37 °C for 30 min at a final compound concentration of 1
c
d
e1
ꢀ
M; dosed at 2 mg/kg, vehicle 100% DMSO; dosed at 5 mg/kg; vehicle 10% Soluplus, 40%
e2
PEG 400, 50% DI water/MSA, pH 2.2; vehicle 15% HPBCD, 2% HPMC, 1% Pluronic F68,
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
f
g
pH 2.2 water/MSA; vehicle 40% PEG 400 in DI Water, pH 2.2 water/MSA; vehicle 1% Tween
h
8
0, 2% HPMC, pH 2.2 water/MSA; NT = not tested.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
From Table 1, in terms of cellular potency and rat PK profile, the best replacement for the
[
5,6]ꢀfused bicycle in 4 is the (R)ꢀ6’ꢀMeꢀ5,6ꢀdihydropyrrolo[3,4ꢀb]pyrrolꢀ4(1H)ꢀone motif in 9.
Further substitution of a C7ꢀF in 9 provided analog 26, which exhibited improved enzyme
potency (IC = 0.1 nM for both Pimꢀ1 and Pimꢀ2) but similar cellular potency (IC = 29 nM),
5
0
50
and rat PK profile (IV CL = 0.5 L/h/kg, %F = 54). Compounds 9 and 26, with DLM of 88 and
1
05 ꢀL/min/mg, respectively, were further profiled in dog IV PK (dosing: 0.3 mg/kg; vehicle:
0% PEG 400 and 50% water). Although 9 (CL = 1.6 L/h/kg, Vss = 1.7 L/h, T1/2 = 0.6 h) and 26
5
(CL = 1.5 L/h/kg, Vss = 1.7 L/h, T1/2 = 1.1 h) had improved dog IV PK properties over
compound 1 (CL = 3.4 L/h/kg, Vss = 8. 7 L/h, T1/2 = 0.6 h), their IV CL values were still high.
Metabolite identification studies of compound 9 in dog hepatocytes showed that there was
extensive hydroxylation on the quinoxaline bicycle. Subsequently, we focused our SAR work on
the leftꢀhand portion of 9, seeking a replacement for the aminoꢀquinoxaline motif to improve in
vivo stability while maintaining the desirable Pim potency. Our strategy was to focus on bicycles
with reduced electron density relative to the aminoꢀquinoxaline scaffold in 9.
Table 2 summarizes our efforts to replace the aminoꢀquinoxaline in compound 9 while
keeping the rightꢀhand portion constant as (R)ꢀ6’ꢀMeꢀ5,6ꢀdihydropyrrolo[3,4ꢀb]pyrrolꢀ4(1H)ꢀ
one. Compared to 9, pyridoꢀpyrimidinone 16 had weaker potency (Pimꢀ2 IC = 2.1 nM, KMSꢀ
5
0
1
2 BM IC50 >2000 nM) and lower solubility in aqueous medium. Interestingly, the
regioisomeric quinazolinone 17 had not only improved potency (Pimꢀ2 IC = 0.2 nM, KMSꢀ12
50
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5
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7
8
9
BM IC = 31 nM) but also greater aqueous solubility. Compared to the C7ꢀH analog 17, the
5
0
C7ꢀF analog 18 displayed marginally improved enzyme potency (IC = 0.1 nM for both Pimꢀ1
50
and Pimꢀ2) but similar cellular potency (IC = 26 nM); on the other hand, the Nꢀ7 analog 19
5
0
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
displayed greatly reduced enzyme (Pimꢀ2 IC = 7.4 nM) and cellular potency (IC = 1642 nM),
5
0
50
as did the CH ꢀlinker analog 20 (Pimꢀ2 IC50 = 4.9 nM, KMSꢀ12 BM IC50 = 5690 nM).
2
Compared to 9, benzoꢀthiadiazineꢀdioxide 21 had improved aqueous solubility but reduced
enzyme potency, with Pimꢀ1 and Pimꢀ2 IC of 6.5 and 5.2 nM, respectively.
5
0
Next we probed the SAR surrounding the tꢀbutylamino motif in compound 17 with
alkylaminoꢀquinazolinones 22ꢀ25 (while keeping cLogP <4). Although the structural difference
was very subtle between 22 and 17, the methylcyclopropylamino 22 had better cellular potency
(IC = 11 nM) than the tꢀbutylamino analog 17 (IC = 31 nM) and was 3 times more soluble in
5
0
50
FaSIF (pH 6.8) medium. Hydroxylation of the tꢀbutyl group in 17 provided compound 23, which
displayed a 4ꢀfold loss of potency in enzymatic and cellular assays. The tertiary amino
quinazolinones, tꢀbutylmethylamino analog 24 and 2,2ꢀdimethylpyrrolidineꢀderived 25 were 5ꢀ
fold and 3ꢀfold weaker, respectively, in both enzymatic and cellular assays than 17.
The compounds in Table 2 displayed a wide range of enzyme (Pimꢀ2 IC ) to cell (KMSꢀ
5
0
12 BM IC ) shift, from 30ꢀfold for compounds 9 and 22 to >1000ꢀfold for compounds 16 and
5
0
20. Compounds 16 and 20 had no aqueous solubility which may explain the extremely large cell
shift. On the other hand, 9 and 22 were among the more aqueous soluble compounds in the
Table. There appeared to be little correlation between cell shift and calculated lipophilicity
(cLogP).
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Potency, cLogP, and aqueous solubility for compounds 9, 16ꢀ25.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
c
IC (nM)
solubility (ꢀM)
5
0
b
cLogP
PBS
pΗ 7.4)
99
FaSIF
(pΗ 6.8)
245
0
cpd
9
Pimꢀ1
Pimꢀ2 KMSꢀ12 BM
(
0.7
3.1
0.3
0.1
4.5
7.3
6.5
0.2
0.7
1.5
1.0
2.1
0.2
0.1
7.4
4.9
5.2
0.4
0.9
1.0
30
>2000
31
4.0
2.8
2.6
2.8
2.1
3.2
2.0
2.3
1.3
2.8
1
6
0
17
60
104
23
1
1
2
2
8
9
0
1
26
14
1642
5960
1142
11
15
40
0
9
111
56
313
344
134
122
22
2
2
3
4
127
167
101
61
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9
2
5
0.9
0.6
88
2.7
20
35
a
Average of at least 2 determinations, for full statistical information, see the Supporting
b
Information; cLogP values were calculated via a Pipeline Pilot Web Port interface (Biovia, Inc.)
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
c
to the Daylight software suite (v. 4.81, Daylight Chemical Information Systems, Inc.); n = 1 for
pH solubility data, PBS = phosphateꢀbuffered saline, FaSIF = fasted state simulated intestinal fluid
.
From Table 2, the aminoꢀquinazolinones 17, 18, and 22, with cellular IC <50 nM, were
5
0
evaluated in a panel of in vitro assays and in vivo pharmacokinetics and drug metabolism
PKDM) tests. The results are shown in Table 3. All 3 compounds had rat plasma protein free
(
unbound fractions of about 6%, and appeared to be Pgp substrates as judged by the efflux ratio
(ER) in LLCꢀPK1 cells overexpressed with rat and human Pꢀglycoproteins. However, 17 stood
ꢀ6
out from the group as having the best permeability (Papp = 44 x 10 cm/s), the lowest rat IV
clearance (0.3 L/h/kg) and the highest oral bioavailability (%F = 39). Therefore, 17 was
subjected to additional PKDM and safety profiling. It had moderate dog IV clearance (0.8
L/h/kg) with Vss of 1 L/kg and T1/2 of 0.7 h, and good oral bioavailability (44%). The dog
plasma protein binding unbound fractions (fu) of 17 was measured (using rapid equilibrium
dialysis) to be 11%. The unbound clearance CL of 17 in dog was 7.3 L/h/kg, which was
u
significantly lower than that of our starting point for this series, the quinoxalineꢀpyrroloꢀ
pyridinone 1 (dog IV CL = 56.6 L/h/kg). Compound 17 had intrinsic clearance (CL ) of 12, 12
u
int
6
and 3 ꢀL/min/10 cells, in rat, dog and human hepatocytes, respectively. With the low turnover
in microsomes and hepatocytes across species (rat, dog and human), along with low IV clearance
in rat and moderate IV clearance in dog, compound 17 had a low projected human clearance
25
(0.034 L/h/kg, ~ 3% of liver blood flow).
In addition, 17 had minimal inhibition towards
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CYPs (with IC values of >50, >50, 31 and >50 ꢀM for 3A4, 1A2, 2C19 and 2D6, respectively),
5
0
2
6
and no activity for human Pregnane X Receptor (hPXR) at 10 ꢀM.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. In vitro and in vivo PKDM data for compounds 17, 18, and 22.
c
d
permeability
PPB
fu (%)
R / H
rat IV /PO PK
R /H LM
CLint
a
Papp
Pgp (ER)
CL
Vss T1/2
F
cpd
b
ꢀ6
(µL/min/mg)
(10 cm/s) Parental / R / H
(L/h/kg) (L/kg) (h) (%)
1
1
7
8
2
44
16
29
1.1 / 7.8 / 8.6
2.4 / 20.3 / 23.3 6.2 / NT
1.2 / 5.9 / 11.8 6.1 / NT
6.1 / 7.9
<14 / <14
16 / <14
49 / 20
0.3
0.4
2.7
1.5
1.4
1.9
4.6 39
4.3 10
e
2
0.6
5
a
Determination of fraction unbound (fu) in plasma protein using rapid equilibrium dialysis (RED
b
(compound concentration @ 5 ꢀM) (n = 1), R = rat, H = human; In vitro microsomal stability (n
=
1) measurements were conducted in the presence of NADPH at 37 °C for 30 min at a final
c
d
compound concentration of 1 ꢀM; dosed at 2 mg/kg, vehicle 100% DMSO; dosed at 5 mg/kg;
e
NT = not tested.
In a hERG binding assay, 17 had an IC of >30 ꢀM. For broader selectivity against
50
receptors and ion channels, 17 was tested at 10 ꢀM against 147 targets provided by Cerep, with
2
7
only one target showing percent of control (POC) <10%. For kinase selectivity, 17 was tested
at 1 ꢀM in a customized KINOMEscan panel containing 100 nonꢀmutant kinases offered by
DiscoveRx. The S(35), S(10), and S(1) selectivity scores were determined to be 0.07, 0.06, and
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9
0
.03, respectively. At 1 ꢀM, 17 hit 6 kinases with POC <10%: CLK4 (0.2%), YSK4 (0.4%),
p38γ (0.9%), Pimꢀ1 (1.3%), CSNK2A1 (3.5%), and DYRK1A (6%). For panꢀPim kinase
activities, the K values of 17 for Pimꢀ1, Pimꢀ2 and Pimꢀ3 were determined by DiscoveRx to be
d
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
2
8
1
.3, 1.7, and 1.2 nM, respectively.
The Xꢀray structure of 17 bound to Pimꢀ1 protein (PDB code 5IPJ) was obtained and is
presented in Figure 2. This structure reveals a number of key interactions between 17 and Pimꢀ1
protein, which contribute to its potency and kinase selectivity. The high kinome selectivity
found in 17 is largely due to the unique tertiary amide Pro123 as the linker residue in Pimꢀ1 as it
accommodates the C5ꢀH in quinazolinone. Two water molecules bridge the carbonyl oxygen
(C4=O) in 17 and the carbonyl oxygen of Pro123, which are 4.7 Å apart. The C6ꢀH in 17 forms a
Hꢀbonding (3.1 Å) interaction with the carbonyl oxygen of Glu121. Additional favorable
hydrophobic contacts are observed between the (R)ꢀC6’ꢀMe in the [5,5]ꢀfused ring with the
Phe49 and Ile185 (shown in Figure 2b); and the tꢀbutyl group fills a small lipophilic pocket
under the Pꢀloop of Pimꢀ1. The carbonyl oxygen (C4’=O) of pyrrolone engages in a Hꢀbonding
(
2.8 Å) with the conserved catalytic Lys67. The dihedral angle between the pyrrole and
quinazolinone is 0 degrees – the combination of two factors, the intramolecular Hꢀbond from the
pyrroleꢀNH to the quinazolinoneꢀN1 and the relatively small size of a 5ꢀmembered pyrrole ring,
helps to keep it coꢀplanar with respect to the quinazolinone scaffold.
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Xꢀray structure of 17/Pimꢀ1 (PDB code 5IPJ). a) Inhibitor 17 occupies the ATPꢀ
binding site; hydrogen bond contacts are shown with dashed lines; the red dots represent water
molecules; the top of the ATPꢀbinding pocket is omitted for clarity. b) CPK (CoreyꢀPaulingꢀ
Koltun) rendering of 17 illustrating that the alkyl substituents form a complementary
hydrophobic surface to Leu44, Gly45, Val52, Phe49, and Ile185. The figures were generated
with the PyMOL Molecular Graphics System, Version 1.7.0.1 Schrödinger, LLC.
In a 3ꢀday luminescent KMSꢀ12 BM cell viability assay, compound 17 had an IC value of
50
260 nM. Given the promising potency, kinase selectivity, and metabolic profile of 17, it was
progressed to a mouse KMSꢀ12 BM xenograft model (Figure 3). Dosed orally once daily (QD)
at 10, 25, and 50 mg/kg, 17 resulted in a doseꢀdependent decrease in tumor growth compared to
the vehicle control group, with 50 mg/kg dose achieving 93% tumor growth inhibition (TGI).
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9
There was no appreciable difference in TGI between 50 and 100 mg/kg doses, probably due to
the solubility limited exposure at the top dose of 100 mg/kg (Figure 3b). No overt toxicity was
observed based on body weight and overall appearance of the treated animals. On the last day of
the xenograft study, a terminal pharmacodynamic (PD) study was conducted to measure the
effect of 17 on the inhibition of BAD phosphorylation. In the PD models, we typically observed
a maximum inhibition of 60-70% pBAD for selective Pim kinase inhibitors; the remainder of
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
8,29
BAD phosphorylation was attributed to AKT (PKB).
00 mg/kg dose, there was a doseꢀ and exposureꢀdependent decrease in pBAD levels, with 50
mg/kg dose achieving nearꢀmaximal suppression for at least 12 h. The EC for pBAD inhibition
As shown in Figure 4, except for the
1
5
0
was determined to be 254 nM of total plasma 17. As the fraction unbound of 17 in mouse plasma
protein is 0.084, this corresponds to an unbound plasma concentration of 21.4 nM, similar to the
measured in vitro KMSꢀ12 BM pBAD IC of 31 nM (Table 2).
5
0
1
0
a)
1200
1000
% TGI
b)
Vehicle
10 mg/kg
30%
65%
93%
95%
1
25 mg/kg
800
50 mg/kg
100 mg/kg
600
0.1
*
4
00
00
0
2
*
*
0.01
R
x
0
5
10
15
20
Time (h)
15
20
25
30
= p < 0.0001
1
0 mg/kg N/A(V1)
0 mg/kg N/A(V1)
25 mg/kg N/A(V1)
100 mg/kg N/A(V1)
Time (day)
*
5
Figure 3. (a) The effect of 17 on tumor growth in mouse KMSꢀ12 BM xenograft model (left);
b) The concentration of 17 in plasma (right). Data represent the mean (n = 10) ± SEM for the
(
xenograft study and (n = 2/time point) for the concentration of 17 in plasma. Statistical
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9
1
1
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
significance (*) was determined by repeated measures analysis of variance (RMANOVA)
followed by Dunnet’s post hoc.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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Figure 4. The effect of 17 on BAD phosphorylation in terminal KMSꢀ12 BM xenograft tumors.
Data represent the mean (n = 2) ± standard deviation. Bars represent levels of pBAD and are
expressed as percent of vehicle (POV) of pBAD/total BAD (tBAD). Red dots represent the
mean total plasma concentration of 17. Statistical significance was evaluated by analysis of
variance (ANOVA), followed by the Bonferroni/Dunn post hoc test. * p <0.0001, ** p <0.001,
*** p <0.05.
CHEMISTRY
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The structures of four commonly used intermediates are shown in Scheme 2: 8ꢀbromoꢀNꢀ
tertꢀbutyl)ꢀ3ꢀmethylquinoxalinꢀ2ꢀamine (AQ1), Nꢀ(tertꢀbutyl)ꢀ3ꢀmethylꢀ8ꢀ(4,4,5,5ꢀtetramethylꢀ
(
1,3,2ꢀdioxaborolanꢀ2ꢀyl)quinoxalinꢀ2ꢀamine
(AQ2),
2ꢀbromoꢀ1ꢀ(3ꢀ(tertꢀbutylamino)ꢀ2ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
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23
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25
26
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52
53
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55
56
57
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59
60
methylquinoxalinꢀ5ꢀyl)ethanone (AQ3), and pyrrolopyrrolone boronic ester BE1. The syntheses
1
8,19
of intermediates AQ1, AQ2, and AQ3 have been disclosed.
For the synthesis of boronic
ester BE1, the chemistry towards pyrrolopyrrolone fused bicycle previously reported by Vanotti
3
0
and coꢀworkers was modified. It is worth pointing out that erosion of chiral purity occurred
during the removal of tosyl protecting group in BE1e using K CO in MeOH. Chiral
2
3
supercritical fluid chromatography (SFC) was used to purify the crude product (74% ee) and
provided pyrrolopyrrolone BE1f in >99% ee. Subsequent C2ꢀH activation of BE1f in the
presence of iridium (I) catalyst and (BPin) afforded the boronic ester BE1, which was used as
2
crude without further purification.
Scheme 2. Structures of aminoꢀquinoxaline intermediates AQ1, AQ2, and AQ3, and synthesis of
boronic ester BE1.
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0
a
Reagents and conditions: (a) 1) CDI, THF; 2) monoꢀtꢀbutyl malonate, MgCl , KOtBu, 75%; (b)
2
NH OAc, NH in MeOH, chloroacetaldehyde, 63%; (c) NaOtBu, pTsCl, THF, 70%; (d) TFA,
4
3
DCM, 50 °C; (e) T3P, DIPEA, EtOAc >95% (2 steps); (f) K CO , MeOH; SFC chiral
2
3
purification, 73%; (g) (BPin) , bis(1,5ꢀcyclooctadiene)diꢀ
ꢀꢀmethoxydiiridium(I), 4,4'ꢀdiꢀtꢀbutylꢀ
2
2,2'ꢀdipyridyl, MTBE.
In Scheme 3, the synthetic routes to compounds 5 and 6 are outlined. Commercially
available 5a was alkylated with 1,2ꢀdibromoethane to give 5b which was treated with (2,4ꢀ
dimethoxyphenyl)methanamine to provide pyrrolopyrazinone 5c. Commercially available 6a
was brominated with 1,3ꢀdibromoꢀ5,5ꢀdimethylhydantoin to afford 2ꢀbromoꢀ6,7ꢀdihydroꢀ1Hꢀ
indolꢀ4(5H)ꢀone (6b) which was treated with hydroxylamine hydrochloride/NaOAc followed by
thionyl chloride to afford the [5,7]ꢀfused bicycle 6c. The Suzuki coupling reactions of boronic
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ester AQ2 (structure shown in Scheme 2) with 5c (followed by protecting group removal) and 6c
provided compounds 5 and 6, respectively.
Scheme 3. Synthesis of compounds 5 and 6.
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60
a
Reagents and conditions: (a) NaH, DMF, 1,2ꢀdibromoethane, 0ꢀ70 °C, 46%; (b) (2,4ꢀ
dimethoxyphenyl)methanamine, DMSO, 150 °C, 85%; (c) AQ2, Pd (dba) , XꢀPhos, K PO , 135
2
3
3
4
°C; (d) TFA, DCM, 22% (2 steps); (e) 1,3ꢀdibromoꢀ5,5ꢀdimethylhydantoin, DMF, 60 °C, 83%;
f) hydroxylamine hydrochloride, NaOAc, MeOH, 50 °C; (g) SOCl , 55% (2 steps); (h) AQ2,
(
2
Pd (dba) , XꢀPhos, K PO , 105 °C, 96%.
2
3
3
4
The general synthetic route to aminoquinoxalineꢀpyrrolopyrrolones 7ꢀ12 is shown in
Scheme 4A. Alkylation of βꢀketo ester 7a and 9aꢀ12a with 2ꢀbromoꢀ1ꢀ(3ꢀ(tertꢀbutylamino)ꢀ2ꢀ
methylquinoxalinꢀ5ꢀyl)ethanone (AQ3) and subsequent ring formation in the presence of
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6
AcONH /HOAc in EtOH afforded pyrroles 7b and 9bꢀ12b, with erosion of chirality for 9b and
4
10b. Saponification of 7b and 9bꢀ12b with LiOH followed by Boc removal with HCl provided
the oꢀaminomethylꢀpyrroleꢀcarboxylic acids 7c and 9cꢀ12c, which were treated with an
activating agent (PyBop or HATU or EDCI) to form the pyrroloꢀpyrrolones 7, 9d, 10d, 11 and
12. Chiral SFC purification of 9d (about 85% ee) provided (S)ꢀMe 8 (minor) and (R)ꢀMe 9
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0
(major). Benzyl ether 10d was converted to amine 10e (>90% ee) in a 4ꢀstep sequence: removal
of benzyl protecting group with BCl followed by conversion of the resulting alcohol to a
3
methanesulfonate, azidation, and reduction with PMe . Amine 10e (about 90% ee) was subjected
3
to chiral SFC purification to afford (R)ꢀCH CH NH 10 (99% ee). Lastly, compound 13 was
2
2
2
derived from the treatment of 9 with Nꢀchlorosuccinimide.
Scheme 4A. Synthesis of aminoquinoxalineꢀpyrrolopyrrolones 7ꢀ13.
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a
o
Reagents and conditions: (a) AQ3, K CO , THF, EtOH, RT; (b) AcONH , EtOH, AcOH, 50 C;
2
3
4
(
c) LiOH then HCl; (d) PyBop or HATU or EDCI, DIPEA; (e) 1) BCl ; 2) MsCl, Et N; 3) NaN ;
3 3 3
4
) PMe ; (f) NCS, CHCl , 70%.
3 3
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6
As the program progressed, we found a more convergent route to prepare the
aminoquinoxalineꢀpyrrolopyrrolones, exemplified by the synthesis of 26 shown in Scheme 4B.
Compound 26 was derived from the Suzuki coupling reaction of chiral boronic ester BE1 and
bromoꢀquinoxaline 26g, which in turn was synthesized from the commercially available 2ꢀ
bromoꢀ1,3ꢀdifluoroꢀ4ꢀnitrobenzene (26a) in six steps.
0
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0
Scheme 4B. Synthesis of aminoquinoxalineꢀpyrrolopyrrolone 26.
a
Reagents and conditions: (a) (NH ) CO , Et N, DMF, 97%; (b) Pt/C, H , EtOH, 99%; (c)
4 2
3
3
2
o
o
o
ethyl 2ꢀbromopropionate, 120 C, 96%; (d) H O , NaOH, 85 C, 80%; (e) POCl , 90 C, 49%; (f)
2
2
3
o
tꢀbutylamine, DMSO, 100 C, 83%; (g) BE1, XPhos Pd G2, K PO , 54%.
3
4
In Scheme 5, 2ꢀformylthiopheneꢀ3ꢀcarboxamide 14a was converted to thienopyrrolone
1
4b via a 3ꢀstep sequence: reduction with NaBH followed by mesylate formation with MsCl
4
and pyrrolone formation in the presence of LiHMDS. Bromination of 14b with NBS followed
by removal of the tꢀbutyl group with acid gave bromoꢀthienopyrrolone 14d. In a similar fashion,
o
1
5d was constructed, except that the removal of tꢀbutyl group required high temperature (130 C
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in microwave).
The Suzuki coupling reactions of boronic ester AQ2 with bromoꢀ
thienopyrrolones 14d and 15d gave quinoxalineꢀthienopyrrolones 14 and 15, respectively.
Scheme 5. Synthesis of compounds 14 and 15.
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12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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O
O
O
O
N
H
c
a
b
N
Br
N
Br
NH
S
S
S
S
CHO
1
4a
14b
14c
14d
O
N
d
N
S
NH
HN
1
4
O
O
O
O
S
S
S
S
N
a
e
f
N
Br
N
Br
NH
H
CHO
1
5a
15b
15c
15d
S
O
N
d
N
NH
HN
15
a
Reagents and conditions: (a) 1) NaBH ; 2) MsCl, DIPEA; 3) LiHMDS, 77% for 14b and 60%
4
for 15b; (b) NBS, 34%; (c) H SO , 25%; (d) AQ2, Pd (dba) , XꢀPhos, K PO , 26% for 14 and
2
4
2
3
3
4
5
2% for 15; (e) Br , HOAc; (f) H SO , 130 °C, 68% for 2 steps.
2 2 4
In Scheme 6, compounds 16, 19, 20 and 21 were derived from the Suzuki coupling
reaction of boronic ester BE1 with bromoꢀpyridopyrimidinone 16b, chloroꢀpyridopyrimidinone
19b, bromoꢀquinazolinone 20b, and bromoꢀbenzothiadiazineꢀ1,1ꢀdioxide 21e, respectively.
Given that boronic ester BE1 decomposed readily at high temperatures, this type of Suzuki
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5
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5
5
5
5
5
6
o
coupling reaction was typically run at 40ꢀ45 C in an oil bath for 1ꢀ3 hour(s) in the presence of
XPhos Pd G2, in yields ranging from 21% to 71%.
Scheme 6. Synthesis of compounds 16, 19, 20 and 21.
0
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9
0
a
o
Reagents and conditions: (a) diethyl methylmalonate, 220 C; (b) BOP, tꢀbutyl amine, DBU,
2
1%; (c) BE1, XPhos Pd G2, K PO , 40% for 16, 21% for 19, 62% for 20, 71% for 21; (d)
3
4
triphosgene, MeNH , 44%; (e) BOP, tꢀbutyl amine, 41%; (f) T3P, CH NH , 62%; (g) tꢀ
2
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2
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o
butylacetyl chloride
, 100 C, quant.; (h) NaNO , concentrated HCl, CuSO ; (i) CH NH , DIPEA, 26%
2
4
3
2
for 2 steps; (j) Pt/C, H , MeOH, 95%; (k) t-butyl nitrite, TMSN , MeCN, 20%; (l) PPh , t
-
2
3
3
o
butyl isocyanate, DBU, 120 C, 45%.
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In Scheme 7, commercially available oꢀaminoꢀbenzoic acids 17a and 18a were converted
to chloroꢀquinazolinones 17d and 18d, respectively, via methyl amide formation in the presence
of T3P, followed by hydroxyꢀquinazolinone formation using triphosgene, and chlorination using
POCl . Displacement of the C2ꢀCl in 17d and 18d with amines gave aminoꢀquinazolinones 17e,
3
18e, and 25e, which were coupled with boronic ester BE1 to afford compounds 17, 18, and 25,
respectively. Alternatively, oꢀaminoꢀbenzoic acid 22a was converted to chloroꢀquinazolinone
22d via sulfinylꢀ/sulfonylꢀquinazolinone formation, followed by hydrolysis with LiOH and
chlorination using POCl . In a similar fashion, displacement of the C2ꢀCl in 22d with amines
3
gave aminoꢀquinazolinones 22e, and 23e, which were coupled with boronic ester BE1 to afford
compounds 22 and 23, respectively. The displacement of C2ꢀCl in 22d with N,2ꢀ
dimethylpropanꢀ2ꢀamine was not successful, probably due to the steric hindrance of the amine.
Therefore, aminoꢀquinazolinone 24f was prepared from the reaction of 22d with tꢀbutylamine
followed by methylation of the resulting 24e with NaH/MeI. Finally, the Suzuki coupling of 24f
with boronic ester BE1 afforded compound 24.
Scheme 7. Synthesis of compounds 17, 18, and 22ꢀ24.
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