Identification of potent and selective MTH1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.02.026

Structure based design of a novel class of aminopyrimidine MTH1 (MutT homolog 1) inhibitors is described. Optimization led to identification of IACS-4759 (compound 5), a sub-nanomolar inhibitor of MTH1 with excellent cell permeability and good metabolic stability in microsomes. This compound robustly inhibited MTH1 activity in cells and proved to be an excellent tool for interrogation of the utility of MTH1 inhibition in the context of oncology.

Full text of DOI:10.1016/j.bmcl.2016.02.026

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of potent and selective MTH1 inhibitors
⇑
y
Alessia Petrocchi , Elisabetta Leo , Naphtali J. Reyna, Matthew M. Hamilton, Xi Shi, Connor A. Parker,
Faika Mseeh, Jennifer P. Bardenhagen, Paul Leonard, Jason B. Cross, Sha Huang, Yongying Jiang,
à
Mario Cardozo , Giulio Draetta, Joseph R. Marszalek, Carlo Toniatti, Philip Jones, Richard T. Lewis
Institute for Applied Cancer Science, MD Anderson Cancer Center, 1901 East Road, Houston (TX) 77054, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure based design of a novel class of aminopyrimidine MTH1 (MutT homolog 1) inhibitors is
described. Optimization led to identification of IACS-4759 (compound 5), a sub-nanomolar inhibitor of
MTH1 with excellent cell permeability and good metabolic stability in microsomes. This compound
robustly inhibited MTH1 activity in cells and proved to be an excellent tool for interrogation of the utility
of MTH1 inhibition in the context of oncology.
Received 15 January 2016
Revised 9 February 2016
Accepted 10 February 2016
Available online xxxx
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
MTH1 inhibitors
Antitumor
IACS-4759
2
-Aminopyrimidine
Å
Reactive oxygen species (ROS), such as hydroxyl radical ( OH),
represent a novel class of anticancer agents with a favorable ther-
apeutic index.
Å
ꢀ
7–9
hydrogen peroxide (H
2
O
2
), and superoxide anion ( O
2
), are cellular
metabolism by-products that can react with and damage cellular
components like proteins, lipids and DNA. The deoxynucleotide
triphosphates (dNTPs) are particularly sensitive to oxidative dam-
age, and incorporation of oxidized nucleotides into DNA can cause
mutations and DNA damage.1 MTH1 (MutT homolog 1) is an
enzyme that prevents the incorporation of oxidized purines into
DNA by preferentially hydrolyzing 8-oxo-dGTP (8-O-G) and 2-
OH-dATP, two of the most abundant oxidative nucleotide lesions,
to their corresponding monophosphates.2
A class of MTH1 inhibitors, as exemplified by TH287 and cyclo-
propyl analog TH588 with nanomolar potency (Fig. 1) against the
recombinant enzyme has been recently described, and reported
to inhibit cancer cells proliferation at micromolar concentrations.9
Here we describe the design and optimization of a novel series
of MTH1 inhibitors with sub-nanomolar inhibitory potency, with
excellent selectivity (with respect to kinase activity), good phy-
sico-chemical properties (including solubility and cell permeabil-
ity), and excellent microsomal stability. These compounds have
been instrumental in allowing us to independently evaluate
MTH1 as an oncology target.
An examination of the binding modes of TH287 and of enzyme
substrate 8-O-G (Scheme 1), led us to hypothesize that a 2-amino
pyrimidine motif would enable successful engagement of the key
interactions made by the guanine motif of the natural substrate.
Molecular modeling, using Schrödinger GLIDE XP docking10 and a
docking grid based on the MTH1 protein crystal structure 3ZR0,
In normal cells, ROS levels are tightly controlled in order to
maintain intracellular redox homeostasis. On the contrary, altered
redox regulation, oxidative stress and increased ROS levels are
commonly observed in cancer cells.3 Recent publications suggest
,4
5
that MTH1 inhibition can specifically kill cancer cells. It has been
proposed that blockade of MTH1 in cancer cells results in abnor-
mally high levels of oxidized bases incorporated into DNA, with a
concomitantly increased mutational burden; resulting in genetic
5
,6
instability, and ultimately triggering of cell death mechanisms.
predicted that the pyrimidine core may participate in p–p interac-
Since normal cells have lower ROS levels, MTH1 inhibition would
not be expected to result in excessive and toxic incorporation of
oxidized bases into their DNA.6 Therefore MTH1 inhibitors might
tions with Trp117 and Phe72. The aminopyrimidine would satisfy a
hydrogen bonding network with Asp119, Asp120, and Asn33, obvi-
ating the need for the additional aminoalkyl substituent present in
,7
1
1
TH287. We also noted that the amino alkyl substituent of TH287
9
⇑
Corresponding author. Tel.: +1 713 794 5263.
E-mail address: apetrocchi@mdanderson.org (A. Petrocchi).
Present address: Cancer Research Technology-Discovery Laboratories, Jonas
appears to be a metabolic soft spot; avoiding the need for this sub-
stituent might also be advantageous from a metabolic stability
standpoint. The 4-hydroxy substituent of the 8-O-G sugar appears
to be within H-bonding distance of the backbone carbonyl of Thr8,
y
Webb Building, Babraham Research Campus, Cambridge CB22 3AT, UK.
à
Present address: Nurix, 1700 Owens St Suite 290, San Francisco, CA 94158, USA.
http://dx.doi.org/10.1016/j.bmcl.2016.02.026
0
960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Petrocchi, A.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.02.026

2
A. Petrocchi et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
Effect on MTH1 inhibition of substituents at 5 or 6 position of the 2-aminopyrimidine
NH
N
NH
N
Entry
Structure
MTH1 IC50 (nM)12
N
Cl
N
Cl
Cl
Cl
H N
H N
N
2
2
3
53 ± 10
H N
N
O
2
TH287
IC50=0.8 nM (literature)
IC50=1.6 nM
TH588
IC50=5 nM (literature)
IC50=10 nM
9
9
N
N
1
2
12
4 (IACS-4619)
0.2 ± 0.04
0.6 ± 0.16
H N
N
N
O
2
Figure 1. Chemical structure of the MTH1 inhibitors TH287 and TH588.
5
6
(IACS-4759)
H N
O
O
O
OH
2
offering a potential additional interaction to engage. These consid-
erations led us to design compound 1 (IC50 = 4 M), which inhib-
l
ited MTH1 in the single-digit micromolar range (Scheme 1).
The addition of a gem-dimethyl group in the middle of the
chain, designed to mitigate the desolvation penalty of the ligand
and engage with the lipophilic cavity (exploited by the dichloro-
phenyl motif of TH287), increased the enzymatic potency 26-fold
N
N
112 ± 21
302 ± 44
H N
N
2
7
(
compound 2, IC50 = 0.14
Removing the terminal hydroxyl group (3, Table 1) resulted in
lM).
H N
N
OH
2
increased potency (IC50 = 53 nM), suggesting that the hydroxyl
group is not a net contributor to the in vitro potency of the
molecule.
12Data are an average of >2 independent determinations.
A significant boost in potency came from the substitution of the
aminopyrimidine ring with a methyl group in position 5: com-
pound 4 (IACS-4619) was identified as a picomolar inhibitor of
MTH1 (IC50 = 0.2 nM). The same outcome was observed for 5
beneficially impact alignment of the aminopyrimidine with
1
0
Asp119 and Asp120.
Examination of substituents at the adjacent 6-position did not
provide the same boost in potency (e.g., compounds 6 (IC50
=
(
IACS-4759 (IC50 = 0.6 nM)), an analog of compound 2.
112 nM) and 7 (IC50 = 302 nM)). It is apparent that the 5-position
offers the optimal trajectory for exploiting this pocket.
A model of the binding mode of 5 (Fig. 2) docked into MTH1
derived from PDB: 3ZR0) indicates that the methyl group effec-
(
Additional exploration of a range of small substituents at the
5-position revealed the methyl to be optimal (Table 2). Chloride
8 (IC50 = 22 nM) and the methyl ether 9 (IC50 = 10 nM) conferred
no advantage. Fused cyclopentane 10 (IC50 = 4.5 nM) displayed a
10-fold loss in intrinsic potency compared with 5.
Intrigued by the influence of the linking atom on preferred con-
formation of the 4-substituent (and on the pKa of the pyrimidine
core), we also explored 4-substituents linked through nitrogen
and carbon (Table 3). Replacement of the alkoxy chain with an
alkyl amine afforded 11 (IC50 = 2980 nM), 12 (IC50 = 332 nM), 13
(IC50 = 120 nM) and 14 (IC50 = 84 nM) which confirmed that the
amino group was tolerated. The SAR of chain modifications in the
diaminopyrimidine series paralleled that of their alkoxy-linked
tively fills a lipophilic pocket formed by the side-chains of residues
Phe27, Phe72, Phe74, Trp117, and Met81. It is possible that the
substantial increase in potency results in part from displacement
of one or more energetically disfavored water molecules from this
region of the protein. Water network analysis using 3D-RISM (as
implemented in MOE 2015.10, Chemical Computing Group) was
used to examine differences in the positions and energetics of
putative water molecules in binding models of compounds 2 and
5
. For compound 2, there is a poorly bound, or energetically unfa-
vorable, water site adjacent to position 5. This water site is not evi-
dent when the analysis is run for compound 5, suggesting the
addition of the methyl group at position 5 has displaced this water
molecule. Furthermore, the inclusion of the methyl group might
counterparts. Introducing
a methyl group in the 5-position
NH
N
Cl
Cl
H N
N
2
Tyr 8
Asp 119
Asp 120
O
NHR
H
TH287
0
IC5 =1.7 nM
O
O
H
N
N
X=NH,O
O-
OH
H
H
H N
N
O
OH
N
H
N
X
R
O
2
N
N
O
1
O
IC50=4 uM
H N
N
N
2
H
N
O
sugar
H
8-O-G-substrate
Asn 33
N
H N
N
O
OH
2
2
IC50=0.14 uM
Scheme 1. Structure based design of a novel class of MTH1 inhibitors.
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A. Petrocchi et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 3
Influence of the heteroatom linker at position 4 of the 2-aminopyrimidine
Entry
Structure
MTH1 IC50 (nM)12
N
N
N
N
N
N
N
N
N
1
1
1
1
1
1
1
2
3
4
5
6
2980 ± 497
332 ± 42
120 ± 20
84 ± 17
H N
N
N
N
N
N
N
N
N
N
N
H
OH
OH
O
2
H N
N
H
2
H N
N
H
2
H N
N
H
2
3.1 ± 1.5
H N
N
H
OH
2
0.5 ± 0.15
0.5 ± 0.12
0.2 ± 0.06
1.8 ± 0.8
H N
N
H
2
Figure 2. Model of compound 5 docked into the crystal structure of MTH1 (PDB:
ZR0). Ligand binding surface is represented by gray mesh. Interatomic distances in
Å are marked in yellow. The 5-methyl substituent effectively fills a lipophilic pocket
formed by the side-chains of residues Phe27, Phe72, Phe74, Trp117, and Met81. The
aminopyrimidine engages Asp119, Asp120, and Asn33.
3
17
H N
N
OH
2
1
8
9
H N
N
2
Table 2
Replacing the methyl group at position 5 of the 2-aminopyrimidine
1
H N
Entry
Structure
MTH1 IC50 (nM)12
2
12Data are an average of >2 independent determinations.
Cl
N
8
22 ± 4
H2N
N
O
O
OH
OH
1
2
OR2
N
NR R
Cl
N
N
N
X
Y
X
Y
X
B
A
N
N
9
10 ± 1
H N
N
O
2
Y
N
NH2
N
NH
2
NH
2
1
2
1
0
4.5 ± 0.9
A = NHR R , TEA, EtOH, 150 °C, MW
2
H N
N
O
OH
B= R OH, NaH, dioxane, 60 °C
2
12Data are an average of >2 independent determinations.
Cl
Z
Z
A or B
C
X
Y
R1
R2
X
Y
N
N
N
generated pyrimidines 15 (IC50 = 3.1 nM) and 16 (IC50 = 0.5 nM) of
comparable potency to the alkoxy derivatives. Interestingly, the
tertiary amines 17 (IC50 = 0.5 nM) and 18 (IC50 = 0.2 nM) were sim-
ilarly potent. Furthermore, the introduction of a pyrrolidine, piper-
idine or a morpholine ring, as alternative replacements for the
alkoxy chain, generated compounds with inhibitory activities in
the low nanomolar range (data not shown).
N
Cl
N
Cl
N
NH2
C= NH
3
in EtOH (100 °C) or IPA (150°C)
Scheme 2. General synthetic pathway to afford 2-aminopyrimidine.
A carbon-linked chain in the 4-position of the aminopyrimidine
was similarly tolerated (e.g., 19 (IC50 = 1.8 nM)).
Further SAR exploration of the alkoxy chain indicated signifi-
cant tolerance for steric bulk and lipophilicity in this region, but
conferred no additional advantage to the compounds.
Scheme 2 outlines the synthesis of the aminopyrimidines.
Starting with the appropriate 2-amino-4-chloropyrimidine, the
alkoxy or the amino moieties were installed via nucleophilic
aromatic substitution under basic conditions. The appropriately
substituted pyrimidines could also be obtained by reaction of the
readily available 2,4-dichloropyrimidines, under conditions A or
B and subsequently heating at reflux in ammoniacal ethanol, to
install the 2-amino functionality. The route to compound 5 shown
in Scheme 3 was found to be more operationally convenient on a
larger scale.
Due to their high enzymatic potency, 4 and 5 were further eval-
uated (Table 4). Compound 5 showed good cell permeability
(assessed in
a confluent MDCK cell monolayer), solubility
(75.6 M in sodium phosphate buffer (pH = 7.0)), and a high free
l
fraction (46%) in human plasma. It was also found to be stable in
rat and human plasma and liver microsomes. In contrast, 4 showed
good permeability but high turnover in liver microsomes. The
hydroxyl functionality clearly confers significantly improved meta-
bolic stability.
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4
A. Petrocchi et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
a) NaH, dioxane, 60 °C
HO OPMB
120
N
O
N
100
H N
N
Cl
N
H
N
O
OPMB
2
b) EtOAc, H2O
80
60
40
20
0
TFA, DCM
Compound 5
Compound 4
Compound 20
NaOH, THF/H2O
O
N
O
5
F
F
N
H
N
O
O
F
0.001 0.01
0.1
1
10
100
1000 10000
nM
Scheme 3. Synthesis of compound 5.13
Figure 3. MTH1-overexpressing U2OS cells were treated in culture with increasing
concentrations of compound (1 pM–10
washed, lysed, and ‘residual’ MTH1 activity was tested with a PPiLight Pyrophos-
phate assay.
l
M) for 1 h at 37 °C. Cells were extensively
TM
Table 4
Comparison between TH287, IACS-4759 (compound 5) and IACS-4619 (compound 4)
Compound
IACS-4619 IACS-4759 TH287
1
20
Enzyme IC50 (nM)
0.2
6.9
0.6
8.0
1.6
5.9
Cmpd 5
LipE (pIC50 ꢀ cLogD)
*
ꢀ6
100
Cmpd 4
TH287
TH588
Permeability (10 cm/s) (efflux ratio) 37 (0.8)
32 (0.8)
54
7.5/5.5
60/1.5
86/58
30 (0.8)
9
Human plasma protein binding (%)
Human Clint (mL min mg /T1/2 h)
Mouse Clint (mL min mg /T1/2 h)
Plasma stability T1/2 h (rat/human)
94
92
ꢀ
1
ꢀ1
9
82/0.5
280/0.3
20/19
54 /—
8
6
4
2
0
0
0
0
0
ꢀ
1
ꢀ1
Cmpd 20
*
Assessed in a confluent monolayer of MDCK cells.
Isothermal Titration Calorimetry (ITC),14 showed a 1:1 stoi-
chiometry of binding for compound 5 with MTH1, with a signifi-
ꢀ
1
cant enthalpic component (
free energy (
D
H = ꢀ16.8 kcal mol ) to the binding
ꢀ1
D
G < ꢀ11.2 kcal mol ), attesting to the efficiency of
0.001 0.01
0.1
1
10
100 1000 10000
the molecular recognition event.
nM
Compound 5 was profiled against a panel of 97 kinases
KinomeEdge panel; DiscoveRx ) at 1 lM test concentration. No
TM
(
off-target kinase activity was evident in this panel.
1000
Specific assessment of endogenous MTH1 target engagement in
intact cells has proved to be challenging.9 As a surrogate, U2OS
cells were modified to overexpress human MTH1 and treated in
culture with increasing concentrations of test compounds. After
,15
100
1
h treatment, cells were extensively washed, lysed, and the
10
remaining MTH1 activity in the lysate was measured by monitor-
ing the hydrolysis of 8-O-G (Fig. 3). MTH1 activity was found to
be significantly inhibited by compounds 4 and 5, indicating that
these inhibitors penetrate intact cells, inhibit MTH1, and display
a prolonged residency time. The durability of the cellular target
engagement, despite the extensive washing protocol, is a testa-
ment to the high enthalpy driven MTH1-binding that characterizes
1
0
.1
0
.1
1
10
100
1000
these compounds. We did not observe MTH1 inhibition in samples
from cells treated with a weaker analog 2016 (IC50 = 850 nM); with
recombinant MTH1 IC50 (nM)
TH287 and TH588 some inhibition was observed only at the high-
est concentrations (data not shown), indicating a potentially faster
rate of dissociation from MTH1 in comparison with compounds 4
and 5.
Figure 4. Lysates from MTH1-overexpressing U2OS were treated with increasing
concentrations of compound (1 pM–10
lM) and tested for MTH1 activity with a
PPiLightTM Pyrophosphate assay.
As a control, lysates of MTH1-overexpressing cells were pre-
pared, and then treated with increasing concentrations of com-
pounds. As shown in Figure 4, all the compounds inhibited MTH1
when added directly to the lysates; we observed an excellent cor-
relation between the IC50 measured in the isolated recombinant
enzyme assay and the potency in cell lysates.
effects of our compounds 4 and 5, in comparison with TH287
and TH588, using a panel of human cancer and normal cell lines.17
We were not able to observe the expected phenotypic responses
for compounds 4 and 5 in any of the tested cells at compound con-
centrations up to 50 lM, even upon prolonged drug treatments
Having established high potency of MTH1 inhibition, excellent
cell penetration, and durability of MTH1 target engagement in
the cellular context, we then evaluated the anti-proliferative
and different assay formats (data not shown). In particular, cell
lines characterized by high levels of intrinsic ROS actually tolerated
these compounds very well, and no cytotoxicity or antiproliferative
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A. Petrocchi et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
phenotype was apparent. In contrast, we were able to observe a
cytotoxic phenotype for TH287 and TH588 at micromolar concen-
trations in these contexts, consistent with the observations previ-
ously reported for these compounds.9
We conclude that selective inhibition of MTH1 by compounds 4
and 5 appears to be insufficient to confer a robust anti-proliferative
phenotype in the contexts we have examined thus far. These
results also suggest that the mode of action of TH287 and TH588
appears to be distinctly different from our compounds, and may
involve other factors.
energy surface shapes are highly similar for each compound pair. This indicates
that the activity improvements are likely not due to differences in
conformational state of the molecules. See also: Friesner, R. A.; Banks, J. L.;
Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E.
H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. J. Med. Chem.
2
004, 47, 1739; Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.;
Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. J. Med. Chem.
006, 49, 6177.
2
11. The aryl appendage of TH287 interacts with Trp117, apparently preventing the
aminopyrimidine from penetrating as deeply into the guanine binding site;
hence the need for a 6-aminoalkyl substituent to fully engage the guanine
binding domain in this class of compounds.
12. Compounds were incubated with MTH1 (0.2 nM final concentration), and
hydrolysis of 8-oxodGTP (30
pyrophosphate was monitored via a coupled enzyme system using PPiLight
Lonza). IC50 values calculated using four-parameter logistic curve fit analysis.
13. Synthesis of compound 5. To a solution of 4-chloro-5-methylpyrimidin-2-amine
500 mg, 3.48 mmol) and 3-((4-methoxybenzyl)oxy)-2,2-dimethylpropan-1-ol
(859 mg, 3.83 mmol) in dioxane (10 ml) were added NaH 60% in mineral oil
279 mg, 6.97 mmol) portion wise and the resulting mixture was stirred at 60 °C
8 h. Additional NaH 60% in mineral oil (1.2 equiv) and after 5 h water was
lM final concentration) to 8-oxodGMP and
In summary, we have developed a novel series of potent and cell
penetrant MTH1 inhibitors with a pharmacological profile that is
distinctively different from those already in the public domain.
TM
(
(
We disclose our compounds as additional tools to further elucidate
the biology and pharmacology of MTH1 inhibition.18
(
1
Acknowledgments
slowly added to the mixture. The layers were separated and the aqueous phase
was extracted with EtOAc, the combined organic layers were washed with brine,
2 4
dried over anhydrous Na SO , filtered and concentrated under reduced pressure.
We thank Barbara Czako, Maria Emilia Di Francesco, Michael
Soth and Wylie Palmer for helpful discussion.
The residue was purified via silica gel chromatography (15–80% EtOAc in
hexanes to give N-(4-(3-((4-methoxybenzyl)oxy)-2,2-dimethylpropoxy)-5-
methylpyrimidin-2-yl)acetamide (896 mg, 2.39 mmol, 69% yield) as a yellow
liquid. To a solution of the above acetamide in DCM (4 mL) were added TFA
References and notes
(
2 mL) and the resulting mixture was stirred at RT for 2 h. The volatiles were
removed under reduced pressure to yield 3-((2-acetamido-5-methylpyrimidin-
4-yl)oxy)-2,2-dimethylpropyl 2,2,2-trifluoroacetate, which was directly used as
such in the next step. The crude residue was dissolved in MeOH (2 mL), THF
(4 mL), water (2 mL) and NaOH (275 mg, 6.87 mmol) was added and the
resulting mixture was stirred at 40 °C for 2 h. The mixture was cooled down in an
ice bath and pH adjusted to 7 with 6 M HCl (1.5 mL). The volatiles were removed
under reduced pressure and the residue was directly purified by mass-triggered
1.
2.
3.
4.
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2
preparative HPLC (Mobile phase: A = 0.1% TFA/H O, B = 0.1% TFA/MeCN;
Furuichi, M.; Nakabeppu, Y.; Karran, P.; Aquilina, G.; Bignami, M. Mol. Cell. Biol.
Gradient: B = 0–40%; 12 min; Column: C18) to give 3-((2-amino-5-
2
004, 24, 465.
methylpyrimidin-4-yl)oxy)-2,2-dimethylpropan-1-ol (425 mg, 2.0 mmol, 88%
+
+
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Nakabeppu, Y. Int. J. Mol. Sci. 2014, 15, 12543.
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yield) as a white powder. MS (ES ) C10
H NMR (600 MHz, DMSO-d ) d ppm 8.14 (br s, 2H), 7.97 (s, 1H), 4.70 (br s, 1H),
6
H
17
N
3
O
2
requires: 211, found: 212 [M+H] .
1
4.13 (s, 2H), 3.26 (s, 2H), 1.97 (s, 3H), 0.93 (s, 6H).
7
8
.
.
Helleday, T. Ann. Oncol. 2014, 25, 1253.
14. ITC was performed at 25 °C in 100 mM Tris Acetate, 40 mM NaCl, 10 mM Mg
Acetate, 0.005% Tween 20, 1 mM Dithiothreitol at pH 7.9. Compound 5 binds
with picomolar affinity to MTH1 and therefore the binding affinity and free
energy could not be directly determined by ITC as this affinity is beyond the
detection limit of the technique. A competition assay was not performed so an
upper limit of the affinity (<9 nM) and free energy is provided.
15. Leo, E.; Petrocchi, A.; Bardenhagen, J.; Alimova, M.; Shi, X.; Parker, C.; Reyna, N.;
Hamilton, M.; Felix, E.; Mazan, A.; Dillon, C.; Mseeh, F.; Marszalek, J. R.;
Toniatti, C.; Draetta, G.; Jones, P.; Lewis, R. T. Poster Presentation: Identification
of Potent, Cell Active MTH1 Inhibitors and Their Use in Target Validation Studies;
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16. The structure of compound 20:
N
H N
N
O
OH
2
1
0. Docking was performed using Glide v 6.3 in extra-precision mode as
1
1
7. U2OS, SaOS2, SW480, HCT116, MDAMB231, HeLa, UOK262, 293T, A549, H460,
H358, WI38, BJ, hMEC, MRC5.
implemented in Maestro 9.8 (Release 2014-2); Schrödinger, LLC, New York,
NY, 2015. [MTH1/8-O-G] PDB 3ZRO; [MTH1/TH588] PDB 4N1U. ACD predicts a
8. During the preparation of this manuscript we became aware of additional data,
independently generated by a group at AstraZeneca using chemical matter that
is diverse from ours, but which supports our observations and conclusions.
Kevin Foote, personal communication: Kettle, J. G.; Alwan, H.; Bista, M.; Breed,
J.; Davies, N. L.; Kay Eckersley, K.; Fillery, S.; Foote, K. M.; Goodwin, L.; Jones, D.
R.; Käck, H.; Lau, A.; Nissink, J. W. M.; Read, J.; Scott, J. S.; Taylor, B.; Walker, G.;
Wissler, L.; Wylot, M. J. Med. Chem. manuscript submitted.
0
a a
.5 pK increase resulting from introduction of the 5-methyl moiety. The pK of
the aminopyrimidine of 5 is estimated to lie between 5.8 and 6.1; docking was
performed in both the neutral and mono-protonated states and overall docking
poses were found to be very similar. Conformational analysis (around ether
and amine dihedral) of compound pairs 2/5 and 12/15 using MMFF94 force
field show that the protein bound model structures are in the low energy well
and very close to global minima in all cases (<2 kcal/mol). As well, the potential
Please cite this article in press as: Petrocchi, A.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.02.026