Discovery of pyrazolopyrimidine derivatives as novel inhibitors of ataxia telangiectasia and rad3 related protein (ATR)

Article abstract of DOI:10.1016/j.bmcl.2017.01.045

The ATR pathway is a critical mediator of the replication stress response in cells. In aberrantly proliferating cancer cells, this pathway can help maintain sufficient genomic integrity for cancer cell progression. Herein we describe the discovery of 19,

Full text of DOI:10.1016/j.bmcl.2017.01.045

Accepted Manuscript
Discovery of pyrazolopyrimidine derivatives as novel inhibitors of ataxia te-
langiectasia and rad3 related protein (ATR)
Sreekanth A. Ramachandran, Pradeep S. Jadhavar, Manvendra P. Singh, Ankesh
Sharma, Gaurav N. Bagle, Kevin P. Quinn, Po-yin Wong, Andrew A. Protter,
Roopa Rai, Son M. Pham, Jeffrey N. Lindquist
PII:
S0960-894X(17)30058-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.01.045
BMCL 24621
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Accepted Date:
20 December 2016
13 January 2017
Please cite this article as: Ramachandran, S.A., Jadhavar, P.S., Singh, M.P., Sharma, A., Bagle, G.N., Quinn, K.P.,
Wong, P-y., Protter, A.A., Rai, R., Pham, S.M., Lindquist, J.N., Discovery of pyrazolopyrimidine derivatives as
novel inhibitors of ataxia telangiectasia and rad3 related protein (ATR), Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.01.045
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Bioorganic & Medicinal Chemistry Letters
Discovery of pyrazolopyrimidine derivatives as novel inhibitors of ataxia
telangiectasia and rad3 related protein (ATR)
a
a
a
a
Sreekanth A. Ramachandran , Pradeep S. Jadhavar , Manvendra P. Singh , Ankesh Sharma , Gaurav N.
a
b
b
c
b
b,*
Bagle , Kevin P. Quinn , Po-yin Wong , Andrew A. Protter , Roopa Rai , Son M. Pham and Jeffrey N.
b,*
Lindquist
a
Integral BioSciences, Pvt. Ltd., C-64, Hosiery Complex Phase II Extension, Noida, Uttar Pradesh, 201306, India
Medivation, now Pfizer Inc., 525 Market Street, 36th Floor, San Francisco, California 94105, United States
b
c
Current address: Palo Alto Pharmaceutical Consulting Service
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
The ATR pathway is a critical mediator of the replication stress response in cells. In aberrantly
proliferating cancer cells, this pathway can help maintain sufficient genomic integrity for cancer
cell progression. Herein we describe the discovery of 19, a pyrazolopyrimidine-containing
inhibitor of ATR via a strategic repurposing of compounds targeting PI3K.
Accepted
Available online
2
016 Elsevier Ltd. All rights reserved.
Keywords:
ATR
pyrazolopyrimidine
DNA damage
PI3K
HTS
Damage to cellular DNA, from intrinsic and extrinsic sources, are dealt with constantly in normal human cells. The DNA-
Damage Response (DDR) is a complex process that has mechanisms specifically tailored for the type of DNA damage
1,2
encountered. These mechanisms may involve activation of checkpoint response, cell-cycle arrest, and DNA-repair or apoptosis in
response to DNA-damage. In normal, healthy cells this response results in either a correction of the damage, or, if the damage is too
extensive to repair, leads to cell death via apoptosis. In cells with oncogenic transformation, the DNA-damage response may be
3
-6
upregulated to enable rapid division and deal with the increased replicative stress of the enhanced rate of cell division. The
7
approval of olaparib (formerly AZD2281) by the US FDA in 2014 for the treatment of certain forms of advanced ovarian cancer
8-
demonstrated the clinical benefit of inhibiting one component of the DDR pathway namely poly(ADP-ribose) polymerase (PARP).
10
Another critical component of the replication stress response is the ataxia telangiectasia and rad-3 related protein (ATR), a
1
1-13
member of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family.
This family also includes the DNA-damage
14
response proteins, ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK). Together all three proteins
maintain genomic surveillance and stability in proliferating cells. ATR is the primary responder to replication stress in cells,
1
5
including replication stress due to treatment with DNA-damaging agents like gemcitabine and hydroxyurea. In this scenario, ATR
activates checkpoint response and regulates the transition of S-phase to G2-phase to allow time for DNA repair and reducing
efficacy of DNA-damaging agents. In addition, in cells lacking proper p53 activity, and thus a proper regulation of G1 to S-phase,
are more dependent upon ATR pathway for survival and proliferation. This makes ATR an attractive target to increase tumor
1
6,17
18
sensitivity to DNA-damaging agents,
combine with other agents targeting DNA-Damage-Repair, such as PARP inhibitors, or
19
as a monotherapy in certain indications/genotypes.
Recent reports have described utilizing homology between ATR and phosphoinositide 3-kinase (PI3K) as a means for guiding
2
0-23
ATR inhibitor design.
Our labs have recently reported the discovery of small-molecule Bruton’s tyrosine kinase (BTK) and
24
PI3Kδ dual inhibitors and we sought to employ this collection to identify inhibitors of ATR. Towards this end, we selected
compounds from our library of BTK and PI3Kδ dual inhibitors and screened them against ATR at 0.5 and 5 μM (Eurofins
Scientific, Dundee, Scotland). The results from this initial screen are graphically illustrated in Figure 1.

Figure 1. ATR percent activity at 0.5 and 5 μM.
Among the 299 compounds tested, 11 compounds inhibited ATR activity to >50% at 5 μM whereas at 0.5 μM only 1 compound
inhibited ATR activity to >50%. Surprisingly, an analysis of ATR and PI3Kα/γ inhibition showed relatively poor correlation of
inhibitory activity between these protein targets, Figure 2. These results suggested that using PI3K homology to guide optimizations
would not be feasible with this chemical series. Therefore we would likely need to construct single-point changes in order to
identify the key binding interactions on ATR as part of a larger effort to improve our hits.
Figure 2. A. ATR % inhibition vs PI3Kα IC50; B. ATR % inhibition vs PI3Kγ IC50
.
To confirm activity as well as benchmark our starting point, we performed dose-response tests on several of our best compounds
which identified one sub-micromolar and three single-digit micromolar hits, Table 1. Compound 1 immediately stood out as a
molecule of interest, not only as it was the most potent from our screening series, but it also lacked significant activity against BTK
and PI3Kδ ((±)-1, BTK inh = 24%, PI3Kδ inh = 52% at 100 nM) compared to the other hits. Moreover, (±)-1 had the largest
lipophilic efficiency (LipE = 3.2) among the best hits.

Table 1. Hits from ATR screen
a
ATR IC50
μM)
ATR
LipE
BTK IC50
(μM)
PI3Kδ IC50
Compound
(
(μM)
b
b
(±)-1
0.212
3.2
ND
ND
2
3
4
1.86
3.56
2.63
1.5
1.4
2.6
0.123
0.012
0.022
0.155
0.010
0.307
a
Compound 1 is racemic.
ND, IC50 not determined.
b
Before embarking on an optimization campaign, we wished to identify the parts of the molecule which were contributing to the
inhibitory activity against ATR. Using 1 as the starting point, we identified possible regions of interaction before setting out to
prepare the various derivatives, Figure 3. As the 4-aminopyrazolopyrimidine scaffold was a ubiquitous motif in this series, we
speculated that the highlighted region was critical for affinity, likely serving as the kinase hinge binder. However, we could not
discount the importance of the azaindolyl group towards ATR affinity at this stage even though no other screening hit contained this
substituent. As compounds 1-4 had different N-1 substituents, we envisioned that this region was more amenable to changes, but
perhaps space-filling would be an important feature.
Figure 3. Possible areas of ATR interaction and molecular targets.
Compounds 5-8 were designed to examine the importance of the hydrogen-bond donor/acceptor motifs of the 4-
aminopyrazolopyrimidine scaffold (5 and 6) and of the azaindolyl substituent (7 and 8). Compounds 9 and 10 tested the significance
of space-filling for groups connected to N-1 and C-3. The syntheses of compounds 5-10 were prepared from commercially available
materials and described in the supplementary material. The ATR inhibition results summarized in Table 2 effectively show a

complete loss in ATR binding when either the 4-amino group (5) or the nitrogen at position-5 (6) are removed. These results
demonstrated that this hydrogen-bond donor/acceptor component is in fact a critical element for ATR binding within this molecule
class. Replacing the C-3 azaindolyl with an indolyl (7) resulted in a more modest 7.5-fold loss in ATR IC50. Substituting the same
azaindolyl with a pyridyl (8) resulted in a complete loss of activity against ATR. Interestingly, aminopyridine-containing derivative
(9) which maintains all the hydrogen-bonding elements of 1 also had diminished activity against ATR albeit with a measurable IC50
up to 30 μM. Together, the results for compounds 7-9 suggest that while the hydrogen-bonding elements of the C-3 substituent may
be beneficial, the space-filling features are critical for activity against ATR. Considering the observed potency of 2 and 3, it’s likely
that the para-bromo substituent occupies sufficient space to achieve low-micromolar activity against the protein target. Substituting
the tetralinyl group with a cyclopentyl group provided 10 which maintained moderate activity against ATR and only a 6.7-fold loss
in activity compared to (±)-1. This result supports our previous supposition that the N-1 position is more amendable to change than
other parts of the molecule.
Table 2. Results for 1, 5-10.
a
Compound
ATR IC50 (μM)
Fold
(
(
(
(
(
(
±)-1
±)-5
±)-6
±)-7
±)-8
±)-9
0.212
>30
1.0
b
ND
b
>30
ND
1.59
>30
7.5
b
ND
19.3
1.42
91
1
0
6.7
a
Fold = compound IC50/(±)-1 IC50
ND, fold not determined.
.
b
Having identified key ATR affinity elements within our molecule, we then turned our attention to exploring the effects of
different groups at C-3. Starting from 4-amino-3-iodopyrazolopyrimidine, I, the desired analogs were prepared by alkylating N-1
with tetraline-2-mesylate and then derivatizing with various boronic acids or boronate esters (Scheme 1) to afford compounds 11-
15, Figure 4.
2 3 2 3 4 2 3
Scheme 1. Reagents and conditions: (a) R-X, Cs CO , DMF, 70-130 °C, 4 h; (b) Ar-B(OR) , Pd(PPh ) , 2 M aqueous Na CO , DMF, 100 °C, 16 h.

Figure 4. Compounds 11-19.
The methylated indole derivative 11 effectively maintained affinity towards ATR and had a slight loss in potency (fold = 2.7)
compared to (±)-1, Table 3. This further supports our hypothesis that the hydrogen-bonding capacity of the C-3 substituent is not an
important factor in ATR binding. Somewhat surprisingly, isomeric indole adduct 12 was attenuated by nearly 10-fold compared to
11 and 22-fold compared to (±)-1 indicating that stereoelectronic effects contributes at least partially towards ATR affinity.
Connecting the azaindolyl group through the five-membered ring at the 3´-position (13) resulted in a complete loss of ATR activity.
It’s likely that this new orientation exceeds the limits of the binding pocket, however unfavorable stereoelectronic features cannot
be discounted at this time. A similar effect was observed with naphthyridin-2-one analogue 14 which was over 100-fold less potent
against ATR compared to (±)-1. We also attempted to improve the ATR affinity of 9 by adding another ring to the 2-aminopyridyl
group, however the resulting analogue (15) was equipotent to 9 and 90-fold less potent relative to (±)-1. Taken together, these
results begin to describe the binding pocket off of the C-3 position of the pyrazolopyrimidine scaffold, but additional studies such as
crystallography would be required to fully define the steric and stereoelectronic features of this region.
Table 3. Results for 11-15.
a
Compound
ATR IC50 (μM)
Fold
(±)-11
(±)-12
(±)-13
(±)-14
(±)-15
0.580
4.62
>30
2.7
22
b
ND
22.4
19.1
106
90
a
c
Fold = compound IC50/(±)-1 IC50
ND, fold not determined.
.
Given that our C-3 modifications did not improve affinity towards ATR, we chose to maintain the azaindolyl group of 1 when
investigating changes at pyrazolopyrimidine N-1. Following Scheme 1, we alkylated iodide I with a variety of electrophiles and
then cross coupled with 7-azaindole-5-boronic acid pinacol ester to provide N-1 analogues 16-19.
Among our first tasks was the resynthesis of (±)-1 and subsequent resolution of its enantiomers (1 and 1´). Although there
appears to be a distinction between the two enantiomers of (±)-1 with respect to ATR affinity, the overall difference is not more
than 2-fold (1 IC50 = 0.304 μM vs. 1´ IC50 = 0.158 μM), Table 4. Indane-containing analogue 16 eliminates the stereogenic center
and slightly alters the trajectory of the N-1 substituent. However, this modification does not improve but rather attenuates potency

against ATR by approximately 3-fold compared to (±)-1. We also employed the N-1 group present in 4. While 17 and 17´ were an
improvement relative to 4, they were essentially equipotent with respect to (±)-1 (17 fold = 1.3, 17´ fold = 2.6). Analogue 18 utilizes
22
a sulfonamide moiety illustrated in a series of ATR inhibitors recently reported by Barsanti et al. However, this analogue
performed no better than our initial hits providing low-micromolar potency against ATR, (18, IC50 = 1.32 μM). We also took
25,26
inspiration from Vertex and employed a phenylsulfonyl group similar to VX970.
Satisfyingly, 19 was our best ATR inhibitor to
date with an IC50 = 0.066 μM which represented a 3-fold improvement over screening hit (±)-1.
Table 4. Results for 1, 17-20
a
Compound
1
ATR IC50 (μM)
0.304
Fold
1.4
1
1
1
1
1
1
´
0.158
0.75
3.1
6
7
7´
8
9
0.654
0.269
1.3
0.547
2.6
1.32
6.2
0.066
0.31
a
Fold = compound IC50/(±)-1 IC50
.
With a double-digit nanomolar ATR inhibitor in hand, we now had a practical analogue with which to obtain a PK-ADME and
27
selectivity profile. To benchmark our development progress, we included clinical candidates VX970 and AZD6738 in our ATR
and in vitro ADME studies alongside 19. In our hands, VX970 had an IC50 = 0.015 μM against ATR and AZD6738 had an IC50
.086 μM, Table 5. It’s worth noting that reported results for both agents were considerably lower (VX970, lit. K < 0.2 nM;
=
0
i
AZD6738, lit. IC50 = 1 nM) compared to our tests which is likely a result of differences in assay methodologies. Nonetheless, 19
appears to be comparable to both clinical candidates from a biochemical potency standpoint. Moreover, the lipophilic efficiency of
19 was calculated to be 5.8 which is comparable if not an improvement to VX970 and AZD9738. The in vitro metabolism assays
were performed with either human, mouse or rat liver microsomes, which were supplemented with NADPH to assess oxidative
stability (hLM, mLM or rLM). The results for all three compounds demonstrate reasonable oxidative stability across the species
tested.
Table 5. ATR IC50’s (μM) and in vitro ADME for 19, VX970 and AZD6738.
ATR IC50
μM)
a
a
a
Cmpd
LipE
hLM
mLM
rLM
(
1
9
0.066
0.015
0.086
5.8
4.7
5.2
83
71
84
79
77
68
63
80
93
VX970
AZD6738
a
Percent parent remaining (%R) after 30 min.
Compound 19 was tested for off-target activity in a panel of 394 kinases at 1 and 10 μM. At 1 μM, 19 inhibited 23 proteins
70% whereas at 10 μM, 76 proteins were inhibited >70%, Figure 5. Under both conditions, 19 more greatly inhibited ATR (inh =
>
96% and 106%, 1 μM and 10 μM, respectively) and breast tumor kinase (BRK, inh = 102%, 1 μM and 10 uM) than any others
tested. Notable kinase hits >70% at 1 μM include B-Raf (WT and V599E), DDR1, cKit (D816H and V560G) and FMS, Table 6. As
the lead compound was derived from a BTK/PI3Kδ dual inhibitor library, we were pleased to observe that 19 only inhibited BTK to
2
(
5% and PI3Kδ to 42% at 1 μM. Regarding kinases relevant to DNA damage repair, 19 appears selective for ATR over both ATM
inh = 28% at 1 μM) and DNA-PK (inh = 59% at 1 μM). Overall, 19 appears to be relatively selective with most activity related to
the tyrosine kinase family.

Figure 5. Kinase map of 19 depicting hits >70% inhibition at 1 μM of 394 kinases tested; atypical protein kinases such as ATR, ATM, mTOR and PRKDC
are not listed. See Supplemental Information for full list of kinase activity.
Table 6. Selected kinase panel results at 1 μM.
Kinase
ATR(h)
Inh (%)
96
Kinase
cKit(V560G)(h)
FMS
Inh (%)
76
BRK(h)
102
93
76
B-Raf(V599E)(h)
DDR1(h)
TGFβR1
76
92
DNA-PK(h)
PI3Kδ(h)
ATM(h)
59
B-Raf(h)
89
42
cKit(D816H)(h)
ALK4(h)
85
28
80
BTK(h)
25
Lastly, we evaluated the in vivo properties of 19 by conducting pharmacokinetic (PK) studies in mice. Compound 19 was dosed
as a solution at 2 mg/kg IV and 10 mg/kg PO. The results are summarized in Table 7. Compound 19 had good clearance in mice,
but had poor oral bioavailability indicating improvements in adsorption might be beneficial for further development candidates.
a
Table 7. In vivo mouse pharmacokinetic properties of 19.
CL (L/h/kg)
.33
V
z
(L/kg)
2.15
AUCPO (μM•h)
PO t1/2 (h)
F (%)
1
0.212
2.28
2.83
a
WinNonlin noncompartmental analysis.
Compound 1 was identified as a hit from an ATR biochemical screen of a BTK/PI3Kδ dual inhibitor library. Systematic
modifications of the scaffold helped to define the binding mode, particularly the importance of the 4-amino group and the 5-position
nitrogen. Variations to the N-1 and C-3 substituents provided additional information, however more detailed studies are required to
fully understand the binding pocket. Further modifications led to the discovery of 19, a novel pyrazolopyrimidine-containing
analogue that: (1) has an ATR IC50 = 66 nM; (2) is selective for ATR over ATM and DNA-PK; (3) has acceptable mouse PK
properties; and (4) represents a candidate for further cellular and in vivo profiling including proof-of-concept (PoC) studies.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. It was
funded by Medivation, now Pfizer Inc.

Acknowledgments
The authors would like to thank Amantullah Ansari and Kakoli Mukherjee for their scientific input and the Integral BioSciences
analytical, bioanalytical and purification teams for their assistance. The kinase map was reproduced courtesy of Cell Signaling
Technology, Inc. (www.cellsignal.com). This research was funded solely by Medivation, Inc.
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Supplementary Material
Supplementary data associated with this article can be found online at doi:

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Discovery of pyrazolopyrimidine derivatives
as novel inhibitors of ataxia telangiectasia
and rad3 related protein (ATR)
Leave this area blank for abstract info.
Sreekanth A. Ramachandran, Pradeep S. Jadhavar, Manvendra P. Singh, Ankesh Sharma, Gaurav N. Bagle,
Kevin P. Quinn, Po-yin Wong, Andrew A. Protter, Roopa Rai, Son M. Pham and Jeffrey N. Lindquist