Investigation of biaryl heterocycles as inhibitors of Wee1 kinase

Article abstract of DOI:10.1016/j.bmcl.2019.04.017

In continuation of our previous research towards the discovery of potent, selective and drug-like Wee1 inhibitors, 2 novel series of biaryl heterocycles were designed, synthesized and evaluated. The new biaryl cores were designed to enable structure?activ

Full text of DOI:10.1016/j.bmcl.2019.04.017

Accepted Manuscript
Investigation of biaryl heterocycles as inhibitors of Wee1 kinase
Anthony Mastracchio, Chunqiu Lai, Maricel Torrent, Kenneth Bromberg, Fritz
G. Buchanan, Debra Ferguson, Velitchka Bontcheva, Eric F. Johnson, Loren
Lasko, David Maag, Nirupama Soni, Alexander R. Shoemaker, Thomas D.
Penning
PII:
S0960-894X(19)30226-4
https://doi.org/10.1016/j.bmcl.2019.04.017
BMCL 26384
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 February 2019
5 April 2019
8 April 2019
Please cite this article as: Mastracchio, A., Lai, C., Torrent, M., Bromberg, K., Buchanan, F.G., Ferguson, D.,
Bontcheva, V., Johnson, E.F., Lasko, L., Maag, D., Soni, N., Shoemaker, A.R., Penning, T.D., Investigation of
biaryl heterocycles as inhibitors of Wee1 kinase, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://
doi.org/10.1016/j.bmcl.2019.04.017
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1
2
Investigation of biaryl heterocycles as inhibitors of Wee1 kinase
3
Anthony Mastracchioa*, Chunqiu Laia, Maricel Torrentb, Kenneth Bromberga, Fritz G. Buchanana,
Debra Fergusona, Velitchka Bontchevaa, Eric F. Johnsona, Loren Laskoa, David Maaga, Nirupama
Sonia, Alexander R. Shoemakera and Thomas D. Penninga
4
5
6
aCancer Research, Discovery, Global Pharmaceutical Research and Development, AbbVie, 1
North Waukegan Road, North Chicago, Illinois 60064
7
8
aMolecular Modeling, Structural Biology, Discovery, Global Pharmaceutical Research and
Development, AbbVie, 1 North Waukegan Road, North Chicago, Illinois 60064
*Corresponding Author: Anthony Mastracchio (anthony.mastracchio@abbvie.com);
Number of figures: 3
9
10
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12
13
14
15
Number of schemes: 3
Number of tables: 1
Key words: Wee1, Cdk1, Cdc2, G1 checkpoint, G2/M checkpoint.
Running title: Investigation of biaryl heterocycles as inhibitors of Wee1 kinase
1

1
2
Wee1 is a key tyrosine kinase that helps control the G2/M checkpoint by preventing entry into
mitosis.1,2 This is of particular interest for cancer therapy since many cancer cells depend more
heavily than do normal cells on a functional G2/M checkpoint for DNA repair.3-5 This suggests
that inhibition of Wee1 could induce premature entry into mitosis of DNA damaged cancer cells
leading to mitotic catastrophe and cell death.6,7 Following on this insight many cancer programs
have focused on the development of selective drug-like Wee1 inhibitors.8–10 AZD1775 (aka MK-
1775, adavosertib), the first selective Wee1 inhibitor reported, is currently in clinical trials for
the treatment of solid tumors. Clinical results for AZD1775 showed on-target efficacy as a single
agent and in combination thus providing additional support for targeting Wee1 in cancer.11–13
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
In a previous account,14 we used a class of Wee1 inhibitors featuring an imino-
dihydropyrimidone (1) that had emerged in the patent literature as a starting point. Then, aided
by molecular modeling, a series of compounds bearing a pyrimidine-based tricyclic scaffold
were design as exemplified by 2 and proved to be potent inhibitors of Wee1 (Figure 1). Our
studies also showed that the fused imidazole ring of 2 occupies a key region within the ATP
binding pocket in Wee1 adjacent to the bicyclic core that was not utilized by 1. Encouraged by
these results we sought to explore a variety of biaryl cores that would 1) possess the key
elements for binding in the active site (i.e. proper hinge binder and pi interactions with phe433)
and 2) accommodate a substituent next to the bicyclic bridge (position C-8 or N-8). This
substituent could in turn be used as a vector for modulation of compound properties and to
further explore this region of the binding pocket. Modeling studies suggested that biaryl system
such as 3 or 4 could fulfill these requirements. Consequently, we sought to explore structure-
2

1
2
3
4
5
6
7
activity relationships for these biaryl cores bearing substituents at C-8 or N-8.15 Building on the
insights we gained during our exploration of tricyclic compounds 2, we selected a bis-Cl aryl
moiety at the C-6 or N-6 position and an arylpiperazine or cyclopropyl tetrahydroisoquinoline in
the amino aryl region as preferred scaffolds to evaluate the various bicyclic cores. Our findings
indicate that both 4-pyridones (3) and 2-pyridones (4) may serve as interesting lead series for
the development of potent and selective Wee1 inhibitors with desirable pharmacokinetic
properties as well as antitumor efficacy in a murine xenograft model.
Previously disclosed work:
Cl
NH
O
N
computer aided
drug design
Ar
O
N
N
N
N
R
N
F
N
N
N
N
H
N
N
H
H
1
2
WO 2008/15307
tricyclic core designed to
take advantage of untapped
1
region by
This work:
Cl
Cl
NH
O
O
computer aided
drug design
Ar
O
N
N
N
N
N
R
1
N
8
N
1
N
8
R
Cl
R
Cl
N
N
N
N
N
H
H
H
H
R1
1
R1
4
3
WO 2008/15307
N
exploration of new biaryl
N
R
heterocycles with vector
exploitable for drug design
R =
or
N
preferred scaffolds for aminoaryl region
8
9
Figure 1. Modification strategies of novel and selective Wee1 inhibitors.
10
11
Schemes 1 & 2 outline the general synthesis of analogs related to biaryls 3 and 4. As seen in
Scheme 1, the known pyrimidine ester 5 was hydrolyzed under standard conditions to give the
3

1
2
3
4
5
6
7
corresponding acid which in turn was treated with thionyl chloride to afford acyl chloride 6.
Next, an in situ generated organocuprate of compound 7 was added to yield ketone 8. At this
point, the desired substituent at R1can be introduced by treatment of 8 with an appropriately
substituted primary amine or aniline followed by condensation/cyclization with DMF−DMA at
elevated temperature. The methyl thioether group of 10 was oxidized by mCPBA, setting the
stage for the final SNAr reaction with the desired aminoaryl substrate to provide analogs of 4-
pyridone 3.
Cl
ZnCl
Cl
Cl
O
O
O
8
a, b
c
N
OEt
N
Cl
N
Cl
S
N
Cl
S
N
Cl
S
N
Cl
6
7
9
Cl
R NH2
d, e
O
N
Cl
O
N
Cl
N
N
N
R
f, g
H
N
11
Cl
Cl
or
N
O
S
N
N
R
10
N
N
Cl
N
N
N
R
H
12
8
9
Scheme 1. Synthesis of 4-pyridone series. Reagents and conditions: (a) 5 molar aqueous NaOH,
THF, 60⁰C, 16 hrs, 89% yield; (b) SOCl2, DMF (cat), 90⁰C, 3 hrs, 99% yield; (c) CuCN, LiCl, THF, –
25⁰C to rt, 79% yield; (d) amine, 60⁰C, IPA, 16 hrs, (for R = Me, 91% yield): (e) DMF-DMA, 150⁰C,
10
11
4

1
2
90 minutes, (for R = Me, 76% yield); (f) m-CPBA, CH2Cl2, 30 minutes, rt; (g) Ar-NH2, TFA, MeCN,
100⁰C, 16 hrs (for R = Me and Ar = tetrahydroisoquinoline analog, two steps 65% yield).
3
4
2-Pyridone analogs of 4 were prepared as outlined in Scheme 2. The synthesis commences with
either commercially available pyrimidine ester 15 or with pyrimidine ester 14, the latter being
obtained through the condensation of ethyl 3-oxopentanoate 12 with methyl
5
6
7
carbamimidothioate 13 and DMF−DMA at elevated temperature. The esters were then
converted to arylamides 16 via a three-step sequence involving saponification, acyl chloride
formation then treatment with 2,6-dichloroaniline. The 2-pyridone ring can then be forged
after treatment of compound 16 with Vilsmeier-Haack reagent 18 in DMF at 60C. For 17b a
methyl group at C-8 was obtained but for 17a an additional molecule of Vilsmeier-Haack
reagent was added at C-8 to yield aldehyde 19. This aldehyde can then act as an ideal functional
handle to introduce various substituents for structure−activity studies near the bridged
pyrimidine region as shown in Scheme 2. Finally the aminoaryl moiety was installed using the
same synthetic sequence we described for the 4-pyridone series.
8
9
10
11
12
13
14
15
5

O
NH
NH2
O
O
N
N
OEt
a
Cl
Cl
O
O
S
OEt
S
S
N
b, c
Me
O
N
N
N
N
13
14
15
H
H
Cl
Cl
S
N
Me
S
N
Me
d
17a
17b
OEt
Me
N
H
Me
H
Me
N +
Me
16
N +
Me
d
commercially
available
Cl
Cl
18
18
Cl
O
Cl
Cl
N
O
O
e, f or g
N
N
N
N
O
N
Cl
or
S
N
Cl
Cl
S
N
19
S
N
20
21
Me
reaction e
Cl
Cl
O
O
N
N
N
N
Cl
Cl
S
N
S
N
22
23
reaction f
OH
F
F
reaction g
Cl
Cl
O
R
O
R
h, i
N
N
N
S
N
N
Cl
N
Cl
N
H
N
NH2
N
26
25
24
1
2
Scheme 2. Synthesis of 2-pyridone series. Reagents and conditions: (a) 13, DMF-DMA, 80⁰C, 10
minutes then methyl carbamimidothioate, reflux, 16 hrs, 52% yield; (b) 1 molar aqueous LiOH,
MeOH, THF, rt, 2 hrs, (for 16, 86% yield; for 15, 74% yield); (c) SOCl2, DMF (cat), dioxane, rt then
2,6-dichloroaniline, 100⁰C, 16 hrs, (for 17a, 40% yield; for 17b, 29% yield); (d) DMF, 60⁰C, 3 hrs,
(for 19, 29% yield, for 20, 43%): (e) Ph3PCH3Br, KOt-Bu, THF, rt, 48 hrs, 62% yield; (f) NaBH4,
CeCl3*7H2O, CH2Cl2/MeOH (3:1), 0⁰C, 30 minutes, 70 % yield; (g) DAST, CH2Cl2, 3 hrs, rt, 61%
3
4
5
6
7
6

1
2
yield; (h) m-CPBA, CH2Cl2, 30 minutes, rt; (i) Ar-NH2, TFA, MeCN, 100⁰C, 16 hrs. (for R = Me, two
steps 81% yield)
3
4
Table 1 shows the structures of the compounds evaluated and their respective affinities
presented as Ki values which was assessed in a routine 6-point TR-FRET binding assay. Activities
in functional antiproliferative cell viability assays (H1299 cell line) and in a pCDK1 ELISA assay
measuring the ability of Wee1 to phosphorylate CDK1 at Tyr 15, both utilizing the NCI-H1299
cell line (human non-small cell lung carcinoma), are also shown. In addition, mouse microsomal
stability data (Cl(mic), L/hr/Kg) are also included in Table 1 for initial assessment of metabolic
clearance.16
5
6
7
8
9
10
11
12
Table 1. Biaryl analogs.17
H1299
EC50
(μM)
pCDK1
EC50
(μM)
CL(mic)
(L/hr/Kg)
Mouse
Ki
(nM)
Compound
Structure
Cl
O
N
N
N
2
1.1
0.3
0.31
0.52
0.060
0.079
38.1
16.2
N
F
N
N
N
H
Cl
Cl
N
O
N
N
N
26
27
Cl
N
N
N
N
O
H
N
N
1.0
0.46
0.022
34.9
Cl
N
H
N
7

Cl
N
O
N
N
N
28
29
30
Cl
1.3
24.4
0.2
1.72
7.65
>10
0.324
2.85
4.02
34.9
118.0
28.9
N
H
N
O
O
Cl
Cl
N
N
N
N
N
N
Cl
Cl
N
H
N
N
N
O
N
N
H
N
OH
Cl
N
O
N
N
N
Cl
31
0.2
0.50
0.246
NA
N
H
N
Cl
O
N
32
33
0.6
0.2
0.33
0.10
0.029
0.011
31.9
24.8
N
Cl
Cl
N
N
N
H
H
Cl
Cl
O
N
N
N
N
N
H
N
N
N
O
34
0.3
0.21
0.019
NA
Cl
N
H
N
8

Cl
Cl
O
N
O
N
35
0.2
0.39
0.031
27.5
N
Cl
N
H
N
N
N
N
36
37
0.5
0.3
0.13
0.08
0.028
0.021
43.6
27.8
N
N
Cl
Cl
N
H
N
N
Cl
O
O
N
N
H
Cl
N
N
N
38
39
0.3
0.6
0.06
0.14
0.025
0.018
19.2
45.2
N
N
Cl
Cl
N
H
N
N
OH
Cl
O
N
F
N
H
F
1
2
3
4
5
6
7
8
Most analogs yielded equal or better binding affinity than the lead molecule 2 suggesting that
1) the proposed cores can retain binding requirements to the protein similar to compound 1
and 2 and that 2) these biaryl systems are likely to participate in productive pi-pi interaction
with Phe 433 as suggested by our docking studies.
Compounds 26-35 explored the effect of substituent variation at N-8 on binding affinity and cell
antiproliferative activity for the 4-pyridone series. Computational modeling of compound 26
9

1
2
predicted that a range of substitutions could be accommodated at N-8 since this vector is
oriented towards an open area between Phe433 located at the base of the ATP binding site,
and Val313 located in the Gly-rich loop (Figure 2).18 The experimental results validated our
computational model and showed that binding affinity remained largely unaffected when
neutral, polar or hydrogen bond donor groups are present (compounds 26-28 & 30-35).
However, when a basic nitrogen was introduced (compound 29) a significant reduction in
binding affinity was observed. One hypothesis for the drop in affinity with amine 29 is that the
amine substituent sits on a large hydrophobic patch with no polar residues to stabilize the net
charge. Exploration of the N-8 substituent did confirm that this vector could be utilized for
modulation of physicochemical properties as a range of cell permeability and microsomal
clearance was obtained with variations of the stereoelectronic nature of the substituent. In
general it was observed that small non-polar alkyl substituents offer the best balance of cell
activity and compound stability. This can be seen with methyl analog 33 and ethyl analog 34
where both compounds compare well with first generation compound 2 in terms of binding
affinity (Ki = 1.1 nM vs 0.3 nM for 33), cell viability (EC50 = 0.31 μM vs 0.10 μM for 33) and in the
PD assay pCDK1 ELISA (EC50 = 0.060 μM vs 0.011 μM for 33). This establishes the 4-pyridone
core as a suitable ring system for productive binding interaction in the active site of Wee1.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
10

1
2
Figure 2. Structure and ligand interaction plot of compound 26 docked into the Wee1 kinase
binding site. The model predicts two key hydrogen bond interactions with the backbone polar
atoms of the hinge (Cys379) and an additional hydrogen bond interaction with the side chain of
gate keeper residue Asn376 . The biaryl core is also predicted to engage in pi-pi interactions
with Phe433.
3
4
5
6
7
8
Given the similar shape and electronic properties between 4-pyridones and 2-pyridones we
decided to evaluate the latter to see if this new ring system could also be used as template for
generation of potent Wee1 inhibitors. We determined that indeed the 2-pyridone analogs
retain good enzymatic potency and cell potency. Both series offered a similar activity profile
and the different chemical structures of the two could be utilized to introduce different yet
complimentary substituents at position 8 of the bicyclic ring system.
9
10
11
12
13
14
15
16
17
Following these results, compound 33 was selected for advanced profiling. The compound
showed desirable PK properties in mouse (iv t1/2 = 2.7 h; iv CL = 1.5 L/h/kg; po AUC = 6.2 μM*h;
po F = 44%) and was thus selected to study its ability to potentiate the efficacy of the DNA-
11

1
2
3
4
5
6
damaging agent irinotecan in BRCA1 deficient, p53 deficient MX-1 TNBC xenograft model. As
shown in Figure 3, the combination of compound 33 with irinotecan achieved 88% tumor
growth inhibition (TGI) when compared to vehicle and 57% TGI when compared to irinotecan
alone on day 43. Moreover, the durability of the combination response was highlighted by 48%
tumor growth delay when compared to irinotecan alone (using 1000 mm3 as an endpoint) and
therefore supports the enhanced efficacy of irinotecan by compound 33 in vivo. 19
3500
Vehicle
3000
Irinotecan (10 mkd, ip, Q4d x4)
2500
33 (25 mkd, po, 2d on, 2d off x4) +
Irinotecan (10 mkd, ip, Q4d x4)
2000
1500
1000
500
0
10
15
20
25
30
35
40
45
50
55
60
65
Irinotecan
Days Post Tumor Cell Inoculation
Compound 33
7
8
Figure 3. Tumor growth inhibition with compound 33 in combination with irinotecan.20 MX-1
brei (homogenized tumor cell suspension) was sub-cutaneously injected into the flank of SCID
mice. After the tumor volume reached ~350 mm3, mice were treated with vehicle (black line),
10 mkd (mg/kg/day) irinotecan Q4Dx4 (closed circles), or 10 mkd irinotecan Q4Dx4 + 25 mkd
compound 33 QD 2 days on 2 day off for 4 cycles (open squares). Dosing began on day 24.
Tumor volumes were measured twice a week.
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11
12
13
14
12

1
2
In summary, we designed and synthesized 3 new series of pyrimidine-based biaryl derivatives
and evaluated their binding affinity and activity in cells. The data provided shows that both 4-
pyridone and 2-pyridone cores are suitable replacements for the tricyclic core and allow for a
new substitution pattern that can be used for evaluating substituents at position C-8 or N-8 of
the bicyclic ring system. Our studies have also identified 4-pyridone 33 as a viable Wee1
inhibitor with improved in vitro activity in cell over benchmark compound 2. This compound
also exhibits good drug-like properties and potentiated the anti-proliferative activity of
irinotecan in vivo when dosed orally in an MX-1 xenograft model. These findings suggest that
pyridinone-based biaryl core such as 3 and 4 may serve as a suitable template for the
development of potent and efficacious Wee1 inhibitors.
3
4
5
6
7
8
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12
13
14
15
16
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18
19
Acknowledgments
We would like to acknowledge the work Nirupama Soni for the generation of the Wee1 TR-FRET
binding assay data.
Disclosures:
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.
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22
23
24
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25
26
27
28
29
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32
33
34
35
36
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43
9.
10.
11.
12.
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14.
15.
14

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2
16.
17.
18.
19.
20.
Mouse microsomal stability was used as a routine assay for translation of clearance into
the efficacy models.
Note that the number of values for all data collected for each compounds were at least
N=2.
The coordinates used for docking were obtained from the Protein Data Bank with the
entry code 1X8B
For evidence of on-target activity see TR-FRET kinome profiling data of compound 33 in
supporting information.
In house tumor efficacy studies showed that Wee1 inhibitor alone, in similar mouse
xenograft tumor studies, shows only modest tumor growth inhibition thus supporting
that Wee1 inhibition synergizes the action of Irinotecan. For an example of such studies
see reference (14).
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15

Proprietary benchmark series
New cores investigated
l
C
l
C
O
l
C
O
O
N
N
N
N
N
N
1
N
8
r
A
8
N
r
A
1
N
l
C
l
C
r
A
N
X
N
N
N
H
H
N
R1
H
R1
X = H, F or Cl
New cores for the development of potent
and efficacious Wee1 inhibitors