Discovery of 8-Methyl-pyrrolo[1,2-a]pyrazin-1(2 H)-one Derivatives as Highly Potent and Selective Bromodomain and Extra-Terminal (BET) Bromodomain Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b01784

The bromodomain and extra-terminal (BET) family proteins have recently emerged as promising drug targets for cancer therapy. In this study, identification of an 8-methyl-pyrrolo[1,2-a]pyrazin-1(2H)-one fragment (47) as a new binder to the BET bromodomains

Full text of DOI:10.1021/acs.jmedchem.9b01784

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Article
Discovery of 8-Methyl-pyrrolo[1,2-a]pyrazin-1(2H)-one
Derivatives as Highly Potent and Selective Bromodomain
and Extra-terminal (BET) Bromodomain Inhibitors
Zizhou Li, Senhao Xiao, Yaxi Yang, Chao Chen, Tian Lu, Zhifeng
Chen, Hualiang Jiang, Shijie Chen, Cheng Luo, and Bing Zhou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01784 • Publication Date (Web): 24 Mar 2020
Downloaded from pubs.acs.org on March 26, 2020
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Discovery of 8-Methyl-pyrrolo[1,2-a]pyrazin-1(2H)-one
Derivatives as Highly Potent and Selective Bromodomain
and Extra-terminal (BET) Bromodomain Inhibitors
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Zizhou Li,a,b, § Senhao Xiao,b,c,d,§ Yaxi Yang,a,b Chao Chen,a,b Tian Lu,b,c Zhifeng
c
c
c
b,c,*
Bing Zhoua,b,*
Chen, Hualiang Jiang, Shijie Chen, Cheng Luo,
a
Department of Medicinal Chemistry, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi
Road, Shanghai 201203, China.
b
University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049,
China
c
Drug Discovery and Design Center, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi
Road, Shanghai 201203, China.
d
School of Life Science and Technology, Shanghai Tech University, Shanghai
2
00031, China.
§
These authors contributed equally.
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Abstract
The bromodomain and extra-terminal (BET) family proteins have recently emerged as
promising drug targets for cancer therapy. In this study, identification of a
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-methyl-pyrrolo[1,2-a]pyrazin-1(2H)-one fragment (47) as a new binder to the BET
bromodomains and subsequent incorporation of fragment 47 to the scaffold of
ABBV-075 which recently entered Phase I clinical trials, enabled the generation of a
series of highly potent BET bromodomain inhibitors. Further druggablity optimization
led to the discovery of compound 38 as a potential preclinical candidate.
Significantly, compared with ABBV-075 which exhibits a 63-fold selectivity for
BRD4(1) over EP300, compound 38 demonstrates an excellent selectivity for the BET
bromodomain family over other bromodomains, with a ~1500-fold selectivity for
BRD4(1) over EP300. Orally administered 38 achieves a complete inhibition of the
tumor growth with a TGI of 99.7% accompanied by a good tolerability.
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INTRODUCTION
Bromodomains are relatively small protein domains consisting of roughly 110
amino acid residues and serve as epigenetic “readers” to regulate gene expression
through binding to acetylated lysines on the histone proteins as well as other
proteins.1,2 The human genome encodes 61 bromodomains that are respectively
expressed in 46 human proteins and these proteins are divided in to eight BRD
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families based on phylogenetic/structural modeling. Among them, the bromodomain
and extra terminal (BET) proteins are the most prominent and consist of BRD2,
BRD3, BRD4 and BRDT, each containing its tandem domains (BD1 and BD2).
Mechanistically, BET proteins phosphorylate the RNA polymerase II and
facilitates pause release and transcript elongation by linking chromatin to the positive
5
transcription elongation factor b (p-TEFb). More importantly, the block of this
critical function of BET proteins leads to attenuated expression of key oncogenes such
as BCL-2 and especially MYC which is proven to be intractable previously.6-8
Recently, BET proteins have emerged as new therapeutic targets for various human
9
10
diseases and conditions, such as NUT midline carcinoma, MLL1-fusion leukemia,
castration-resistant prostate cancer,11 triple-negative breast cancer,12 inflammatory
1
3
14
15
diseases, lung fibrosis, and acute heart failure.
JQ-1 (1) was the first reported BET bromodomain inhibitor.9 This
groundbreaking discovery demonstrated the high druggability of the BET
bromodomains and motivated further drug development efforts in this area. Since
then, several BET bromodomain inhibitors with different chemotypes have been
discovered (Figure 1), as exemplified by the JQ-1 derivatives 3-6,16-20
,5-dimethylisoxazoles 710,21 and 10 , pyrimidone 8,
22
23,24
and ABBV075 (9) with
3
pyrrolopyridone binding motif.25,26 More significantly, some of them have progressed
into clinical trials, such as compounds TEN-010 (3), OTX-015 (4), I-BET762 (5),
CPI-0610 (6), RVX-208 (8) and ABBV-075 (9). Despite great progress, some BET
bromodomain inhibitors have exhibited a moderate selectivity profile over members
of other subfamilies, which may lead to a potential safety issue. For example,
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ABBV075 (9) that currently is undergoing Phase I clinical trials, is to date one of the
most potent BET bromodomain inhibitors with K value of 1.5 nM to BRD4.
d
However, ABBV075 has also exhibited a high off-target binding affinity to EP300
bromodomain with K value of 87 nM, presumably because these bromodomains
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possess long ZA loops with similar residues facing the KAc binding site. Therefore,
there remains a continuing need in the design of alternative BET bromodomain
inhibitors with higher selectivity for BET bromodomain family over other
bromodomains, and also with good drug-like properties.
N
N
N
N
N
N
O
N
N
N
N
S
S
R
N
R
MeO
N
NH
N
NH2
N
O
O
O
O
Cl
Cl
5 (I-BET762)
Cl
6 (CPI-0610)
Cl
1 (JQ-1), R = O Bu
(MS417), R = Me
t
3 (TEN-010), R = NH2
4 (OTX-015), R = HN
2
BRD4 (1) Kd = 50-61 nM
BRD4 (1) IC50 = 39 nM
OH
BRD4 (1) IC50 = 77 nM
BRD4 IC50 = 92-112 nM
N
N
O
O
H
H
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O
N
O
N
N
OH
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O
OMe
OMe
HN
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NH
O
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NH
N
O
O
S
N
F
F
O
N
H
N
10 (CF53)
8
(RVX-208)
9 (ABBV-075)
7
(I-BET151)
BRD4 (1) Kd = 2.2 nM
EP300 Kd = 110 nM
CREBBP Kd = 47 nM
BRD4 (2) IC50 = 40 nM
BRD4 (1) Kd = 1.5 nM
EP300 Kd = 87nM
BRD4 (1) IC50 = 794 nM
Figure 1. Representative BET inhibitors. The cores of compounds are marked in red
as the acetylated lysine mimetics.
In this study, by screening novel fragments as new binders to the BET
bromodomains and subsequent incorporation of the discovered fragment to the
scaffold of ABBV-075, we developed a series of novel BET bromodomain inhibitors
with new binding motifs. Further optimization led to the discovery of compound 38 as
a potential preclinical candidate. Compound 38 has excellent potency, good oral
pharmacokinetics properties, excellent in vivo antitumor efficacy, and especially a
high selectivity for BET bromodomain family over non-BET bromodomains.
RESULTS AND DISCUSSION
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Lead Generation.
Our drug discovery commenced with a screening of ∼1000 low molecular weight
compounds by using a thermal shift assay. The compounds resulting in a thermal
o
transition with the △T value of >1 C were subsequently tested in an AlphaScreen
m
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assay to evaluate their potency (Figure 2). Among these compounds, fragment 47
o
favored protein folding and induced a positive T shift for 1.9 C and showed a
m
moderate inhibitory activity with IC values of 19.4 μM for BRD4(1) and 3.6 μM for
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BRD4(2). Therefore, fragment 47 was selected as the starting point for further
optimization. Through the predicted binding mode analysis of fragment 47 by
molecular docking, 8-methyl-pyrrolo[1,2-a]pyrazin-1(2H)-one moiety fits well into
the KAc binding pocket and forms a bidentate interaction between the pyrazin core
and Asn433. Based on an overlay of our modeled structure of fragment 47 and an
analogue of ABBV075 in a complex with BRD4 (2) (Figure 3), we hypothesized that
incorporation of a 2-phenoxyaryl ring in the fragment 47 might further provide some
valuable interactions with the WPF pocket. To this end, several biaryl ether
derivatives were synthesized. As shown in Table 1, to our delight, a 4-fold
improvement in potency was observed in compound 12 when incorporating a
2-phenoxyphenyl group into the fragment 47. More importantly, introduction of a
sulfonamide group at the para-position of the central phenyl ring resulted in
compound 13, which has an IC value of 15.0 nM. More significantly, compound 13
50
exhibits a very weak inhibitory activity of EP300 bromodomain with an IC value of
50
2
5.1 µM and thus shows a >1500-fold selectivity for BRD4 (1) over EP300.
O
HN
N
Screening of
overlay of fragment 47 and
an analogue of ABBV075
docking-guided
optimization
X
R1
~1000 compounds
R2
8
-methyl-pyrrolo[1,2-a]
pyrazin-1(2H)-one derivatives
New binding motif
fragment 47
Tm = 1.9 o
C
△
BRD4(1) IC50 = 19.4 μ M
BRD4(2) IC50 = 3.6 μ M
Figure 2. Flowchart for the discovery and optimization strategy for novel BET
bromodomain inhibitors. Crystal structure of BRD4(2) with an analogue of ABBV075
(PDB code 5UVX) was used for model construction.
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Figure 3. The overlay of our modeled structure of fragment 47 and an analogue of
ABBV075 in a complex with BRD4 (2). Tyr432 was removed for clarity.
Table 1. Structure−Activity Relationships of Compounds 12-15 and 47..
O
O
HN
O
O
N
N
N
O
HN
HN
O
O
N
N
HN
O
O
O
Structure
N
S
N
O
O
O
H
Br
S
S
N
N
O
H
O
H
Cpd
47
12
13
14
15
BRD4 (1)
1
9.4
4.5
0.015
0.026
0.076
0.13
34.7
>2
a
IC (μΜ)
5
0
BRD4 (2)
3.6
1.6
>2
a
IC (μΜ)
5
0
EP300
N.D.b
N.D.b
25.1
N.D.b
a
IC (μΜ)
5
0
a
b
IC values are means of two measurements. N.D. means not determined.
5
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The cocrystal structure of BRD4(1) with compound 13 was determined at 2.44 Å
resolution (Figure 4). The pyrazin-1(2H)-one core engaged in a very productive
bidentate hydrogen-bonding interaction with the crucial Asn140, along with a third
water-mediated hydrogen bond to Tyr97. The 8-methyl group of pyrrolo[1,2-a]
pyrazin-1(2H)-one core fitted well into the amphipathic water pocket and the phenyl
group occupied the hydrophobic WPF pocket. In addition, the co-crystal structure
showed that one of oxygen atoms of the sulfonamide group formed another hydrogen
bond with the Asp88 moiety. Changing the core to pyrrolo[1,2-a]pyrazin-1(2H)-
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one,3,4-dihydro moiety gave compound 14, which is 5 times less potent than 13. As
expected, N-methyl pyrrolo[1,2-a]pyrazin-1(2H)-one derivative 15 resulted in a
dramatic loss in potency, presumably due to the destruction of the crucial bidentate
hydrogen-bonding interaction with the crucial Asn140.
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Figure 4. Co-crystal structures of compound 13 with BRD4(1). (A) 13 binding with BRD4(1)
(resolution = 2.44 Å, PDB code 6KEF). Ala89, Leu94 and Tyr139 were removed for clarity. (B)
The protein is shown as a cartoon and the ligands are shown in sticks. (C) 2D diagrams of
interaction between compound 13 and BRD4(1). Ligands and residues are shown in sticks,
hydrophobic are shown in the shape of eyelashes. Water molecules which participate in hydrogen
bond formation are shown in blue spheres.
Lead to Candidate.
With an effective and potent 8-methyl-pyrrolo[1,2-a]pyrazin-1(2H)-one core in
hand, SAR studies at additional sites was performed. Both of well characterized
clinical candidates OTX015 and ABBV075 were included as reference compounds to
validate all the experimental procedures. First, a variety of analogues with different
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substituents pointing to the WPF shelf region were prepared to explore the chemical
space and assess the DMPK properties. As shown in Table 2, compared to 13, the
phenylamino derivative 16 lost substantial potency in both the biochemical and
cellular assays and replacement of the phenylether moiety with the benzyl group (17)
and phenylthio group (18) dramatically decreased the potency. At this point, we
proceeded to continue with the ether as linker, because the above results indicated that
the ether linker is crucial for making sure that the attached substituents occupy the
WPF shelf.
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Next, replacement of the phenyl group with a polar 2-pyridine group yielded 19,
which led to a 10-fold loss in cellular potency compared to 13, despite a similar
biochemical activity. 4-Cl derivative (20) also resulted in 5-10-fold decreased
potency. To our delight, a small fluorine substituent (21) at the para- and
ortho-position of the phenyl ring was tolerated, demonstrating a slight decrease in
potency compared to compound 13 but, more importantly, a substantial increase in rat
liver microsomal stability (Table 3). In addition to aryl group, compounds with alkyl
groups (22-25) were also examined. While 22, 24 and 25 decreased potency,
cyclohexyl ether 23 had a 2-4-fold increase in potency, but exhibited a very poor
stability in mouse, rat and human liver microsomes. On the basis of results from our
SAR study on potency and microsomal stability (Tables 2 and 3), 2,4-difluorophenyl
ether moiety (21) showed the best combination of microsomal stabilities and potency,
and was chosen as the preferred moiety to occupy the WPF shelf for future analogues.
Table 2. Structure−Activity Relationships of Compounds 16-25.a
O
HN
N
X
R
O
S
N
H
O
BRD4(1)
IC (nM)
BRD4(2)
EP300
MV4-11
Cpd
X
-
R
-
IC (nM)
IC (nM) IC (nM)
5
0
50
50
50
OTX-015
40.8
197
>33000
46.3
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ABBV-075
-
O
-
1.0
15.1
2.7
26.0
63
1.9
20.4
256.9
N.D.b
N.D.b
211
1
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1
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0
1
Ph
25100
> 33000
15600
>100000
12600
57000
17800
NH
CH2
S
Ph
Ph
68.0
28.4
> 1000
> 1000
27.3
> 1000
> 1000
25.9
1
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Ph
O
2-pyridine
4-chloro-phenyl
2,4-difluorophenyl
O
128
178
118
O
48.3
11.7
36.6
2
2
O
78.6
77.2
25100
185
4.9
2
2
2
3
4
5
O
O
O
cyclohexyl
4-tetrahydropyran
CH C(CH )
9.5
93.7
67.3
669
5400
>33000
26900
33.6
387
N.D.b
> 1000
2
3 3
a
b
IC values are means of two measurements. N.D. means not determined.
5
0
Table 3. In Vitro Metabolic Stability in Liver Microsomes. a
HLM
clearance
RLM
clearance
mLM
clearance
cpds
T1/2
T1/2
T1/2
(mL/min/g)
(min)
(mL/min/g)
(min)
(mL/min/g)
(min)
13
21
23
74.0
28.4
58.1
2.7
3039
0.7
6.4
0.9
116
18.1
14.9
6.3
36.2
752
327
141
333
2137
aHLM, RLM and mLM represent the human, rat and mouse liver microsomes,
respectively.
To further improve the potency and microsomal stability, the next part for SAR
study was the sulfonamide moiety (Table 4). Considering that the hydrogen bond
between sulfonamide and Asp88 is important for the potency, a variety of analogs
were designed that contained a hydrogen bond accepting group, such as ethyl
sulfonamide (26), sulfone (27), reversed sulfonamides (28 and 29), amide (30), and
methylenesulfones (31 and 32). The ethyl sulfonamide 26 gave a 2-3-fold increase in
cellular potency and maintained similar microsomal stability with methyl sulfonamide
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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
2
1 (Table 5). Unfortunately, the sulfone 27, reversed sulfonamides (28 and 29), and
amide (30) resulted in a dramatic loss in potency, presumably due to the inability of
forming the crucial hydrogen bond with Asp88. Methylenesulfone derivatives 31 and
3
2 offered a better profile with improved potency and, more importantly, an improved
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
microsomal stability in rat liver microsome (Table 5). However, the stability of
compounds 31 and 32 was still poor in mouse liver microsomes.
We speculated that the poor microsomal stabilities of compounds 21, 26, 31, and 32
were presumably owing to the oxidative metabolism of the central phenyl group.
Thus, a variety of compounds (33-38) containing a central pyridine ring were
prepared (Table 4). Interestingly, the sulfonamide 36 with a central pyridine ring
result in a decrease in inhibitory and cellular potency. In contrast, the
methylenesulfone derivatives 37 and 38 led to a substantial improvement in potency,
with compound 38 being the best (IC = 2.6 nM for BRD4(1) in AlphaScreen assay,
5
0
IC = 2.4 nM in the MV-4-11 cell line). More significantly, both compounds 37 and
5
0
38 had a substantial increase in mouse liver microsomal stability compared to 31 and
32 (Table 5). Notably, BET bromodomain inhibitor 38 are remarkably selective
(~1500-fold) over EP300.
Table 4. Structure−Activity Relationships of Compounds 26-38.a
O
HN
F
N
O
F
X
R
BRD4(1)
IC (nM)
BRD4(2)
EP300
MV4-11
cpd
R
-
X
-
IC (nM)
IC (nM)
IC (nM)
5
0
50
50
50
OTX-015
ABBV-07
5
40.8
1.0
197
2.7
>33000
63
46.3
1.9
-
-
2
1
-NHSO Me
CH
48.3
11.7
17800
36.6
2
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2
2
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3
3
3
3
3
3
3
6
7
8
9
0
1
2
3
4
5
6
7
8
-NHSO Et
CH
CH
CH
CH
CH
CH
CH
N
10.0
134
438
198
219
4.2
13.0
117
420
71.3
97.9
7.0
4000
> 33000
> 100000
> 100000
> 100000
16500
14.5
N.D.b
N.D.b
N.D.b
N.D.b
14.8
2
-SO Me
2
-SO NHC(CH )
2
3 3
-SO N(CH )
2
3 2
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
-NHC(O)Me
-CH SO Me
2
2
-CH SO Et
4.7
7.4
18600
6.5
2
2
-SO Me
288
740
788
76.6
7.4
229
772
478
16.2
3.5
> 33000
> 100000
> 100000
> 5000
N.D.b
N.D.b
N.D.b
537
2
-SO N(CH )
N
2
3 2
-SO NHC(CH )
N
2
3 3
-NHSO Me
N
2
-CH SO Me
N
2200
14.2
2
2
-CH SO Et
N
2.6
2.2
3857
2.4
2
2
a
b
IC values are means of two measurements. N.D. means not determined.
5
0
Table 5. In Vitro Metabolic Stability in Liver Microsomes.a
HLM
clearance
RLM
clearance
mLM
clearance
cpd
T1/2
T1/2
T1/2
(mL/min/g)
(min)
(mL/min/g)
(min)
(mL/min/g)
(min)
2
3
3
3
3
6
1
2
7
8
62.0
33.9
18.2
46.3
80.4
172
264
7.9
229
9.1
115
46.0
26.1
12.2
112
61.9
64.6
64.8
18.8
33.9
32.5
32.4
380
175
5.5
12.0
58.5
83.0
35.7
25.4
aHLM, RLM and mLM represent the human, rat and mouse liver microsomes,
respectively.
To understand the structural basis for the high binding affinity of the two
compounds 37 and 38, we determined the cocrystal structure of BRD4(1) with 37
(
Figure 5). The pyrazin-1(2H)-one core formed a productive bidentate
hydrogen-bonding interaction with the important Asn140, along with a third
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
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5
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5
5
5
5
5
6
water-mediated hydrogen bond to Tyr97. The 2,4-difluorophenyl ether moiety
occupied the hydrophobic WPF pocket and the methylenesulfone moiety formed
another productive hydrogen bond with Asp88. This co-crystal structure afforded the
structural basis for the high-affinity binding of compounds 37 and 38.
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 5. Co-crystal structures of compound 37 with BRD4(1). (A) 37 binding with BRD4(1)
(resolution = 2.12 Å, PDB code 6KEE). Ala89, Leu94 and Tyr139 were removed for clarity. (B)
The protein is shown as a cartoon and the ligands are shown in sticks. (C) 2D diagrams of
interaction between compound 37 and BRD4(1). Ligands and residues are shown in sticks,
hydrophobic are shown in the shape of eyelashes. Water molecules which participate in hydrogen
bond formation are shown in blue spheres.
Next, the pharmacokinetic properties of 37 and 38 were examined in male SD rats
(Table 6). While compound 37 achieves an AUC value of 520 ng·h/mL with 3 mg/kg
oral administration and a clearance of 37.5 mL/min/kg, compound 38 exhibits a better
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oral exposure with an AUC value of 884 ng·h/mL and a lower clearance of 21.5
mL/min/kg. Given the good pharmacokinetic property of 38 in rat, the
pharmacokinetics of 38 in mice were further evaluated. With 3 mg/kg oral
administration, compound 38 has a Cmax of 399 ng/mL and an oral bioavailability of
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
5
2.3%. The above data demonstrated that 38 was preferred over 37 based on its 7-fold
improvement in cellular activity together with its better oral exposures and lower
clearance. Thus, ethyl sulfonamide 38 was selected for further evaluation in
subsequent experiments.
Table 6. Pharmacokinetic (PK) Study of Compounds 37 and 38.a
T
1/2
Vdss
C
max
AUC0-t
CL
F
cpd
species
route (dose)
(h)
1.5
(mL/kg) (ng/mL)
160
(ng·h/mL)
(mL/min/kg)
(%)
3
3
3
3
3
3
7
7
8
8
8
8
rat
rat
PO (3 mg/kg)
IV (1 mg/kg)
PO (3 mg/kg)
IV (1 mg/kg)
PO (3 mg/kg)
IV (3 mg/kg)
520
457
37.9
34.7
52.3
0.8
3.6
1.3
1.3
0.9
1902
37.5
21.5
15.3
rat
159
884
rat
1464
848
mouse
mouse
399
1710
3270
782
a
Three animals was used for each dose group.
Bromodomain Selectivity.
To further evaluate the binding affinities of 38 to the BET bromodomain family
and its selectivity profile, compound 38 was also assessed in the commercially
available, phage-based, multiplexed bromodomain displacement assay (BROMOscan,
DiscoverX). As shown in the Table 7, 38 binds to BRD2(2), BRD3(2), BRD4(1),
BRD4(2) and BRDT(2) with high affinities (K = 1−3 nM). In addition, single point
d
measurements at 1 μM demonstrated that compound 38 exhibited a high selectivity
for BET bromodomain family over non-BET bromodomains (Figure 6).
Table 7. Comparative Binding Data for 38 within BET Family.
BRD4(1)
BRD4(2)
BRD2(2)
BRD3(2)
BRDT(2)
cpd
K (nM)
d
K (nM)
d
K (nM)
d
K (nM)
d
K (nM)
d
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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
3
8
3.0
1.6
1.3
1.0
2.1
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 6. Selectivity of 38 against a panel of bromodomains by using a DiscoverX
BROMOscan Platform at 1 μM concentration. The % Ctrl = [(test compound signal −
positive control signal)/(negative control signal − positive control signal)] × 100.
Cellular Activities and Mechanism Studies.
In addition to acute myeloid leukemia cell line MV-4-11, other acute leukemia cell
lines such as Kasumi-1 and RS-4-11, and multiple myeloma cancer cell line MM1.S
were also used to evaluate the antiproliferative activities of compound 38. As shown
in Table 8, 38 strongly inhibited the growth of Kasumi-1, RS-4-11, and MM1.S cells
with IC values of 4.8, 17.6 and 15.1 nM, respectively, exhibiting a ~10-20-fold
50
more potent antiproliferative activities than OTX-015.
Table 8. Inhibitory activities of compounds 38 and OTX-015 against several cell
lines.
MV4-11
Kasumi-1
RS-4-11
MM1.S
cpd
IC (nM)
IC (nM)
IC (nM)
IC (nM)
5
0
50
50
50
3
8
2.4
7.4
135
6.3
22.1
416
6.4
15.9
137
6.5
OTX015
46.3
1.9
ABBV-075
Previous studies demonstrated that the BRD4 inhibition can reduce the expression
10
of c-Myc, BCL-2 and CDK6, and up-regulate the expression of p21. Next, the effect
of 38 on the expression of c-Myc and p21 in the MV4-11 cells were examined by
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using western blotting and the results showed that 38 is highly effective in inducing
dose-dependent inhibition on c-Myc expression and up-regulation of p21 levels
(Figure 7). Moreover, while OTX-015 gave an obvious down-regulation of c-Myc
levels at 500 nM, compound 38 showed a very profound down-regulation on the
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
expression of c-Myc even at as low as 31 nM. In addition, real-time quantitative PCR
(RT-qPCR) assays were also performed to examine the mRNA expression of c-Myc,
BCL-2 and CDK6. Figure 8 shows that 38 robustly reduced the expression of c-Myc,
BCL-2 and CDK6 and exhibited a much better inhibitory effect than OTX-015.
Figure 7. Western blot analysis of 38 and OTX-015 on the expression of c-Myc and
p21.
Figure 8. The effect of 38 or OTX-015 on c-Myc, BCL-2, and CDK-6 mRNA levels
by RT-qPCR.
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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
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5
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5
5
5
5
5
6
Previous studies demonstrated that BRD4 has an essential regulatory function both
for proliferation and cell cycle progression and BRD4 inhibition results in cell cycle
2
7
arrest at G0/G1 phase. To further elaborate on the anti-proliferation mechanism of
3
8, flow cytometric analysis was performed to elucidate the effects of 38 on cell cycle
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
and the results showed that compound 38 even at 31 nM led to more pronounced
G1-phase cell cycle arrest compared with OTX-015 at 125 nM (Figure 9).
Figure 9. Flow cytometric analysis on the cell cycle of 38 or OTX-015.
In Vitro Metabolic Stability and Cytochrome P450 Enzyme Inhibition Studies.
Besides the mouse, rat, and human microsomes, the stabilities of 38 in dog and
monkey microsomes were further examined and the results showed that compound 38
has a moderate to good stability in all five microsomes (Table 9). In addition,
compound 38 exhibited a negligible inhibition on five cytochrome P450 enzymes,
indicating a low potential for drug−drug interactions (Table 10).
Table 9. In Vitro Metabolic Stability of compound 38 in Liver Microsomes.
T1/2 (min)
Clint In Vitro (mL/min/g)
cpd
human rat mouse dog monkey
human
12.2
rat mouse dog monkey
3
8
172
32.4
83.0
83.4
49.0
64.8
25.4 25.2
42.7
Table 10. Cytochrome P450 Enzyme Inhibition of compound 38.
1A2
2C19
2C9
2D6
3A4
cpd
IC (μM)
IC (μM)
IC (μM)
IC (μM)
IC (μM)
5
0
50
50
50
50
38
>20
>20
>20
>20
>20
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hERG Study.
The inhibitory activity of 38 on the human Ether-a-go-go Related Gene (hERG)
was also evaluated and 38 did not exhibit inhibitory activity on hERG even at 40 μM,
indicating a low risk of cardiac side effects.
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
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9
0
1
2
3
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5
6
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8
9
0
1
2
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4
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In Vivo Antitumor Efficacy.
The effect of 38 on tumor growth was subsequently evaluated by utilizing an
MV4-11 mouse xenograft model (Figure 10). OTX015 was used as the control
compound as OTX015 entered into phase II clinical trials for the treatment of
hematologic malignancies such as acute myeloid leukemia. At one fourth of the
dosage of OTX-015, compound 38 exhibited stronger antitumor activities than
OTX-015 and completely inhibit the growth of tumor with a tumor growth inhibition
(TGI) of 99.7% at 12.5 mg/kg. In comparison, OTX015 at 50 mg/kg exhibited only
6
2.3% TGI. More importantly, even at one eighth of the dosage of OTX-015,
compound 38 also exhibited a better antitumor effect than OTX-015, with a TGI of
76.4% at 6.25 mg/kg. Significantly, 38 did not induce weight loss and was well
tolerated in the treated mice.
(A)
(B)
Figure 10. Antitumor efficacy of 38 and OTX015. (A) The tumor growth curve of
vehicle control, OTX-015 (50 mg/kg, qd), 38 (12.5 mg/kg, qd), and 38 (6.25 mg/kg,
qd). Each group contained six animals. (B) Animal weight change.
Chemistry
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4
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As shown in the Scheme 1, hydrolysis of commercially available 1H-Pyrrole-2-
carboxylic acid, 3-methyl-, ethyl ester gave 39 which was coupled with
2
-chloroethylamine to provide 40. Cyclization of 40 and subsequent bromination of
the resulting 41 with NBS delivered 42. Direct coupling of 1H-Pyrrole-2-carboxylic
acid, 3-methyl-, ethyl ester with bromoacetaldehyde diethyl acetal produced 43 which
was subsequently hydrolyzed to give acid 44. Condensation of 44 with ammonium
chloride and subsequent intramolecular cyclization gave 8-methyl-pyrrolo[1,2-a]
pyrazin-1(2H)-one 46. Direct bromination of 46 with NBS provided fragment 47
which was alkylated with methyl iodide to deliver 48.
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
Scheme 1. Synthetic Route of Intermediates 42, 47 and 48.a
O
O
O
O
O
Cl
iv
HN
i
ii
N
iii
HN
OEt
OH
N
H
N
NH
NH
NH
Br
39
40
41
42
v
O
O
O
O
O
O
OEt
OH
NH2
i
N
vi
vii
HN
iv
HN
viii
N
N
N
N
N
N
EtO
EtO
EtO
Br
Br
OEt
OEt
OEt
44
45
46
47
48
43
a
Reagents and conditions: (i) LiOH·H O, EtOH, H O, 75°C; (ii) 2-chloroethylamine
2
2
hydrochloride, HATU, DIPEA, DCM; (iii) NEt , DCM; (iv) NBS, TFA, DCM, 0°C;
3
(
v) bromoacetaldehyde diethyl acetal, NaH, NMP, 130°C; (vi) NH Cl, HATU,
4
DIPEA, DCM, 80%; (vii) AcOH, 105°C; (viii) CH I, NaH, THF.
3
In Scheme 2A and 2B, S Ar displacement of commercially available
N
3
-bromo-4-fluoronitrobenzene or 3-bromo-2-fluoro-5-nitropyridine by different
phenol, aniline, and thiophenol, further reduction of the nitro group, and subsequent
functionalization of the resulting aniline followed by coupling with
bis(pinacolato)diboron under the palladium catalyst delivered 79-88 and 96-98.
Scheme 2. Synthetic Route of Intermediates 79-88 and 96-98.a
1
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9
Br
Br
Br
O
O
Br
(
A)
B
X
d
F
X
b
X
c
R
a
R
R
O
X
O
R
S
N
S
O2N
O2N
H2N
O
H
N
O
H
4
5
5
5
5
5
9; X = O, R = Ph
0; X = NH, R = Ph
1; X = S, R = Ph
2; X = O, R = 2-pyridine
3; X = O, R = 4-chloro-phenyl
4; X = O, R = 2,4-difluorophenyl
55; X = O, R = cyclopropylmethylene
6; X = O, R = cyclohexyl
57; X = O, R = 4-tetrahydropyran
8; X = O, R = CH2C(CH3)3
59; X = O, R = Ph
0; X = NH, R = Ph
61; X = S, R = Ph
2; X = O, R = 2-pyridine
63; X = O, R = 4-chloro-phenyl
4; X = O, R = 2,4-difluorophenyl
5; X = O, R = cyclopropylmethylene
6; X = O, R = cyclohexyl
69; X = O, R = Ph
70; X = NH, R = Ph
71; X = S, R = Ph
72; X = O, R = 2-pyridine
73; X = O, R = 4-chloro-phenyl
74; X = O, R = 2,4-difluorophenyl
75; X = O, R = cyclopropylmethylene
76; X = O, R = cyclohexyl
77; X = O, R = 4-tetrahydropyran
78; X = O, R = CH2C(CH3)3
7
8
8
8
8
8
8
8
8
8
9; X = O, R = Ph
0; X = NH, R = Ph
1; X = S, R = Ph
2; X = O, R = 2-pyridine
3; X = O, R = 4-chloro-phenyl
4; X = O, R = 2,4-difluorophenyl
5; X = O, R = cyclopropylmethylene
6; X = O, R = cyclohexyl
7; X = O, R = 4-tetrahydropyran
8; X = O, R = CH2C(CH3)3
6
6
6
6
6
6
6
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
5
7; X = O, R = 4-tetrahydropyran
8; X = O, R = CH2C(CH3)3
5
O
O
Br
Br
F
(B)
Br
F
Br
F
B
F
F
f
O
e
O
b
O
d
O
X
X
O2N
X
X
RHN
F
R
X
O2N
F
H2N
F
N
F
H
9
9
9
6; X = CH, R = SO2Et
7; X = CH, R = C(O)CH3
8; X = N, R = SO2Me
X = CH or N
89; X = CH
0; X = N
91; X = CH
92; X = N
9
3; X = CH, R = SO2Et
94; X = CH, R = C(O)CH3
5; X = N, R = SO2Me
9
9
a
Reagents and conditions: (a) R-X, Cs CO , DMSO, 110 °C, 1 h; (b) Fe, NH Cl,
2
3
4
EtOH, H O, 100 °C, 2 h; (c) MeSO Cl, pyridine, rt, 2 h; (d) bis(pinacolato)diboron,
2
2
Pd(dppf)Cl , KOAc, 1,4-dioxane, 100 °C, 14 h; (e) 2,4-difluorophenol, Cs CO ,
2
2
3
DMSO, 110 °C, 1 h; (f) RCl, pyridine, rt, 2 h;
In Scheme 3, treatment of the commercially available 99 with MeSO Cl, and
2
subsequent coupling with bis(pinacolato)diboron yielded 101.
Scheme 3. Synthetic Route of Intermediates 101.a
Br
O
O
Br
B
a
O
S
b
O
O
S
O
N
H2N
H
N
H
9
9
100
101
a
Reagents and conditions: (a) MeSO Cl, pyridine, rt, 2 h; (b) bis(pinacolato)diboron,
2
Pd(dppf)Cl , KOAc, 1,4-dioxane, 100 °C, 14 h.
2
In Scheme 4, treatment of the commercially available 102 or 103 with
2,4-diflourophenol, and subsequent coupling with bis(pinacolato)diboron delivered
106, and 107.
Scheme 4. Synthetic Route of Intermediates 106 and 107.a
Br
Br
F
O
O
N
R
O
B
F
a
b
O
O
S
O
O
S
X
X
F
O
S
O
F
102, X = CH, R = F
03, X = N, R = Cl
104, X = CH
105, X = N
O
1
1
06, X = CH
07, X = N
1
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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
a
Reagents and conditions: (a) 2,4-difluorophenol, Cs CO , DMSO, 100 °C, 2 h; (b)
2
3
bis(pinacolato)diboron, Pd(dppf)Cl , KOAc, 1,4-dioxane, 100 °C, 14 h;
2
In Scheme 5, treatment of the commercially available 3-bromo-4-
fluoro-benzenesulfonyl chloride or 5-bromo-6-fluoro-3-pyridinesulfonyl chloride with
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
different amine, and subsequent S Ar displacement with 2,4-diflourophenol, followed
N
by coupling with bis(pinacolato)diboron provided 116-119.
Scheme 5. Synthetic Route of Intermediates 116-119.a
Br
F
O
O
X
Br
Br
B
F
O
F
F
c
O
a
b
O
O
O
R1
X
O
X
X
S
F
S
R1
S
N
R1
F
S
Cl
N
R2
O
N
O
O
O
R2
R2
108; X = CH, R1 = H, R = C(CH3)3
2
112; X = CH, R1 = H, R2 = C(CH3)3
116; X = CH, R1 = H, R2 = C(CH3)
3
117; X = CH, R1 = R2 = CH3
X = CH or N
113; X = CH, R1
R2 = CH3
114; X = N, R1
R2 = CH3
=
1
1
1
09; X = CH, R1
R2 = CH3
10; X = N, R1
R2 = CH3
11; X = N, R1 = H, R2 = C(CH3)3
=
=
118; X = N, R1 =
R2 = CH3
119; X = N, R1 = H, R2 = C(CH3)
=
115; X = N, R1 = H, R2 = C(CH3)3
3
a
1
2
Reagents and conditions: (a) NHR R , Et N, DCM, rt, 2 h; (b) 2,4-difluorophenol,
3
Cs CO , DMSO, 110 °C, 1 h; (c) bis(pinacolato)diboron, Pd(dppf)Cl , KOAc,
2
3
2
1
,4-dioxane, 100 °C, 14 h;
In Scheme 6, S Ar displacement of commercially available 3-bromo-4-
N
chloro-benzaldehyde
or
5-bromo-6-chloro-3-pyridinecarboxylic
acid
by
2
,4-diflourophenol, then reduction of the aldehyde and carboxylic acid group, and
subsequent bromination of the resulting alcohol delivered 124 and 125. Treatment 124
and 125 with sodium thiomethoxide or sodium ethanethiolate, and subsequent
oxidation of the resulting thioether followed by further coupling with
bis(pinacolato)diboron yielded 134-137.
Scheme 6. Synthetic Route of Intermediates 134-137.a
2
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8
9
Br
F
Br
F
Br
O
O
F
b
a
d
H
HO
Br
F
H
F
F
O
O
120
121
O
Br
X
F
Br
F
Br
F
d
Br
124;
125; X = N
X = CH
O
O
c
Cl
a
HO
N
HO
N
HO
N
F
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
O
O
123
1
22
Br
F
O
O
Br
F
B
F
O
O
g
f
O
S
O
e
R
O
S
X
R
S
X
F
X
R
F
O
F
O
126; X = CH, R = Me
130; X = CH, R = Me
131; X = CH, R = Et
132; X = N, R = Me
133; X = N, R = Et
134; X = CH, R = Me
135; X = CH, R = Et
136; X = N, R = Me
137; X = N, R = Et
1
1
1
27; X = CH, R = Et
28; X = N, R = Me
29; X = N, R = Et
a
Reagents and conditions: (a) 2,4-difluorophenol, Cs CO , DMSO, 100 °C, 2 h; (b)
2
3
NaBH , EtOH, THF, rt, 1 h; (c); BH -THF, THF, 50 °C, 1h; (d) phosphorus
4
3
®
tribromide, DCM, rt, 3h; (e) RSNa, DMF, rt, 6 h; (f) OXONE , MeOH, rt, 1h; (g)
bis(pinacolato)diboron, Pd(dppf)Cl , KOAc, 1,4-dioxane, 100 °C, 14 h;
2
As shown in Schemes 7 and 8, generally, the mixture of intermediates 47, 42, or 48
and the corresponding boronic acid pinacol ester were heated in the presence of
o
palladium catalyst in 1,4-dioxane at 110 C under argon for 3 h gave the compounds
13−38.
Scheme 7. Synthetic Route of Compounds 13-25.a
1
1
3; X = O, R = Ph
6; X = NH, R = Ph
17; X = CH2, R = Ph
18; X = S, R = Ph
19; X = O, R = 2-pyridine
0; X = O, R = 4-chloro-phenyl
21; X = O, R = 2,4-difluorophenyl
22; X = O, R = cyclopropylmethylene
23; X = O, R = cyclohexyl
X = O, R = 4-tetrahydropyran
5; X = O, R = CH C(CH )
O
HN
O
O
O
N
B
i
X
R
HN
+
X
2
O
R
N
O
S
Br
N
S
O
N
H
H
O
47
79-88,101
13, 16-25
24;
2
2
3 3
O
O
O
O
N
N
HN
HN
O
O
B
N
N
N
N
O
Ph
O
O
Ph
42
Br
Ph
48 Br
O
S
O
O
i
N
O
i
S
S
H
N
N
H
H
O
O
7
9
15
14
a
Reagents and conditions: (i) Pd(dppf)Cl , Na CO , dioxane, 110 °C.
2
2
3
2
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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
Scheme 8. Synthetic Route of Compounds 26-38.a
2
6; X = CH, R = NHSO Et
2
27; X = CH, R = SO Me
2
2
2
3
8; X = CH, R = SO NHC(CH )
9; X = CH, R = SO N(CH )
0; X = CH, R = NHC(O)Me
2
3 3
O
2
3 2
HN
F
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
31; X = CH, R = CH SO Me
2 2
32; X = CH, R = CH2SO2Et
33; X = N, R = SO Me
34; X = N, R = SO2N(CH3)2
O
O
O
X
N
B
F
i
O
HN
O
F
2
+
N
X
3
3
3
3
5; X = N, R = SO NHC(CH )
6; X = N, R = NHSO Me
7; X = N, R = CH SO Me
8; X = N, R = CH SO Et
2
3 3
Br
R
F
R
2
9
6-98, 106-107,
26-38
47
2
2
116-119, 134-137
2
2
a
Reagents and conditions: (i) Pd(dppf)Cl , Na CO , dioxane, 110 °C.
2
2
3
CONCLUSIONS
In summary, by identifying a 8-methyl-pyrrolo[1,2-a]pyrazin-1(2H)-one
fragment (47) as new acetyl-lysine mimic and subsequently incorporating it into the
scaffold of ABBV075, we synthesized a class of highly potent BET bromodomain
inhibitors, exemplified by compound 38 which is highly potent in both biochemical
and cellular assays (BRD4(1) IC = 2.6 nM, MV4-11 IC = 2.4 nM), and exhibits
50
50
good oral pharmacokinetics and good therapeutic effects in the MV4-11 mouse
xenograft model. At one fourth of the dosage of clinical candidate OTX-015,
compound 38 was found to be much more effective than OTX-015 in the inhibition of
tumor growth and achieved a complete inhibition of the tumor growth with a TGI of
9
9.7% accompanied by a good tolerability. Moreover, compound 38 is remarkably
selective for BET bromodomain family over EP300 (~1500-fold selectivity) and
exhibits no appreciable binding affinity on other 26 non-BET bromodomains at 1000
nM, suggesting that compound 38 is a highly promising preclinical candidate.
EXPERIMENTAL SECTION
Chemistry
Unless otherwise noted, commercial solvents and reagents were used without
further purification. Silica gel 60H (200-300 mesh) manufactured by Qingdao
Haiyang Chemical Group Co. (China) was used for general chromatography. The
final compounds were all purified by C18 reverse phase preparative HPLC column
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with H O and MeCN as eluents. The purity of all the final compounds was confirmed
2
to be > 95% by HPLC analysis with Agilent ZORBAX SB-C18 reversed-phase
column (250 mm × 4.60 mm, 5 μm). Conditions were as follows: flow rate = 1
mL/min, CH CN/H O eluent (containing 0.1% trifluoroacetic acid); gradient, 40%
3
2
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
CH CN to 100% CH CN; 10 min; monitored by UV absorption at 210 and 254 nm.
3
3
Reactions were monitored by thin-layer chromatography (TLC) or LC-MS. LC-MS
was performed by Waters UPLC H-Class with ACQUITY UPLC BEH C18
reversed-phase column (2.1 mm × 50 mm, 1.7 μm, flow rate = 0.5 mL/min). Mass
1
analysis was performed by waters SQD2 single quadrupole mass detection system. H
13
NMR and C NMR spectra were determined with BRUKER AVANCE II 400M or
BRUKER AVANCE III 500M NMR spectrometer. High resolution ESI mass analysis
was performed by Agilent 1290-6545 UHPLC-QTOF high resolution mass
spectrometer.
8-methyl-6-(2-phenoxyphenyl)pyrrolo[1,2-a]pyrazin-1(2H)-one
(12).
Compound 47 (45 mg, 0.2 mmol) and 2-phenoxyphenylboronic acid (86 mg, 0.4
mmol) was dissolved in the mixture of dioxane (6 mL) and 2 M aqueous sodium
carbonate
solution
(2
mL).
1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (29 mg, 0.04 mmol) was
added under the protection of argon. The reaction mixture was heated to 110 °C under
argon and stirred for 3 h. After the reaction mixture was cooled to ambient
temperature, water (25 mL) was added. The mixture was extracted with ethyl acetate
(25 mL × 3) and the combined organic layers were dried over anhydrous sodium
sulfate and concentrated in vacuum. The residue was purified by reverse phase HPLC
(gradient 40% to 70%, CH CN/H O with 0.1% TFA) to afford the title compound 12
3 2
1
(
21 mg, 33%) as a white solid. H NMR (400 MHz, DMSO-d ) δ 10.23 (d, J = 5.4 Hz,
6
1
H), 7.50 – 7.40 (m, 2H), 7.36 – 7.29 (m, 2H), 7.26 (td, J = 7.5, 1.1 Hz, 1H), 7.09 (t, J
=
7.4 Hz, 1H), 6.97 (d, J = 8.2 Hz, 1H), 6.95 – 6.90 (m, 2H), 6.84 (d, J = 5.3 Hz, 1H),
13
6
1
1
.43 (t, J = 5.7 Hz, 1H), 6.39 (s, 1H), 2.43 (s, 3H). C NMR (125 MHz, DMSO-d ) δ
6
57.2, 156.0, 154.3, 132.1, 130.3, 129.9, 125.4, 123.8, 123.6, 122.0, 121.9, 120.3,
+
+
18.6, 118.4, 114.5, 113.8, 12.2. HRMS (M + H) calculated for C H N O
2
2
0
17
2
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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
3
17.1285, found 317.1278. HPLC retention time 9.27 min, > 98% pure.
N-(3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-phenoxyphenyl
)methanesulfonamide (13). Compound 13 was prepared according to the reaction
reported for compound 12 replacing 2-phenoxyphenylboronic acid with 79. White
solid, 52%yield. 1H NMR (400 MHz, Chloroform-d) δ 9.92 (s, 1H), 7.50 – 7.45 (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
4
1
1
1
4
H), 7.42 – 7.36 (m, 1H), 7.09 (d, J = 5.9 Hz, 1H), 6.45 (s, 1H), 6.39 (t, J = 5.5 Hz,
1
3
H), 2.64 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 157.1, 156.5, 150.3, 134.2,
6
29.8, 124.8, 123.5, 123.2, 123.0, 122.3, 121.9, 120.5, 120.4, 117.8, 114.5, 113.9,
+
+
05.8, 24.9, 12.2. HRMS (M + H) calculated for C H N O S 410.1169, found
2
1
20
3
4
10.1163. HPLC retention time 9.28 min, > 99% pure.
N-(3-(8-methyl-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-6-yl)-4-phenoxy
phenyl)methanesulfonamide (14). 42 (45 mg, 0.2 mmol) and 79 (155 mg, 0.4 mmol)
was dissolved in the mixture of dioxane (6 mL) and 2 M aqueous sodium carbonate
solution (2 mL). 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (29 mg,
0.04 mmol) was added under the protection of argon. The reaction mixture was heated
to 110 °C under argon and stirred for 3 h. After the reaction mixture was cooled to
ambient temperature, water (25 mL) was added. The mixture was extracted with ethyl
acetate (25 mL × 3) and the combined organic layers were dried over anhydrous
sodium sulfate and concentrated in vacuum. The residue was purified by reverse
phase HPLC (gradient 40% to 70%, CH CN/H O with 0.1% TFA) to afford the title
3
2
1
compound 14 (20.5 mg, 25%) as white solid. H NMR (400 MHz, Chloroform-d) δ
9
.70 (s, 1H), 7.41 (d, J = 2.7 Hz, 1H), 7.33 – 7.26 (m, 3H), 7.03 (t, J = 7.4 Hz, 1H),
.95 (d, J = 8.8 Hz, 1H), 6.93 – 6.87 (m, 3H), 6.45 (s, 1H), 4.10 – 3.98 (m, 2H), 3.71
6
1
3
–
3.58 (m, 2H), 3.07 (s, 3H), 2.43 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 161.1,
6
1
56.8, 149.9, 134.3, 129.9, 129.4, 124.4, 124.2, 123.5, 123.0, 121.9, 120.8, 120.5,
+
+
1
17.4, 112.0, 42.7, 12.2. HRMS (M + H) calculated for C H N O S 412.1326,
21 22 3 4
found 412.1331. HPLC retention time 5.90 min, > 98% pure.
N-(3-(2,8-dimethyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-phenoxyph
enyl)methanesulfonamide (15). 48 (48mg, 0.2 mmol) and 79 (155 mg, 0.4 mmol)
was dissolved in the mixture of dioxane (6 mL) and 2 M aqueous sodium carbonate
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solution (2 mL). 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (29 mg,
0
.04 mmol) was added under the protection of argon. The reaction mixture was heated
to 110 °C under argon and stirred for 3 h. After the reaction mixture was cooled to
ambient temperature, water (25 mL) was added. The mixture was extracted with ethyl
acetate (25 mL × 3) and the combined organic layers were dried over anhydrous
sodium sulfate and concentrated in vacuum. The residue was purified by reverse
phase HPLC (gradient 40% to 70%, CH CN/H O with 0.1% TFA) to afford the title
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
3
2
1
compound 15 (33.8 mg, 40%) as white solid. H NMR (400 MHz, DMSO-d ) δ 9.82
6
(s, 1H), 7.33 – 7.26 (m, 4H), 7.09 – 7.04 (m, 1H), 7.03 – 7.00 (m, 1H), 6.97 (d, J =
6
.0 Hz, 1H), 6.93 – 6.89 (m, 2H), 6.70 (d, J = 6.0 Hz, 1H), 6.41 (s, 1H), 3.28 (s, 3H),
13
3
.03 (s, 3H), 2.43 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 156.4, 150.3, 134.2,
6
1
29.8, 124.4, 123.4, 123.3, 122.7, 122.3, 121.7, 120.2, 120.2, 118.7, 117.9, 117.5,
+
+
1
14.9, 105.9, 33.8, 12.2. HRMS (M + H) calculated for C H N O S 424.1326,
22 22 3 4
found 424.1342. HPLC retention time 7.17 min, > 95% pure.
N-(3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-(phenylamino)
phenyl)methanesulfonamide (16). Compound 47 (45 mg, 0.2 mmol) and 80 (155
mg, 0.4 mmol) was dissolved in the mixture of dioxane (6 mL) and 2 M aqueous
sodium
carbonate
solution
(2
mL)
under
argon.
1
,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (29 mg, 0.04 mmol) was
added under the protection of argon. The reaction mixture was heated to 110 °C under
argon and stirred for 3 h. After the reaction mixture was cooled to ambient
temperature, water (25 mL) was added. The mixture was extracted with ethyl acetate
(25 mL × 3) and the combined organic layers were dried over anhydrous sodium
sulfate and concentrated in vacuum. The residue was purified by reverse phase HPLC
(
gradient 40% to 70%, CH CN/H O with 0.1% TFA) to afford the title compound 16
3
2
1
(
29.4 mg, 36%) as white solid. H NMR (500 MHz, DMSO-d ) δ 10.15 (d, J = 5.3
6
Hz, 1H), 9.56 (s, 1H), 7.31 (d, J = 8.8 Hz, 1H), 7.27 (s, 1H), 7.22 (dd, J = 8.7, 2.5 Hz,
1
H), 7.16 – 7.10 (m, 3H), 6.94 (s, 1H), 6.93 (s, 1H), 6.75 (t, J = 7.3 Hz, 1H), 6.68 (d,
13
J = 5.8 Hz, 1H), 6.40 (s, 1H), 6.35 (t, J = 5.6 Hz, 1H), 2.98 (s, 3H), 2.47 (s, 3H). C
NMR (126 MHz, DMSO-d ) δ 157.3, 143.9, 138.7, 131.6, 128.9, 126.4, 124.7, 122.8,
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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5
5
5
5
5
5
5
6
1
22.3, 122.1, 120.4, 119.6, 116.8, 114.4, 113.8, 105.7, 12.4. HRMS (M + H)+
+
calculated for C H N O S 409.1329, found 409.1331. HPLC retention time 7.17
2
1
21
4
3
min, > 95% pure.
Following similar procedures to that for compound 16 gave compounds 17−38.
N-(4-benzyl-3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)phenyl)
methanesulfonamide (17). White solid, 52% yield. 1H NMR (400 MHz,
Chloroform-d) δ 9.33 (d, J = 5.2 Hz, 1H), 8.46 (s, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.36
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
–
5
3
1
7.29 (m, 3H), 7.24 (dd, J = 12.8, 7.2 Hz, 3H), 7.18 (d, J = 1.6 Hz, 1H), 6.69 (d, J =
.9 Hz, 1H), 6.46 (s, 1H), 6.32 (d, J = 5.8 Hz, 1H), 4.01 (s, 2H), 3.13 (s, 3H), 2.48 (s,
13
H). C NMR (126 MHz, DMSO-d ) δ 157.3, 140.8, 139.1, 134.0, 131.7, 129.8,
6
28.7, 128.5, 126.1, 125.8, 125.5, 121.8, 120.2, 114.8, 114.0, 105.2, 40.5, 40.1, 12.3.
+
+
HRMS (M + H) calculated for C H N O S 408.1376, found 408.1394. HPLC
2
2
22
3
3
retention time 6.14 min, > 95% pure.
N-(3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-(phenylthio)ph
1
enyl)methanesulfonamide (18). White solid, 45% yield. H NMR (400 MHz,
Methanol-d4) δ 7.35 (d, J = 8.6 Hz, 1H), 7.29 (dd, J = 8.6, 2.5 Hz, 1H), 7.23 – 7.17
(m, 4H), 7.12 – 7.07 (m, 2H), 6.67 (d, J = 5.9 Hz, 1H), 6.38 (d, J = 5.9 Hz, 1H), 6.29
1
3
(s, 1H), 3.02 (s, 3H), 2.50 (s, 3H). C NMR (126 MHz, DMSO-d ) δ 157.1, 138.2,
6
1
34.9, 133.5, 133.1, 130.2, 129.9, 129.3, 127.1, 126.7, 121.9, 121.6, 120.5, 120.0,
+
+
1
14.5, 114.2, 105.1, 12.2. HRMS (M + H) calculated for C H N O S 426.0941,
21 20 3 3 2
found 426.0962. HPLC retention time 7.32 min, > 99% pure.
N-(3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-(pyridin-3-ylox
1
y)phenyl)methanesulfonamide (19). White solid, 57% yield. H NMR (400 MHz,
DMSO-d ) δ 10.27 (d, J = 5.3 Hz, 1H), 9.90 (s, 1H), 8.25 (dd, J = 3.5, 2.3 Hz, 1H),
6
8
1
.20 (s, 1H), 7.32 (dd, J = 8.7, 2.7 Hz, 1H), 7.30 – 7.24 (m, 3H), 7.14 (d, J = 8.7 Hz,
H), 6.88 (d, J = 5.8 Hz, 1H), 6.46 (t, J = 5.7 Hz, 1H), 6.38 (s, 1H), 3.05 (s, 3H), 2.40
1
3
(s, 3H). C NMR (126 MHz, DMSO-d ) δ 157.1, 153.1, 149.4, 144.2, 139.8, 135.0,
6
1
24.5, 124.4, 124.3, 123.3, 123.2, 122.2, 122.0, 120.8, 120.6, 114.6, 114.1, 105.7,
+
+
1
2.1. HRMS (M + H) calculated for C H N O S 411.1122, found 411.1144.
20 19 4 4
HPLC retention time 2.78 min, > 95% pure.
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N-(4-(4-chlorophenoxy)-3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6
1
-
yl)phenyl)methanesulfonamide (20). White solid, 51% yield. H NMR (400 MHz,
Chloroform-d) δ 9.31 (d, J = 4.8 Hz, 1H), 8.60 (s, 1H), 7.41 – 7.33 (m, 2H), 7.21 –
7
6
1
1
.13 (m, 2H), 7.02 (d, J = 9.0 Hz, 1H), 6.87 (d, J = 5.9 Hz, 1H), 6.78 – 6.72 (m, 2H),
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
1
3
.41 – 6.32 (m, 2H), 3.08 (s, 3H), 2.53 (s, 3H). C NMR (126 MHz, DMSO-d ) δ
6
57.1, 155.5, 149.7, 134.7, 129.6, 126.8, 124.5, 123.3, 123.2, 122.2, 121.9, 120.9,
+
20.5, 119.2, 114.6, 114.0, 105.7, 40.1, 12.2. HRMS (M + H) calculated for
+
C H ClN O S 444.0779, found 444.0779. HPLC retention time 7.09 min, > 95%
2
1
19
3
4
pure.
N-(4-(2,4-difluorophenoxy)-3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazi
1
n-6-yl)phenyl)methanesulfonamide (21). White solid, 44% yield. H NMR (400
MHz, Chloroform-d) δ 9.47 (d, J = 4.3 Hz, 1H), 8.76 (s, 1H), 7.37 (d, J = 2.5 Hz, 1H),
7
.32 (dd, J = 8.8, 2.6 Hz, 1H), 6.98 – 6.85 (m, 3H), 6.79 (t, J = 10.0 Hz, 2H), 6.46 (s,
1H), 6.37 (t, J = 5.6 Hz, 1H), 3.05 (s, 3H), 2.56 (s, 3H). 13C NMR (125 MHz,
DMSO-d ) δ 158.1 (dd, J = 243.1, 10.6 Hz), 157.1, 153.1 (dd, JC-F = 249.5, 12.9
6
C-F
Hz), 150.9, 139.1 (dd, JC-F = 11.4, 3.4 Hz), 134.0, 124.4, 123.8, 122.5, 122.5, 122.0,
121.4, 120.6, 117.4, 114.7, 114.0, 112.0 (dd, JC-F = 22.9, 3.3 Hz), 105.7, 105.6 (dd,
+
+
JC-F = 27.3, 22.6 Hz), 40.1, 12.2. HRMS (M + H) calculated for C H F N O S
21
18
2
3
4
4
46.0981, found 446.0988. HPLC retention time 6.36 min, > 95% pure.
N-(4-(cyclopropylmethoxy)-3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazi
1
n-6-yl)phenyl)methanesulfonamide (22). White solid, 63% yield. H NMR (400
MHz, Chloroform-d) δ 9.67 (s, 1H), 8.40 (s, 1H), 7.36 (d, J = 8.7 Hz, 1H), 7.29 (s,
1
H), 6.95 (d, J = 8.8 Hz, 1H), 6.75 (d, J = 5.7 Hz, 1H), 6.41 (s, 1H), 6.34 (t, J = 5.3
Hz, 1H), 3.78 (d, J = 6.7 Hz, 2H), 3.01 (s, 3H), 2.61 (s, 3H), 1.18 – 1.04 (m, 1H), 0.51
1
3
(
d, J = 7.5 Hz, 2H), 0.21 (d, J = 4.6 Hz, 2H). C NMR (125 MHz, DMSO-d ) δ
6
1
1
3
57.3, 153.0, 131.0, 126.2, 124.8, 123.2, 121.8, 120.3, 120.1, 114.2, 113.6, 113.3,
+
+
06.6, 72.4, 38.8, 12.3, 9.9, 3.0. HRMS (M + H) calculated for C H N O S
1
9
22
3
4
88.1326, found 388.1352. HPLC retention time 5.42 min, > 98% pure.
N-(4-(cyclohexyloxy)-3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)
1
phenyl)methanesulfonamide (23). White solid, 57% yield. H NMR (400 MHz,
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1
1
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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
Methanol-d4) δ 7.34 – 7.29 (m, 1H), 7.23 (d, J = 2.7 Hz, 1H), 7.10 (d, J = 8.9 Hz,
1
3
H), 6.80 (d, J = 5.9 Hz, 1H), 6.41 (d, J = 5.8 Hz, 2H), 4.31 – 4.22 (m, 1H), 2.94 (s,
1
3
H), 2.57 (s, 3H), 1.80 – 1.72 (m, 2H), 1.54 – 1.23 (m, 8H). C NMR (150 MHz,
DMSO-d ) δ 157.3, 151.8, 130.9, 126.4, 124.8, 123.2, 121.8, 121.0, 120.2, 115.2,
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
+
1
14.3, 113.2, 106.7, 75.2, 38.9, 31.0, 25.0, 22.7, 12.3. HRMS (M + H) calculated for
+
C H N O S 416.1639, found 416.1666. HPLC retention time 7.03 min, > 98%
2
1
24
3
4
pure.
N-(3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-((tetrahydro-2
1
H-pyran-4-yl)oxy)phenyl)methanesulfonamide (24). White solid, 59% yield. H
NMR (400 MHz, Methanol-d4) δ 7.32 (dd, J = 8.8, 2.8 Hz, 1H), 7.25 (d, J = 2.7 Hz,
1
H), 7.13 (d, J = 8.9 Hz, 1H), 6.80 (d, J = 5.9 Hz, 1H), 6.42 (s, 1H), 6.39 (d, J = 5.9
Hz, 1H), 4.51 – 4.44 (m, 1H), 3.61 – 3.53 (m, 2H), 3.50 – 3.40 (m, 2H), 2.95 (s, 3H),
.58 (s, 3H), 1.90 – 1.82 (m, 2H), 1.62 – 1.52 (m, 2H). 13C NMR (150 MHz,
2
DMSO-d ) δ 157.3, 151.4, 131.4, 126.2, 124.6, 123.0, 121.9, 121.3, 120.3, 115.7,
6
+
114.3, 113.3, 106.6, 72.2, 63.9, 40.0, 38.9, 31.3, 12.3. HRMS (M + H) calculated for
+
C H N O S 418.1431, found 418.1459. HPLC retention time 4.08min, > 98% pure.
2
0
24
3
5
N-(3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazin-6-yl)-4-(neopentyloxy)
1
phenyl)methanesulfonamide (25). White solid, 64% yield. H NMR (400 MHz,
Chloroform-d) δ 7.40 – 7.36 (m, 2H), 6.79 (d, J = 8.8 Hz, 1H), 6.18 (s, 1H), 3.59 (s,
1
3
2
1
1
4
H), 2.92 (s, 3H), 1.34 (s, 12H), 1.07 (s, 9H). C NMR (126 MHz, DMSO-d ) δ
6
57.2, 153.7, 131.0, 125.9, 124.5, 123.3, 121.6, 120.2, 120.1, 114.2, 113.6, 113.4,
+
+
06.4, 78.1, 38.8, 31.5, 26.2, 12.3. HRMS (M + H) calculated for C H N O S
2
0
26
3
4
04.1639, found 404.1671. HPLC retention time 6.94 min, > 98% pure.
N-(4-(2,4-difluorophenoxy)-3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1,2-a]pyrazi
1
n-6-yl)phenyl)ethanesulfonamide (26). White solid, 51% yield. H NMR (400 MHz,
Chloroform-d) δ 9.12 (s, 1H), 8.28 (s, 1H), 7.35 (d, J = 2.7 Hz, 1H), 7.29 (dd, J = 8.8,
2
.7 Hz, 1H), 6.97 – 6.87 (m, 3H), 6.83 – 6.79 (m, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.47
(s, 1H), 6.36 (t, J = 5.7 Hz, 1H), 3.15 (q, J = 7.6 Hz, 2H), 2.59 (s, 3H), 1.40 (t, J = 7.4
13
Hz, 3H). C NMR (150 MHz, DMSO-d ) δ 158.1 (dd, J = 243.2,10.5 Hz), 157.2,
6
C-F
1
53.1 (dd, JC-F = 249.5, 13.0 Hz), 150.7, 139.0 (dd, JC-F = 11.4, 3.4 Hz), 134.0, 124.5,
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23.3, 122.5 (d, JC-F = 9.5 Hz), 122.0, 121.4, 120.6, 117.4, 114.7, 114.1, 112.0 (dd,
JC-F = 23.0,2.8 Hz), 105.7, 105.7 (dd, JC-F = 27.4, 22.5 Hz), 12.2, 8.0. HRMS (M +
+
+
H) calculated for C H F N O S 460.1137, found 460.1161. HPLC retention time
2
2
20
2
3
4
6
.82 min, > 95% pure.
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
6
-(2-(2,4-difluorophenoxy)-5-(methylsulfonyl)phenyl)-8-methylpyrrolo[1,2-a]p
1
yrazin-1(2H)-one (27). White solid, 45% yield. H NMR (400 MHz, Methanol-d4) δ
8
7
6
.04 (d, J = 2.3 Hz, 1H), 8.00 (dd, J = 8.7, 2.4 Hz, 1H), 7.28 (td, J = 9.0, 5.4 Hz, 1H),
.20 (ddd, J = 11.3, 8.6, 3.0 Hz, 1H), 7.07 – 7.01 (m, 2H), 6.99 (d, J = 5.9 Hz, 1H),
13
.61 (s, 1H), 6.51 (d, J = 5.9 Hz, 1H), 3.19 (s, 3H), 2.60 (s, 3H). C NMR (125 MHz,
DMSO-d ) δ 159.3 (dd, J = 244.5, 10.8 Hz), 158.3, 157.2, 153.6 (dd, JC-F = 250.3,
6
C-F
1
3.0 Hz), 137.1 (dd, J C-F= 11.8, 3.4 Hz), 135.4, 131.2, 129.6, 124.5 (d, JC-F = 10.0
Hz), 123.5, 122.2, 121.1, 120.7, 115.3, 115.1, 114.2, 112.6 (dd, JC-F = 23.0, 2.7 Hz),
+
1
06.0 (dd, JC-F = 27.7, 22.5 Hz), 105.9, 43.6, 12.2. HRMS (M + H) calculated for
+
C H F N O S 431.0872, found 431.0903. HPLC retention time 6.58 min, > 98%
2
1
17
2
2
4
pure.
N-(tert-butyl)-4-(2,4-difluorophenoxy)-3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1
1
,2-a]pyrazin-6-yl)benzenesulfonamide (28). White solid, 56% yield. H NMR (400
MHz, Methanol-d ) δ 7.95 (d, J = 2.4 Hz, 1H), 7.91 (dd, J = 8.7, 2.4 Hz, 1H), 7.27 –
4
7
1
1
1
.12 (m, 2H), 7.04 – 6.90 (m, 3H), 6.54 (s, 1H), 6.50 (d, J = 5.9 Hz, 1H), 2.59 (s, 3H),
1
3
.23 (s, 9H). C NMR (125 MHz, DMSO-d ) δ 159.1 (dd, J = 244.9, 10.8 Hz),
6
C-F
57.2, 157.1, 153.6 (dd, JC-F = 250.1, 12.9 Hz), 138.9, 137.4 (dd, JC-F = 11.8, 3.7 Hz),
30.3, 128.8, 124.3 (d, JC-F = 9.8 Hz), 123.7, 122.3, 121.0, 120.4, 115.0, 114.3, 112.5
(dd, JC-F = 23.0, 3.0 Hz), 106.0 (dd, JC-F = 27.5, 22.3 Hz), 105.6, 53.4, 29.7, 12.2.
+
+
HRMS (M + H) calculated for C H F N O S 488.1450, found 488.1483. HPLC
2
4
24
2
3
4
retention time 8.40 min, > 99% pure.
4
-(2,4-difluorophenoxy)-N,N-dimethyl-3-(8-methyl-1-oxo-1,2-dihydropyrrolo[1
1
,
2-a]pyrazin-6-yl)benzenesulfonamide (29). White solid, 57% yield. H NMR (400
MHz, Methanol-d ) δ 7.83 – 7.80 (m, 2H), 7.29 – 7.14 (m, 2H), 7.04 – 7.00 (m, 2H),
4
6
3
.96 (d, J = 5.9 Hz, 1H), 6.56 (s, 1H), 6.49 (d, J = 5.9 Hz, 1H), 2.72 (s, 6H), 2.58 (s,
13
H). C NMR (125 MHz, DMSO-d ) δ 159.6 (dd, J = 245.0,11.1 Hz), 158.4,
6
C-F
2
9
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