Synthesis and biological evaluation of novel pyrazolo[1,5-a]pyrimidines: Discovery of a selective inhibitor of JAK1 JH2 pseudokinase and VPS34

Article abstract of DOI:10.1016/j.bmcl.2019.126813

A series of novel 3,6-di-substituted or 3-substituted pyrazolo[1,5-a]pyrimidines were prepared via a microwave-assisted approach that generated a broad array of derivatives in good yields (20–93%, ave. = 59%). The straightforward synthesis involved sequential treatment of commercially-available acetonitrile derivatives with DMF-dimethylacetal (120 °C, 20 min), followed by treatment with NH₂NH₂·HBr (120 °C, 20 min), and 1,1,3,3-tetramethoxypropane or 2-aryl-substituted malondialdehdyes (120 °C, 20 min). Compounds were screened for antimitotic activities against MCF7 breast cancer and/or A2780 ovarian cancer cell lines in vitro. The most active compounds exhibited EC₅₀ values ranging from 0.5 to 4.3 μM, with the 3-(4-(trifluoromethyl)phenyl)-6-[4-(2-(piperidin-1-yl)ethoxy]phenyl analogue (34e) and the 3-(2-fluorophenyl)-6-[4-(2-(4-methylpiperizin-1-yl)ethoxy]phenyl analogue (35a) being two to three fold more active than Compound C (Dorsomorphin) in A2780 and MCF7 assays, respectively. Importantly, a monosubstituted 3-(benzothiazol-2-yl) derivative (13) was equipotent with the more synthetically challenging 3,6-disubstituted derivatives (34a–e and 35a–e), and exhibited a promising and unique selectivity profile when screened against a panel consisting of 403 protein kinases (Kinomescan selectivity score = 0.005, K_d = 0.55 ± 0.055 μM and 0.410 ± 0.20 μM for JAK1 JH2 pseudokinase and VPS34, respectively).

Full text of DOI:10.1016/j.bmcl.2019.126813

Journal Pre-proofs
Synthesis and biological evaluation of novel pyrazolo[1,5-a]pyrimidines: Dis-
covery of a selective inhibitor of JAK1 JH2 pseudokinase and VPS34
Justin D. Singleton, Reuben Dass, Nathaniel R. Neubert, Rachel M. Smith, Zak
Webber, Marc D.H. Hansen, Matt A. Peterson
PII:
S0960-894X(19)30782-6
DOI:
https://doi.org/10.1016/j.bmcl.2019.126813
BMCL 126813
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Bioorganic & Medicinal Chemistry Letters
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23 September 2019
1 November 2019
6 November 2019
Please cite this article as: Singleton, J.D., Dass, R., Neubert, N.R., Smith, R.M., Webber, Z., Hansen, M.D.H.,
Peterson, M.A., Synthesis and biological evaluation of novel pyrazolo[1,5-a]pyrimidines: Discovery of a selective
inhibitor of JAK1 JH2 pseudokinase and VPS34, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://
doi.org/10.1016/j.bmcl.2019.126813
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Bioorganic & Medicinal Chemistry Letters
Synthesis and biological evaluation of novel pyrazolo[1,5-a]pyrimidines: Discovery
of a selective inhibitor of JAK1 JH2 pseudokinase and VPS34
Justin D. Singletona , Reuben Dassa , Nathaniel R. Neubertb, Rachel M. Smithb, Zak Webberb, Marc D. H.
Hansenb, and Matt A. Petersona,*
a Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, 84602 USA
b Department of Physiology & Developmental Biology, Brigham Young University, Provo, UT, 84602 USA
 Corresponding author. Tel.: +0-801-422-6843; fax: +0-801-422-0153; email:matt_peterson@byu.edu
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of novel 3,6-di-substituted or 3-substituted pyrazolo[1,5-a]pyrimidines were prepared
via a microwave-assisted approach that generated a broad array of derivatives in good yields
(20–93%, ave. = 59%). The straightforward synthesis involved sequential treatment of
commercially-available acetonitrile derivatives with DMF-dimethylacetal (120 °C, 20 min),
.
Available online
followed by treatment with NH2NH2 HBr (120 °C, 20 min), and 1,1,3,3-tetramethoxypropane
or 2-aryl-substituted malondialdehdyes (120 °C, 20 min). Compounds were screened for
antimitotic activities against MCF7 breast cancer and/or A2780 ovarian cancer cell lines in
vitro. The most active compounds exhibited EC50 values ranging from 0.5-4.3 M, with the 3-
(4-(trifluoromethyl)phenyl)-6-[4-(2-(piperidin-1-yl)ethoxy]phenyl analogue (34e) and the 3-(2-
fluorophenyl)-6-[4-(2-(4-methylpiperizin-1-yl)ethoxy]phenyl analogue (35a) being two to three
fold more active than Compound C (Dorsomorphin) in A2780 and MCF7 assays, respectively.
Importantly, a monosubstituted 3-(benzothiazol-2-yl) derivative (13) was equipotent with the
more synthetically challenging 3,6-disubstituted derivatives (34a–e and 35a–e), and exhibited a
promising and unique selectivity profile when screened against a panel consisting of 403 protein
kinases (KinomescanTM selectivity score = 0.005, Kd = 0.55 ± 0.055 M and 0.410 ± 0.20 M
for JAK1 JH2 pseudokinase and VPS34, respectively).
Keywords:
Pyrazolo[1,5-a]pyrimidines
Microwave assisted synthesis
JAK1 JH2 pseudokinase inhibitor
VPS34 kinase inhibitor
.
2009 Elsevier Ltd. All rights reserved.
The pyrazolo[1,5-a]pyrimidine scaffold is a privileged scaffold
in medicinal chemistry (Figure 1).1 Derivatives from this class of
compounds exhibit biological activities as anticancer, anti-
inflammatory, antiprotozoal, antifungal, antibacterial, and/or
between Dorsomorphin and several anticancer drugs has also
been noted .16
We hypothesized that new analogues of Dorsomorphin might
have improved activities relative to the parent compound, as has
been observed with halogen-substituted analogues of a variety of
antiviral agents.
Pyrazolo[1,5-a]pyrimidines have also found
use as synthetic dyes, protein kinase (and/or other enzyme)
inhibitors, antagonists for a variety of receptors, solid-tumor
imaging agents, analgesics, antiepileptics, and sedative-hypnotic
other biologically active substrates.
Examples of such
improvements in the activities of lead structures have been
reviewed,17 and include enhancements attributable to halogen
bonding with chlorine or bromine,18 hydrogen bonding or
multipolar interactions in fluorine,19 or tetrel bonding with CF3.20
We reasoned that appropriately placed halogens or CF3 moieties
on a C-3 phenyl analogue of Dorsomorphin could alter binding
interactions with target proteins and enhance activity.
Additionally, key heterocyclic C-3 substitutions to the
pyrazolo[1,5-a]pyrimidine scaffold would enter currently
unexplored chemical space21 and such substituents could be
easily introduced via readily available commercial starting
materials. We therefore set out to systematically define an SAR
by screening novel analogues with both C-3 and C-6 substitutions
for anticancer activity. Our basic synthetic approach provided
agents, etc.1
One well-studied example of this class of
compounds is Compound C, or Dorsomorphin (1) (Figure 1).
First discovered as an AMPK inhibitor,2 Dorsomorphin was later
shown to also inhibit VEGFR2,3 BMPR1b,4 and indeed a wide-
variety of other tyrosine and serine-threonine protein kinases.5
Dorsomorphin exhibits antiproliferative activities against several
human cancer types, including multiple myeloma,6 B-acute
lymphoblastic leukemia,7 cholangiocarcinoma,8 glioma,9
melanoma,10 and ovarian,11 MCF breast,12 and colorectal
cancer.13 It also exhibits antiproliferative effects against cultured
vascular smooth muscle cells and fibroblasts,14 as well as cancer
cell lines A549, SMMC-7721, and HeLa.15 A synergistic effect

target compounds in good to excellent yields after only 60
minutes total reaction time (Scheme 1), significant
JAK1 and VPS34 have been shown to play key roles in numerous
cancers including breast,32 ovarian,33 melanoma,34 pancreatic,35
non-small lung,36 and colorectal cancer.37 Importantly, 13 did not
inhibit binding to JAK1 JH1 kinase domain, suggesting that 13
may prove useful as a novel biochemical tool for dissecting the
distinct roles played by JAK1 JH1 and JH2 domains in
JAK/STAT signaling. JH2 pseudokinase domains have been
shown to play key roles in regulating JH1 kinase domain
a
improvement over non-microwave-assisted approaches reported
earlier.22 Thus, the majority of targets could be readily prepared
in only 3-steps with an average yield of 59% (Tables 1–2).
Another series of derivatives was designed to enhance aqueous
solubility by incorporating a tertiary amine feature (Scheme 2).23
Novel compounds 5–35 were screened for antimitotic activity
using ovarian A2780 cancer cells at a single 10 M compound
concentration (Figure 2).24 Compounds 13, 34a–e and 35a–e
gave results comparable to those of Dorsomorphin, whereas a
majority of the other analogues were considerably less active.
This is remarkable considering compound 13 is much more
synthetically tractable than either compounds 34a–e, 35a–e, or
Dorsomorphin. Compounds 13, 34a–e, and 35a–e were next
tested in multi-dose inhibition assays in order to determine their
potency (Table 3). The most potent compound in the Ovarian
A2780 assay was compound 35a, which was approximately two-
fold more active than Dorsomorphin. Most of the other
analogues on the other hand exhibited potency equal to, or
approximately half that of Dorsomorphin. Compound 13 was
nearly equipotent with Dorsomorphin (EC50 0.90 and 1.1 M,
respectively). Compounds 34a–e were also tested for antimitotic
activity against MCF7 breast cancer cells. A majority were more
potent than Dorsomorphin, with the most active compound (34e)
being approximately 3-fold more active (EC50 = 4.9 and 1.6 M,
respectively).
activities in other members of Janus Kinase (JAK) family.38
A
limited number of compounds that exhibit binding affinities for
JAK1 JH2 have been reported in the literature. To date,
quantitative binding analyses for only two such compounds have
been reported (BMS-986165 and (R)-N-(1-(3-(8-methyl-5-
(methylamino)-8H-imidazo[4,5-d]thiazolo[5,4-b]pyridin-2-
yl)phenyl)ethyl)-2-(methylsulfonyl)benzamide)).39
Neither of
these compounds was selective for JAK1 JH2, with the former
being five-fold more selective39a for TYK2 JH2 and the latter
binding TYK2 JH2 and JAK1 JH2 with equal potency.39b In
contrast, compound 13 was six-fold more selective for JAK1 JH2
compared to TYK JH2 (see Supplementary Material).
In summary, we have prepared a series of novel pyrazolo[1,5-
a]pyrimidine derivatives and shown them to be approximately
equipotent with Dorsomorphin in inhibiting proliferation in
Ovarian A2780 and Breast MCF7 cell lines in vitro. One of the
derivatives, compound 13, showed remarkable selectivity when
screened in the KinomescanTM competitive binding assay, giving
an S(1) selectivity score = 0.005 with only two kinases (JAK1
JH2 pseudokinase and VPS34) inhibited >99% of control (POC =
0.8 and 0.9%, respectively).25 Such unique selectivity is highly
desirable, suggesting that compound 13 may be useful as a
biochemical probe and/or as a productive lead in future drug-
discovery efforts. We are currently pursuing this line of research
and will report our findings as they become available.
The unexpected potency of compound 13, especially
compared to closely-related derivatives such as 9–12, 14, 20, and
29, prompted us to analyze its selectivity in a comprehensive
kinase competitive binding inhibition assay (KinomescanTM).25, 26
For comparison, compound 34b was also subjected to
KinomescanTM analysis.27 Both compounds were tested at 10 M
concentration. The resulting data were used to calculate
selectivity scores (S-scores), which are defined as the number of
kinases targeted by a given compound divided by the total
number of kinases screened in the assay.28 Specific selectivity
scores were calculated with kinase binding activities set at
standard Percent-of-Control (POC) threshold levels (e.g., S(1):
POC <1%; S(10): POC <10%; and S(35): POC <35%).25
Compound 13 showed remarkable selectivity. While compound
34b strongly inhibited binding in 22 of 403 kinases (S(1) =
0.055; POC = 0–0.7%), compound 13 inhibited binding of only
two of the 403 distinct, non-mutant kinases tested (S(1) = 0.005;
POC = 0.8–0.9% ). Less stringent selectivity scores showed a
similar pattern: S(10) and S(35) selectivity scores for 13 were
0.012 and 0.077, compared to 0.156 and 0.313 for compound
Acknowledgments
Generous support from the Simmons Cancer Research Center
and College of Physical and Mathematical Sciences at Brigham
Young University is gratefully acknowledged.
Supplementary Material
Supplementary data (detailed experimental procedures, NMR
data for all new compounds, and KinomescanTM data) can be
found in the online version at doi:xxxxxxxxx.
34b,
respectively.
Taken together, compound 13 was
approximately one order of magnitude more selective than 34b in
this assay. Of further interest are the identities of the two kinases
affected most by 13: JAK1 JH2 pseudokinase domain29,30 (POC =
0.8%) and VSP3431 (POC = 0.9%). Compound 34b was inactive
against VSP34 (POC = 100%), and only weakly inhibited
binding of JAK1 JH2 (POC = 41%). There were only 5 kinases
inhibited with POC <10% for compound 13, whereas there were
63 kinases inhibited by compound 34b at the same POC level.
The 5 kinases inhibited by 13 with POC <10% were: EPHB6,
TYK2 (JH2 pseudokinase domain), MEK5, VPS34, and
JAK1(JH2domain-pseudokinase); with POC = 9.9, 5.0, 3.9, 0.9,
and 0.8%, respectively). The only kinases inhibited with POC
<10% by both compound 13 and compound 34b were MEK5 and
EPHB6, further emphasizing the difference in selectivity between
these two compounds. The Kd values for compound 13 were
determined to be 0.55 ± 0.055 M and 0.410 ± 0.20 M for
JAK1 JH2 pseudokinase domain and VPS34, respectively. Both

Figure 1
Scheme 1.
Scheme 2. Reagents and conditions: (a) HBr, AcOH, 130
°C, 2h; (b) ClCH2CH2NR2, Cs2CO3, NaI, DMF, 60°C, 24h; (c)
HOCH2CH2NR2, PPh3, DIAD, THF, 0°C –> RT, 72 h.

µ
Ovarian A2780 Single Dose Assay (10 M)
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Figure 2. Single Dose ATP Lite Luminescence Assay.24

.

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NMR and HRMS and gave spectra consistent with the
reported structures. Compounds 5–33 were dissolved in
DMSO and used directly in the biological assays,
whereas compounds 34a–e and 35a–e were converted to
their CH3SO3H salts in order to maximize solubility.
25 The KinomescanTM assay is a competitive binding
assay which measures the inhibition of binding of a 453-
member panel of kinases to an immobilized ATP-
binding site ligand, as affected by the sample
compound. Compounds for which there is no inhibition
of binding will give POC = 100%, whereas compounds
that bind more tightly will give lower POC values.
Thus, for a compound that inhibits binding by 99.9%,
the POC value will be 0.1%, etc. (See supplementary
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