Design and Optimization Leading to an Orally Active TTK Protein Kinase Inhibitor with Robust Single Agent Efficacy

Article abstract of DOI:10.1021/acs.jmedchem.8b01869

Triple negative breast cancer (TNBC) is an aggressive disease with high relapse rates and few treatment options. Outlined in previous publications, we identified a series of potent, dual TTK/CLK2 inhibitors with strong efficacy in TNBC xenograft models. P

Full text of DOI:10.1021/acs.jmedchem.8b01869

Subscriber access provided by IDAHO STATE UNIV
Article
Design and optimization leading to an orally active TTK
protein kinase inhibitor with robust single agent efficacy
Jennifer R. Riggs, Jan Elsner, Dan Cashion, Dale Robinson, Lida Tehrani, Mark Nagy, Kimberly Fultz,
Rama Krishna Narla, Xiaohui Peng, Tam Tran, Ashutosh Kulkarni, Sogole Bahmanyar, Kevin Condroski,
Barbra Pagarigan, Gustavo Fenalti, Laurie LeBrun, Katerina Leftheris, Dan Zhu, and John Boylan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01869 • Publication Date (Web): 18 Apr 2019
Downloaded from http://pubs.acs.org on April 18, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Design and optimization leading to an orally active
TTK protein kinase inhibitor with robust single
agent efficacy
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Jennifer R. Riggs*, Jan Elsner, Dan Cashion, Dale Robinson, Lida Tehrani, Mark Nagy, Kimberly
E. Fultz, Rama Krishna Narla, Xiaohui Peng, Tam Tran, Ashutosh Kulkarni, Sogole Bahmanyar,
Kevin Condroski, Barbra Pagarigan, Gustavo Fenalti, Laurie LeBrun, Katerina Leftheris, Dan Zhu
and John F. Boylan
Celgene Corporation, 10300 Campus Point Drive, Suite 100, San Diego, California 92121.
Keywords: TTK, Mps1, monopolar spindle 1, spindle assembly checkpoint, mitotic exit, TNBC,
oncology, oral bioavailability
Abstract.
Triple negative breast cancer (TNBC) is an aggressive disease with high relapse rates and
few treatment options. Outlined in previous publications, we identified a series of a potent, dual
TTK/CLK2 inhibitors with strong efficacy in TNBC xenograft models. PK properties and kinome
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 41
1
2
3
4
5
6
7
8
9
selectivity were optimized resulting in the identification of a new series of potent, selective and
orally bioavailable TTK inhibitors. We describe here the structure-activity relationship (SAR) of
the 2,4-disubstituted-7H-pyrrolo[2,3-d]pyrimidine series leading to significant single agent
efficacy in a TNBC xenograft model without body weight loss. The design effort evolving an iv-
dosed TTK/CLK2 inhibitor to an orally bioavailable TTK inhibitor is described.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
2

Page 3 of 41
Journal of Medicinal Chemistry
1
2
3
4
Introduction.
5
6
7
8
In our previous work, we reported a dual inhibitor of TTK protein kinase (monopolar
spindle 1, Mps1), and CDC2-like kinase (CLK2), identified from a TNBC phenotypic screen.1-2
This series was optimized to provide an inhibitor with significant single agent efficacy upon once
weekly iv dosing and ultimately lead to the nomination of CC-671 for IND-enabling studies.
Although once weekly administration allows for an iv dosing paradigm, the flexibility of an oral
dosing regimen is an important clinical consideration. As TNBC remains a high unmet medical
need with few treatment options, we used CC-671 as a starting point to develop an oral, TTK
selective inhibitor.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cellular mechanism of action studies confirmed inhibition of both TTK and CLK2 by CC-
671.2 Significant single agent in vivo efficacy of this compound was attributed to increased
apoptosis from accelerated mitotic exit, mechanistically linked to TTK inhibition. TTK is a dual
serine/threonine kinase that controls the progression of cells through mitosis by regulating the
spindle assembly checkpoint (SAC).3-4 TTK has been shown to be a promising target for TNBC.
TTK knockout by siRNA induced apoptosis in TNBC cell lines by accelerating mitotic
progression.5-6 Elevated TTK expression in TNBC tumors correlated with a poor patient
prognosis.6 Due to the high interest in the target, a number of potent TTK inhibitors have been
described with five reportedly entering Phase I clinical trials.5, 7-18
Here we describe the identification of orally efficacious TTK inhibitors derived from CC-
671 (1, Figure 1) and the SAR optimization leading to good oral bioavailability. Upon oral
administration, lead compounds were well tolerated and produced significant single agent efficacy
in a TNBC xenograft study with good PK/PD correlation. This efficacy and tolerability profile was
superior to the standard of care in TNBC, Docetaxel.
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 41
1
2
3
4
5
6
7
8
9
N
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
N
N
N
H
N
N
H
H
O
1
Figure 1. Structure of iv starting point CC-671
Results and Discussion
Scheme 1 details the synthesis of 2,4-disubstituted-7H-pyrrolo[2,3-d]pyrimidines. SEM-
protection of 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine provided intermediate 2. Alcohol
displacement of the C-4 chloride afforded intermediates 3a-h. Pd-catalyzed C-N cross-coupling
was utilized to install the C-2 substituent. Final analogs (4-20) were obtained after facile
deprotection using either TBAF or TFA followed by ammonium hydroxide-assisted cleavage of
the hemiaminal intermediate in 2 steps.
Scheme 1. Synthesis of compounds 4-20
ACS Paragon Plus Environment
4

Page 5 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
Cl
R1
Cl
R1
C4
N
c
N
a
b
N
N
N
N
N
Cl
N
N
R2
Cl
N
N
Cl
N
C2
H
SEM
SEM
7
SEM
8
9
2
3a-3h
4-20 SEM
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
d, e
or f
R1
N
N
N
R2
H
4-20
Reagents and conditions: (a) SEM-Cl, NaH, DMF, 0-22 °C; (b) R1-alcohol, 1,4-dioxane,
NaOtBu, 70-90 °C; (c) R2-amine, Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane, 110 °C; (d) TFA,
DCM; (e) NH4OH, MeOH; (f) TBAF, THF, 50 °C.
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 41
1
2
3
4
5
6
7
8
9
To identify orally bioavailable TTK inhibitors, our strategy was to retain TTK inhibitory
activity and TNBC sensitivity of CC-671 (1) while focusing on improving the physicochemical
properties of analogs in an effort to obtain good oral exposure. The antiproliferative potency in
CAL-51, a TNBC cell line, was utilized to evaluate analogs and drive the SAR. Compound 1 has
poor oral exposure (0.9 %F, Supporting information, Table 1), which could be attributed in part to
the high molecular weight, polar surface area and cLogP of 1 (Table 1). Utilizing 1 as a starting
point, the C-5 substituent was truncated, removing the acid-labile benzoxazole group. In addition,
the amide NH at C-2 was methylated, removing one H-bond donor, providing 4 (Table 1).
Although the potency of 4 is reduced by 10-fold in the TNBC cell assay, the improved
physicochemical properties of 4 provide a more efficient starting point for optimization of potency
and oral exposure.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Modification of 1 leads to improved physicochemical properties
N
O
O
O
N
H
O
O
N
N
N
N
N
H
N
N
N
N
H
H
H
H
O
O
4
1
CAL-51 potency
Analog
Mol Wt
PSAb
cLogPb
IC50 (M)a
1
4
0.028
0.497
513
395
127
92
5.0
3.9
amean reported, SEM available in Supporting information, Table 2,
bpipeline pilot calculation
ACS Paragon Plus Environment
6

Page 7 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The SAR at the C-4 position were explored with the goal of improving potency and kinase
selectivity while maintaining drug-like properties (Table 2). A subset of kinases were used as a
representation of overall selectivity against a larger kinase panel (Supporting information, Table
3). Replacement of the cyclopentyl as in oxetane 5 or isopropyl 6 resulted in a loss of CAL-51
potency. Cyclohexyl 7 and THP 8 improved cellular potency as compared to the cyclopentyl 4,
despite equivalent TTK enzyme potency. Considerable potency gains were obtained with
cyclohexanols 9 and 10, but at the expense of kinase selectivity, which could be contributing to
the observed cellular efficacy. Notably, gem-dimethyl substituted THP 11 affords considerable
kinase selectivity compared to cyclohexanols 9 and 10 and favorable potency. The gem-dimethyl
substituted THP at C-4 was held constant to explore the C-6 para-position SAR with the goal of
optimizing potency (Table 3). In parallel, we held the C-4 methyl cyclohexanol constant to explore
the C-6 ortho-position SAR to optimize selectivity (Table 4). Overall, the TTK enzyme potency
correlated with the CAL-51 proliferation assay, although the dynamic range of the enzyme assay
for these compounds was far narrower. For this reason, antiproliferative activity in CAL-51 was
used as the primary potency measure to drive the SAR, together with overall kinase selectivity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. C-4 position SAR
C4
O
O
N
N
N
N
N
H
H
O
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 41
1
2
3
4
5
6
7
8
TTK enzyme
CAL-51 potency
# kinases
Analog
C4
Mol Wtb
PSAb
IC50 (M)a
IC50 (M)a
>80% inhc
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
5
6
0.0013
0.497
1.959
1.265
395
383
369
92
101
92
15/46
12/46
11/46
O
0.0072
0.0032
0.0013
7
0.190
409
92
10/46
0.198d
0.069
0.044
411
440
440
101
101
112
12/46
25/46
21/46
O
8
9
0.001
0.0006
0.0007
OH
OH
10
11
0.0010
0.131
440
101
8/46
O
amean reported, SEM available in Supporting information, Table 2,
bpipeline pilot calculation, csingle point assessment at 3 M, d n=1
We then explored para-substitution (R3) on the C-2 aryl group (Table 3). Based on
structural data previously reported, many substituents should be well tolerated in this solvent-
exposed region.1 The oral exposure and selectivity of 11 were high, serving as a starting point for
ACS Paragon Plus Environment
8

Page 9 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
further SAR with the goal of improving potency and properties. Morpholine amide 12 resulted in
a potency loss. Deletion of the amide carbonyl providing direct-linked morpholine (13) resulted in
a potency gain as compared to both 11 and 12. Both hydrogen bond donors and acceptors were
tolerated at this position (13 and 14) and resulted in a modest potency increase as compared to
amide 11. The additional hydrogen bond donors/acceptors and increased molecular weight of 14
and 15 lead to a decrease in oral exposure as compared to 11. Oxetane 16 had increased oral
exposure as compared to 14 and 15, and a small increase in potency compared to 11. All analogs
made in this C-2 exploration maintained reasonable kinase selectivity similar to 11, likely due to
the C-4 gem-dimethyl THP.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. C-2 position SAR
O
O
R3
N
N
N
N
H
H
O
CAL-51
potency
Rat PO PK
AUC (Mh)c >80% inhd
# kinases
Analog
11
R3
Mol Wt
PSAb
IC50 (M)a
O
0.131
440
101
30e
8/46
N
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 41
1
2
3
4
O
5
6
7
12
0.406
481
101
ND
10/46
N
O
8
9
N
13
14
0.053
0.071
454
482
93
14.7f
11.0f
7/46
8/46
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
N
104
HO
N
15
16
0.037
0.084
511
509
125
97
9.5e
24f
10/46
7/46
N
HO
O
N
N
O
amean reported, SEM available in Supporting information, Table 2, pipeline pilot
b
calculation, cdose 10 mg/kg, dsingle point assessment at 3 M, d n=1, eformulated in
f
CMCT (0.5% CMC/0.25%tween-80), formulated in 5%TPGS (D-α-tocopheryl
polyethylene glycol 1000 succinate), 10% captisol, 50 mM citrate buffer
While the gem-dimethyl THP at C-4 afforded improved kinase selectivity, this was often
at the expense of potency (Table 2, 11 compared to 10). Compound 10 had attractive potency, but
poor selectivity. Using available structural information, we knew that TTK would tolerate larger
substituents at the ortho-position of C-2 and this was a potential selectivity handle.1 To optimize
for selectivity, we kept the methyl cyclohexanol constant and explored a variety of ortho-
substituents on C-2 (Table 4). Replacing the methoxy (10) with ethoxy (17) resulted in an
improvement in potency and kinase selectivity. The trifluoroethyl 18 further improved potency
and enhanced kinase selectivity. Stereoselective addition of a methyl substituent to 18 resulted in
ACS Paragon Plus Environment
10

Page 11 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a highly selective and potent analog 19. Additional potency was gained by substituting C-5 of 19
with a Cl (20). These changes had a relatively minor effect on oral exposure despite increased
molecular weight.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. C-2 and C-5 position SAR
HO
O
O
N
C5
N
N
N
N
H
H
O
R4
CAL-51
potency
Rat PO PK
# kinases
Analog
R4
C5
Mol Wtb PSAb
AUC (Mh)c
>80% inhd
IC50 (M)a
0.044
10
17
Me
Et
H
H
440
453
112
112
21.8e
ND
21/46
15/46
0.026
18
19
H
H
0.015
0.024
508
521
112
112
16.1f
30e
11/46
8/46
F3C
F3C
F3C
10/46
20
Cl
0.010
556
112
26e
10/258
amean reported, SEM available in Supporting information, Table 2, pipeline pilot
b
calculation, cdose 10 mg/kg, dsingle point assessment at 3 M, eformulated in CMCT
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 41
1
2
3
4
5
6
(0.5% CMC/0.25%tween-80), fformulated in 5%TPGS (D-α-tocopheryl polyethylene
glycol 1000 succinate), 10% captisol, 50 mM citrate buffer
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compounds 15, 16, and 20 showed nanomolar potency against the TTK enzyme, reduction of the
cellular substrate (p-TTK), and strong antiproliferative activity (Table 5). Compound 20 exhibited
the most potent activity across multiple assays. The correlation of the cellular biomarker p-TTK
and proliferation in the CAL-51 cell line was good for the series (Figure 2).
Table 5. TTK enzyme, CAL-51 p-TTK and proliferation data for 15, 16, and 20
TTK enzyme
CAL-51 p-TTK CAL-51 potency
# kinases
Analog
IC50 (M)a
IC50 (M)a
IC50 (M)a
0.037
>80% inhb
0.016
15
16
0.0013
0.0025
10/46
0.044
0.009
0.084
7/46
10/46
20
0.0007
0.010
10/258
b
amean reported, SEM available in Supporting information, Table 2, single point
assessment at 3 M
ACS Paragon Plus Environment
12

Page 13 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. CAL-51 proliferation compared to CAL-51 p-TTK
To further characterize the compounds effect on the TTK pathway in cells, inhibition of
phosphorylation of KNL1, a direct TTK substrate, in addition to TTK autophosphorylation, was
measured as a cellular pharmacodynamic marker. Western blot analysis was conducted using
CAL-51 cells treated with increasing concentrations of compound 20 for 1 h (Figure 3). Compound
20 potently inhibits phosphorylation of both TTK substrates with a p-TTK IC50 of 11 nM,
(approximately 5-fold more potent than our starting point 1, IC50 = 57 nM) and p-KNL1 IC50 of
30 nM.
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 41
1
2
3
4
Figure 3. TTK substrate phosphorylation inhibition post treatment with compound 20 as measured
5
6
by Western blot
7
8
9
A co-crystal structure of 20 with TTK was obtained at 2.4 Å resolution (Figure 4).19 The
crystal structure reveals a ligand-dependent, induced-fit conformation of the TTK catalytic kinase
domain (Supporting information, Figure 1).20 The pyrrolopyrimidine core interacts with the ATP
binding pocket and forms three hydrogen bonds to the hinge region. The pyrrole NH (N-7)
hydrogen bonds to the backbone carbonyl of Glu603 and the aminopyrimidine forms two hydrogen
bond interactions with Gly605. The methyl cyclohexanol at C-4 binds in the ribose pocket, and
the chlorine at C-5 binds deeper in the pocket in proximity of catalytic Lys553 and gate-keeper
residue Met602. Both substituents at C-4 and C-5 are stabilized by extensive hydrophobic and
van der Waals interactions in the TTK binding site (Supporting information, Figure 1).21 The C-2
aniline substituent binds in the solvent pocket, and the ortho- (R)-(1,1,1-trifluoropropan-2-yl)oxy
substituent forms stabilizing polar and hydrophobic interactions in a pocket formed by G-loop
residues Glu541, Lys529, and Ile531, along with hinge residue Cys604.22 This unique
arrangement of residues allow for this bulky substituent to be tolerated in the hinge region of TTK
and contribute to broad kinome selectivity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
14

Page 15 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Co-crystal structure of 20 bound in the ATP pocket of TTK. Residues within 4.0Å of
the ligand are shown (PDB ID 6N6O). The activation loop (residues 670 – 684) are disordered
and not resolved in the crystal structure (some residues eliminated for clarity).
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 41
1
2
3
4
5
6
7
8
Selectivity of compound 20 was further assessed against 258 kinases. Only 9 kinases other
than TTK were strongly inhibited (Supporting information, Table 4).23 Four kinases had IC50
values within 100-fold of TTK; LRRK2 G2019S, LRRK2, JNK1 and 2 (Table 5).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 5. 10/259 kinases inhibited > 80% after 3 M treatment with compound 20 (IC50 (M))
LRRK2
TTK
LRRK2 JNK1
JNK2 DYRK3 JNK3 TSSK1 CLK2
Ltk
G2019S
0.001
0.0007
0.001
0.032
0.059
0.104
0.107
0.217
0.261
0.567
Our SAR optimization resulted in the identification of multiple analogs with good potency,
selectivity and oral exposure in rat. Mouse PK studies were used to select the top candidates for in
vivo xenograft studies. Three representative compounds are detailed in Table 6 along with their
oral PK in rat and mouse. Compounds 15, 16, and 20 afforded good oral exposure in mouse with
Cmax values between 5 and 10 µM and AUC values from 17 to 35 µM·h when dosed at 30 mg/kg.
Compound 20 had an oral bioavailability of 60% in mouse (Supporting information, Table 5).
Table 6. PK properties of selected analogs
Rat PO PKb,c,d
Mouse PO PKb,c,e
ACS Paragon Plus Environment
16

Page 17 of 41
Journal of Medicinal Chemistry
1
2
3
4
Human S9
Cmax
Cmax
5
6
7
8
Analog Met. Stabilitya
(M)
AUC (M·h)
(M)
AUC (M·h)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
15
16
20
79
85
65
2.53
4.43f
4.35
9.52
24f
9.86
11.8
5.2
17.6
35.3
22.5
26.3
a% remaining at 60 min, bmean reported, SEM available in Supporting information, Table
2, cformulated as a solution in CMCT (0.5% CMC/0.25%tween-80), dose 10 mg/kg,
d
edose 30 mg/kg in SCID mice, formulated in 5%TPGS (D-α-tocopheryl polyethylene
f
glycol 1000 succinate), 10% captisol, 50 mM citrate buffer
Compound 20 exhibited promising cellular potency, selectivity, and acceptable oral rat and
mouse PK. In vivo efficacy assessment was performed using a CAL-51 mouse xenograft model.
The compound was administered every seven days (QW) for 16 days measuring tumor growth
compared to vehicle control and body weight loss (Figure 6). Compound 20 demonstrated a dose-
dependent increase in efficacy from 30-120 mg/kg QD QW. With a tumor growth inhibition (TGI)
of 59%, only the 30 mg/kg QW dose failed to produce significant TGI as defined by (vehicle -
treated / vehicle) x 100%) of > 60% (Figure 6a). Cmax and AUC values were dose proportional
through 90 mg/kg. We sought to explore whether efficacy was predominantly Cmax or AUC-driven.
One dose group of BID QW was added to assess the effect of sustained AUC over the IC50.
Interestingly, the 30 mg/kg BID QW dose group produced 84% TGI, while the equivalent overall
dose, 60 mg/kg QD QW, produced a lower 65% TGI. This data suggests the efficacy is
predominantly AUC-driven. All dosing groups were well tolerated without appreciable BWL.
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 41
1
2
3
4
5
6
7
8
9
Although LRRK is also likely inhibited at these doses, LRRK is not expressed in this cell line and
therefore not contributing to efficacy. The potential pulmonary toxicity highlighted in the literature
from LRRK inhibition requires additional in vivo assessment.24 BID and QD QW oral dosing of
compound 20 were generally more efficacious and better tolerated than Docetaxel (BWL in
Supporting information, Figure 2). Docetaxel was administered at 5 mg/kg, Q4D, the maximum
tolerated dose leading to 20% BWL. Suspended dosing of Docetaxel was necessary to allow for
recovery and provided a modest TGI of 61%. In the CAL-51 xenograft model, compound 20 has
a superior efficacy and tolerability profile compared to Docetaxel, the current standard of care for
TNBC.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1750
Vehicle (QW)
120 mg/kg, QW
90 mg/kg, QW
1500
1250
1000
750
500
250
0
60 mg/kg, QW
30 mg/kg, QW
30 mg/kg, BID-QW
Docetaxel (5 mg/kg, IV, Q4D)
Docetaxel (Q4D)
QW
10 12 14 16 18 20 22 24 26 28 30 32
Days After Tumor Cell Inoculation
ACS Paragon Plus Environment
18

Page 19 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 6. CAL-51 tumor xenograft studies with BID and QD once weekly dosing of compound
20. On day 31, all treatment groups were significantly (*p<0.001, one way ANNOVA) different
compared to vehicle treated group.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
800
700
600
500
400
300
200
100
0
10
9
8
7
6
5
4
3
2
1
0
pHH3
Tumor
Plasma
***
***
****
****
****
****
****
****
Vehicle1 h 3 h 6 h 24 h48 h
1 h 3 h 6 h 24 h48 h
10 mg/kg
30 mg/kg
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 41
1
2
3
4
5
6
7
8
9
25000
20000
15000
10000
5000
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
****
****
****
*
0
.
e
h
h
h
h
h
h
h
h
h
h
cl
1
3
6
1
3
6
i
24
48
24
48
h
Ve
30 mg/kg
10 mg/kg
Figure 7. Single dose study with compound 20 measuring PK, pHH3, and cleaved caspase in
CAL-51 tumors (*p<0.05, *** p<0.001, ****p<0.0001 one way ANNOVA).
Compound 20 was also assessed in a single dose biomarker study measuring the mitotic
cell cycle marker, phosphohistone H3 (pHH3), and tumor cell apoptosis marker, cleaved caspase-
3. A single dose of 10 or 30 mg/kg in mice caused considerable reduction in pHH3 after 1 h,
maintained through 48 h, for the 30 mg/kg dose. Significant cleaved caspase-3 induction occurred
at both 10 and 30 mg/kg as compared to vehicle (159% and 250%, respectively). The maximal
ACS Paragon Plus Environment
20

Page 21 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
amount of caspase-3 induction was achieved at 48 h post dose. This data confirms proximal on-
target biomarker reduction (pHH3) and apoptosis.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Conclusion
In summary, we have detailed the optimization and SAR of the 2,4-disubstituted-7H-
pyrrolo[2,3-d]pyrimidine series of oral TTK inhibitors. Starting from our lead iv preclinical
candidate, we successfully identified potent, selective and orally bioavailable TTK inhibitors.
Truncation of the iv lead 1 provided starting points with improved physicochemical properties.
SAR optimization, starting from compound 4, lead to improved selectivity and potency with good
oral exposure in the series. Interestingly, the overall physiochemical property space occupied by
compound 20 is not significantly different from compound 1, but the oral exposure of compound
20 and similar analogs is far superior. These compounds occupying “unideal property space” while
maintaining good oral exposure support the notion that strict adherence to the rule of five (Ro5) is
not always necessary.25 Compound 20 showed significant reduction in pHH3 and induction of
cleaved caspase-3 in an in vivo tumor biomarker study. Apoptotic induction produced significant
efficacy in CAL-51 cell lines in vitro and in vivo in the xenograft model. Compound 20 represents
a highly competitive, oral TTK inhibitor with strong single agent efficacy in a TNBC xenograft
model.
Experimental Section
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 41
1
2
3
4
5
6
7
8
9
Chemistry – Compounds were named using ChemDraw Ultra. All materials were obtained from
commercial sources unless otherwise noted and used without further purification. Chromatography
solvents were HPLC grade. Air-sensitive reactions were performed under a positive pressure of an
inert nitrogen atmosphere. 1H NMR spectra were obtained on 300, 400, or 500 MHz
spectrometers. Chemical shifts are relative to tetramethylsilane (TMS) as the internal standard or
relative to a residual solvent peak. Chemical shifts () are reported in ppm and coupling constants
(J) are given in Hz. LCMS analysis was performed on a PE Sciex ESI MS or Agilent 1100 MS.
Semi-preparative reverse phase HPLC was performed on a Shimadzu system utilizing a
Phenomenex 15 micron C18 column (250 x 10 mm). Preparative reverse phase HPLC was
performed on a Shimadzu system equipped with a Phenomenex 15 micron C18 column (250 x 50
mm). Thin Layer Chromatography (TLC) analysis was performed on Whatman thin layer plates.
Compounds were analyzed for purity by either: (A) Gradient (0-75% acetonitrile + 0.1% formic
acid in water + 0.1% formic, over 7 min, followed by 75% acetonitrile + 0.1% formic acid for 2
min); Flow Rate 1 mL/min, Column Phenomenex Gemini-NX 5u C18 110A (50 x 4.60mm); (B)
Gradient (0-75% acetonitrile + 0.1% formic acid in water + 0.1% formic, over 20 min, followed
by 75% acetonitrile + 0.1% formic acid for 5 min); Flow Rate 1 mL/min, Column Phenomenex
Gemini-NX 5u C18 110A (250 x 4.60mm). The purity of all final tested compounds was
determined to be >95% by HPLC using an Agilent 1100 system with a reverse phase C18 column
and diode array detector. Reported yields are unoptimized. Elemental analysis was performed at
Robertson Microlit Laboratories, Ledgewood, New Jersey.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthetic procedures for all intermediates 2, 3a-3h, and 4-20 SEM are included in the
Supporting information.
ACS Paragon Plus Environment
22

Page 23 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
General Procedure for the Synthesis of Compounds 4-20. The SEM protected
pyrrolo[2,3-d]pyrimidine intermediate was treated with TFA (5-500 equiv.) in DCM (1-5
times the volume of TFA). The mixture was stirred at room temperature for 1-24 h. The
solvent was removed and the residue was stirred with aqueous ammonium hydroxide (10-
30% solution, 2-100 equiv.) in methanol or 1,4-dioxane (1-5 times the volume of ammonium
hydroxide solution) with or without DCM (0.5-2.5 times the volume of methanol) at room
temperature to 60 °C for 1-24 h. In cases where the compound contained aqueous acid
sensitive functionalities, the residue was treated with an excess of methanolic ammonia (2-
7 N) prior to addition of the aqueous ammonium hydroxide. The final product was either
collected by filtration or purified using standard techniques.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4-((4-(Cyclopentyloxy)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N,N-
1
dimethylbenzamide (4). Yield 0.109 g, 0.28 mmol, 69%, HPLC purity >95%. H NMR (400
MHz, DMSO-d6)  11.50-11.59 (m, 1H), 8.59 (d, J=8.31 Hz, 1H), 7.60 (s, 1H), 7.04 (br d, J=13.94
Hz, 3H), 6.26-6.32 (m, 1H), 5.55-5.63 (m, 1H), 3.93 (s, 3H), 2.99 (s, 6H), 1.97-2.10 (m, 2H), 1.70-
1.86 (m, 4H), 1.54-1.69 (m, 2H). MS (ESI) m/z 396.2 [M+1]+.
3-Methoxy-N,N-dimethyl-4-((4-(oxetan-3-yloxy)-7H-pyrrolo[2,3-d]pyrimidin-2-
yl)amino)benzamide (5). To a solution of 3-methoxy-N,N-dimethyl-4-((4-(oxetan-3-yloxy)-7-
((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)benzamide (5 SEM)
(0.240 g, 0.467 mmol) in THF (2.3 ml) was added TBAF (2.3 ml, 2.3 mmol). The mixture was
placed under nitrogen and heated to 50 °C for 18 h. The volatiles were removed by rotary
evaporation. The residue was purified by semi-preparative HPLC to obtain the title compound
(0.027 g, 0.068 mmol, 15% yield, HPLC purity > 96%). 1H NMR (400 MHz, DMSO-d6) δ 0.87 -
0.99 (m, 2 H) 1.22 - 1.38 (m, 1 H) 1.47 - 1.63 (m, 1 H) 2.90 - 3.06 (m, 4 H) 3.10 - 3.22 (m, 1 H)
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 41
1
2
3
4
5
6
3.24 - 3.45 (m, 2 H) 3.91 (s, 2 H) 4.65 (dd, J=7.81, 5.08 Hz, 1 H) 4.95 (t, J=7.23 Hz, 1 H) 5.69 -
5.81 (m, 1 H) 6.36 (d, J=3.12 Hz, 1 H) 7.01 - 7.12 (m, 2 H) 7.62 (s, 1 H) 8.46 (d, J=8.59 Hz, 1 H)
7
8
9
MS (ESI) m/z 384.1 [M+1]+.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4-((4-Isopropoxy-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N,N-
1
dimethylbenzamide (6). Yield 0.11 g, 0.288 mmol, 62%, HPLC purity > 95%. H NMR (400
MHz, DMSO-d6) δ 11.46 - 11.62 (m, 1 H), 8.58 (d, J=8.31 Hz, 1 H), 7.52 - 7.64 (m, 1 H), 6.96 -
7.12 (m, 3 H), 6.30 (dd, J=3.42, 1.96 Hz, 1 H), 5.42 - 5.55 (m, 1 H), 3.93 (s, 3 H), 2.99 (s, 5 H),
2.94 (s, 1 H), 1.38 (d, J=6.11 Hz, 6 H). MS (ESI) m/z 370.2 [M+1]+.
4-((4-(Cyclohexyloxy)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N,N-
dimethylbenzamide (7). Yield 0.054 g, 0.13 mmol, 23% over two steps, HPLC purity > 97%. 1H
NMR (400 MHz, DMSO-d6) δ 11.48-11.55 (m, 1H), 8.52-8.57 (m, 1H), 7.58-7.63 (m, 1H), 7.05-
7.08 (m, 1H), 6.99-7.05 (m, 2H), 6.28-6.33 (m, 1H), 5.18-5.29 (m, 1H), 3.92 (s, 3H), 2.99 (s, 6H),
2.00-2.11 (m, 2H), 1.73-1.83 (m, 2H), 1.28-1.63 (m, 6H). MS (ESI) m/z 410.2 [M+1]+.
3-Methoxy-N,N-dimethyl-4-((4-((tetrahydro-2H-pyran-4-yl)oxy)-7H-pyrrolo[2,3-
d]pyrimidin-2-yl)amino)benzamide (8). Yield 0.092 g, 0.225mmol, 35%, HPLC purity > 98%.
1H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 8.55-8.53 (d, J=8.4, 1H), 7.66 (s, 1H), 7.07-7.03
(m, 3H),6.34-6.33 (d, J=2, 1H), 5.43-5.41 (m, 1H), 3.92-3.90 (d, J=9.6, 5H), 3.58-3.53 (m, 2H),
2.99 (s, 6H), 2.11-2.08 (m, 2H), 1.75-1.70 (m, 2H). MS (ESI) m/z 412.2 [M+1]+.
4-((4-(((1r,4r)-4-Hydroxy-4-methylcyclohexyl)oxy)-7H-pyrrolo[2,3-d]pyrimidin-2-
yl)amino)-3-methoxy-N,N-dimethylbenzamide (9). Yield 0.046 g, 0.105 mmol, 22% over 2
steps, HPLC purity > 99%. 1H NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 8.55 (d, J=8.31 Hz,
1H), 7.61 (s, 1H), 6.99-7.09 (m, 3H), 6.31 (dd, J=1.96, 3.42 Hz, 1H), 5.30-5.37 (m, 1H), 4.24 (s,
ACS Paragon Plus Environment
24

Page 25 of 41
Journal of Medicinal Chemistry
1
2
3
4
1H), 3.92 (s, 3H), 2.99 (s, 6H), 1.95-2.06 (m, 2H), 1.61-1.76 (m, 4H), 1.44-1.55 (m, 2H), 1.18 (s,
5
6
3H). MS (ESI) m/z 440.2 [M+1]+.
7
8
9
4-((4-(((1s,4s)-4-Hydroxy-4-methylcyclohexyl)oxy)-7H-pyrrolo[2,3-d]pyrimidin-2-
yl)amino)-3-methoxy-N,N-dimethylbenzamide (10). Yield 0.171 g, 0.39 mmol, 59%, HPLC
purity >98%. 1H NMR (400 MHz, DMSO-d6) δ 11.53 (s, 1H), 8.55 (d, J = 8.4 Hz, 1H), 7.60 (s,
1H), 7.06-7.01 (m, 3H), 6.31-6.30 (m, 1H), 5.19-5.15 (m, 1H), 4.21 (s, 1H), 3.92 (s, 3H), 2.99 (s,
6H), 1.89-1.84 (m, 4H), 1.67-1.64 (m, 2H), 1.52-1.45 (m, 2H), 1.16 (s, 3H). MS (ESI) m/z 440.3
[M+1]+.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(S)-4-(4-(2,2-Dimethyltetrahydro-2H-pyran-4-yloxy)-7H-pyrrolo[2,3-d]pyrimidin-2-
ylamino)-3-methoxy-N,N-dimethylbenzamide (11). Yield after chiral separation 0.82 g, 0.187
mmol, 20%, HPLC purity > 99 %, ee >95 %. 1H NMR (400 MHz, CD3OD) δ 8.65 (d, J = 8.4 Hz,
1H), 7.08 (d, J = 2.0 Hz, 1H), 7.04 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 6.94 (d, J = 3.6 Hz, 1H), 6.35 (d,
J = 3.6 Hz, 1H), 5.70-5.63 (m, 1H), 3.98 (s, 3H), 3.94-3.82 (m, 2H), 3.10 (s, 6H), 2.17-2.12 (m,
2H), 1.81-1.74 (m, 1H), 1.70-1.64 (m, 1H), 1.36 (s, 3H), 1.31 (s, 3H). MS (ESI) m/z 440.2 [M+1]+.
Anal.Calcd for (%) C23H29N5O4-0.4H2O: C, 61.84; H, 6.72; N, 15.68. Found: C, 61.81; H, 6.84;
N, 15.62.
(S)-(4-((4-((2,2-Dimethyltetrahydro-2H-pyran-4-yl)oxy)-7H-pyrrolo[2,3-d]pyrimidin-2-
yl)amino)-3-methoxyphenyl)(morpholino)methanone (12). To a vial was added (S)-4-((4-
((2,2-dimethyltetrahydro-2H-pyran-4-yl)oxy)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-
methoxybenzoic acid (0.100 g, 0.242 mmol), morpholine (0.027 ml, 0.315 mmol), HATU (0.111
g, 0.291 mmol), DMF (1.5 ml), and DIEA (0.169 ml, 0.970 mmol). The mixture was stirred at
room temperature for 2 hours. The mixture was diluted with DMSO (1 ml) and purified by semi-
preparative HPLC to obtain the title compound (0.073 g, 0.152 mmol, 63% yield, HPLC purity >
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 41
1
2
3
4
5
6
7
8
9
99%). 1H NMR (500 MHz, DMSO-d6) δ 11.50 - 11.58 (m, 1H), 8.53 (d, J = 8.20 Hz, 1H), 7.67 (s,
1H), 7.07 (dd, J = 2.68, 14.97 Hz, 2H), 7.01 (dd, J = 1.58, 8.20 Hz, 1H), 6.32 (d, J = 3.47 Hz, 1H),
5.54 - 5.63 (m, 1H), 3.94 (s, 3H), 3.78 - 3.86 (m, 1H), 3.69 - 3.76 (m, 1H), 3.62 (br. s., 4H), 3.54
(br. s., 3H), 2.03 - 2.12 (m, 2H), 1.58 - 1.69 (m, 1H), 1.50 - 1.56 (m, 1H), 1.28 (s, 3H), 1.24 (s,
3H). MS (ESI) m/z 482.2 [M+1]+.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(S)-4-((2,2-Dimethyltetrahydro-2H-pyran-4-yl)oxy)-N-(2-methoxy-4-morpholinophenyl)-
7H-pyrrolo[2,3-d]pyrimidin-2-amine (13). Yield 0.058 g, 0.127 mmol, 79%, HPLC purity
>99%. 1H NMR (500 MHz, DMSO-d6) δ 11.32 (s, 1 H), 8.01 (d, J=8.83 Hz, 1 H), 7.37 (s, 1 H),
6.94 (dd, J=3.47, 2.21 Hz, 1 H), 6.66 (d, J=2.52 Hz, 1 H), 6.48 (dd, J=8.83, 2.84 Hz, 1 H), 6.24
(dd, J=3.47, 1.89 Hz, 1 H), 5.42 - 5.55 (m, 1 H), 3.85 (s, 2 H), 3.74 - 3.78 (m, 3 H), 3.65 - 3.81 (m,
5 H), 3.05 - 3.12 (m, 3 H), 2.03 (dd, J=12.30, 4.41 Hz, 1 H), 1.55 - 1.63 (m, 1 H), 1.50 (dd, J=12.61,
10.40 Hz, 1 H), 1.23 (d, J=4.73 Hz, 4 H). MS (ESI) m/z 454.2 [M+1]+.
(S)-1-(4-((4-((2,2-dimethyltetrahydro-2H-pyran-4-yl)oxy)-7H-pyrrolo[2,3-d]pyrimidin-2-
yl)amino)-3-methoxyphenyl)-4-methylpiperidin-4-ol (14). Yield 0.075 g, 0.16 mmol, 43% over
1
two steps, HPLC purity >95%. H NMR (400 MHz, DMSO-d6) δ 11.30 (br s, 1H), 7.93 (d, J=8.80
Hz, 1H), 7.34 (s, 1H), 6.89-6.94 (m, 1H), 6.62 (d, J=2.20 Hz, 1H), 6.47 (dd, J=2.08, 8.68 Hz, 1H),
6.22 (dd, J=1.90, 3.36 Hz, 1H), 5.44-5.52 (m, 1H), 4.25 (s, 1H), 3.75-3.85 (m, 4H), 3.61-3.75 (m,
1H), 3.17-3.27 (m, 2H), 3.02-3.17 (m, 2H), 2.02 (br dd, J=3.55, 11.98 Hz, 2H), 1.45-1.64 (m, 6H),
1.21 (d, J=3.79 Hz, 6H), 1.13-1.18 (m, 3H). MS (ESI) m/z 481.9 [M+1]+.
(S)-1-(4-(4-((4-((2,2-Dimethyltetrahydro-2H-pyran-4-yl)oxy)-7H-pyrrolo[2,3-d]pyrimidin-2-
yl)amino)-3-methoxyphenyl)piperazin-1-yl)-2-hydroxyethanone (15). To a solution of 2-
hydroxyacetic acid (0.672 g, 8.84 mmol) in DMF (20 mL) was added HATU (3.7 g, 9.72 mmol),
ACS Paragon Plus Environment
26

Page 27 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
DIEA (4.63 mL, 26.5 mmol) and (S)-4-((2,2-dimethyltetrahydro-2H-pyran-4-yl)oxy)-N-(2-
methoxy-4-(piperazin-1-yl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-amine (4 g, 8.84 mmol)
sequentially. The mixture was stirred at room temperature 4 h. After the reaction was complete, as
monitored by LCMS, the mixture was diluted with ethyl acetate, washed with saturated aqueous
sodium chloride and dried over anhydrous sodium sulfate. Concentrated and purified by semi-
preparative HPLC to give the title compound (1.3 g, 2.55 mmol, 29% yield, HPLC purity > 99%).
1H NMR (400 MHz DMSO-d6) δ 11.3-11.4 (m, 1H), 7.9-8.1 (m, 1H), 7.3-7.4 (m, 1H), 6.9-7.0 (m,
1H), 6.6-6.7 (m, 1H), 6.5-6.6 (m, 1H), 6.2-6.3 (m, 1H), 5.4-5.6 (m, 1H), 4.6-4.7 (m, 1H), 4.1-4.2
(m, 2H), 3.8-3.9 (m, 3H), 3.7-3.8 (m, 1H), 3.6-3.7 (m, 3H), 3.5-3.6 (m, 2H), 3.09 (br d, 4H, J=3.2
Hz), 2.0-2.1 (m, 2H), 1.4-1.7 (m, 2H), 1.1-1.3 (m, 6H). MS (ESI) m/z 511.5 [M+1]+. Anal. Calcd
for (%) C26H34N6O5-0.2H20: C, 61.16; H, 6.71; N, 16.46; Found: C, 60.7; H, 6.43; N, 16.01.
4-[(4S)-2,2-Dimethyltetrahydropyran-4-yl]oxy-N-[2-methoxy-4-[4-(oxetan-3-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
yl)piperazin-1-yl]phenyl]-7H-pyrrolo[2,3-d]pyrimidin-2-amine (16). Yield 3.78 g, 7.4
1
mmol, 74%, HPLC purity > 99%. H NMR (400 MHz, CDCl3) δ 9.38 (s, 1H), 8.30 (d, J =
8.8 Hz, 1H), 7.22 (s, 1H), 6.71-6.70 (m, 1H), 6.62 (d, J = 2.4 Hz, 1H), 6.40-6.39 (m, 1H),
5.68-5.60 (m, 1H), 4.76-4.69 (m, 4H), 3.99-3.83 (m, 5H), 3.61 (t, J = 6.4 Hz, 1H), 3.21 (t, J
= 4.8 Hz, 4H), 2.56 (t, J = 5.2 Hz, 4H), 2.15-2.09 (m, 2H), 1.75-1.70 (m, 2H), 1.36 (d, J =
6.8 Hz, 6H). MS (ESI) 510.4 m/z [M+1]+. Anal.Calcd for (%) C27H36N6O4-0.47H2O: C,
62.72; H, 7.20; N, 16.25. Found: C, 62.7; H, 6.92; N, 16.31.
3-Ethoxy-4-((4-(((1s,4s)-4-hydroxy-4-methylcyclohexyl)oxy)-7H-pyrrolo[2,3-d]pyrimidin-2-
yl)amino)-N,N-dimethylbenzamide (17). Yield 0.126 g, 0.28 mmol, 34%, HPLC purity >99%.
1
H NMR (400 MHz, DMSO-d6) δ 11.46 - 11.58 (m, 1 H), 8.48 - 8.61 (m, 1 H), 7.54 - 7.62 (m, 1
H), 6.93 - 7.10 (m, 3 H), 6.28 - 6.35 (m, 1 H), 5.06 - 5.20 (m, 1 H), 4.21 - 4.25 (m, 1 H), 4.12 -
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 41
1
2
3
4
4.21 (m, 2 H), 2.89 - 3.05 (m, 6 H), 1.79 - 1.94 (m, 4 H), 1.59 - 1.72 (m, 2 H), 1.45 - 1.54 (m, 2
5
6
H), 1.33 - 1.45 (m, 3 H), 1.09 - 1.21 (m, 3 H). MS (ESI) m/z 454.3 [M+1]+.
7
8
9
4-((4-(((1s,4s)-4-Hydroxy-4-methylcyclohexyl)oxy)-7H-pyrrolo[2,3-d]pyrimidin-2-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
yl)amino)-N,N-dimethyl-3-(2,2,2-trifluoroethoxy)benzamide (18). Yield 0.076 g, 0.15 mmol,
1
52%, HPLC purity >99%. H NMR (500 MHz, DMSO-d ) δ 11.42 - 11.63 (m, 1 H), 8.47 - 8.57
6
(m, 1 H), 7.50 - 7.63 (m, 1 H), 7.22 - 7.31 (m, 1 H), 7.09 - 7.15 (m, 1 H), 6.98 - 7.06 (m, 1 H), 6.29
- 6.41 (m, 1 H), 5.05 - 5.20 (m, 1 H), 4.84 - 4.98 (m, 2 H), 4.15 - 4.24 (m, 1 H), 2.99 (s, 6 H), 1.80
- 1.97 (m, 4 H), 1.60 - 1.71 (m, 2 H), 1.40 - 1.52 (m, 2 H), 1.17 (s, 3 H). MS (ESI) m/z 508.2
[M+1]+.
4-((4-(((1s,4s)-4-Hydroxy-4-methylcyclohexyl)oxy)-7H-pyrrolo[2,3-d]pyrimidin-2-
yl)amino)-N,N-dimethyl-3-(((R)-1,1,1-trifluoropropan-2-yl)oxy)benzamide (19). Yield 0.073
1
g, 0.14 mmol, 39% yield, HPLC purity >99%. H NMR (500 MHz, DMSO-d6) δ 11.41 - 11.74
(m, 1 H), 8.45 - 8.71 (m, 1 H), 7.47 - 7.55 (m, 1 H), 7.27 - 7.32 (m, 1 H), 7.08 - 7.17 (m, 1 H), 6.98
- 7.07 (m, 1 H), 6.27 - 6.35 (m, 1 H), 5.27 - 5.42 (m, 1 H), 5.03 - 5.18 (m, 1 H), 4.13 - 4.26 (m, 1
H), 2.98 (s, 6 H), 1.81 - 1.97 (m, 4 H), 1.58 - 1.75 (m, 2 H), 1.39 - 1.52 (m, 5 H), 1.04 - 1.21 (m,
3 H). MS (ESI) m/z 522.2 [M+1]+. Anal. Calcd for (%) C25H30F3N5O4: C, 57.57; H, 5.80; N,
13.43. Found: C, 57.28; H, 5.77; N, 13.37.
4-[[5-Chloro-4-(4-hydroxy-4-methyl-cyclohexoxy)-7H-pyrrolo[2,3-d]pyrimidin-2-
yl]amino]-N,N-dimethyl-3-[(1R)-2,2,2-trifluoro-1-methyl-ethoxy]benzamide
(20).
1
Yield 3.44 g, 6.2 mmol, 47%, HPLC purity >99%. H NMR (400 MHz DMSO-d6) δ 11.74
(s, 1H), 8.42 (d, J = 8.4 Hz, 1H), 7.62 (s, 1H), 7.32 (s, 1H), 7.16-7.11 (m, 2H), 5.36-5.33
(m, 1H), 5.13 (s, 1H), 4.23 (s, 1H), 2.98 (s, 6H), 1.86-1.87 (m, 4 H), 1.69-1.66 (m, 2H),
ACS Paragon Plus Environment
28

Page 29 of 41
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1.48-1.43 (m, 5H), 1.16 (s, 3H). MS (ESI) m/z 556.3 [M+1]+. Anal.Calcd for (%)
C25H29N5O4-0.5H2O: C, 53.68; H, 5.29; N, 12.52. Found: C, 53.49; H, 5.29; N, 12.24.
In Vitro kinase selectivity profiling. The kinase selectivity profile of compound 20 was assessed
at 3 μM against 255 kinases at Invitrogen using one of the following protocols: Z ′-Lyte®
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
protocol, ADAPTA® or the Lantha Binding Assay. The TTK binding affinity was measured at
Invitrogen using the LanthaScreen ™ Eu Kinase Binding Assays. The LanthaScreen ™ Eu Kinase
Binding Assays are based on the binding and displacement of a proprietary, Alexa Fluor® 647-
labeled, ATP-competitive kinase inhibitor scaffold (kinase tracer).
Immunoblot analyses. Cells were lysed in RIPA buffer (Thermos Scientific, Catalog # 89900).
The protein concentration of the lysates was determined by the Bio-Rad protein assay (Bio-Rad,
Catalog # 500-0001). Thirty μg of protein from each lysate were loaded onto NuPAGE Novex 4-
12% Bis-Tris gels (Invitrogen #EA0375) and run in MES SDS running buffer. Protein was then
transferred and blotted with primary antibodies. Primary antibodies used were as follows:
Phospho-TTK T686 (custom made rabbit monoclonal antibody), p-KNL1 T943 (custom-made
polyclonal antibody). Goat anti-rabbit AlexaFluor680 (Invitrogen Cat#A21076) and goat anti-
mouse IRDye800 (Rockland Immunochemicals Cat#610-132-121) were then used to detect the
protein bands. The membranes were scanned using the Odyssey Infrared Imaging System (LI-
COR Biosciences).
Cellular proliferation assays. CAL-51 was purchased from DSMZ. The cell line was maintained
in growth media as recommended by the vendors. The cells were incubated with compound for 3
days. CellTiter-Glo reagent was added to each well and the luminescence was measured on the
ACS Paragon Plus Environment
29