Identification of pyrimidine derivatives as hSMG-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.08.107

hSMG-1 kinase plays a dual role in a highly conserved RNA surveillance pathway termed nonsense-mediated RNA decay (NMD) and in cellular genotoxic stress response. Since deregulation of cellular responses to stress contributes to tumor growth and resistance to chemotherapy, hSMG-1 is a potential target for cancer treatment. From our screening efforts, we have identified pyrimidine derivatives as hSMG-1 kinase inhibitors. We report structure-based optimization of this pan-kinase scaffold to improve its biochemical profile and overall kinome selectivity, including mTOR and CDK, to generate the first reported selective hSMG-1 tool compound.

Full text of DOI:10.1016/j.bmcl.2012.08.107

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6636–6641
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of pyrimidine derivatives as hSMG-1 inhibitors
Ariamala Gopalsamy a, , Eric M. Bennett b, Mengxiao Shi a, Wei-Guo Zhang c, Joel Bard b, Ker Yu c
⇑
a Worldwide Medicinal Chemistry, Pfizer, 200 Cambridgepark Drive, Cambridge, MA 02140, USA
b Global Biotherapeutics Technologies, Pfizer, 87 Cambridgepark Drive, Cambridge, MA 02140, USA
c Oncology Research Unit, Pfizer, 401 N. Middletown Road, Pearl River, NY 10965, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2012
Revised 23 August 2012
Accepted 28 August 2012
Available online 7 September 2012
hSMG-1 kinase plays a dual role in a highly conserved RNA surveillance pathway termed nonsense-med-
iated RNA decay (NMD) and in cellular genotoxic stress response. Since deregulation of cellular responses
to stress contributes to tumor growth and resistance to chemotherapy, hSMG-1 is a potential target for
cancer treatment. From our screening efforts, we have identified pyrimidine derivatives as hSMG-1
kinase inhibitors. We report structure-based optimization of this pan-kinase scaffold to improve its bio-
chemical profile and overall kinome selectivity, including mTOR and CDK, to generate the first reported
selective hSMG-1 tool compound.
Keywords:
hSMG-1 inhibitor
Pyrimidine
mTOR
Ó 2012 Elsevier Ltd. All rights reserved.
Mammalian cells express five members of an unusual family of
protein kinases termed PIKKs on the basis of their limited homol-
ogy to the phosphoinositide 3-kinases. The PIKKs function in di-
verse cellular pathways of mitogenic- and stress-induced
signaling, which are often deregulated in disease states.1 Because
of their functional importance and the distinct catalytic site, PIKKs
are emerging targets for disease intervention.2–4 mTOR, a member
of the PIKK family, is a clinically validated target for cancer and
immunosuppressive therapy.5–7 Our laboratory has generated sev-
eral small molecule inhibitors of mTOR.8–10 Small molecule inhib-
itors of other PIKKs, such as DNA-PK and ATM, have also shown
anticancer efficacy in preclinical models.11,12
hSMG-1, a 400 kDa serine/threonine kinase, is the latest member
of the PIKK family to be identified as a therapeutic intervention tar-
get. It plays a critical role in a highly conserved RNA surveillance
pathway termed nonsense-mediated RNA decay (NMD), which en-
surestheintegrityoftranslatedproteinsbyeliminatingmRNAsbear-
ing a premature termination codon (PTC).13–16 This activity may
contribute to certain genetic disease phenotypes by preventing the
production of truncated, but still partially functional, proteins.17,18
Components of the NMD machinery also regulate RNA process-
ing, storage, transport, and translation. More recently, studies have
found roles for NMD machinery in DNA metabolism, genome sta-
bility, and cell cycle regulation. UPF1, an essential NMD factor
which interacts with ribosomes in yeast, is a physiological sub-
strate of hSMG-1 and is required for S phase progression.19,20
hSMG-1 intersects with other genome maintenance pathways
through its phosphorylation of p53. Depletion of hSMG-1 in certain
cancer cells induces c-H2AX foci and decreases cell survival in re-
sponse to ionizing radiation.21,22 In head and neck carcinomas,
HPV-positive tumors show reduced hSMG-1 (but not ATR or
ATM) expression, which correlates with higher recurrence-free pa-
tient survival after radiation treatment.23 hSMG-1 is also involved
in cellular responses to granzyme B- and hyperoxia-induced
stress.24,25 hSMG-1 may therefore play a general and prominent
role in mitogenic signaling, and in safeguarding both the genome
and transcriptome.
Because deregulation of the cellular response to various types of
stress contributes to tumor growth and resistance to chemother-
apy, hSMG-1 is a potential survival kinase that may be particularly
relevant for cancer cells due to a deregulated high demand for RNA
and protein synthesis. Therefore, modulation of hSMG-1 activity is
a potential anticancer strategy. In order to identify small molecule
inhibitors of hSMG-1, we expressed and purified highly active re-
combinant hSMG-1 enzyme, and established a homogeneous high
throughput assay.26 Initial attempts to engineer selectivity into a
previously reported mTOR inhibitor scaffold9 were unsuccessful;
of ꢀ300 such compounds tested against both hSMG-1 and mTOR,
none were selective for hSMG-1 (data not shown). Testing a small
panel of known kinase scaffolds identified the pyrimidine deriva-
tive 1 as a sub-micromolar inhibitor (Fig. 1). Most compounds in
the panel were also nonselective relative to mTOR,27 but this com-
pound had ꢀ8-fold selectivity, providing a starting point for fur-
ther optimization. However, 1 is also a potent CDK inhibitor
(Table 1), and in our in-house kinase selectivity panel28 profiling,
the compound showed less than 10-fold selectivity over 90% of
the kinases in the panel. Thus, our challenge was not only to attain
selectivity over the closely related mTOR kinase, but to introduce
overall kinome selectivity as well.
⇑
Corresponding author.
E-mail address: ariamala.gopalsamy@pfizer.com (A. Gopalsamy).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.107

A. Gopalsamy et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6636–6641
6637
Analogs required for SAR studies were prepared as detailed be-
low in Schemes 1–3. As shown in Scheme 1 and 2-chloroisonicot-
inoyl chloride 3 was converted to Weinreb’s amide 4 and treated
with methyl magnesium bromide to give the intermediate 4-acet-
yl-2-chloropyridine 5. Reaction of 5 with DMF–DMA provided 6,
which was condensed with appropriate guanidines 7 to generate
the chloro intermediate 8. Chloro displacement with primary or
secondary amines of choice afforded target molecules 9 described
in Table 1. The required guanidines 7 were readily prepared as
shown in Scheme 2, from the corresponding anilines 10 by reacting
with cyanamide under acidic conditions.
The phenyl urea analogs 11 described in Table 2 were prepared
as shown in Scheme 3 by coupling the advanced chloro intermedi-
ate 8 with boronates followed by urea formation.
A detailed knowledge of the protein/inhibitor interactions is
very useful when optimizing selectivity among closely related ki-
nases. Available crystal structures showed that this class of inhib-
O
H
N
N
N
OH
N
database
mining
N
N
HN
HN
N
Cl
SO2NH2
1
2
hSMG-1 IC50: 0.11 µM
mTOR IC50: 0.80 µM
hSMG-1 IC50: 0.45 µM
mTOR IC50: 0.89 µM
Figure 1. hSMG-1 hits identified from screening and database mining.
Table 1
Optimization of the pyridyl region (R1) and phenyl substituent (R2) of the HTS hit 1 to improve hSMG-1 activity
N
N
R1
N
R2
N
H
Example
R1
R2
hSMG-126 IC50
(lM)
mTOR27 IC50
1.5
(
lM)
Fold Selective (mTOR/hSMG-1)
8
CDK1/CDK228 IC50
<0.0052/<0.0052
(lM)
H
N
1
OH
OH
m-Cl
0.18
H
N
9a
9b
m-SO2NH2
m-Cl
0.022
0.32
>20
15
0.012/.0067
O
O
O
O
O
O
O
O
O
0.28
0.011
1.0
>71
0.035/0.0089
N
9c
9d
9e
9f
m-SO2NH2
p-Cl
0.61
>20
>20
>20
1.7
56
0.055/0.019
N
N
N
N
N
N
N
N
>19
>54
>61
7
ND
p-SO2NH2
m-CH3
0.37
0.33
0.25
1.1
ND
0.10/0.046
9g
9h
9i
m-CF3
ND
m-OCH3
m-CN
4.6
4
ND
0.08
1.5
>4
>50
>3
ND
9j
H
>4
ND
9k
9l
m-SO2NH2
m-SO2NH2
0.003
0.025
0.11
3.2
37
0.0074/<0.0052
0.10/0.031
N
O
130
N
(continued on next page)

6638
A. Gopalsamy et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6636–6641
Table 1 (continued)
Example
R1
R2
hSMG-126 IC50
0.006
(
lM)
mTOR27 IC50
(l
M)
Fold Selective (mTOR/hSMG-1)
33
CDK1/CDK228 IC50
0.048/0.021
(lM)
O
N
9m
m-SO2NH2
0.2
N
N
N
9n
9o
m-SO2NH2
m-SO2NH2
0.31
3.3
10
47
ND
0.003
0.14
0.036/0.016
N
Cl
NH
N
Cl
N
Cl
NH2
a
b
HN
NH2
a
R2
R2
O
N
O
O
Cl
3
O
4
5
10
7
NH
N
Cl
Scheme 2. Reagents and conditions: (a) NH2CN, Conc. HNO3, H2O/EtOH.
c
d
HN
NH2
+
and clearly making a bidentate interaction with the hinge region
(Fig. 2a). The sulfonamide makes no hydrogen bonds. Docking34
modified compounds to the hSMG-1 homology model suggested
that moving the sulfonamide to the meta position could allow
hydrogen bonding with residues 883–886 possibly increasing po-
tency. Multiple hydrogen bonding arrangements are possible; in
Figure 2b, the sulfonamide of 9o interacts with the backbones of
Gly884 and Ala885. Synthesis and testing of 9a resulted in a ꢀ8-
fold potency increase over 1, with a m-chloro substituent on the
phenyl ring; a similar trend held for compounds 9b and 9c. Consis-
tent with the predicted loss of hydrogen bonds to the protein, the
p-chloro or p-sulfonamide analogs 9d and 9e were much less
potent.
R2
O
N
6
7
N
R1
N
N
Cl
e
N
N
R2
R2
N
H
N
N
H
9
8
Other meta substituents on the phenyl ring were explored as
exemplified by compounds 9f–9i and the m-sulfonamide group
was more favorable for hSMG-1 potency. Elimination of the substi-
tuent from the aromatic ring (compound 9j) leads to significant
loss of potency. Fixing the m-sulfonamide and optimizing the R1
substituent of the pyridyl region revealed significant flexibility
accommodating aliphatics, acyclics, and bridged bicyclics (com-
pounds 9k–9o). Most of these analogs improved the hSMG-1 activ-
ity compared to the original hit 1 while maintaining or slightly
improving the selectivity over mTOR. However, none of these mod-
ifications gave significant improvements in CDK selectivity.
From our earlier work9 on PI3K and mTOR it was clear that a
urea group bridging Asp841 and the catalytic Lys833 can signifi-
cantly increase potency of other inhibitor scaffolds (Fig. 3). We
investigated whether the same group could increase the potency
of our scaffold towards hSMG-1 and increase the selectivity win-
Scheme 1. Reagents and conditions: (a) (OMe)MeNH, (i-Pr)2EtN, CH2Cl2, 0 °C to rt
95%; (b) MeMgBr, THF, 0 °C to rt, 85%; (c) DMF–DMA, 95%; (d) NaOEt/DMF, 75%; (e)
1° or 2° amines, microwave, 150 °C, 15 min.
itor can adopt two general binding modes. The first mode, exempli-
fied by imatinib,29,30 is monodentate at the hinge with a hydrogen
bond acceptor on the ring directly attached to the pyrimidine ring.
This mode often occurs when the kinase adopts a DFG-out confor-
mation. The second mode is bidentate,31 where the aminopyrimi-
dine moiety donates and accepts one hydrogen bond from the
hinge. SAR data and molecular modeling using a hSMG-1 homol-
ogy model32 were both more consistent with bidentate binding,
but not conclusive. Therefore, we confirmed the binding mode by
crystallography.
Database mining around the original hit afforded compound 2
which was soaked into crystals of PI3K-
c. The 2.5 Å X-ray com-
plex33 showed the scaffold adopting a U-shaped conformation
dow. Superposition of PI3K-
c
crystal structure complexes with
H
N
H
N
N
N
Cl
NH2
N
O
a, b
+
N
R2
B
N
O
O
R2
N
H
N
H
N
8
11
Scheme 3. Reagents and conditions: (a) Pd(Ph3P)4, 2 M aqNa2CO3, toluene, microwave, 20 min, 140 °C; (b) triphosgene, Et3N, THF, MeNH2., 0 °C to rt.

A. Gopalsamy et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6636–6641
6639
Table 2
Optimization of the phenyl sulfonamide region (R2 and R3) to improve selectivity over CDKs
H
N
H
N
N
O
R3
R2
N
N
H
N
Example
R2
R3
hSMG-1 IC50
(l
M)
mTOR IC50
(lM)
Fold Selective (mTOR/hSMG-1)
CDK1/CDK2 IC50
(
lM)
PI3Ka/c IC50 (lM)
11a
11b
11c
11d
11e
11f
11g
11h
11i
SO2NH2
H
H
H
CH3
CH3
CH3
CH3
Cl
<0.00005
0.0001
0.0002
<0.00005
<0.00005
<0.00005
0.0002
<0.00005
<0.00005
0.00011
0.018
0.015
0.025
0.016
0.045
0.018
0.088
0.0077
0.012
0.05
>360
>150
125
>320
>900
>350
440
>154
>244
455
0.062/0.023
0.21/0.10
3.2/0.56
ND
1.2/0.46
0.23/0.11
14.0/3.0
0.32/0.14
1.4/0.52
32/7.1
0.044/0.091
0.080/0.082
0.078/0.059
ND
0.061/0.072
0.043/ 0.063
0.13/0.093
ND
SO2NH(CH3)
SO2N(CH2CH3)
SO2NH2
2
SO2N(CH3)
2
SO2NH(CH3)
SO2N(CH2CH3)
SO2NH(CH3)
SO2N(CH3)
SO2N(CH2CH3)
2
2
Cl
Cl
0.033/0.054
0.092/0.060
2
11j
both scaffolds (PDB entries 3IBE and 4FUL) suggested the R1 posi-
tion of the pyridyl ring (Table 1) was the most appropriate substi-
tution site to install a phenyl urea. As shown in Figure 3, the
inhibitor in 3IBE does not adopt the same U-shaped conformation
as 2, so the R1 equivalent substituent is oriented in a different
direction. Synthesis and testing of this designer analog 11a in-
creased hSMG-1 potency below 1 nM, with a smaller increase in
mTOR potency and no significant change in CDK inhibition
(Table 2).
Analysis of the sulfonamide region of a similar inhibitor crystal-
lized in complex with CDK (PDB entries 1OIT31 and 2FVD35
)
revealed that bulky substituents at the meta and para positions
might not be readily accommodated due to unfavorable steric con-
tacts with CDK residue Lys89 (PI3K-
c Lys890). This position has
been previously exploited to achieve selectivity among CDK iso-
forms.36 In 1OIT, the sulfonamide sulfur is ꢀ4 Å from the lysine
side chain, which is well ordered, and in particular it is only
4.5 Å from the Lys
a
-carbon. Unlike both mTOR and PI3K-
c,
hSMG-1 has a Gly, and therefore no
a-carbon, at this position.
Figure 2. Binding of pyrimidine inhibitors to PIKKs. (A) Co-crystal structure of 2
(stick rendering) with PI3K-c, showing a bidentate interaction with the hinge
Val882. Electron density for the piperidine ring was weak, but the carbonyl appears
to interact with the catalytic Lys833. Loop residues 804–810 were observed, but are
omitted from this figure for clarity. (B) Docking model of 9o interacting with a
homology model of hSMG-1, showing one possible way a m-sulfonamide may form
hydrogen bonds with the protein. For clarity, hSMG-1 residues are numbered
Figure 3. Overlay of PI3K crystal structure complexes of a potent, selective mTOR
inhibitor (from 3IBE, purple) and the nonselective hSMG-1 inhibitor 2 (orange). Key
active site residues and distances from 3IBE are shown with green carbons.
according to the corresponding residue in PI3K-c.

6640
A. Gopalsamy et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6636–6641
The off-target selectivity was assessed in the cellular setting as
well. Compounds 9e, 9o and 11j with a range of potency and
selectivity ratios were evaluated in MDA 361 cells (Fig. 5).
Weaker compound 9e (hSMG-1 IC50: 0.37
inhibition of hSMG-1 phosphorylation of UPF1 even at 3
More potent analog 9o (IC50: 0.003 M) prevented UPF1 phos-
lM), showed minimal
lM.
l
phorylation in a dose dependent manner. However, 9o also
clearly reduced phosphorylation of p70S6K and AKT, which are
targets of mTOR and PI3K, indicating that ꢀ47-fold in vitro selec-
tivity did not translate to functional selectivity in cells. The best
compound in the set 11j (IC50: 0.0001
lM) eliminated UPF1 phos-
phorylation at 1 M and significantly reduced it at 0.3
l
lM. But it
had little apparent effect on phospho p70s6 K and phospho AKT,
targets of mTOR and PI3K. The compound 11j also showed good
inhibition in an MDA468 cell proliferation assay with an IC50 of
75 nM, providing a tool compound to probe hSMG-1 mediated
tumor biology.
In summary, we have discovered the first general class of small
molecule hSMG-1 inhibitors, with selectivity over a broad panel of
kinases including PI3K and mTOR. The compounds are also
selective in a cellular setting and are able to inhibit cell growth.
These inhibitors should be useful for further probing the role of
hSMG-1 in tumor biology. We also identified key residues
in the hSMG-1 binding site which may be exploited to improve
kinome selectivity of other chemical scaffolds targeting this
enzyme.
Figure 4. Cutaway model of 11j (gray carbons) in the CDK active site (based on PDB
entry 2FVD). Selected key CDK residues are shown with green carbons. The diethyl
functionality on the sulfonamide can only be accommodated at the expense of
unfavorable electrostatic repulsion between the backbone carbonyl of His84 and a
sulfonamide carbonyl. Allowing flexibility in the protein34 was insufficient to
relieve this bad contact, which was not present for 11a. Residues shown as tubes
were unrestrained during energy minimization, while residues shown in ball and
stick form were restrained at 1.0 kJ/mol-A2.
Acknowledgements
The authors thank Dr. John Ellingboe, Dr. Tarek Mansour and
Dr. Bob Abraham for their support of this work. We also thank
the Wyeth Chemical Technologies group for analytical data.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
08.107. These data include MOL files and InChiKeys of the most
important compounds described in this article.
References and notes
Figure 5. Inhibition of kinase activity in MDA361 cells. Phosphorylation targets of
hSMG-1, mTOR, and PI3K were detected by immunostaining protein isolated from
cells after a 6-h treatment with various inhibitors. Actin was monitored as a control
for total cellular protein.
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26. Gopalsamy, A.; Shi, M.; Bennett, E. M.; Zask, A.; Curran, K. J.; Venkatesan, A. M.
International patent publication WO 2010/120991 A1, 2010.
27. Yu, K.; Toral-Barza, L. Methods Mol. Biol. 2012, 821.
28. Assays for kinases other than PI3K, mTOR, and h-SMG1 were performed using
kits from Caliper Life Sciences (Hopkinton, MA).
29. Namboodiri, H. V.; Bukhtiyarova, M.; Ramcharan, J.; Karpusas, M.; Lee, Y.;
Springman, E. B. Biochemistry 2010, 49, 3611.
34. Molecular modeling methods: CDK modeling used PDB entry 2FVD. After
processing with the Schrodinger Protein Preparation tool, 11a was placed in
the CDK binding site by superposition of a PI3K-docked model of 11a. The
binding site was relaxed to
a
gradient of 0.05 kJ/mol-A2 using the PRCG
minimizer with OPLS-2005 and GB/SA solvation. Residues 33, 52–56, 64, 78–
84, 134, 144–148, and the inhibitor were unrestrained. Other atoms were
restrained (10 kJ/mol-Å2) and several interatomic contact distances were
restrained. The resulting model (RMSD 0.24 Å relative to the PDB coordinates)
was used to dock 11a and 11j using 150,000 steps of the LMCS+MCMM
protocol in MacroModel with the MOLS method providing small rotations/
translations (up to 15° and 0.25 Å in each coordinate) in the binding site. The
ligand and residues 10–12, 18, 32–35, 52, 63–66, 69, 76–86, 89, 125, 134, 144–
148 were free to move, residues within 4.0 Å of these were restrained at 1 kJ/
mol-Å2, and remaining atoms were frozen. Ligand/protein interaction distance
restraints (10 kJ/mol-Å2) were applied to favor optimization of the general
binding mode observed by crystallography: 3.0 0.3Å for hinge backbone
interactions, 3.1 0.5Å for the urea carbonyl/Lys33, and 4.4 0.5Å for the
sulfonamide sulfur to Lys89 NZ. For the lowest energy structure of 11j, shown
in Figure 4, none of the distance restraints were triggered. Docking to hSMG-1
homology models followed a similar protocol, except that docking performed
prior to determining the X-ray complex with 2 used larger MOLS translations/
rotations (typically up to 180° or 2.0 Å in each coordinate) to probe the
compound orientation in the binding site.
30. Seeliger, M. A.; Nagar, B.; Frank, F.; Cao, X.; Henderson, M. N.; Kuriyan, J.
Structure 2007, 15, 299.
31. Anderson, M.; Beattie, J. F.; Breault, G. A.; Breed, J.; Byth, K. F.; Culshaw, J. D.;
Ellston, R. P.; Green, S.; Minshull, C. A.; Norman, R. A.; Pauptit, R. A.; Stanway, J.;
Thomas, A. P.; Jewsbury, P. J. Bioorg. Med. Chem. Lett. 2003, 13, 3021.
32. Homology modeling methods: hSMG-1 models were constructed using
modeller (Eswar, N.; Marti-Renom, M. A.; Webb, B.; Madhusudhan, M. S.;
Eramian, D.; Shen, M.; Pieper, U.; Sali, A. Curr. Protocols Bioinform. Suppl.
2006, 15, 5.6.1) with various PI3K structures as templates. Active site side
chain positions were then reoptimized in the presence of an
aminopyrimidine inhibitor using MacroModel with protocols similar to
those described for docking. Initial models used unpublished PI3K-
c crystal
structures from the same inhibitor series as PDB entry 3IBE. After solving
4FUL, models were rebuilt using 4FUL and 2RD0 as templates, and these
models gave better results (tighter conformer clusters closer to the position
observed for 2 bound to PI3K), possibly due to adjustments in the hinge and
35. Chu, X. J.; DePinto, W.; Bartkovitz, D.; So, S. S.; Vu, B. T.; Packman, K.; Lukacs, C.;
Ding, Q.; Jiang, N.; Wang, K.; Goelzer, P.; Yin, X.; Smith, M. A.; Higgins, B. X.;
Chen, Y.; Xiang, Q.; Moliterni, J.; Kaplan, G.; Graves, B.; Lovey, A.; Fotouhi, N. J.
Med. Chem. 2006, 49, 6549.
36. Pratt, D. J.; Bentley, J.; Jewsbury, P.; Boyle, F. T.; Endicott, J. A.; Noble, M. E. J.
Med. Chem. 2006, 49, 5470.
C-helix between 3IBE and 4FUL. Relative to PI3K-
residue insertion in the kinase domain. The models did not include this
region, but covered 220 residues of the kinase domain, of which 14 were
c, hSMG-1 contains a ꢀ60-