Discovery and optimization of 3-(2-(Pyrazolo[1,5- a ]pyrimidin-6-yl) ethynyl)benzamides as novel selective and orally bioavailable discoidin domain receptor 1 (DDR1) inhibitors

Article abstract of DOI:10.1021/jm301824k

Discoidin domain receptor 1 (DDR1) is an emerging potential molecular target for new anticancer drug discovery. We have discovered a series of 3-(2-(pyrazolo[1,5-a]pyrimidin-6-yl) ethynyl)benzamides that are selective and orally bioavailable DDR1 inhibitors. The two most promising compounds (7rh and 7rj) inhibited the enzymatic activity of DDR1, with IC₅₀ values of 6.8 and 7.0 nM, respectively, but were significantly less potent in suppressing the kinase activities of DDR2, Bcr-Abl, and c-Kit. Further study revealed that 7rh bound with DDR1 with a K_d value of 0.6 nM, while it was significantly less potent to the other 455 kinases tested. The S(35) and S(10) selectivity scores of 7rh were 0.035 and 0.008, respectively. The compounds also potently inhibited the proliferation of cancer cells expressing high levels of DDR1 and strongly suppressed cancer cell invasion, adhesion, and tumorigenicity. Preliminary pharmacokinetic studies suggested that they possessed good PK profiles, with oral bioavailabilities of 67.4% and 56.2%, respectively.

Full text of DOI:10.1021/jm301824k

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Article
Discovery and Optimization of 3-(2-(Pyrazolo[1,5-a]pyrimidin-6-
yl)ethynyl)benzamides as Novel Selective and Orally
Bioavailable Discoidin Domain Receptor 1 (DDR1) Inhibitors
Ke Ding, Mingshan Gao, Duan Lei, Jinfeng Luo, Lianwen Zhang, Xiaoyun
Lu, Yan Zhang, zhang zhang, zhengchao Tu, Yong Xu, and Xiaomei Ren
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301824k • Publication Date (Web): 22 Mar 2013
Downloaded from http://pubs.acs.org on April 1, 2013
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Discovery and Optimization of 3ꢀ(2ꢀ(Pyrazolo[1,5ꢀ
a]pyrimidinꢀ6ꢀyl)ethynyl)benzamides as Novel Selective
and Orally Bioavailable Discoidin Domain Receptor 1
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(DDR1) Inhibitors
Mingshan Gao, ω
,
ξ,,
δ
Lei Duan, ω Jinfeng Luo, Lianwen Zhang, ω Xiaoyun Lu, Yan Zhang, Zhang
,
δ
ω
ω
ω
Zhang, ω Zhengchao Tu, ω Yong Xu, Xiaomei Ren, and Ke Ding
*
ω
ω
ω*
ω
Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of
Sciences, #190 Kaiyuan Avenue, Guangzhou 510530, China
ξ
University of Chinese Academy of Sciences, # 19 Yuquan Road, Beijing 100049, China
δ
These authors contributed equally to this work.
*Corresponding authors: Tel: +86ꢀ20ꢀ32015276. Fax: +86ꢀ20ꢀ32015299.
EꢀMail: ding_ke@gibh.ac.cn or tu_zhengchao@gibh.ac.cn
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
ABSTRACT. Discoidin Domain Receptor 1 (DDR1) is an emerging potential molecular target for new
anticancer drug discovery. We have discovered a series of 3ꢀ(2ꢀ(pyrazolo[1,5ꢀa]pyrimidinꢀ6ꢀyl)
ethynyl)benzamides that are selective and orally bioavailable DDR1 inhibitors. The two most promising
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compounds (7rh and 7rj) inhibited the enzymatic activity of DDR1 with IC values of 6.8 and 7.0 nM,
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respectively, but were significantly less potent in suppressing the kinase activities of DDR2, BcrꢀAbl
and cꢀKit. Further study revealed that 7rh bound with DDR1 with K value of 0.6 nM, while was
d
significantly less potent to the other 455 kinases tested. The S(35) and S(10) selectivity scores of 7rh
were 0.035 and 0.008, respectively. The compounds also potently inhibited the proliferation of cancer
cells expressing high levels of DDR1 and strongly suppressed cancer cell invasion, adhesion and
tumorigenicity. Preliminary pharmacokinetic studies suggested that they possessed good PK profiles,
with oral bioavailabilities of 67.4% and 56.2%, respectively.
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KEYWORDS: Discoidin Domain Receptor 1, inhibitor, invasion, adhesion, tumorigenicity
INTRODUCTION
Discoidin Domain Receptors (DDRs) are members of the transmembrane receptor tyrosine kinases
(RTKs) superꢀfamily. They are distinguished from other RTKs by the presence of a discoidin motif in
1
, 2, 3
the extracellular domain.
Unlike typical RTKs that use peptideꢀlike growth factors as ligands,
DDRs are activated by various types of tripleꢀhelical collagens, which are key components of the
4
, 5
extracellular matrix (ECM).
Two types of DDRs (DDR1 and DDR2) have been identified with
distinct expression profiles and ligand specificities. DDR1 is widely expressed in epithelial cells in lung,
kidney, colon and brain, whereas DDR2 is primarily expressed in mesenchymal cells including
fibroblasts, myofibroblasts, smooth muscle and skeletal in kidney, skin, lung, heart and connective
tissues. DDR1 is activated by all collagens tested to date (types I, II, III, IV, V, VIII and XI), while
3
, 4, 5, 6
DDR2 is only activated by fibrillar collagens (collagen types I and III in particular).
Collective
evidences imply that that DDR1 and DDR2 play crucial roles in the regulation of fundamental cellular
4, 7, 8
process, such as proliferation, survival, differentiation, adhesion, and matrix remodeling.
Dysregulation of DDR1 and DDR2 has been linked to a number of human diseases, including fibrotic
9
, 10, 11
disorders, atherosclerosis and cancer.
For instance, overꢀexpression of DDR1 is associated with
12
the poor prognosis in nonꢀsmall cell lung cancer (NSCLC) and is implicated in cell survival and
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invasiveness in hepatocellular carcinoma, pituitary adenoma and prostate cancer.
High
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expression levels and/or mutation of DDRs are also frequently detected in the cancer cell lines and
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primary tumor tissues from lung, breast, brain, ovary, head and neck, liver, pancreas and
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prostate. Inhibition of DDR1 by small interfering RNA (siRNA) has been demonstrated to suppress
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tumorigenicity, inhibit lung cancer bone metastasis and increase cancer cell chemosensitivity.
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Selective suppression of DDR2 also displayed promising antitumor activity in mouse xenografts of
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squamous lung cancer cells harboring a “gainꢀof function” mutation of DDR2. Thus, DDR1 and
DDR2 are considered as novel potential molecular targets for anticancer drug discovery.
23, 25, 27
Several small molecule BcrꢀAbl inhibitors, imatinib (1), nilotinib (2), dasatinib (3),
bafetinib (4)
2
6
24, 27
and ponatinib (5),
have been reported to potently suppress the kinase activity of DDR1 and
DDR2. These compounds also strongly inhibited the collagenꢀinduced activation of DDR1 and DDR2 in
24
HEK 293 cells, with IC values in low nanomolar ranges. Given its promising therapeutic effect in
5
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mouse xenograft models, drug 3 has been selected for clinical investigation to treat squamous lung
2
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cancer (SCC) patients with mutated DDR2 (NCT01514864). Sorafenib, a multipleꢀtarget drug used in
the treatment of liver and renal cancer, was also reported to bind DDR1/DDR2 with nanomolar IC50
2
7, 28
values.
A thienopyridine derivative, LCB 03ꢀ0110, was recently found to inhibit collagenꢀinduced
29
autophosphorylation of DDR1 and DDR2, with IC values of 164 and 171 nM, respectively. However,
50
all of the reported DDR inhibitors displayed broad inhibition against a panel of many other kinases.
New selective DDR1 and/or DDR2 inhibitors are highly desirable for further validating DDRs as drug
targets.
In this paper, we report a series of 3ꢀ(2ꢀ(pyrazolo[1,5ꢀa]pyrimidinꢀ6ꢀyl)ethynyl)benzamides as novel
selective DDR1 inhibitors. The compounds potently suppressed the enzymatic activities of DDR1 with
low nanomolar IC values but were markedly less potent against BcrꢀAbl, DDR2 and cꢀKit kinases.
5
0
These compounds also strongly suppressed the proliferation of human cancer cells expressing high
levels of DDR1, with IC50 values in the low micromolar range. Furthermore, the representative
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compounds 7rh and 7rj potently suppressed cancer cell invasion, adhesion and tumorigenicity,
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indicating their potential to serve as new lead compounds for further anticancer drug discovery.
CF3
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STI-571, imatinib
2 nilotinib
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OH
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Cl
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dasatinib
4 bafetanib, INNO-406
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OH
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H
N
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O
N
CF3
O
5 ponatinib, AP24534
6 LCB 03-0110
Figure 1. Chemical structures of reported nonꢀselective DDR1 and DDR2 inhibitors.
CHEMISTRY
The new inhibitors were readily prepared using palladiumꢀcatalyzed Sonogashira coupling as the key
30
steps (Scheme 1). Briefly, commercially available or newly prepared methyl 3ꢀiodoꢀbenzoates (8)
were treated with ethynyltrimethylsilane under palladium catalysis to afford the Sonogashira coupling
products, which were deprotected to produce the terminal alkynes 9. The coupling of 9 with 6ꢀ
bromopyrazolo[1,5ꢀa]pyrimidine under Sonogashira conditions afforded the key intermediates 10. The
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esters 10 were hydrolyzed under basic conditions to yield the corresponding carboxylic acids 11, which
were coupled with various aryl amines to yield the desired inhibitors 7.
Scheme 1. Syntheses of 3ꢀ(2ꢀ(pyrazolo[1,5ꢀa]pyrimidinꢀ6ꢀyl)ethynyl)benzamide derivatives as new
DDR inhibitors.
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OH
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Reagents and conditions: (a) i) trimethylsilyl acetylene, CuI, PdCl (PPh ) , TEA, EtOAc, overnight,
2
3 2
~
85.0%; ii) K CO , MeOH, 5 mins, 90.0ꢀ92.0%%; (b) CuI, PdCl (PPh ) , NMP, 80 ℃, overnight, 80.0ꢀ
2 3 2 3 2
8
5.0%; (c) NaOH, CH OH, H O, 50℃, 6 hrs, ~90.0%; (d) PyBOP, DIPEA, DCM, overnight, 40.0ꢀ
3 2
90.0%.
RESULTS AND DISCUSSION
Almost all previously reported DDR inhibitors were originally developed as BcrꢀAbl kinase
suppressers. Furthermore, sequence alignment of human DDR1 and DDR2 demonstrated that they share
2
5d
~61% sequence identity with BcrꢀAbl in the ATP binding domain. Therefore, we initiated an effort to
identify new DDR inhibitors by screening a library of approximately 2000 compounds that were
originally designed as inhibitors of BcrꢀAbl and other RTKs. Inhibition of DDR1 and DDR2 was first
3
1
evaluated using the wellꢀestablished LANCE ULTRA kinase assay. For compounds with good
inhibitory activity (IC below 100 nM) against DDR1 and/or DDR2, the inhibition of BcrꢀAbl and cꢀKit
5
0
kinases was determined to investigate selectivity. Two reported inhibitors of both DDRs and BcrꢀAbl
(nilotinib (2) and dasatinib (3)) were included to validate the screening conditions. Under the
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experimental conditions, 3 potently inhibited DDR1, DDR2 and BcrꢀAbl with IC values of 4.8, 11.7
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and 0.26 nM, respectively; these values were similar to previously reported data (Table 1).
The
similar IC50 values of compound 2 with respect to previously reported data further confirmed the
25
reliability of our screening methods.
A number of compounds were found to exhibit strong inhibition of DDR1 and DDR2: Nꢀisopropylꢀ4ꢀ
methylꢀ3ꢀ(2ꢀ(pyrazolo[1,5ꢀa]pyrimidinꢀ6ꢀyl)ethynyl) benzamide (7a) stood out because of its good
activity and relatively high selectivity over BcrꢀAbl. This compound potently inhibited the kinase
activity of DDR1 with an IC value of 39.6 nM but did not detectably inhibit BcrꢀAbl and DDR2 at 1.0
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ꢀM. However, further evaluation showed that this compound also strongly suppressed the activity of cꢀ
Kit, with an IC value of 67 nM.
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Taking 7a as a lead molecule, extensive structural optimization was conducted to improve its DDRꢀ
inhibitory activity and its selectivity over BcrꢀAbl and cꢀKit kinases. The results are summarized in
Table 1 and Table 2. We found that the isopropyl group in 7a could be replaced with an nꢀbutyl (7b),
cyclohexyl (7d) or cyclopentyl (7e) moiety without reducing the inhibitory activity against DDR1. For
instance, compound 7d (with a cyclohexyl moiety) displayed an IC value of 29.1 nM against DDR1,
5
0
which was similar to that of 7a, but its inhibitory activity against cꢀKit decreased approximately 3ꢀfold.
Interestingly, when the isopropyl group in 7a was changed to an isobutyl moiety (7c), the inhibitory
activity was dramatically decreased for all 4 of the tested kinases. Further investigation revealed that the
isopropyl group in 7a could also be replaced with a phenyl substituent (7f), which maintained strong
inhibition of DDR1 and reduced the inhibition of cꢀKit. Thus, compounds 7f and 7a displayed almost
identical IC values against DDR1. However, the selectivity of 7f for DDR1 over cꢀKit kinase was
50
approximately 12 times greater than that of 7a.
Systematic structural optimization of 7f revealed that substituents on the Nꢀterminal phenyl ring
dramatically impacted DDR1 kinase inhibition. For instance, when a methyl group was introduced at R3
(
7h), the IC was approximately 5.96 nM against DDR1, or 6.5 times more potent than 7f. Although a
50
methyl group at R (7i) did not greatly influence the potency against DDR1, the R ꢀmethylated
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compound 7g was over 25ꢀfold less potent than 7f. The compounds featuring R ꢀchloro (7k) or methoxy
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(
7n) substituents were also significantly more potent than 7f or the corresponding R ꢀ or R ꢀsubstituted
2 4
analogues (7j, 7l, 7m and 7o). However, none of these compounds displayed marked selectivity for
DDR1 over cꢀKit kinase. Further studies suggested that the R position could also tolerate Br (7p), I
3
(7q), isoꢀpropoxyl (7t), dimethylamino (7u), or 1ꢀimidazolyl (7v) substituents while retaining good or
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moderate inhibition against DDR1. However, when a 1ꢀ(4ꢀmethyl)imidazolyl (7w) or (4ꢀ
methylpiperazinꢀ1ꢀyl)methyl (7x) moiety was introduced at R , the potency was significantly decreased.
3
Encouragingly, when R was a CF group, the resulting compound 7r displayed a 6.14 nM IC value
3
3
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against DDR1 and 12–35ꢀfold selectivity over DDR2, BcrꢀAbl and cꢀKit. By contrast, CF substitution
3
at R (7s) almost totally abolished the inhibition of the 4 evaluated kinases, which confirmed that R is
4
3
the optimal position for structural optimization.
a, b
Table 1 In vitro inhibitory activities of compounds 7a-7x against DDR1, DRR2, BclꢀAbl and cꢀKit.
N
R4
N
O
N
N
R3
H
R2
7
Kinase inhibition (IC , nM)
5
0
Compound
R2
R3
R4
DDR1
39.63
DDR2
BcrꢀAbl
>1ꢁM
cꢀKit
67
N
N
O
N
7
7
a
>1ꢁM
N
H
N
N
O
N
b
65.14
>1ꢁM
>1ꢁM
16.7
N
H
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N
O
N
7
c
>1ꢁM
29.15
>1ꢁM
>1ꢁM
>1ꢁM
497
200
N
H
N
N
O
N
7
d
>1ꢁM
>1ꢁM
N
H
0
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0
N
N
O
N
7e
7f
39.82
>1ꢁM
12.5
N
H
H
Me
H
H
H
H
H
38.90
>1ꢁM
5.96
>1ꢁM
>1ꢁM
>1ꢁM
>1ꢁM
>>1ꢁM
>1ꢁM
>1ꢁM
>1ꢁM
>1ꢁM
>1ꢁM
>1ꢁM
>1ꢁM
73.84
>1ꢁM
>1ꢁM
>1ꢁM
>1ꢁM
>1ꢁM
>1ꢁM
>1ꢁM
>1ꢁM
>1ꢁM
>1ꢁM
275.42
>1ꢁM
97.44
457
>1ꢁM
3.03
2.61
8320
4.89
29.6
82.3
1.9
7
7
g
h
Me
H
H
7i
H
Me
H
47.75
>1ꢁM
2.29
7
j
Cl
H
H
7
k
Cl
H
H
7l
H
Cl
H
>1ꢁM
470
7
m
MeO
H
H
7
n
MeO
H
H
4.04
7o
H
MeO
H
22.36
16.64
19.75
6.14
9.28
99.5
254
7
7
p
q
H
Br
I
H
H
7r
H
CF
3
H
214
7
s
H
H
CF
3
>1ꢁM
35.02
>1ꢁM
>1ꢁM
>1ꢁM
>1ꢁM
>1ꢁM
117
7
t
H
iꢀPrO
H
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
7
u
v
H
H
H
H
H
H
9.72
19.03
386.5
>1ꢁM
>1ꢁM
2270
310.63
198
18.8
82.4
284
7
159
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
7w
7
x
H
H
324.1
>1ꢁM
105
504.0
43.5
0.26
257
N.D.
2.38
32.6
2
c
c
(43)
(55)
4
.80
11.7
3
c
c
(0.5)
(1.4)
a
DDR1 and DDR2 experiments were performed using LANCE ULTRA kinase assay according to the
manufacturer’s instructions. The data are means from at least 2 independent experiments. BcrꢀAbl and
cꢀKit activity experiments were performed using the FRETꢀbased Z’ꢀLyte assay according to the
b
c
manufacturer’s instructions. The data are means from at least 3 independent experiments. Reported
2
4, 25
data.
A series of R , R ꢀdisubstituted compounds was also designed and synthesized (Table 2). R , R ꢀ
3
5
3
5
Dimethyl compound 7y displayed similar potency against DDR1 to that of the monoꢀmethyl compound
7h (Table 1), but its selectivity was improved. The R ꢀmethyl, R ꢀchloro compound 7z also potently
3
5
inhibited DDR1, with an IC of 29.8 nM. These results strongly suggested that one hydrophobic group
5
0
in R (or R ) would be sufficient to interact with DDR1, whereas the other group might be located
3
5
outside of the binding pocket, providing an ideal position for a pharmaceutically acceptable hydrophilic
group to improve the hydrophilicꢀlipophilic balance of the molecule. Therefore, compounds 7ra, 7re,
7
rf and 7rg, which featured hydrophilic 1ꢀ(4ꢀmethyl)imidazolyl, morpholinomethyl, piperidinꢀ1ꢀ
ylmethyl or (pyrrolidinꢀ1ꢀyl)methyl groups, respectively, were designed and synthesized. All
compounds showed comparable inhibition of DDR1 to the monoꢀsubstituted lead compound 7r. For
instance, 7ra displayed an IC of 8.93 nM against DDR1, while 7r had a value of 6.14 nM. However,
5
0
introduction of a hydrophilic group seemed to increase inhibition of BcrꢀAbl, causing a significant loss
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Journal of Medicinal Chemistry
Page 10 of 50
of DDR1 selectivity. Compounds 7rb, 7rc and 7rd were also obvious less selective than the
corresponding monoꢀsubstituted compounds 7h, 7k and 7n, although their potencies against DDR1 were
not significantly changed.
1
2
3
4
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
a, b
Table 2 In vitro inhibitory activities of compounds 7y-7rj against DDR1, DRR2, BclꢀAbl and cꢀKit.
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
N
R5
N
O
N
N
R3
H
R1
7
Kinase inhibition (IC , nM)
5
0
Compound
R1
R3
R5
DDR1
6.34
DDR2
116.0
>1ꢁM
BcrꢀAbl
>1ꢁM
cꢀKit
185
7y
Me
Me
Me
Me
Me
Cl
7z
29.81
>1ꢁM
>1ꢁM
7
ra
Me
Me
Me
Me
CF
CH
Cl
3
8.93
100.56
37.1
16.2
756
6.71
38.9
274
7rb
3
13.94
103
125
59.5
128
407
413
7
rc
13.3
7rd
OMe
7re
7rf
Me
Me
CF
3
17.49
37.58
775.9
214
129.1
115
>1ꢁM
>1ꢁM
CF
3
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
7
rg
Me
Et
CF
3
12.47
6.811
131.36
101.4
49.04
355
741
7rh
CF
3
>10ꢁM
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
7
ri
rj
Cl
H
CF
3
10.05
7.02
172.65
93.65
54.7
447
1500
243
7
CF
3
a
DDR1 and DDR2 experiments were performed using LANCE ULTRA kinase assay according to the
b
manufacturer’s instructions. The data are means from at least 2 independent experiments. BcrꢀAbl and
cꢀKit activity experiments were performed using the FRETꢀbased Z’ꢀLyte assay according to the
manufacturer’s instructions. The data are means from at least 3 independent experiments.
Prior studies suggested that the “flagꢀmethyl” R group was important for AP24534 (5) and other
1
3
2
inhibitors to maintain strong binding to BcrꢀAbl kinase. We hypothesized that BcrꢀAbl inhibition
might be decreased or eliminated by modifying the R substituent. Indeed, although the R ꢀCl compound
1
1
7ri showed similar potency and selectivity to that of 7ra, we were pleased to find that the BcrꢀAbl
inhibitory potencies were significantly decreased by replacing the R ꢀmethyl group in 7ra with an ethyl
1
group (7rh) or a hydrogen atom (7rj); the activities against DDR1 and DDR2 remained unchanged. For
instance, compound 7rh inhibited DDR1 with an IC of 6.81 nM, while the IC values for DDR2, Bcrꢀ
5
0
50
Abl and cꢀKit were 101.4, 355 and over 10,000 nM, respectively. Compound 7rj also displayed 12–64ꢀ
fold selectivity for DDR1 over the other 3 kinases evaluated. Thus, compounds 7rh and 7rj represented
the two most potent and selective DDR1 inhibitors for further investigation.
The direct binding affinity of compound 7rh with DDR1 kinase was determined by using an activeꢀ
siteꢀdependent competition binding assay (conducted by Ambit Bioscience, San Diego, USA). It was
shown that compound 7rh tightly bound to the ATPꢀbinding sites of DDR1 with K values of 0.6 nM.
d
We further profiled the compound against a panel of 456 kinases (including 395 nonꢀmutated kinases)
using the Ambit Kinome screening platform to investigate the selectivity of the new DDR1 inhibitor.
The screening concentration was 100 nM which was about 160 times higher than its K values with
d
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Page 12 of 50
DDR1. The results clearly revealed that compound 7rh displayed an excellent selective profile on
DDR1 (Supporting Information and Table 3.). For instance, compound 7rh showed almost 100%
competition rate (99.5% inhibition, Ctrl% = 0.05) with DDR1 at 100 nM, while it only displayed
obvious binding (inhibition rate >65%, or Ctrl% < 35%) with 14 of the other 395 nonꢀmutated kinases
evaluated. These kinases included Abl1, BꢀRaf (V600E), DDR2, EPHA8, HCK, LOK, MAK, PDGFRβ,
Tie2, TRKb, TRBc and ZAK etc. The S(35) and S(10) selectivity scores were 0.035 and 0.008,
respectively.
1
2
3
4
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
Table 3 Hits with Ctrl% < 35% in a selectivity profiling study of compound 7rh against 396 nonꢀ
a
mutated kinases at 100 nM.
Kinases
Ctrl% @ 100 M
27
Sꢀscore
S(35)
0.035
(S10)
0.008
Nonꢀ
phosphorylated
Ablꢀ1
BꢀRaf (V600E)
DDR1
DDR2
EPHAꢀ8
HCK
29
0.05
28
21
30
GCN2
LOK
25
1.6
13
MAK
PDGFRβ
Tie2
31
31
TRBb
8.7
12
TRBc
ZAK
22
a
The binding rates of 7rh with different kinases were determined by using an activeꢀsiteꢀdependent
competition binding assay (conducted by Ambit Bioscience, San Diego, USA). The results were
reported as “control%” (ctrl%), where lower numbers indicate stronger binding. Sꢀcore = number of hits
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Page 13 of 50
Journal of Medicinal Chemistry
/
–
numbers of assays. Ctrl% = (test compound signal – positive control signal) / (negative control signal
positive control signal) ×100. Wherein, negative control = DMSO (Ctrl% = 100%); positive control =
1
2
3
4
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
control compound (Ctrl% = 0%). Sꢀscore (selectivity score) = number of hits / number of assays. S(35)
= (number of nonꢀmutated kinases with ctrl% < 35%) / (number of nonꢀmutated kinases tested). S(10) =
(
number of nonꢀmutated kinases with ctrl% < 10%) / (number of nonꢀmutated kinases tested).
To further validate these new DDR1 inhibitors, we examined the effects of the representative
compounds 7rh and 7rj on the activation of DDR1 and downstream signals in NCIꢀH23 nonꢀsmall cell
lung cancer cells (NSCLC) expressing high level of DDR1; the results are summarized in Figure 2. It
was clear that both compounds inhibited phosphorylation of DDR1 in a doseꢀdependent manner. It has
previously been demonstrated that DDR1 activation can trigger proꢀsurvival Ras/Raf/Erk and PI3K/Akt
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
3
signals, resulting in enhanced expression of DDR1 and the antiꢀapoptotic BclꢀxL. Not surprisingly,
the inactivation of DDR1 by compounds 7rh and 7rj induced significant decrease of total protein levels
of DDR1 and BclꢀxL. Prior studies have also revealed that DDRs stimulate the production of matrix
3
3
metalloproteinase (MMP). Indeed, inhibition of DDR1 by 7rh and 7rj caused a significant reduction
in the level of MMPꢀ2. However, the effect on MMPꢀ9 was less obvious.
Figure 2. Compounds 7rh and 7rj inhibit both the expression and phosphorylation of DDR1 and
downstream signaling in a doseꢀdependent manner. A) Compound 7rh inhibits the expression and
phosphorylation of DDR1 and downstream signaling in NCIꢀH23 NSCLC cells; B) Compound 7rj
inhibits the expression and phosphorylation of DDR1 and downstream signaling in NCIꢀH23 NSCLC
cells.
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The antiꢀproliferative effects of compounds 7rh and 7rj were also investigated using a panel of cancer
cell lines expressing high levels of DDR1 (Table 4). The drugs 2 and 3 were included as positive
controls because of their strong DDR1/DDR2 inhibitory activities. The cancer cell lines included A549,
NCIꢀH23 and NCIꢀH460 human NSCLC cells; MDAꢀMBꢀ435S, MCFꢀ7 and T47D human breast cancer
cells; HCT116 human colon cancer cells; and K562 chronic myelogenous leukemia cells. It was also
1
2
3
4
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
noteworthy that the cells have been successfully used as models for the functional investigation on
23, 39c, 39d, 43
DDR1.
As shown in Table 3, compounds 7rh and 7rj potently inhibited proliferation, with
IC values in the low ꢀM range. For instance, compound 7rh suppressed the growth of A549, NCIꢀH23
5
0
and NCIꢀH460 human NSCLC cells with IC values of 2.7, 2.1 and 3.0 ꢀM, respectively, while the
5
0
value in K562 human CML cells was approximately 0.038 ꢀM. The strong inhibition of 7rh and 7rj on
K562 cell growth might be, at least in part, due to the high level of activated DDR1 (Supporting
Information). Further investigation also revealed that the compounds induced apoptosis of NCIꢀH23
NSCLC cells in a doseꢀdependent manner (see Supporting Information).
Table 4. The antiꢀproliferative effects of compounds 7rh and 7rj on a panel of cancer cells harboring
high levels of DDR1.
a
Antiꢀproliferative activity (IC , ꢀM)
5
0
compound
b
b
b
b
c
A549
NCIꢀb
H23
NCIꢀb
H460
MDAꢀMBꢀ
MCFꢀ7
T47D
HCT116
K562
b
435S
7
rh
2.74±0 2.08±0 2.98±0.5
22 .12
2.22±0.05
2.86±0.07
2.15±0.04 1.88±0.22
2.82±0.18 3.13±0.14
1.13±0.18
2.41±0.18
0.038±0.
011
.
7
7rj
2
11.73± 11.91± 7.77±0.9
0.49 0.28
0.059±0.
005
5
6
.63±0 3.05±0 14.41±2.
0.015±0.
2.66±0.211
6.92±1.03 6.08±0.01
2.39±0.02
2.30±0.01
d
.
45
.45
31
003
3
.90±0.05
2.57±0.08 0.90±0.20
1
2.66± 2.27±0
8.99±0.7
1
0.0007±0
3
1.12 e
.44 e
e
e
e
d
e
(7.78)
（12.4）
(0.45)
(4.45)
.0001
(>1.0) (1.02)
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Page 15 of 50
Journal of Medicinal Chemistry
a
b
Data are means from at least 4 independent experiments. The antiꢀproliferative activities of the
1
c
compounds were evaluated using an MTT assay. IC values were determined by using the cell
5
0
2
3
4
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
d
counting kit (CCKꢀ8) assay. T4h2e potent antiproliferative activity might be due to strong inhibition
e
against BcrꢀAbl. Reported data.
MMP2 and MMP9 are key enzymes mediating the degradation of extracellular matrix (ECM), which
is a critical step in metastasis. Increased levels of MMP2 and MMP9 have been associated 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
3
5, 36
metastasis of different types of solid tumor.
Collectively, the studies demonstrate that DDR1
4
, 34
stimulates the production of MMPs.
Our results clearly demonstrate that 7rh and 7rj potently
decrease MMPs (Figure 2), suggesting that DDR1 inhibitors may inhibit the migration and invasion of
cancer cells. Therefore, we investigated the effects of 7rh and 7rj on the invasiveness of NCIꢀH23
3
7
NSCLC cells using a Boyden chamber assay. Matrigel was applied to the filter membrane, and the
number of NCIꢀH23 cells that penetrated the matrigel and membrane was quantified. As shown in
Figure 3, compounds 7rh and 7rj inhibited the invasiveness of NCIꢀH23 cells in a doseꢀdependent
manner. Treatment with 7rh at 1.0, 2.0 or 5.0 ꢀM for 24 hours inhibited invasion by ~30%, ~37% or
~82%, respectively (P<0.01, compared to vehicle control; see Figure 3A and Supporting Information).
The corresponding numbers for 7rj were 36%, 39% and 57%, respectively (Figure 3B and Supporting
Information). These results strongly suggested that the compounds may suppress the metastasis of
NSCLC cancers.
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Journal of Medicinal Chemistry
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1
2
3
4
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 3. Compounds 7rh and 7rj inhibit the invasion of NCIꢀH23 NSCLC cells in a dose dependent
manner. A) Compound 7re inhibits the invasion of NCIꢀH23 NSCLC cells in a Boyden chamber assay.
B) Compound 7rj inhibits the invasion of NCIꢀH23 NSCLC cells in a Boyden chamber assay. The
results are representative of two independent experiments.
Cell adhesion involves a number of regulatory processes, including growth, differentiation,
proliferation, migration and regeneration. Adhesion is crucial in the formation and maintenance of
38
coherent multicellular structures. DDR1 has been implied as one of the key regulators of cell adhesion.
3
9
Therefore we investigated the impact of 7rh and 7rj on cellꢀmatrix adhesion using a wellꢀestablished
4
0
crystal violet adhesion assay. NCIꢀH23 NSCLC cells adhering to the preꢀcoated BD Matrigel were
treated with 7rh and 7rj in different concentrations. As shown in Figure 4, compounds 7rh and 7rj
potently inhibited cellꢀmatrix adhesion in a doseꢀdependent manner. Treatment with 7rh at 5.0, 10.0 or
2
0.0 ꢀM for 2 hours inhibited cellꢀmatrix adhesion by ~17%, ~40% or ~88%, respectively, compared to
vehicle control (Figure 4A)
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
A
B
110
00
110
100
90
80
70
60
50
40
30
20
10
0
1
90
80
70
60
*
*
*
*
*
50
40
30
20
10
**
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
**
0
0
1
5
10
(ꢀM
20
0
1
5
7.5
10
Co
ncent
r
a
t
ion(
))
Concentration(ꢀM)
Figure 4. Compounds 7rh and 7rj inhibit the cellꢀmatrix adhesion of NCIꢀH23 cells in a crystal violet
adhesion assay. A) Compound 7rh inhibits the cellꢀmatrix adhesion of NCIꢀH23 cells in a dose
dependent manner. B) Compound 7rj inhibits the cellꢀmatrix adhesion of NCIꢀH23 cells in a dose
dependent manner. The results are representative of 2 independent experiments. (* p<0.05, **p<0.01)
The effects of 7rh and 7rj on the tumorigenicity of NCIꢀH23 cancer cells were also examined using
an in vitro colony formation assay, as well as anchorageꢀindependent growth in soft agarose. As shown
in Figures 5A and 5B, the compounds inhibited colony formation by NCIꢀH23 NSCLC cells in a doseꢀ
dependent manner, with IC50 values of 0.56 and 3.29 ꢀM, respectively (Supporting Information).
Anchorageꢀindependent growth of cells in soft agar is one of the hallmarks of cellular transformation
and uncontrolled cell growth. Therefore, the antiꢀtumorigenic effects of 7rh and 7rj on NCIꢀH23 cancer
cells were further evaluated using a 3ꢀdimensional (3D) softꢀagar assay, which is closer to the in vivo
cellular environment. The results (Figure 5C) reveal that the compounds potently reduce the number
and size of colonies, suggesting strong inhibition of cancer cell transformation.
Given their promising in vitro biological activities, preliminary in vivo pharmacokinetic (PK) studies
of 7rh and 7rj were also conducted in rats. The results are summarized in Table 5. Compounds 7rh and
7
rj displayed good pharmacokinetic properties, with oral bioavailability of 67.4% and 56.2%,
respectively. 7rh and 7rj also possessed reasonable halfꢀlives of 15.5 and 9.8 hours, respectively, after
oral administration.
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Figure 5. Compounds 7rh and 7rj suppress NCIꢀH23 cell tumorigenicity. A) Compound 7rh inhibits
NCIꢀH23 cancer cell colony formation in a colongenic assay. B) Compound 7rj inhibits NCIꢀH23
cancer cell colony formation in a clonogenic assay. C) 7rh and 7rj reduce the number and size of
colonies of NCIꢀH23 cancer cells in a soft agar cell transformation assay. The results are representative
of 2 independent experiments.
a
Table 5. Compounds 7rh and 7rj display a good pharmacokinetic profile in rats.
7rh
7rj
Oral (25 mg/kg)
37587.54±3453.28
15.53±1.58
i.v. (5 mg/kg)
11156.31±921.89
13.21±1.48
Oral (25 mg/kg)
24706.67±4079.71
9.80±4.72
i.v. (5 mg/kg)
8799.33±2550.47
7.55±3.47
AUC(0ꢀ∞) (ꢀg/L*h)
T1/2 (h)
Tmax (h)
4.25±1.26
0.033
4.00±1.41
0.033
Cmax (ꢀg/L)
1867.50±118.43
67.4%
1122.50±97.40
1767.50±216.39
56.2%
1235.00±68.56
F (%)
a
SD rats (male, 4 animals per group) weighted 180~220g were used for the study.
In summary, a series of 3ꢀ(2ꢀ(pyrazolo[1,5ꢀa]pyrimidinꢀ6ꢀyl)ethynyl)benzamides were found to be
selective small molecule DDR1 inhibitors. The compounds potently inhibited DDR1 but were
significantly less potent against many other kinases such as DDR2, Abl and cꢀKit. The most promising
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Journal of Medicinal Chemistry
compounds 7rh and 7rj displayed nanomolar IC50 values against DDR1 and potently inhibited the
proliferation of a panel of cancers cell lines expressing high levels of DDR1, including A549, NCIꢀH23,
NCIꢀH460 human NSCLC cells; MDAꢀMBꢀ435S, MCFꢀ7, T47D human breast cancer cells; HCT116
colon cancer cells; and K562 CML cells. These compounds also potently suppressed the activation of
DDR1 and downstream signaling. Further investigation demonstrated that compounds 7rh and 7rj
strongly inhibited invasiveness, cellꢀmatrix adhesion and tumorigenicity in NCIꢀH23 human NSCLC
cells. Moreover, in preliminary in vivo pharmacokinetic studies, 7rh and 7rj displayed excellent profiles.
Our study provides new research probes and lays a basic foundation for further validation of DDR1 as a
potential target for anticancer drug development.
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EXPERIMENTAL
Chemistry. Reagents and solvents were obtained from commercial suppliers and used without further
purification. Flash chromatography was performed using silica gel (300ꢀ400 mesh). All reactions were
1
monitored by TLC, silica gel plates with fluorescence F254 were used and visualized with UV light. H
1
3
and C NMR spectra were recorded on a Bruker AVꢀ400 spectrometer at 400 MHz and Bruker AVꢀ500
spectrometer at 125 MHz, respectively. Coupling constants (J) are expressed in hertz (Hz). Chemical
shifts (δ) of NMR are reported in parts per million (ppm) units relative to internal control (TMS). The
low or high resolution of ESIꢀMS was recorded on an Agilent 1200 HPLCꢀMSD mass spectrometer or
Applied Biosystems QꢀSTAR Elite ESIꢀLCꢀMS/MS mass spectrometer, respectively. The purity of
compounds was determined to be over 95% (>95%) by reverseꢀphase high performance liquid
chromatography (HPLC) analysis. HPLC instrument: DIONEX SUMMIT HPLC (Column: Diamonsil
C18, 5.0 µm, 4.6 x 250 mm(Dikma Technologies); Detector: PDAꢀ100 Photodiode Array; Injector:
ASIꢀ100 Autoinjector; Pump: pꢀ680A.). Elution: 85% MeOH in water; flow rate, 1.0 mL/min.
Methyl 3-ethynyl-4-methylbenzoate (9a) To a solution of methyl 3ꢀiodoꢀ4ꢀmethylbenzoate (27.61 g,
100 mmol) in EtOAc (300 mL) was added Pd(PPh ) Cl (0.70 g, 1 mmol), CuI (0.19 g, 1 mmol), and
3
2
2
triethylamine (30.4 g, 300 mmol). The mixture was stirred at room temperature overnight under argon
atmosphere. The reaction mixture was filtered through a pad of celite. The filtrate was concentrated and
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Journal of Medicinal Chemistry
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the resulting residue was purified by flash chromatography to give methyl 4ꢀmethylꢀ3ꢀ
1
2
3
4
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
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5
5
5
5
5
5
5
6
1
(
(trimethylsilyl)ethynyl)benzoate as a white solid (20.9 g, 85.0%). H NMR (400 MHz, DMSOꢀd ) δ
6
7.89 (d, J = 1.6 Hz, 1 H), 7.83 (dd, J = 8.0, 1.6 Hz, 1 H), 7.42 (d, J = 8.0 Hz, 1 H), 3.83 (s, 3 H), 2.42 (s,
13
3
1
H), 0.24(s, 9 H). C NMR (100 MHz, DMSOꢀd ) δ 165.1, 145.1, 132.0, 129.8, 129.0, 127.4, 122.4,
6
+
02.4, 99.2, 51.8, 20.0, ꢀ0.4. LCꢀMS: m/z 247 [M+H] . To a solution of methyl 4ꢀmethylꢀ3ꢀ
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
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6
7
8
9
0
1
2
3
4
5
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7
8
9
0
(
(trimethylsilyl)ethynyl)benzoate (19.7 g, 80 mmol) in CH OH (300 mL), and treated with K CO (16.6
3 2 3
g, 120 mmol), and the mixture was stirred at room temperature for 5 minutes. The solvents were
evacuated and EtOAc and H O were added to the residue. The organic layer was separated, and the
2
aqueous layer was exacted with EtOAc (3×100 mL). The combined layers were dried over Na SO and
2
4
concentrated. The resulting crude was further purified by flash chromatography to give the product as a
1
white solid(12.5 g, 90.0%). H NMR (400 MHz, DMSOꢀd ) δ 7.93 (d, J = 1.2 Hz, 1 H), 7.85 (dd, J =
6
1
3
8
.0, 1.6 Hz, 1 H), 7.44 (d, J = 8.4 Hz, 1 H), 4.49 (s, 1H), 3.84 (s, 3 H), 2.44 (s, 3 H). C NMR (100
MHz, DMSOꢀd ) δ 165.2, 145.3, 132.3, 129.9, 129.0, 127.4, 121.9, 85.1, 80.9, 51.9, 20.1. LCꢀMS: m/z
6
+
ꢀ
1
75 [M+H] ; 173 [MꢀH] .
Methyl 4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzoate (10a) To a solution of 9a (10.45 g,
60 mmol) in NMP (120 mL), 6ꢀbromopyrazolo[1,5ꢀa]pyrimidine (11.88 g, 60 mmol), N, Nꢀ
diisopropylethylamine (7.76 g, 180 mmol), Pd(PPh ) Cl (0.42 g, 0.6 mmol), and CuI (0.12 g, 0.6 mmol)
3
2
2
was placed in a vial with rubber septum. The mixture underwent 3 cycles of vacuum/filling with Ar.
The mixture was stirred at 80 ℃ overnight and then quenched with H O. EtOAc and more H O were
2
2
added for extraction. The combined organic layer was dried over Na SO , filtered, concentrated, and the
2
4
resulting residue was purified by chromatography, giving the title compound as a white solid (14.0 g,
1
8
0.0%). H NMR (400 MHz, DMSOꢀd ) δ 9.56 (s, 1H), 8.70 (s, 1H), 8.32 (s, 1H), 8.08 (s, 1H), 7.89 (d,
6
1
3
J= 7.6 Hz, 1H), 7.50 (d, J= 8.0 Hz, 1H), 6.82 (s, 1H), 3.86 (s, 3H), 2.56 (s, 3H). C NMR (125 MHz,
DMSOꢀd ) δ 165.4, 150.9, 146.5, 146.3, 145.3, 138.2, 132.2, 130.3, 129.5, 127.6, 122.0, 104.6, 97.3,
6
+
9
0.2, 87.8, 52.2, 20.5. LCꢀMS: m/z 292 [M+H] .
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Journal of Medicinal Chemistry
4
-Methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzoic acid (11a) To a solution of 10a (11.65 g,40
1
2
3
4
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
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3
3
3
3
3
3
4
4
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4
4
4
4
4
4
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5
6
mmol) in methanol (120 mL) was added NaOH (3.2 g, 80 mmol). The mixture was stirred at 60 ℃
overnight. The mixture was acidified to pH 4 in ice bath by 5% HCl. The precipitates were filtered,
washed with H O, and further purified by recrystallization to give the title compound as a offꢀwhite
2
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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7
8
9
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7
8
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2
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8
9
0
solid (10.1 g, 91.0%). H NMR (400 MHz, DMSOꢀd ) δ 13.08 (br s, 1H), 9.57 (s, 1H), 8.71 (s, 1H),
6
8
.32 (s, 1H), 8.08 (s, 1H), 7.89 (d, J= 8.0 Hz, 1H), 7.49 (d, J= 8.0 Hz, 1H), 6.83 (s, 1H), 2.57 (s, 3H).
1
3
C NMR (125 MHz, DMSOꢀd ) δ 167.0, 151.5, 147.0, 146.8, 145.3, 138.7, 132.9, 130.6, 130.3, 129.3,
6
+
ꢀ
1
22.4, 105.3, 97.8, 90.9, 88.1, 21.0. LCꢀMS: m/z 278 [M+H] ; 276 [MꢀH] .
N-Isopropyl-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7a) To the suspension of
4
ꢀmethylꢀ3ꢀ(pyrazolo[1,5ꢀa]pyrimidinꢀ6ꢀylethynyl)benzoic acid (0.28 g, 1 mmol) in CH Cl (3 mL) was
2 2
added isopropylamine (0.059 g, 1 mmol), N, Nꢀdiisopropylethylamine (0.39 g, 3 mmol), and PyBOP
0.62 g, 1.2 mmol). The mixture was stirred at room temperature for 6 hours. CH Cl and H O were
(
2
2
2
added, the organic layers were separated and the aqueous was extracted with CH Cl (5×100 mL). The
2
2
combined organic layer was dried over Na SO , filtered, and concentrated. The resulting residue was
2
4
purified by flash chromatography and recrystallization, giving the title compound as a white solid (0.26
1
g, 81.7%). H NMR (500 MHz, DMSOꢀd ) δ 9.56 (s, 1H), 8.71 (d, J = 1.5 Hz, 1H), 8.33 (d, J = 2.0 Hz,
6
1
H), 8.32 (d, J = 7.5 Hz, 1H), 8.05 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 6.84 (d, J
1
3
=
1.5 Hz, 1H), 4.11 (sext, J = 7.0 Hz, 1H), 2.55 (s, 3H), 1.17 (d, J = 6.5 Hz, 6H). C NMR (100 MHz,
DMSOꢀd ) δ 164.1, 150.6, 146.4, 146.0, 142.6, 137.7, 132.6, 130.2, 129.4, 127.8, 121.1, 104.7, 97.1,
6
+
ꢀ
9
0.8, 86.9, 40.9, 22.1 (2C), 20.0. LCꢀMS (ESI): m/z 319 [M+H] ; 317 [MꢀH] . HRMS (ESI): m/z calcd
+
for C H N O [M+H] , 319.1553; found 319.1553. HPLC analysis: 5.23 min, 98.0%.
19 19
4
1
N-Butyl-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7b) Yield, 75.0%. H NMR
(500 MHz, DMSOꢀd ) δ 9.56 (s, 1H), 8.70 (d, J = 2.0 Hz, 1H), 8.51 (t, J = 5.5 Hz, 1H), 8.33 (d, J = 2.5
6
Hz, 1H), 8.04 (s, 1H), 7.82 (d, J = 7.5 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 6.84 (d, J = 2.0 Hz, 1H), 3.26
(q, J = 6.5 Hz, 1H), 2.54 (s, 3H), 1.51 (quint, J = 7.5 Hz, 2H), 1.34 (sext, J = 7.5 Hz, 2H), 0.90 (t, J =
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1
3
7
.5 Hz, 3H). C NMR (100 MHz, DMSOꢀd ) δ 164.9, 150.7, 146.4, 146.0, 142.6, 137.7, 132.5, 130.2,
6
1
2
3
4
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
+
1
29.5, 127.7, 121.2, 104.7, 97.1, 90.8, 87.0, 31.0, 20.0, 19.4, 13.4. LCꢀMS (ESI): m/z 333 [M+H] ; 331
ꢀ
+
[MꢀH] . HRMS (ESI): m/z calcd for C H N O [M+H] , 333.1710; found 333.1708. HPLC analysis:
2
0
21
4
5
.98 min, 98.6%.
N-Isobutyl-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7c) Yield, 70.5%.
NMR (500 MHz, DMSOꢀd ) δ 9.56 (d, J = 1.0 Hz, 1H), 8.70 (d, J = 1.5 Hz, 1H), 8.54 (t, J = 4.5 Hz,
1
H
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
1
H), 8.33 (d, J = 2.0 Hz, 1H), 8.05 (d, J = 0.5 Hz, 1H), 7.83 (dd, J = 8.0, 1.0 Hz, 1H), 7.45 (d, J = 8.0
Hz, 1H), 6.83 (d, J = 2.0 Hz, 1H), 3.08 (t, J = 6.0 Hz, 2H), 2.55 (s, 3H), 1.85 (heptet, J = 7.0 Hz, 1H),
1
3
0
1
.90 (d, J = 6.5 Hz, 6H). C NMR (100 MHz, DMSOꢀd ) δ 165.1, 150.7, 146.4, 146.0, 142.7, 137.8,
6
32.6, 130.2, 129.5, 127.8, 121.2, 104.7, 97.1, 90.8, 87.0, 46.6, 27.8, 20.1, 20.0 (2C). LCꢀMS (ESI): m/z
+
ꢀ
+
333 [M+H] ; 331 [MꢀH] . HRMS (ESI): m/z calcd for C H N O [M+H] , 333.1710; found 333.1709.
2
0
21
4
HPLC analysis: 5.96 min, 99.0%.
1
N-Cyclohexyl-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7d) Yield, 67.0%. H
NMR (500 MHz, DMSOꢀd ) δ 9.56 (d, J = 0.5 Hz, 1H), 8.71 (d, J = 1.5 Hz, 1H), 8.34 (d, J = 2.5 Hz,
6
1
H), 8.30 (d, J = 8.0 Hz, 1H), 8.05 (s, 1H), 7.82 (dd, J = 8.0, 1.0 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 6.84
(
(
d, J = 2.0 Hz, 1H), 3.80ꢀ3.72 (m, 1H), 2.55 (s, 3H), 1.86ꢀ1.70 (m, 4H), 1.64ꢀ1.58 (m, 1H), 1.35ꢀ1.26
1
3
m, 4H), 1.18ꢀ1.08 (m, 1H). C NMR (100 MHz, DMSOꢀd ) δ 164.1, 150.7, 146.4, 146.0, 142.6, 137.8,
6
1
32.7, 130.3, 129.4, 127.9, 121.2, 104.7, 97.1, 90.8, 87.0, 48.3, 32.2 (2C), 25.1, 24.7 (2C), 20.1. LCꢀMS
+
ꢀ
+
(ESI): m/z 359 [M+H] ; 357 [MꢀH] . HRMS (ESI): m/z calcd for C H N O [M+H] , 359.1866; found
2
2
23
4
3
59.1865. HPLC analysis: 6.98 min, 99.1%.
N-Cyclopentyl-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7e) Yield, 66.5%. H
NMR (400 MHz, DMSOꢀd ) δ 9.55 (d, J = 1.2 Hz, 1H), 8.70 (d, J = 2.0 Hz, 1H), 8.37 (d, J = 7.2 Hz,
1
6
1
H), 8.33 (d, J = 2.4 Hz, 1H), 8.05 (d, J = 1.2 Hz, 1H), 7.82 (dd, J = 8.0, 1.6 Hz, 1H), 7.43 (d, J = 8.0
Hz, 1H), 6.83 (d, J = 1.6 Hz, 1H), 4.26ꢀ4.20 (m, 1H), 2.54 (s, 3H), 1.92ꢀ1.88 (m, 2H), 1.70ꢀ1.68 (m,
1
3
2
1
H), 1.58ꢀ1.53 (m, 4H). C NMR (100 MHz, DMSOꢀd ) δ 164.7, 150.7, 146.4, 146.0, 142.6, 137.8,
6
32.6, 130.3, 129.4, 127.9, 121.2, 104.7, 97.1, 90.9, 87.0, 50.9, 32.0 (2C), 23.5 (2C), 20.1. LCꢀMS
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+
ꢀ
+
(
ESI): m/z 345 [M+H] ; 343 [MꢀH] . HRMS (ESI): m/z calcd for C H N O [M+H] , 345.1710; found
21 21 4
1
2
3
4
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
3
45.1710. HPLC analysis: 6.15 min, 97.3%.
4-Methyl-N-phenyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7f) Yield, 83.0%. H NMR
400 MHz, DMSOꢀd ) δ10.31 (s, 1H), 9.58 (dd, J = 1.6, 0.4 Hz, 1H), 8.72 (d, J = 2.4 Hz, 1H), 8.34 (d, J
1
(
6
=
2.4 Hz, 1H), 8.18 (d, J = 1.6 Hz, 1H), 7.95 (dd, J = 8.0, 1.6 Hz, 1H), 7.80 (s, 1H), 7.78 (s, 1H), 7.53
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
(d, J = 8.0 Hz, 1H), 7.36 (t, J = 7.6 Hz, 2H), 7.11 (t, J = 7.2 Hz, 1H), 6.84 (dd, J = 2.4, 0.8 Hz, 1H), 2.59
13
(s, 3H). C NMR (125 MHz, DMSOꢀd ) δ 164.4, 150.9, 146.5, 146.3, 143.6, 139.0, 138.1, 132.7,
6
130.8, 129.9, 128.6 (2C), 128.4, 123.7, 121.6, 120.4 (2C), 104.8, 97.3, 90.8, 87.5, 20.4. LCꢀMS (ESI):
+
ꢀ
+
m/z 353 [M+H] ; 351 [MꢀH] . HRMS (ESI): m/z calcd for C H N O [M+H] , 353.1397; found
2
2
17
4
3
53.1395. HPLC analysis: 6.23 min, 99.1%.
4-Methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)-N-(o-tolyl)benzamide (7g) Yield, 63.0%.
NMR (400 MHz, DMSOꢀd ) δ 9.97 (s, 1H), 9.58 (d, J = 1.2 Hz, 1H), 8.72 (d, J = 2.0 Hz, 1H), 8.34 (d, J
1
H
6
=
2.4 Hz, 1H), 8.19 (d, J = 1.2 Hz, 1H), 7.96 (dd, J = 7.6, 1.2 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.34 (d,
J = 7.2 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H), 7.24ꢀ7.21 (m, 1H), 7.20ꢀ7.16 (m, 1H), 6.84 (d, J = 1.6 Hz,
13
1
1
8
H), 2.59 (s, 3H), 2.24 (s, 3H). C NMR (125 MHz, DMSOꢀd ) δ 164.3, 150.9, 146.5, 146.3, 143.5,
6
38.1, 136.3, 133.8, 132.4, 130.8, 130.3, 129.9, 128.3, 126.6, 126.0, 125.9, 121.6, 104.8, 97.3, 90.8,
+
ꢀ
7.4, 20.4, 17.9. LCꢀMS (ESI): m/z 367 [M+H] ; 365 [MꢀH] . HRMS (ESI): m/z calcd for C H N O
2
3
19
4
+
[
M+H] , 367.1553; found 367.1559. HPLC analysis: 5.80 min, 97.9%.
4-Methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)-N-(m-tolyl)benzamide (7h) Yield, 87.5%.
1
H
NMR (400 MHz, DMSOꢀd ) δ 10.23 (s, 1H), 9.58 (d, J = 1.2 Hz, 1H), 8.72 (d, J = 2.0 Hz, 1H), 8.34 (d,
6
J = 2.0 Hz, 1H), 8.17 (d, J = 1.2 Hz, 1H), 7.94 (dd, J = 8.0, 1.2 Hz, 1H), 7.63 (s, 1H), 7.59 (d, J = 8.0
Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.23 (t, J = 8.0 Hz, 1H), 6.94 (d, J = 7.6 Hz, 1H), 6.84 (d, J = 2.0 Hz,
13
1
1
8
H), 2.59 (s, 3H), 2.31 (s, 3H). C NMR (125 MHz, DMSOꢀd ) δ 164.3, 150.9, 146.5, 146.3, 143.5,
6
38.9, 138.1, 137.7, 132.7, 130.7, 129.8, 128.4, 128.3, 124.4, 121.5, 120.9, 117.6, 104.8, 97.3, 90.8,
+
ꢀ
7.4, 21.2, 20.4. LCꢀMS (ESI): m/z 367 [M+H] ; 365 [MꢀH] . HRMS (ESI): m/z calcd for C H N O
2
3
19
4
+
[
M+H] , 367.1553; found 367.1554. HPLC analysis: 7.43 min, 97.2%.
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Journal of Medicinal Chemistry
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1
4
-Methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)-N-(p-tolyl)benzamide (7i) Yield, 90.0%. H NMR
1
2
3
4
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
(
400 MHz, DMSOꢀd ) δ 10.23 (s, 1H), 9.58 (d, J = 0.8 Hz, 1H), 8.73 (d, J = 2.0 Hz, 1H), 8.34 (d, J =
6
2.0 Hz, 1H), 8.17 (d, J = 1.2 Hz, 1H), 7.94 (dd, J = 8.0, 1.6 Hz, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.52 (d, J
13
=
8.0 Hz, 1H), 7.17 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 2.0 Hz, 1H), 2.59 (s, 3H), 2.28 (s, 3H). C NMR
(125 MHz, DMSOꢀd ) δ 164.1, 150.9, 146.5, 146.3, 143.4, 138.1, 136.5, 132.8, 132.7, 130.7, 129.8,
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
1
29.0 (2C), 128.3, 121.5, 120.4 (2C), 104.8, 97.3, 90.8, 87.5, 20.5, 20.4. LCꢀMS (ESI): m/z 367
+
ꢀ
+
[
M+H] ; 365 [MꢀH] . HRMS (ESI): m/z calcd for C H N O [M+H] , 367.1553; found 367.1558.
23 19 4
HPLC analysis: 7.19 min, 99.4%.
N-(2-Chlorophenyl)-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7j) Yied, 52.0%.
1
H NMR (400 MHz, DMSOꢀd ) δ 10.18 (s, 1H), 9.59 (d, J = 1.6 Hz, 1H), 8.73 (d, J = 2.0 Hz, 1H), 8.34
6
(d, J = 2.4 Hz, 1H), 8.20 (d, J = 1.2 Hz, 1H), 7.97 (dd, J = 8.0, 1.6 Hz, 1H), 7.59ꢀ7.56 (m, 2H), 7.55 (d,
J = 8.0 Hz, 1H), 7.40 (td, J = 7.6, 1.2 Hz, 1H), 7.31 (td, J = 7.6, 1.2 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H),
13
2
.60 (s, 3H). C NMR (125 MHz, DMSOꢀd ) δ 164.4, 150.9, 146.5, 146.3, 143.8, 138.1, 134.9, 131.8,
6
130.9, 130.0, 129.6, 129.5, 128.5, 128.3, 127.5, 127.4, 121.7, 104.7, 97.3, 90.7, 87.5, 20.4. LCꢀMS
+
ꢀ
(ESI): m/z 387 (100%), 389 (32%) [M+H] ; 385 (100%), 387 (32%) [MꢀH] . HRMS (ESI): m/z calcd for
+
C H ClN O [M+H] , 387.1007; found 387.1006. HPLC analysis: 7.16 min, 98.8%.
22 16
4
N-(3-Chlorophenyl)-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7k) Yield, 67.0%.
1
H NMR (400 MHz, DMSOꢀd ) δ 10.47 (s, 1H), 9.59 (d, J = 1.6 Hz, 1H), 8.73 (d, J = 1.6 Hz, 1H), 8.34
6
(d, J = 2.4 Hz, 1H), 8.17 (d, J = 2.0 Hz, 1H), 7.98 (t, J = 2.0 Hz, 1H), 7.95 (dd, J = 8.0, 1.6 Hz, 1H),
.74 (dd, J = 8.4, 1.2 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.39 (t, J = 8.4 Hz, 1H), 7.18 (dd, J = 8.0, 1.6
7
13
Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 2.59 (s, 3H). C NMR (100 MHz, DMSOꢀd ) δ 164.4, 150.6, 146.4,
6
146.0, 143.6, 140.4, 137.8, 132.8, 132.2, 130.6, 130.0, 129.7, 128.2, 123.2, 121.5, 119.7, 118.5, 104.6,
+
9
7.1, 90.6, 87.4, 20.1. LCꢀMS (ESI): m/z 387 (100%), 389 (32%) [M+H] ; 385 (100%), 387 (32%) [Mꢀ
ꢀ
+
H] . HRMS (ESI): m/z calcd for C H ClN O [M+H] , 387.1007; found 387.1006. HPLC analysis: 9.01
22 16
4
min, 97.8%.
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Journal of Medicinal Chemistry
N-(4-Chlorophenyl)-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7l) Yield, 72.0%.
1
2
3
4
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
1
H NMR (400 MHz, DMSOꢀd ) δ 10.43 (s, 1H), 9.58 (d, J = 1.6 Hz, 1H), 8.73 (d, J = 2.0 Hz, 1H), 8.34
6
(d, J = 2.0 Hz, 1H), 8.17 (d, J = 1.6 Hz, 1H), 7.95 (dd, J = 8.0, 1.6 Hz, 1H), 7.84 (d, J = 8.8 Hz, 2H),
13
7
.54 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 2.0 Hz, 1H), 2.59 (s, 3H). C NMR (125
MHz, DMSOꢀd ) δ 164.5, 150.9, 146.5, 146.3, 143.7, 138.1, 138.0, 132.4, 130.7, 129.9, 128.5 (2C),
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
1
28.4, 127.3, 121.9 (2C), 121.6, 104.7, 97.3, 90.7, 87.5, 20.4. LCꢀMS (ESI): m/z 387 (100%), 389
+
ꢀ
+
(
32%) [M+H] ; 385 (100%), 387 (32%) [MꢀH] . HRMS (ESI): m/z calcd for C H ClN O [M+H] ,
22 16 4
387.1007; found 387.1005. HPLC analysis: 8.83 min, 98.3%.
N-(2-Methoxyphenyl)-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7m) Yield,
1
6
5.0%. H NMR (400 MHz, DMSOꢀd ) δ 9.58 (d, J = 1.2 Hz, 1H), 9.56 (s, 1H), 8.73 (d, J = 2.0 Hz,
6
1H), 8.34 (d, J = 2.4 Hz, 1H), 8.16 (d, J = 1.6 Hz, 1H), 7.94 (dd, J = 8.0, 1.6 Hz, 1H), 7.73 (dd, J = 8.0,
1
.2 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.20 (td, J = 7.2, 1.6 Hz, 1H), 7.11 (d, J = 1.6 Hz, 1H), 6.97 (td, J
1
3
=
7.6, 0.8 Hz, 1H), 6.84 (d, J = 1.6 Hz, 1H), 3.84 (s, 3H), 2.59 (s, 3H). C NMR (100 MHz, DMSOꢀd )
6
δ 163.8, 151.4, 150.7, 146.4, 146.0, 143.3, 137.8, 132.4, 130.6, 129.7, 127.8, 126.7, 125.5, 124.1, 121.5,
+
ꢀ
1
20.0, 111.4, 104.7, 97.1, 90.7, 87.3, 55.6, 20.1. LCꢀMS (ESI): m/z 383 [M+H] ; 381 [MꢀH] . HRMS
+
(
ESI): m/z calcd for C H N O [M+H] , 383.1503; found 383.1501. HPLC analysis: 7.63 min, 99.0%.
23 19
4
2
N-(3-Methoxyphenyl)-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7n) Yield,
1
7
2.0%. H NMR (400 MHz, DMSOꢀd ) δ 10.28 (s, 1H), 9.58 (dd, J = 2.0, 0.8 Hz, 1H), 8.73 (d, J = 2.0
6
Hz, 1H), 8.34 (d, J = 2.4 Hz, 1H), 8.17 (d, J = 1.6 Hz, 1H), 7.94 (dd, J = 8.0, 1.6 Hz, 1H), 7.53 (d, J =
8
.0 Hz, 1H), 7.47 (t, J = 2.0 Hz, 1H), 7.40 (dd, J = 8.0, 0.8 Hz, 1H), 7.26 (t, J = 8.0 Hz, 1H), 6.85 (dd, J
1
3
=
2.0, 0.8 Hz, 1H), 6.70 (dd, J = 8.0, 2.4 Hz, 1H), 3.76 (s, 3H), 2.59 (s, 3H). C NMR (100 MHz,
DMSOꢀd ) δ 164.2, 159.3, 150.7, 146.4, 146.0, 143.3, 140.0, 137.8, 132.6, 130.6, 129.6, 129.1, 128.2,
6
+
1
21.4, 112.5, 109.1, 106.1, 104.6, 97.1, 90.7, 87.3, 54.9, 20.1. LCꢀMS (ESI): m/z 383 [M+H] ; 381 [Mꢀ
ꢀ
+
H] . HRMS (ESI): m/z calcd for C H N O [M+H] , 383.1503; found 383.1500. HPLC analysis: 6.73
23 19
4
2
min, 98.4%.
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Page 26 of 50
N-(4-Methoxyphenyl)-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7o) Yield,
1
2
3
4
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
1
8
6.0%. H NMR (400 MHz, DMSOꢀd ) δ 10.19 (s, 1H), 9.58 (d, J = 1.2 Hz, 1H), 8.72 (d, J = 2.0 Hz,
6
1H), 8.34 (d, J = 2.4 Hz, 1H), 8.16 (d, J = 1.6 Hz, 1H), 7.94 (dd, J = 8.0, 1.6 Hz, 1H), 7.69 (d, J = 8.8
Hz, 2H), 7.52 (d, J = 8.0 Hz, 1H), 6.95 (d, J = 9.2 Hz, 2H), 6.85 (d, J = 2.0 Hz, 1H), 3.75 (s, 3H), 2.58
1
3
(
s, 3H). C NMR (100 MHz, DMSOꢀd ) δ 163.8, 155.5, 150.7, 146.4, 146.0, 143.1, 137.8, 132.7,
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
1
32.0, 130.5, 129.6, 128.1, 121.9 (2C), 121.4, 113.6 (2C), 104.7, 97.1, 90.7, 87.2, 55.0, 20.1. LCꢀMS
+
ꢀ
+
(
ESI): m/z 383 [M+H] ; 381 [MꢀH] . HRMS (ESI): m/z calcd for C H N O [M+H] , 383.1503; found
23 19 4 2
383.1498. HPLC analysis: 6.21 min, 99.1%.
N-(3-Bromophenyl)-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7p) Yield, 53.0%.
1
H NMR (400 MHz, DMSOꢀd ) δ 10.44 (s, 1H), 9.58 (d, J = 1.2 Hz, 1H), 8.73 (d, J = 2.0 Hz, 1H), 8.34
6
(d, J = 2.4 Hz, 1H), 8.17 (d, J = 1.6 Hz, 1H), 8.11 (s, 1H), 7.95 (dd, J = 8.0, 1.6 Hz, 1H), 7.79 (d, J = 7.6
13
Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.35ꢀ7.29 (m, 2H), 6.85 (d, J = 1.6 Hz, 1H), 2.59 (s, 3H). C NMR
(
125 MHz, DMSOꢀd ) δ 164.4, 150.9, 146.5, 146.3, 143.9, 140.7, 138.1, 132.3, 130.7, 130.6, 130.0,
6
128.4, 126.3, 122.6, 121.6, 121.4, 119.0, 104.7, 97.3, 90.7, 87.6, 20.4. LCꢀMS (ESI): m/z 431, 433
+
ꢀ
+
[
M+H] ; 429, 431 [MꢀH] . HRMS (ESI): m/z calcd for C H BrN O [M+H] , 431.0502; found
22 16 4
4
31.0491. HPLC analysis: 8.45 min, 97.9%.
N-(3-Iodophenyl)-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7q) Yield, 60.0%.
1
H NMR (400 MHz, DMSOꢀd ) δ 10.37 (s, 1H), 9.57 (d, J = 1.2 Hz, 1H), 8.72 (d, J = 2.0 Hz, 1H), 8.34
6
(d, J = 2.4 Hz, 1H), 8.26 (s, 1H), 8.17 (d, J = 1.2 Hz, 1H), 7.93 (dd, J = 7.6, 1.2 Hz, 1H), 7.82 (d, J = 8.0
Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.16 (t, J = 8.0 Hz, 1H), 6.84 (d, J = 1.6 Hz,
1
3
1
H), 2.58 (s, 3H). C NMR (100 MHz, DMSOꢀd ) δ 164.5, 150.9, 146.5, 146.3, 143.8, 140.5, 138.1,
6
132.3, 132.2, 130.7, 130.6, 129.9, 128.4, 121.6, 119.4, 104.7, 97.3, 94.3, 90.7, 87.5, 20.4. LCꢀMS (ESI):
+
ꢀ
+
m/z 479 [M+H] ; 477 [MꢀH] . HRMS (ESI): m/z calcd for C H IN O [M+H] , 479.0363; found
25
16
4
4
79.0357. HPLC analysis: 9.98 min, 98.7%.
4
-Methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)-N-(3-(trifluoromethyl)phenyl)benzamide
(7r)
1
Yield, 46.0%. H NMR (400 MHz, DMSOꢀd ) δ 10.61 (s, 1H), 9.59 (dd, J = 2.0, 0.8 Hz, 1H), 8.73 (d, J
6
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Page 27 of 50
Journal of Medicinal Chemistry
=
2.0 Hz, 1H), 8.34 (d, J = 2.4 Hz, 1H), 8.25 (s, 1H), 8.20 (d, J = 1.6 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H),
1
2
3
4
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
7
.97 (dd, J = 8.0, 2.0 Hz, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 8.0 Hz,
13
1H), 6.85 (dd, J = 2.4, 0.8 Hz, 1H), 2.60 (s, 3H). C NMR (125 MHz, DMSOꢀd ) δ 164.8, 150.9, 146.5,
6
1
46.3, 144.0, 139.8, 138.1, 132.2, 130.8, 130.0, 129.8, 129.6 (q, J = 31.4 Hz), 128.5, 124.1 (d, J = 270.8
Hz), 123.7, 121.6, 120.0 (q, J = 3.8 Hz), 116.4 (q, J = 3.9 Hz), 104.7, 97.3, 90.7, 87.6, 20.4. LCꢀMS
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
+
ꢀ
+
(ESI): m/z 421 [M+H] ; 419 [MꢀH] . HRMS (ESI): m/z calcd for C H F N O [M+H] , 421.1271;
23 16 3 4
found 421.1264. HPLC analysis: 8.51 min, 99.4%.
4-Methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)-N-(4-(trifluoromethyl)phenyl)benzamide
(7s)
1
Yield, 51.0%. H NMR (400 MHz, DMSOꢀd ) δ 10.63 (s, 1H), 9.58 (d, J = 1.2 Hz, 1H), 8.73 (d, J = 2.0
6
Hz, 1H), 8.34 (d, J = 2.4 Hz, 1H), 8.20 (d, J = 1.6 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.96 (dd, J = 8.0,
2.0 Hz, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 1.6 Hz, 1H), 2.60 (s, 3H).
13
C NMR (125 MHz, DMSOꢀd ) δ 164.8, 150.9, 146.5, 146.3, 144.0, 142.7, 138.1, 132.2, 130.8, 129.9,
6
1
28.5, 125.9 (d, J = 6.0 Hz, 2C), 124.3 (d, J = 269.5 Hz), 123.7 (d, J = 31.9 Hz), 121.6, 120.1 (2C),
+
ꢀ
104.7, 97.3, 90.6, 87.6, 20.4. LCꢀMS (ESI): m/z 421 [M+H] ; 419 [MꢀH] . HRMS (ESI): m/z calcd for
+
C H F N O [M+H] , 421.1271; found 421.1275. HPLC analysis: 9.10 min, 98.8%.
23
16
3
4
N-(3-Isopropoxyphenyl)-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7t) Yield,
1
7
5.0%. H NMR (400 MHz, DMSOꢀd ) δ 10.24 (s, 1H), 9.57 (d, J = 1.2 Hz, 1H), 8.72 (d, J = 1.2 Hz,
6
1
H), 8.33 (d, J = 2.0 Hz, 1H), 8.16 (d, J = 1.2 Hz, 1H), 7.93 (dd, J = 8.0, 1.6 Hz, 1H), 7.52 (d, J = 8.0
Hz, 1H), 7.45 (s, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.22 (t, J = 8.0 Hz, 1H), 6.84 (d, J = 1.6 Hz, 1H), 6.66
1
3
(dd, J = 8.0, 1.6 Hz, 1H), 4.56 (heptet, J = 6.0 Hz, 1H), 2.58 (s, 3H), 1.28 (d, J = 6.0 Hz, 6H). C NMR
(100 MHz, DMSOꢀd ) δ 164.2, 157.5, 150.6, 146.4, 146.0, 143.3, 140.1, 137.8, 132.6, 130.6, 129.6,
6
129.1, 128.1, 121.4, 112.4, 111.0, 107.8, 104.7, 97.1, 90.7, 87.3, 69.2, 21.7 (2C), 20.1. LCꢀMS (ESI):
+
ꢀ
+
m/z 411 [M+H] ; 409 [MꢀH] . HRMS (ESI): m/z calcd for C H N O [M+H] , 411.1816; found
25
23
4
2
4
11.1815. HPLC analysis: 8.24 min, 99.3%.
N-(3-(Dimethylamino)phenyl)-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide
(7u)
1
Yield, 72.0%. H NMR (400 MHz, DMSOꢀd ) δ 10.10 (s, 1H), 9.57 (d, J = 1.2 Hz, 1H), 8.72 (d, J = 2.0
6
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Hz, 1H), 8.34 (d, J = 2.0 Hz, 1H), 8.17 (d, J = 1.6 Hz, 1H), 7.94 (dd, J = 8.0, 1.6 Hz, 1H), 7.52 (d, J =
.4 Hz, 1H), 7.20ꢀ7.18 (m, 2H), 7.13 (t, J = 8.0 Hz, 1H), 6.84 (d, J = 1.6 Hz, 1H), 6.50ꢀ6.48 (m, 1H),
1
2
3
4
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
8
1
3
2.90 (s, 6H), 2.58 (s, 3H). C NMR (100 MHz, DMSOꢀd ) δ 164.3, 150.6, 146.4, 146.0, 143.6, 140.3,
6
1
37.8, 132.2, 132.0, 130.6, 130.4, 129.7, 128.4, 128.2, 121.5, 119.4, 104.6, 97.1, 93.9, 90.6, 87.3, 20.1.
+
ꢀ
+
LCꢀMS (ESI): m/z 396 [M+H] ; 394 [MꢀH] . HRMS (ESI): m/z calcd for C H N O [M+H] ,
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
24 22
5
3
96.1819; found 396.1817. HPLC analysis: 7.20 min, 98.5%.
N-(3-(1H-Imidazol-1-yl)phenyl)-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7v)
1
Yield, 71.0%. H NMR (400 MHz, DMSOꢀd ) δ 10.51 (s, 1H), 9.58 (d, J = 1.6 Hz, 1H), 8.72 (d, J = 2.0
6
Hz, 1H), 8.34 (d, J = 2.4 Hz, 1H), 8.20 (d, J = 1.6 Hz, 1H), 8.19 (s, 1H), 8.06 (t, J = 2.0 Hz, 1H), 7.97
(dd, J = 8.0, 1.6 Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.67 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.51 (d, J = 8.0
1
3
Hz, 1H), 7.39 (dd, J = 8.0, 1.6 Hz, 1H), 7.14 (s, 1H), 6.85 (d, J = 1.6 Hz, 1H), 2.60 (s, 3H). C NMR
(
125 MHz, DMSOꢀd ) δ 164.6, 150.9, 146.5, 146.3, 143.9, 140.3, 138.1, 137.1, 135.5, 132.3, 130.7,
6
1
30.1, 130.0, 129.9, 128.4, 121.6, 118.7, 118.1, 115.9, 112.5, 104.7, 97.3, 90.7, 87.6, 20.4. LCꢀMS
+
ꢀ
+
(ESI): m/z 419 [M+H] ; 417 [MꢀH] . HRMS (ESI): m/z calcd for C H N O [M+H] , 419.1615 ; found
2
5
19
6
4
19.1613. HPLC analysis: 5.99 min, 99.9%.
4-Methyl-N-(3-(4-methyl-1H-imidazol-1-yl)phenyl)-3-(pyrazolo[1,5-a]pyrimidin-6-
1
ylethynyl)benzamide (7w) Yield, 70.0%. H NMR (400 MHz, DMSOꢀd ) δ 10.49 (s, 1H), 9.59 (s, 1H),
6
8
.73 (d, J = 2.0 Hz, 1H), 8.34 (d, J = 2.4 Hz, 1H), 8.20 (s, 1H), 8.05 (s, 1H), 8.03 (s, 1H), 7.97 (d, J =
8.0 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.36 (s, 1H),
1
3
7
.34 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 1.6 Hz, 1H), 2.60 (s, 3H), 2.18 (s, 3H). C NMR (125 MHz,
DMSOꢀd ) δ 164.6, 150.9, 146.5, 146.3, 143.9, 140.3, 138.5, 138.1, 137.2, 134.6, 132.4, 130.7, 130.0,
6
129.9, 128.4, 121.6, 118.3, 115.4, 114.2, 112.0, 104.7, 97.3, 90.7, 87.6, 20.4, 13.5. LCꢀMS (ESI): m/z
+
ꢀ
+
4
33 [M+H] ; 431 [MꢀH] . HRMS (ESI): m/z calcd for C H N O [M+H] , 433.1771; found 433.1769.
26 21 6
HPLC analysis: 6.86 min, 95.6%.
4
-Methyl-N-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(pyrazolo[1,5-a]pyrimidin-6-
1
ylethynyl)benzamide (7x) Yield, 67.0%. H NMR (400 MHz, DMSOꢀd ) δ 10.29 (s, 1H), 9.58 (d, J =
6
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Journal of Medicinal Chemistry
1
.2 Hz, 1H), 8.73 (d, J = 1.6 Hz, 1H), 8.34 (d, J = 2.4 Hz, 1H), 8.19 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H),
.74ꢀ7.72 (m, 2H), 7.53 (d, J = 8.0 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 7.03 (d, J = 7.6 Hz, 1H), 6.85 (d, J
1
2
3
4
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
7
1
3
= 1.6 Hz, 1H), 3.44 (s, 2H), 2.59 (s, 3H), 2.37 (br s, 4H), 2.33 (br s, 4H), 2.15 (s, 3H). C NMR (125
MHz, DMSOꢀd ) δ 164.3, 150.9, 146.5, 146.3, 143.5, 139.0, 138.8, 138.1, 132.7, 130.7, 129.8, 128.4,
6
1
28.3, 124.2, 121.5, 120.7, 119.0, 104.8, 97.3, 90.8, 87.4, 62.2, 54.7 (2C), 52.6 (2C), 45.7, 20.4. LCꢀMS
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
+
ꢀ
+
(ESI): m/z 465 [M+H] ; 463 [MꢀH] . HRMS (ESI): m/z calcd for C H N O [M+H] , 465.2397; found
28 29 6
4
65.2385. HPLC analysis: 5.82 min, 99.5%.
N-(3,5-Dimethylphenyl)-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide (7y) Yield,
1
8
1
5.5%. H NMR (400 MHz, DMSOꢀd ) δ 10.14 (s, 1H), 9.58 (d, J = 1.2 Hz, 1H), 8.72 (d, J = 2.4 Hz,
6
H), 8.34 (d, J = 2.0 Hz, 1H), 8.16 (d, J = 1.6 Hz, 1H), 7.93 (dd, J = 8.0, 2.0 Hz, 1H), 7.52 (d, J = 8.0
1
3
Hz, 1H), 7.42 (s, 2H), 6.85 (d, J = 1.6 Hz, 1H), 6.75 (s, 1H), 2.58 (s, 3H), 2.27 (s, 6H). C NMR (125
MHz, DMSOꢀd ) δ 164.2, 150.9, 146.5, 146.3, 143.5, 138.8, 138.1, 137.5 (2C), 132.8, 130.7, 129.8,
6
1
28.3, 125.2, 121.5, 118.1 (2C), 104.8, 97.3, 90.8, 87.4, 21.1 (2C), 20.3. LCꢀMS (ESI): m/z 381
+
ꢀ
+
[M+H] ; 379 [MꢀH] . HRMS (ESI): m/z calcd for C H N O [M+H] , 381.1710; found 381.1707.
2
4
21
4
HPLC analysis: 8.83 min, 95.2%.
N-(3-Chloro-5-methylphenyl)-4-methyl-3-(pyrazolo[1,5-a]pyrimidin-6-ylethynyl)benzamide
(7z)
1
Yield, 65.0%. H NMR (400 MHz, DMSOꢀd ) δ 10.37 (s, 1H), 9.58 (dd, J = 2.0, 0.8 Hz, 1H), 8.72 (d, J
6
=
2.0 Hz, 1H), 8.34 (d, J = 2.4 Hz, 1H), 8.16 (d, J = 2.0 Hz, 1H), 7.94 (dd, J = 8.0, 2.0 Hz, 1H), 7.77 (s,
1H), 7.56 (s, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.01 (s, 1H), 6.85 (d, J = 1.6 Hz, 1H), 2.59 (s, 3H), 2.31 (s,
1
3
3
H). C NMR (100 MHz, DMSOꢀd ) δ 164.4, 150.6, 146.4, 146.0, 143.6, 140.1, 139.8, 137.8, 132.5,
6
1
32.2, 130.6, 129.7, 128.2, 123.7, 121.5, 119.1, 116.9, 104.6, 97.1, 90.6, 87.3, 20.7, 20.1. LCꢀMS (ESI):
+
ꢀ
m/z 401 (100%), 403 (32%) [M+H] ; 399 (100%), 401 (32%) [MꢀH] . HRMS (ESI): m/z calcd for
+
C H ClN O [M+H] , 401.1164; found 401.1159. HPLC analysis: 9.86 min, 98.3%.
23
18
4
4
-Methyl-N-(3-((4-methylpiperazin-1-yl)methyl)-5-(trifluoromethyl)phenyl)-3-(pyrazolo[1,5-
1
a]pyrimidin-6-ylethynyl)benzamide (7ra) Yield, 40.0%. H NMR (400 MHz, DMSOꢀd ) δ 10.58 (s,
6
1
H), 9.58 (d, J = 1.2 Hz, 1H), 8.73 (d, J = 2.0 Hz, 1H), 8.34 (d, J = 2.0 Hz, 1H), 8.21 (d, J = 6.4 Hz,
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