Pyrimidine-based tricyclic molecules as potent and orally efficacious inhibitors of wee1 kinase

Article abstract of DOI:10.1021/ml5002745

Aided by molecular modeling, compounds with a pyrimidine-based tricyclic scaffold were designed and confirmed to inhibit Wee1 kinase. Structure-activity studies identified key pharmacophores at the aminoaryl and halo-benzene regions responsible for binding affinity with sub-nM K_i values. The potent inhibitors demonstrated sub-μM activities in both functional and mechanism-based cellular assays and also possessed desirable pharmacokinetic profiles. The lead molecule, 31, showed oral efficacy in potentiating the antiproliferative activity of irinotecan, a cytotoxic agent, in a NCI-H1299 mouse xenograft model.

Full text of DOI:10.1021/ml5002745

Letter
pubs.acs.org/acsmedchemlett
Pyrimidine-Based Tricyclic Molecules as Potent and Orally Efficacious
Inhibitors of Wee1 Kinase
Yunsong Tong,*,† Maricel Torrent,‡ Alan S. Florjancic,† Kenneth D. Bromberg,† Fritz G. Buchanan,†
Debra C. Ferguson,† Eric F. Johnson,† Loren M. Lasko,† David Maag,† Philip J. Merta,∥
Amanda M. Olson,§ Donald J. Osterling,§ Nirupama Soni,† Alexander R. Shoemaker,†
and Thomas D. Penning†
‡
§
∥
†Cancer Research, Molecular Modeling, Pharmacology, and High Throughput Biology, Research and Development, AbbVie,
1 North Waukegan Road, North Chicago, Illinois 60064-6114, United States
S
* Supporting Information
ABSTRACT: Aided by molecular modeling, compounds with
a pyrimidine-based tricyclic scaffold were designed and
confirmed to inhibit Wee1 kinase. Structure−activity studies
identified key pharmacophores at the aminoaryl and halo-
benzene regions responsible for binding affinity with sub-nM
Ki values. The potent inhibitors demonstrated sub-μM activities in both functional and mechanism-based cellular assays and also
possessed desirable pharmacokinetic profiles. The lead molecule, 31, showed oral efficacy in potentiating the antiproliferative
activity of irinotecan, a cytotoxic agent, in a NCI-H1299 mouse xenograft model.
KEYWORDS: Wee1 kinase inhibitors, antitumor
hinge region and therefore exploitable for drug design
purposes. Taking advantage of this untapped region by 2, our
de novo design and modeling studies suggested that molecules
with a tricyclic core, as displayed in 3, would possess key
elements for interacting with the active site of the Wee1 protein
(see Figure 1A for more details), potentially resulting in
improved Wee1 inhibition. In this letter, we demonstrate that
not only do such molecules show potent activity in enzymatic
and cellular assays but they also exhibit desirable pharmacoki-
netic properties as well as oral antitumor efficacy in a murine
xenograft model.
ells utilize G1 and G2/M checkpoints within cell cycle to
Censure genomic integrity. The fact that cancer cells are
often defective in the G1 checkpoint due to frequent p53
mutations has led to interest in developing anticancer agents
that abrogate G2/M arrest.1,2 One of the key players in this
arena is the Wee1 kinase, an atypical tyrosine kinase that
phosphorylates Cdk1 (also called Cdc2) on tyrosine 15 (Y15)
resulting in functional inactivation.3 Cdk1 recruits Cyclin A and
Cyclin B to initiate mitosis. Wee1 negatively regulates Cdk1
and enables repair of damaged DNA in G2 phase prior to
mitosis. Therefore, inhibition of Wee1 forces tumor cells into
premature mitosis, leading to mitotic catastrophe and cell
death.4,5 Since Wee1 is overexpressed in certain malignan-
cies,6−8 Wee1 inhibitors have the potential to sensitize p53
deficient tumors to DNA-damaging treatments.
Recently, a few classes of Wee1 inhibitors have been
disclosed.9−11 Among them is a selective inhibitor, MK-1775
(1, 2-allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-((4-(4-
methylpiperazin-1-yl)phenyl)amino)-1H-pyrazolo[3,4-d]-
pyrimidin-3(2H)-one).9 MK-1775 exhibited antitumor activity
in various preclinical studies in potentiating chemo- and
radiotherapy and is currently in phase I/II human clinical
trials.12 In addition, a different class of Wee1 inhibitors
featuring an imino-dihydropyrimidinone pyrimidine core (2)
has emerged in patent literature.13 Computer-assisted evalua-
tion of the Wee1 kinase binding site, using public X-ray
structures of bound Wee1 inhibitors as starting points,14,15
revealed a key region within the ATP binding pocket in Wee1
adjacent to the bicyclic core not utilized by inhibitors such as 2.
This region is generally occupied by the pyridyl moiety of MK-
1775 (1) and was deemed to be accessible from the ligand
Special Issue: New Frontiers in Kinases
Received: July 2, 2014
Accepted: August 4, 2014
Published: August 6, 2014
© 2014 American Chemical Society
58
dx.doi.org/10.1021/ml5002745 | ACS Med. Chem. Lett. 2015, 6, 58−62

ACS Medicinal Chemistry Letters
Letter
The general synthesis of analogs presented in this letter is
outlined in Scheme 1. The known amino pyrimidine 4 was
a
Scheme 1
Figure 1. (A) Computational molecular model of compound 10
(shown in cyan, stick) bound to the Wee1 kinase binding site (shown
in brown, ribbon). Key residues are shown in stick. The ligand is
predicted to make three H-bond interactions with the protein (black
dashes). Two of these interactions are made with backbone polar
atoms in the hinge. The third interaction is made with the side chain of
gatekeeper residue Asn376 (shown in orange, stick). The 5-membered
ring added to the original bicyclic core (leading to the novel tricyclic
core) is predicted to be sitting in a sweet spot right between Phe433
located at the base of the ATP binding site, and Val313 located in the
Gly-rich loop. Purple dashes indicate the extent of the “sandwich”
made by these two residues. (B) Close fit of benzene ring (part of R
group) with surrounding protein walls, defined by residues Ile305
(Gly-rich loop) and Gly382 (extended hinge). (C) Halogenated
phenyl ring uses small pocket next to gatekeeper residue Asn376
(shown in orange), while sitting perpendicular to tricyclic core. Green
arrow indicates unutilized space.
a
Reagents and conditions: (i) NaH, 1-Cl-2-isocyanatobenzene, 98%;
(ii) POCl3, DIEA, 84%; (iii) 2,2-dimethoxyethanamine; (iv) HCl, 59%
(2 steps); (v) mCPBA, 92%; (vi) amine.
treated with NaH and a halo-substituted isocyanatobenzene to
afford the bicyclic intermediate 5.16 The pyrimidinedione
moiety was chlorinated in the presence of POCl3 and DIEA. An
SNAr addition using 2,2-dimethoxyethanamine led to 7, which
was subsequently cyclized under acid conditions. The methyl
thioether group of 8 was oxidized by mCPBA, setting the stage
for the final SNAr reaction with a desired amino substrate to
provide 10−25.17 Compounds 26 to 31 were synthesized in the
same fashion using the corresponding bis-halo isocyanatoben-
zene in the first step.
The binding affinity of all compounds, presented as Ki values,
was assessed in a routine 6-point TR-FRET binding assay. A
functional antiproliferative cell viability assay and a mechanism-
based pCDK1 ELISA assay, both utilizing the NCI-H1299 cell
line (human nonsmall cell lung carcinoma) to provide EC50
values, helped to access cellular potency of any potent binders.
It was gratifying to find that the first analogue synthesized, 10,
was active in the biochemical assay (Ki = 2.3 nM) and also
possessed reasonable cellular activity in both the cell viability
and ELISA assays (EC50 = 1.0 and 0.37 μM, respectively).
According to the model of compound 10 bound to the Wee1
active site (Figure 1A), one of the pyrimidine nitrogens and the
tethered amino group form hydrogen-bond interactions to the
backbone NH and backbone carbonyl O of residue Cys379,
respectively (i.e., a two-hinge interaction motif). The model
also predicted a third, nonhinge, H-bond interaction between
the side-chain N of Wee1 gatekeeper residue Asn376 and the
amide carbonyl O in compound 10. This third H-bond
interaction was believed to be critical for good kinase
selectivity18 since human kinases rarely have a hydrogen-bond
donor (HBD) residue such as Asn in the gatekeeper position
(<0.5% of ∼500 human kinases based on internal studies).
Encouraged by these early results, we proceeded to map the
structure−activity relationship (SAR) of the aminoaryl group
attached to the pyrimidine ring. Removing the terminal piper-
azine on 10 led to about 10-fold loss in potency (11), while the
importance of the aminoaryl moiety is clearly demonstrated
with 12 being inactive (Ki >3140 nM) (Table 1). The reason
for loss of activity by replacing the benzene with cyclohexane is
likely due to the nature of the “gate” region leading to the ATP
binding site (Figure 1B). Aromatic rings such as benzene are
the ideal fit for this region in the protein, i.e., they are properly
“sandwiched” between residue Gly382 (located in the extended
hinge region) and residue Ile305 (located in the Gly-rich loop).
Pi−sigma interactions between the aromatic ring in the ligand
and aliphatic C−H bonds in the protein (alpha-C of Gly382
and beta-C of Ile305) are the main drivers behind the optimal
59
dx.doi.org/10.1021/ml5002745 | ACS Med. Chem. Lett. 2015, 6, 58−62

ACS Medicinal Chemistry Letters
Letter
in Figure 1A. Once the ligand tail starts to project into solvent,
R groups with a larger polar surface area (PSA), such as those
in 14−16, are much better choices. Moving the piperazine to
the meta position of the benzene maintained the potency (17,
Ki = 6.2 nM); however, placing it at the ortho position was not
tolerated (18). Piperazine at the meta position confers the
ligand a global conformation that still matches the V-shape/C-
shape needed to complement the kinase binding site.
Unfortunately, piperazine at the ortho position locks the ligand
in a new conformation that is no longer compatible with the
shape of the target binding site. Next, we investigated the SAR
of bicyclic scaffolds with a saturated ring fused to the benzene.
Once again, the presence of a nitrogen atom on the saturated
ring was desirable for enzymatic potency (19 vs 20−22).
Among these analogues, the tetrahydroisoquinoline inhibitor,
20, exhibited much better cellular activities and was the focus of
further optimization. Installing a cyclopropyl group at the 4-
position of the isoquinoline of 20 led to 23, another analogue
not only with high enzymatic potency (Ki =1.2 nM) but also
Table 1. Mono-Cl Analogues
possessing sub-micromolar activity in the cell viability (EC50
=
0.69 μM) and pCDK1 ELISA (EC50 = 0.10 μM) assays.
Acylation of the tetrahydroisoquinoline nitrogen brought a
slight deterioration in activity (24). The gem-dimethyl
analogue, 25, showed overall potent activity comparable to
23. Four representative compounds (10, 20, 23, and 25) were
evaluated for their pharmacokinetic (PK) profile in mice (Table
2). Even though they all demonstrated desirable profiles, 25
a
Table 2. Mouse PK for Mono-Cl Analogues
ID
iv t1/2 (h)
iv CL (L/h/kg)
po AUC (μM·h)
po F (%)
10
20
23
25
1.9
1.5
2.3
2.8
1.8
2.2
1.5
1.4
8.4
72
52
70
80
5.2
9.8
12.3
a
CD-1 mouse. iv dosing: 3 mg/kg. po dosing: 10 mg/kg.
appeared to have the best overall properties. The iv half-life
reached 2.8 h, while clearance was modest (1.4 L/h/kg) with
oral biovailability of 80%.
Having gained much insight in the SAR of the aminoaryl
region, we turned our attention to the modification of the
chloro-benzene ring (Table 3). Modeling suggested that the
ortho-Cl group is essential for situating the phenyl moiety
orthogonal to the tricyclic core (Figure 1C). Not only is the
intramolecular arrangement optimal when the phenyl group is
rotated almost 90° with respect to the plane of the tricyclic
core, but the space available in the pocket (adjacent to
gatekeeper residue Asn376) is also limited, and it can only
accommodate such a large ring if placed “facing” the gatekeeper
residue. The benefit of adding an extra halo substitution at the
other ortho position is 2-fold. On one hand, the 2,6-
disubstitution pattern reinforces the 90° rotation needed for
optimal complementarity with the immediate surroundings. On
the other hand, the second halogen fills the remaining portion
of the pocket (otherwise empty, i.e., with only a monosub-
stituted phenyl), increasing van der Waals contacts with the
protein, and further increasing compound affinity/potency. Not
surprisingly, all bis-halo analogues (26 to 31) demonstrated
potent activity in the biochemical assay with Ki values below the
lower detection limit for all except 27. The potent binding
affinity was presumably responsible for the generally superior
cellular activities possessed by this group of compounds. Four
a
b
Cell viability assay. ELISA assay in H1299 cells. All data were an
average of at least two measurements except for those with a qualifier
or high micromolar values. N.T.: not tested.
protein−ligand complementarity observed in this region.
Unlike benzene, saturated rings such as cyclohexane are too
voluminous to fit well in this somewhat narrow region of the
kinase binding site. At least one nitrogen atom of the piperazine
on 10 was needed to maintain a Ki value below 10 nM (13−
15), although reducing the basicity of the terminal nitrogen
through acylation had little impact on Wee1 binding (16).
Highly hydrophobic groups such as the cyclohexyl on 13 do
not match well with the local environment in this solvent
exposed region of the kinase, dominated by polar residues
Ser383 (ribose residue) and Asp386 (ribose + 3 residue) shown
60
dx.doi.org/10.1021/ml5002745 | ACS Med. Chem. Lett. 2015, 6, 58−62

ACS Medicinal Chemistry Letters
Letter
Table 3. Bis-Halo Analogues
Figure 2. Pharmacodynamic profiles of compound 31. Biomarker
effects of 31 on expression of pY15 in H1299 tumor bearing mice after
po dosing (100 mkd) as analyzed by Western blot. Loading control: α-
tubulin.
a
Chart 1. Tumor Growth Inhibition
a
b
Average of two measurements. Values of 0.013 and <0.010 μM.
compounds (26 and 29−31) were chosen for mouse PK
studies with comparably favorable properties to that observed
for the mono-Cl compounds (Table 4). The best compound,
31, achieved an adequate iv half-life (2.8 h), low clearance (0.55
L/h/kg), and excellent oral biovailability (F = 94%).
a
a
Table 4. Mouse PK for Bis-Halo Analogues
Mouse xenograft (H1299 cell) data with tumor volume 52 days post-
tumor inoculation. Compound 31 was orally dosed QD ×14 from day
28 to day 41. Irinotecan was dosed IP at days 28, 32, 36, and 40.
ID
iv t1/2 (h)
iv CL (L/h/kg)
po AUC (μM·h)
po F (%)
26
29
30
31
1.7
2.6
3.9
2.8
2.0
6.5
12
55
68
49
94
1.1
alone (p < 0.01). All dosing regimens were well tolerated.
Furthermore, the efficacy of 31 to sensitize irinotecan appeared
to be dose responsive.19 Together, these results highlight the
combination efficacy of 31 with irinotecan in vivo.
0.97
0.55
9.8
24
a
CD-1 mouse. iv dosing: 3 mg/kg. po dosing: 10 mg/kg.
As disclosed in this letter, compounds with a pyrimidine-
based tricyclic core were suggested by design/modeling and
supported by their binding and cellular activity as potent Wee1
inhibitors. These molecules are generally selective against other
kinases.18 SAR studies in the aminoaryl and halo-benzene
regions led to compounds with sub-nM Ki values in the
enzymatic assay and sub-μM EC50 values in both functional and
mechanism-based cell assays. Representative inhibitors also
exhibited desirable pharmacokinetic profiles. Compound 31
demonstrated oral in vivo efficacy in a mouse H1299 xenograft
model and potentiated the antiproliferative activity of
irinotecan. These results suggest that this class of compounds
may have potential as therapeutic agents for anticancer
indications.
Compound 31 was dosed orally at 100 mg/kg/day (mkd) in
H1299 tumor bearing mice (n = 4), utilizing inhibition of pY15
Cdk1/2 as an in vivo pharmacodynamics marker of Wee1
inhibition. The resulting Western immunoblotting analysis
(Figure 2) demonstrated a nearly complete depletion of the
pY15 bands at both 6 and 10 h after dosing. The concentration
of 31 in plasma decreased 32% between these two time points,
however, the tumor exposure levels remained at high
throughout (47 and 53 μM).
Compound 31 was further evaluated for its activity in
potentiating the efficacy of the cytotoxic agent, irinotecan, in a
mouse xenograft model (H1299). As presented in Chart 1, 31
achieved a modest tumor growth inhibition (TGI) of 43% (p <
0.001) after oral dosing at 50 mkd on day 41. Meanwhile,
irinotecan alone reached a TGI of 67% (p < 0.001) at 10 mkd.
The combination of 31 and irinotecan achieved an 83% TGI (p
< 0.001). On day 52, the combination group showed a 58%
TGI when compared to irinotecan alone (p < 0.05). The
durability of the combination response was highlighted by a
25% tumor growth delay (TGD) when compared to irinotecan
ASSOCIATED CONTENT
* Supporting Information
■
S
Assay protocols, kinome selectivity profile, modeling protocols,
efficacy study protocols, and synthetic procedures. This
material is available free of charge via the Internet at http://
pubs.acs.org.
61
dx.doi.org/10.1021/ml5002745 | ACS Med. Chem. Lett. 2015, 6, 58−62

ACS Medicinal Chemistry Letters
Letter
kinase: an atypical tyrosine kinase with a key role in CDK1 regulation.
Structure 2005, 13, 541−550.
AUTHOR INFORMATION
■
Corresponding Author
*(Y.T.) Phone: 1-847-935-1252. Fax: 1-847-935-5165. E-mail:
yunsong.tong@abbvie.com.
(15) Smaill, J. B.; Baker, E. N.; Booth, R. J.; Bridges, A. J.; Dickson, J.
M.; Dobrusin, E. M.; Ivanovic, I.; Kraker, A. J.; Lee, H. H.; Lunney, E.
A.; Ortwine, D. F.; Palmer, B. D.; Quin, J., III; Squire, C. J.;
Thompson, A. M.; Denny, W. A. Synthesis and structure-activity
relationships of N-6 substituted analogues of 9-hydroxy-4-
phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-diones as inhibitors of
Wee1 and Chk1 checkpoint kinases. Eur. J. Med. Chem. 2008, 43,
1276−1296.
Notes
The authors declare the following competing financial
interest(s): All authors are employees of AbbVie. The design,
study conduct, and financial support for this research were
provided by AbbVie. AbbVie participated in the interpretation
of data, review, and approval of the publication.
(16) Bamba, M.; Sunami, S. Dihydropyrimidopyrimidine derivatives.
WO 2010/067888, June 17, 2010.
(17) Synthesis of 20 involved the addition of tert-butyl 7-amino-3,4-
dihydroisoquinoline-2(1H)-carboxylate followed by Boc removal and
methylation on the isoquinoline nitrogen. Synthesis of 24 involved the
addition of t-butyl 7′-amino-1′H-spiro[cyclopropane-1,4′-isoquino-
line]-2′(3′H)-carboxylate followed by Boc removal and acylation on
the isoquinoline nitrogen. See Supporting Information for detailed
procedures.
(18) Kinome profiling of a few key inhibitors against 84 kinases is
provided in the Supporting Information.
(19) Efficacy of 31 at a higher dose and more detailed biology studies
will be published elsewhere.
REFERENCES
■
(1) Ma, C. X.; Janetka, J. W.; Piwnica-Worms, H. Death by releasing
the breaks: CHK1 inhibitors as cancer therapeutics. Trends Mol. Med.
2011, 17, 88−96.
(2) Do, K.; Doroshow, J. H.; Kummar, S. Wee1 kinase as a target for
cancer therapy. Cell Cycle 2013, 12, 3159−3164.
(3) Parker, L. L.; Piwnica-Worms, H. Inactivation of the p34cdc2-
cyclin B complex by the human WEE1 tyrosine kinase. Science 1992,
257, 1955−1957.
(4) Stumpff, J.; Duncan, T.; Homola, E.; Campbell, S. D.; Su, T. T.
Drosophila Wee1 kinase regulates Cdk1 and mitotic entry during
embryogenesis. Curr. Biol. 2004, 14, 2143−2148.
(5) Vriend, L. E. M.; De Witt Hamer, P. C.; Van Noorden, C. J. F.;
Wuerdinger, T. WEE1 inhibition and genomic instability in cancer.
̈
Biochim. Biophys. Acta 2013, 1836, 227−235.
(6) Iorns, E.; Lord, C. J.; Grigoriadis, A.; McDonald, S.; Fenwick, K.;
MacKay, A.; Mein, C. A.; Natrajan, R.; Savage, K.; Tamber, N.; Reis-
Filho, J. S.; Turner, N. C.; Ashworth, A. Integrated functional, gene
expression and genomic analysis for the identification of cancer targets.
PLoS One 2009, 4, e5120.
(7) Mir, S. E.; De Witt Hamer, P. C.; Krawczyk, P. M.; Balaj, L.;
Claes, A.; Niers, J. M.; Van Tilborg, A. A. G.; Zwinderman, A. H.;
Geerts, D.; Kaspers, G. J. L.; Peter Vandertop, W.; Cloos, J.; Tannous,
B. A.; Wesseling, P.; Aten, J. A.; Noske, D. P.; Van Noorden, C. J. F.;
Wuerdinger, T. In silico analysis of kinase expression identifies wee1 as
̈
a gatekeeper against mitotic catastrophe in glioblastoma. Cancer Cell
2010, 18, 244−257.
(8) Magnussen, G. I.; Holm, R.; Emilsen, E.; Rosnes, A. K. R.;
Slipicevic, A.; Floerenes, V. A. High expression of Wee1 is associated
with poor disease-free survival in malignant melanoma: potential for
targeted therapy. PLoS One 2012, 7, e38254.
(9) Mizuarai, S.; Yamanaka, K.; Itadani, H.; Arai, T.; Nishibata, T.;
Hirai, H.; Kotani, H. Discovery of gene expression-based pharmacody-
namic biomarker for a p53 context-specific anti-tumor drug Wee1
inhibitor. Mol. Cancer 2009, 8, 34.
(10) Palmer, B. D.; Thompson, A. M.; Booth, R. J.; Dobrusin, E. M.;
Kraker, A. J.; Lee, H. H.; Lunney, E. A.; Mitchell, L. H.; Ortwine, D. F.;
Smaill, J. B.; Swan, L. M.; Denny, W. A. 4-Phenylpyrrolo[3,4-
c]carbazole-1,3(2H,6H)-dione inhibitors of the checkpoint kinase
wee1. Structure−activity relationships for chromophore modification
and phenyl ring substitution. J. Med. Chem. 2006, 49, 4896−4911.
(11) Palmer, B. D.; Smaill, J. B.; Rewcastle, G. W.; Dobrusin, E. M.;
Kraker, A.; Moore, C. W.; Steinkampf, R. W.; Denny, W. A.
Structure−activity relationships for 2-anilino-6-phenylpyrido[2,3-d]-
pyrimidin-7(8H)-ones as inhibitors of the cellular checkpoint kinase
Wee1. Bioorg. Med. Chem. Lett. 2005, 15, 1931−1935.
(12) For a recent review, see De Witt Hamer, P. C.; Mir, S. E.;
Noske, D.; Van Noorden, C. J. F.; Wuerdinger, T. WEE1 kinase
̈
targeting combined with dna-damaging cancer therapy catalyzes
mitotic catastrophe. Clin. Cancer Res. 2011, 17, 4200−4207.
(13) Bamba, M.; Furuyama, H.; Niiyama, K.; Sakamoto, T.; Sunami,
S.; Takahashi, K.; Yamamoto, F.; Yoshizumi, T. Bicycloaniline
derivative. WO 2008/15307, December 18, 2008.
(14) Squire, C. J.; Dickson, J. M.; Ivanovic, I.; Baker, E. N. Structure
and inhibition of the human cell cycle checkpoint kinase, wee1a
62
dx.doi.org/10.1021/ml5002745 | ACS Med. Chem. Lett. 2015, 6, 58−62