The Discovery of a Dual TTK Protein Kinase/CDC2-Like Kinase (CLK2) Inhibitor for the Treatment of Triple Negative Breast Cancer Initiated from a Phenotypic Screen

Article abstract of DOI:10.1021/acs.jmedchem.7b01223

Triple negative breast cancer (TNBC) remains a serious unmet medical need with discouragingly high relapse rates. We report here the synthesis and structure-activity relationship (SAR) of a novel series of 2,4,5-trisubstituted-7H-pyrrolo[2,3-d]pyrimidines with potent activity against TNBC tumor cell lines. These compounds were discovered from a TNBC phenotypic screen and possess a unique dual inhibition profile targeting TTK (mitotic exit) and CLK2 (mRNA splicing). Design and optimization, driven with a TNBC tumor cell assay, identified potent and selective compounds with favorable in vitro and in vivo activity profiles and good iv PK properties. This cell-based driven SAR produced compounds with strong single agent in vivo efficacy in multiple TNBC xenograft models without significant body weight loss. These data supported the nomination of CC-671 into IND-enabling studies as a single agent TNBC therapy.

Full text of DOI:10.1021/acs.jmedchem.7b01223

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Article
The discovery of a dual TTK protein kinase/ CDC2-
like kinase (CLK2) inhibitor for the treatment of triple
negative breast cancer initiated from a phenotypic screen
Jennifer R. Riggs, Mark Nagy, Jan Elsner, Paul Erdman, Dan Cashion, Dale Robinson, Roy
Harris, Dehua Huang, Lida Tehrani, Gordafaried Deyanat-Yazdi, Rama Krishna Narla, Xiaohui
Peng, Tam Tran, Leo Barnes, Terra Miller, Jason Katz, Yang Tang, Ming Chen, Mehran F.
Moghaddam, Sogole Bahmanyar, Barbra Pagarigan, Silvia L Delker, Laurie LeBrun, Philip Paul
Chamberlain, Andrew Calabrese, Stacie S. Canan, Katerina Leftheris, Dan Zhu, and John F. Boylan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01223 • Publication Date (Web): 09 Oct 2017
Downloaded from http://pubs.acs.org on October 9, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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The discovery of a dual TTK protein kinase/ CDC2-
like kinase (CLK2) inhibitor for the treatment of
triple negative breast cancer initiated from a
phenotypic screen
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Jennifer R. Riggs*, Mark Nagy, Jan Elsner, Paul Erdman, Dan Cashion, Dale Robinson, Roy
Harris, Dehua Huang, Lida Tehrani, Gordafaried Deyanat-Yazdi, Rama Krishna Narla, Xiaohui
Peng, Tam Tran, Leo Barnes, Terra Miller, Jason Katz, Yang Tang, Ming Chen, Mehran F.
Moghaddam, Sogole Bahmanyar, Barbra Pagarigan, Silvia Delker, Laurie LeBrun, Philip P.
Chamberlain, Andrew Calabrese, Stacie S. Canan, Katerina Leftheris, Dan Zhu and John F.
Boylan
Celgene Corporation, 10300 Campus Pointe Drive, Suite 100, San Diego, California 92121.
Keywords: TTK, Mps1, monopolar spindle 1, CLK2, CDC like kinase 2, spindle assembly
checkpoint, mitotic exit, mRNA splicing, TNBC, oncology
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Abstract.
Triple negative breast cancer (TNBC) remains a serious unmet medical need with
discouragingly high relapse rates. We report here the synthesis and structure-activity relationship
(SAR) of a novel series of 2,4,5-trisubstituted-7H-pyrrolo[2,3-d]pyrimidines with potent activity
against TNBC tumor cell lines. These compounds were discovered from a TNBC phenotypic
screen and possess a unique dual inhibition profile targeting TTK (mitotic exit) and CLK2
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(mRNA splicing). Design and optimization, driven with a TNBC tumor cell assay, identified
potent and selective compounds with favorable in vitro and in vivo activity profiles and good iv
PK properties. This cell-based driven SAR produced compounds with strong single agent in vivo
efficacy in multiple TNBC xenograft models without significant body weight loss. These data
supported the nomination of CC-671 into IND-enabling studies as a single agent TNBC therapy.
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Introduction.
Phenotypic based drug discovery has proven successful in the generation of novel, first-
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in-class drugs as compared to target-based approaches. Although triple negative breast cancer
TNBC) patients have a reasonable response rate to current standard of care chemotherapy, the
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(
relapse rates are high and treatment options remain limited. Because of the heterogeneous nature
of TNBC, attempts have been made to further categorize TNBC into as many as 10 different
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subtypes. Because of this heterogeneity and lack of well-defined clinical targets, we used a
phenotypic approach to identify a novel treatment for TNBC. By targeting preferential induction
of apoptosis in TNBC cell lines over luminal breast cancer cell lines, we identified a series of
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2,4,5-trisubstituted-7H-pyrrolo[2,3-d]pyrimidines with the desired phenotypic profile.
During the phenotypic SAR chemistry campaign, we established two kinases that were
selectively inhibited by our molecules and likely responsible for the cellular activity: monopolar
spindle 1 (Mps1), also known as TTK protein kinase, and CDC2-like kinase (CLK2). TTK is a
dual serine/threonine kinase that regulates the spindle assembly checkpoint (SAC), thereby
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controlling the progression of cells through mitosis. TTK siRNA knockout accelerates mitotic
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progression and induces cell death in TNBC cell lines.
TTK also phosphorylates key
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oncogenic proteins such as p53, CHK2, and MDM2, critical to DNA damage repair.
TTK
has been shown in the literature to be a promising TNBC targeting opportunity. TTK mRNA and
protein are overexpressed in a TNBC cohort of breast cancer primary tumors, and high TTK
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expression in TNBC patients is linked to a poor patient prognosis. These data provided
sufficient rationale to gain the interest of a number of pharmaceutical companies and academic
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, 13-23
institutions leading to the publication of multiple, potent TTK inhibitors.
Thus far, four
TTK inhibitors have reportedly entered Phase I clinical trials. BAY 1161909 (clinical trials.gov
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ID: NCT02138812), BAY 1217389 (clinical trials.gov ID: NCT02366949), S 81694 (EudraCT
no: 2014-002023-10), and CFI-402257 (clinicaltrials.gov ID: NCT02792465) (Figure 1). While
all are potent TTK inhibitors, they differ in several aspects including kinase selectivity profiles,
route of administration, pre-clinical efficacy model type, ability to produce single agent efficacy
with tolerated doses, dosing paradigm, PK, and likely mean resonance time on the target protein.
The novel kinase profile, the long time on target which allows for intermittent dosing, and strong
single agent efficacy provide possible competitive advantages for our series of TTK inhibitors.
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Figure 1. Structures of TTK inhibitors reportedly in Phase I clinical trials. The structure of S
81694 has not been published.
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SR specific kinases (SRPKs) and CLK2 phosphorylate serine and arginine-rich proteins
SR) within the spliceosome and modulate mRNA splicing activity and consequently protein
(
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synthesis.
The high level of cancer associated mutations or overexpression across the
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splicing process highlights the growth and survival advantage obtained when tumor cells co-opt
this pathway. Downregulation of CLK2 alters splicing patterns and inhibits breast cancer growth
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in vitro and in vivo. Consistent with these data, small molecule CLK2 inhibition alters
expression of SR proteins changing the splicing pattern in mRNAs involved in growth and
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survival leading to cancer cell apoptosis.
A dual TTK/CLK2 inhibitor would produce mitotic acceleration and RNA splicing
modification. Given the rapid adaptation and growth requirements of a tumor cell, this unique
kinase combination profile should prove impactful in a number of cancer subtypes including
high unmet medical need indications such as TNBC. Here we describe the optimization of a
phenotypic hit (1, Figure 2) and SAR exploration leading to favorable in vivo iv PK profiles and
the selection of 4-((4-(cyclopentyloxy)-5-(2-methylbenzo[d]oxazol-6-yl)-7H-pyrrolo[2,3-
5
d]pyrimidin-2-yl)amino)-3-methoxy-N-methylbenzamide (CC-671) for IND-enabling studies.
Figure 2. Structure of phenotypic hit 1
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Chemistry
The synthesis of 2,4,5-trisubstituted-7H-pyrrolo[2,3-d]pyrimidines is outlined in Scheme
and 2. Commercially available 2,4-dichloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine was
1
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converted to the SEM-protected intermediate 2 using 2-(trimethylsilyl)ethoxymethyl chloride
(Scheme 1). The C-4 substituent was introduced by displacement of the chloride with appropriate
alcohols to give 3a-3c. Suzuki-Mukiyama cross coupling reaction of iodide 3 with aryl boronic
acids or esters resulted in disubstituted pyrrolopyrimidines 4a-4u. Alternatively, the C-5
substituent could be installed first from intermediate 2. Suzuki-Mukiyama coupling with
appropriate aryl boronic esters or acids affords 5. Chloride displacement at C-4 with alkyl and
cycloalkyl alcohols provides disubstituted pyrrolopyrimidines 6a-6g.
Scheme 1. Synthesis of disubstituted pyrimidine intermediates 4a-4u and 6a-6g
Cl
Cl
N
I
I
O
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R1
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b
c
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Cl
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Cl
H
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N
SEM
SEM
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2
3a-3c
4a-4u
c
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N
O
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Cl
N
b
R2
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N
Cl
Cl
N
SEM
SEM
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Reagents and conditions: (a) SEM-Cl, NaH, DMF, 0-22 °C; (b) R -alcohol, 1, 4-dioxane,
2
NaOtBu, 70-90 °C; (c) R -boronate ester or acid, sodium carbonate in water, PdCl (dppf)-
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CH Cl or Pd(PPh ) Cl 1, 4-dioxane, 70-90 °C.
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The C-2 substituent was installed using Pd-catalyzed Buchwald coupling to afford 7-44
SEM (Scheme 2). Final analogs (7-44) were obtained after deprotection using either TBAF or a
-step process of TFA followed by NH OH-assisted cleavage of the hemiaminal intermediate.
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Scheme 2. Synthesis of 2,4,5-trisubstituted-7H-pyrrolo[2,3-d]pyrimidines 7-44
Reagents and conditions: (a) R -amine, Pd (dba) Xanphos, Cs CO , 1,4-dioxane, MW 150
3
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3,
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°
C; (b) TFA, DCM; (c) NH OH, MeOH; (d) TBAF, THF, 50 °C.
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Results and Discussion
Our desired profile for TNBC therapy from this phenotypic approach was to retain TNBC
sensitivity while sparing luminal cancer cell lines to assure a lack of general cytotoxicity. With
this in mind, two cellular assays, Cal-51, a TNBC cell line, and BT-474, a luminal breast cancer
cell line were utilized to evaluate analogs. Potency in the Cal-51 assay was primarily used to
drive the SAR. While the phenotypic activity of hit 1 exhibited the desired phenotypic profile, it
was metabolically unstable in liver S9 fractions. Glucuronidation and sulfonation of the phenol
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were the major metabolites. Our first goal was to replace the metabolically labile phenol at C-5
of 1. The contribution of the phenol moiety to the metabolic instability of 1 is further illustrated
by the significant improvement in S9 stability of the unsubstituted phenyl 7 (Table 1). The
dramatic loss in potency of 7 suggests the phenol in 1 is making a key interaction, contributing to
the observed cellular effect. We further examined the hydrogen bonding requirements and the
positioning of the phenol. The m-phenol 8 suffered from a slight reduction in potency as
compared to the p-phenol 1. Replacement of the phenol with an unsubstituted 4 or 3-pyridyl (9
and 10), afforded analogs with similar potency to their respective phenol counterparts (9 vs 1 and
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10 vs 8), suggesting the hydrogen bond donor is not necessary, but a hydrogen bond acceptor is
contributing significantly to potency. Five-membered heterocycles were tolerated as in pyrazole
12, but isomeric pyrazole 11 resulted in a loss of potency suggesting the correct orientation of
the H-bond acceptor is critical. Interestingly, capping the phenol to give the p-methoxy 13
resulted in a loss in potency. Extending the alcohol by adding a methylene as in 14 and 15 was
tolerated, and the proper positioning of the H-bond acceptor (15) resulted in improved potency.
The p-carboxamide, 16, was equipotent to the original hit 1.
In an effort to replace the carboxamide, substituting the aryl ring at the para position with
5-membered heterocycles was explored. Again, the positioning of the H-bond acceptor in the
pyrazole, imidazole, and triazole compounds (17-19) impacted the potency. Fused 6,5-
heterocycles 20-23 gave mixed results, as expected from the H-bond acceptor SAR presented
thus far. Depending on the position of the nitrogen H-bond acceptor, analogs either maintained
potency (20, 23), or lost potency (21, 22). Based on the overall profile of potency in Cal-51,
selectivity over the luminal line BT-474, and high S9 stability, the 2-methyl benzoxazole 23 was
held constant at C-5 in the exploration of C-4 and C-2.
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Table 1. C-5 position potency and metabolic stability SAR
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Cal-51 potency BT-474 potency
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Rat S9
Met. Stability
Analog
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C5
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IC (ꢀM)
IC (ꢀM)
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OH
0.013 ± 0.006
0.496 ± 0.136
3.32 ± 2.28
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86 ± 9
c
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0.017 ± 0.010
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59 ± 8
53 ± 5
36 ± 3
76 ± 7
18 ± 1
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0.460 ± 0.153
0.009 ± 0.007
0.491 ± 0.087
0.089 ± 0.035
10.0 ± 0.0
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10.0 ± 0.0
NH
N
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13
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15
0.030 ± 0.017
0.011 ± 0.010
9.99 ± 0.020
0.739 ± 0.207
54 ± 3
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0.061 ± 0.058
0.008 ± 0.007
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0.017 ± 0.010
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0.158 ± 0.016
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86 ± 10
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6.97 ± 2.77
a
b
c
mean± SEM, % remaining at 60 min, not determined,
d
n=2
Our exploration at C-4 revealed a general tolerance for a variety of cycloalkyl and
oxygen-containing functional groups for Cal-51 potency. We first explored contraction of the
cyclopentyl ring resulting in equipotent cyclobutyl 24 and cyclopropyl 25. Small alkyl
substituents such as isopropyl (26), hydroxy-ethyl (27) or methoxy-ethyl (28) also maintained
Cal-51 potency although a general erosion of selectivity was seen in BT-474 with the smaller
cyclopropyl and alkyl substituents. The free OH at C-4, 29, caused a complete loss in potency.
Introduction of heteroatoms into the cyclic C-4 substituent generally led to a slight improvement
in Cal-51 potency (30 vs 24 and 31 vs 23). The 6-membered THP and cyclohexanols 32-34
generally led to an increase in potency as compared to the cyclopentyl 23. Overall, the C-4
position was tolerant of various groups with respect to potency and reasonable rat S9 stability
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was maintained (data not shown). We felt this position could be used to improve properties, such
as lowering lipophilicity. Considering the potency, selectivity against BT-474, and synthetic
ease, we chose the cyclopentyl at C-4 to hold constant for SAR studies at C-2.
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. C-4 position potency SAR
Cal-51 potency
BT-474 potency
Analog
23
C4
a
IC (ꢀM)
50
a
IC (ꢀM)
50
0.060 ± 0.028
0.047 ± 0.022
0.031 ± 0.010
0.040 ± 0.012
0.056 ± 0.029
0.147 ± 0.146
10.0 ± 0.0
6.97 ± 2.77
10.0 ± 0.0
2
2
2
2
2
4
5
6
7
8
1.35 ± 0.260
0.946 ± 0.385
0.694 ± 0.118
3.76 ± 4.51
10.0 ± 0.0
29
30
H
0.008 ± 0.006
0.017 ± 0.008
0.243 ± 0.025
3.76 ± 0.982
31
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9
3
2
3
0.020 ± 0.011
0.015 ± 0.005
10.0 ± 0.0
3
0.893 ± 0.517
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
3
4
0.008 ± 0.004
10.0 ± 0.0
a
mean± SEM
On the C-2 aryl group, we extensively explored the para substituents (R4) while holding
the ortho-methoxy constant (R5). A subset of these analogs are shown in Table 3. Removal of
the R4 substituent (35) resulted in a loss of potency as compared to the methyl carboxamide 23.
The dimethyl tertiary carboxamide 36 maintained potency. Cyclic tertiary amides such as
piperazine 37 and morpholine 38 were explored with mixed results. Piperazine 37 maintained
good Cal-51 potency while the corresponding morpholine 38 was ~6 fold less potent. Secondary
amides (oxetane 39 and alcohols 40-42), explored to decrease lipophilicity and increase
solubility, generally maintained potency.
SAR at R5 of the C-2 aryl group was also explored. Hydrogen substitution at this position
led to an erosion of kinase selectivity (data not shown) therefore small changes were assessed.
While replacement of the methoxy with a methyl (43) was not well tolerated with a ~6 fold loss
in potency, the corresponding chloro analog (44) provided an equipotent analog to 23. Extending
the alkyl chain, such as ethoxy at R5, resulted in a loss of potency (data not shown).
Table 3. C-2 position potency SAR
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N
O
O
R4
N
N
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
H
R5
Cal-51 potency
a
BT-474 potency
Analog
23
R4
R5
a
IC (ꢀM)
50
IC (ꢀM)
5
0
OMe
OMe
OMe
0.060 ± 0.028
0.771 ± 0.088
0.113 ± 0.079
6.97 ± 2.77
10.0 ± 0.0
8.31 ± 2.36
3
5
H
36
37
OMe
OMe
0.037 ± 0.019
0.351 ± 0.134
3.21 ± 1.57
6.39 ± 1.46
3
8
3
4
4
9
0
1
OMe
OMe
OMe
OMe
Me
0.123 ± 0.059
0.077 ± 0.034
0.061 ± 0.058
0.109 ± 0.027
0.349 ± 0.171
0.067 ± 0.022
10.0 ± 0.0
10.0 ± 0.0
10.0 ± 0.0
42
6.09 ± 2.69
9.61 ± 0.733
9.91 ± 0.237
4
4
3
4
Cl
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a
mean± SEM
With multiple analogs in hand that met the desired TNBC potency, selectivity against
BT-474, and metabolic stability, the iv PK properties of a number of compounds were assessed.
The results of both rat and mouse iv PK are provided in Table 4. Comparing analog 23 to analog
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
1
6, the clearance in both rat and mouse are lower for the C-5 methyl benzoxazole (23) vs methyl
amide (16), leading to a longer MRT. Compounds containing C-4 alkyl and cycloalkyl
substituents 23, 25, 26, 31, 32, 34 have slow to moderate clearance in rat, but THP 32 and
methylcyclohexanol 34 suffer from rapid clearance in mouse. Substitution of the amide at C-2
with the solubilizing hydroxy-ethyl group (40) showed increased clearance as compared to 23 in
both rat and mouse. The oxetane 39 displayed low clearance in both rat and mouse resulting in a
longer MRT.
Table 4. PK properties of selected analogs
b,c
b,d,e
h
Rat IV PK
Mouse IV PK
Rat S9
Met.
Stability
Human S9
Met.
f
g
h
f
Analog
16
a
a
CL
Vss
MRT
CL
MRT
Stability
i
i
i
87 ± 10
98 ± 6
63 ± 8
69 ± 6
62 ± 1
75 ± 7
76 ± 2
91 ± 8
79 ± 13
91 ± 11
75 ± 9
78 ± 4
62 ± 3
70 ± 4
65 ± 1
79 ± 7
61.3
4.0
1.1
63.1
0.36
j
j
2
2
2
3
3
3
3
3
5
6
1
2
4
9
9.30 ± 1.31 2.3 ± 0.22
4.1 ±0.3
8.48
10.4
7.71
19.1
67.6
52.2
14.3
5.2
i
i
i
11.4
1.5
2.5
3.1
4.8
33.9 ±5.4
7.2 ±1.2
3.5 ±0.3
i
i
i
27.4
1.6
1.0
0.73
1.8
i
i
i
22.6
3.8
2.9
17.6 ±3.4
13.0 ± 1.8
2.9±1.2
2.9 ± 1.5
4.7 ±0.8
1.0
3.7 ± 0.8
1.4
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
a
0
80 ± 1
90 ± 3
50.8 ± 9.8
3.9 ± 0.6
1.3 ± 0.1
31.6
0.59
b
c
%
remaining at 60 min formulated as a solution in 15% DMA, 50% PEG, 35% D5W, dose
d
e
f
g
2
mg/kg, dose 10 mg/kg in SCID mice, SD not available with composite PK, mL/min/kg,
h
i
j
L/kg, h, SD not available n=2, dose 5 mg/kg
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Although the project was initiated on a phenotypic premise and TNBC Cal-51/Luminal
BT-474 cell assays drove the SAR exploration, target identification was critical in supporting
potential clinical advancement. In particular, a target biomarker would be required to guide dose
and schedule during development. The pyrrolo[2,3-d]pyrimidine core along with the C-2 amine
arranged a network of hydrogen bonds in a donor-acceptor-donor pattern resembling the hinge-
binding motif of known kinase inhibitors. We therefore profiled project compounds in a
2
8
commercial panel of >250 kinases. Based on the comparison of results for TNBC selective
and non-selective compounds, a small set of candidate targets emerged. For example when 23
was assessed in a panel of 255 kinases, only 7 were inhibited by more than 80% at 3 ꢀM
(
Supplemental Table 1). IC values for the 7 kinases are shown in Table 5, with only TTK and
50
CLK2 having single digit nanomolar potency.
Table 5. The IC (ꢀM) of the 7 kinases that were > 80% inhibition at 3 ꢀM of 23.
5
0
TTK
.005
CLK2
0.006
DYRK3 DYRK1A
0.099 0.104
PHKG
0.136
DYRK1B
0.157
CLK1
0.30
0
HCT-116 cell lysates were treated with compound 23 at 3 µM for 1 hour and tested in the
ActivX KiNative™ profiling assay. Only 4 kinases showed cellular binding of 75% or more
including CLK2, CAMKK2, PIP4K22, and JNK (TTK not offered, full data in supplemental
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table 2). The IC values for the 4 kinases were determined in the HCT-116 cell line (Table 6).
5
0
CLK2 was determined to be the most potent with an IC value of 15 nM. From the 7 kinases
50
that were identified in the biochemical kinase screen, only one additional kinase (PHKgamma-1)
was monitored in the HCT-116 KiNative™ kinase panel, and PhKgamma-1 only showed 26%
binding in the 3 µM screen.
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 6. Kinase binding in the HCT-116 Cell Line
Kinase
CLK2
JNK1,2,3
CAMKK2
PIP4K2C
IC (ꢀM)
5
0
0.015
0.68
1.10
1.30
Inhibition of TTK and CLK2 phosphorylation substrates was monitored to confirm the
cellular inhibitory mechanism of action. Cal-51 cells were treated with compound 23 for 1 hr and
analyzed by Western blot using antibodies against the autophosphorylation of TTK at T686 or
phosphorylation of SR protein 75, a substrate of CLK2. Quantification of the Western blots
enabled IC determinations. The p-TTK and p-SRp75 potency of 23 was shown to be 0.057 ꢀM
5
0
and 0.549 ꢀM, respectively (Figure 3a). To assess the scope of the TNBC sensitivity versus
Luminal BC resistance to compound 23, we chose 2 additional cell lines in each category. As
seen in Figure 3b, the 3 TNBC lines are highly sensitive to Compound 23 while the 3 Luminal
BC lines are resistant.
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1
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2
2
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2
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2
2
2
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a.
Biochemical
Kinase
Cellular
Kinase
Biomarker
0
1
2
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9
0
1
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0
1
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1
2
3
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9
0
1
2
3
4
5
6
7
8
9
0
IC (ꢀM)
IC (ꢀM)
5
0
50
TTK
CLK2
0.005
0.0026
0.057 (p-TTK)
0.549 (p-SRp75)
b.
Figure 3. a). Biochemical and cellular biomarker potency of Compound 23 confirming inhibition
of TTK and CLK2 and phosphorylation of downstream substrates. b). Compound 23
demonstrated preferential anti-proliferative activities in TNBC lines.
The identification of the key targets responsible for TNBC cellular activity, namely TTK
and CLK2, allowed us to initiate crystallography efforts to enable SBDD. We obtained a crystal
structure of compound 23 with TTK at 2.99Å resolution (Figure 4). A detailed view of 23 bound
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in the TTK binding pocket is shown in Figure 4a. When compared to the apo structure of TTK,
an induced fit of the Glycine-rich loop (G-loop) is observed (see supplemental Figure 1a and 1b).
As anticipated, the pyrrolopyrimidine core of 23 forms 3 hydrogen bonds to the hinge region of
the TTK ATP binding pocket. The pyrrole NH (N7) forms a hydrogen bond to backbone
carbonyl of Glu603. The amino-pyrimidine moiety of the core forms two additional hydrogen
bond interactions with the hinge residue Gly605.
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23
24
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45
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The C5 methyl benzoxazole binds deep in the ATP binding pocket and forms favorable
van der Waals interactions with the gate-keeper residue Met602, the sidechain carbons of
catalytic residue Lys553, and the sidechain of activation loop residue Met671. In addition, N3 of
the methyl benzoxazole forms a direct hydrogen bond with the sidechain amine of Lys553. The
C4 cyclopentyl substituent is enclosed in the ribose pocket by a portion of the activation loop
where residues Met671, Gln672, Pro673, and Asp674 are ordered in the crystal structure (Figure
4b). This conformation of the activation loop has been observed in other crystal structures of
2
1
TTK. The ordered portion of the activation loop and the G-loop appear to interact though a
network of hydrogen bonds (see supplemental Figure 1a and 1b for additional views). This
arrangement of the activation loop residues encapsulates the cyclopentyl ring in a hydrophobic
pocket. The C2 aniline binds towards the ‘solvent’ pocket. The ortho-methoxy substituent binds
in a pocket formed by hinge residue Cys604 and G-loop residue Gln541. Cys604 is a small and
2
9
fairly unique residue at the hinge important for obtaining kinase selectivity in this series.
The
para amide substituent of the C-2 aniline is within 3.3Å of Asp608 but does not appear to form
specific hydrogen bonds. Based on modeling and docking studies, Compound 23 binds similarly
in the CLK2 binding pocket (supplemental Figure 2a). The overall residue homology of CLK2
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4
4
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5
5
5
5
5
5
5
5
5
5
6
and TTK is low, however there are similarities in the binding pocket that allows both kinases to
accommodate Compound 23 (supplemental Figure 2b).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
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2
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b.
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45
46
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48
49
50
51
52
53
54
55
56
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Figure 4. a) Crystal structure of 23 bound in the TTK ATP pocket and interactions with residues
within 4.0Å of the ligand are shown (PDB code 6B4W). b) TTK ATP binding pocket with the
ordered portion of the activation loop shown in green.
With numerous potent and selective compounds demonstrating good rat and mouse iv PK
in hand, we set out to assess the time and exposure necessary to illicit an anti-proliferative effect.
Cal-51 cells were treated in cellular wash-out experiments with compound 23 for 6, 12, 24, 48,
and 72 h followed by a wash out. Antiproliferative activity was measured at 3 days post washout.
From the data represented in Figure 5, at time points > 12 h, a considerable left shift in the
potency is observed. This in vitro data suggests that >12 h coverage by the compound is
necessary to give maximal antiproliferative activity.
Figure 5. In vitro wash out data in Cal-51 cell line of compound 23.
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
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3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Compound 23 was chosen for in vivo efficacy assessment based on potency, selectivity,
slow clearance, and long MRT in both rat and mouse species. We sought to investigate the
optimal in vivo dosing schedule and finalize the target product profile including route of
administration. Based on the washout data presented in Figure 5, it is likely that daily dosing
would be required. It is plausible that with the TTK mechanism, <24 h exposure could be
sufficient to commit cells to apoptosis, although likely longer exposure might be necessary to
capture all cycling cells in various phases of mitosis. To investigate this hypothesis, compound
23 was administered iv every 3 days (q3d) or every seven days (q7d) in a Cal-51 mouse
xenograft model (Figure 6). Animals were treated for 16 days and tumor growth compared to
vehicle control and body weight loss (BWL) were measured on the day following the final day of
dosing. Compound 23 demonstrated significant tumor growth inhibition (TGI) (vehicle - treated /
vehicle) x 100%) of 71% at both 10 and 20 mg/kg on a q3d dosing schedule (Figure 6a). The
BWL in the 20 mg/kg group was higher than in the 10 mg/kg group (17% vs 5%). In the q7d
dosing paradigm, 20 mg/kg was well tolerated, without appreciable BWL and compound 23
demonstrated 70% TGI (Figure 6b). Both dosing schedules were better tolerated and more
efficacious than Taxotere. Taxotere was administered at maximum tolerated dose (5 mg/kg,
q4d). The BWL of Taxotere in this model was significant (20%) and required suspended dosing
to allow for recovery. These data suggest that q7d dosing was sufficient to provide significant
tumor volume reduction with improved tolerability over a more frequent dosing schedule, and
thus an iv route of administration was acceptable. Compound 23 has improved efficacy and
better tolerability in the Cal-51 xenograft than Taxotere, the current standard of care for TNBC.
0
1
2
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9
0
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0
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0
1
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0
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a.
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4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 6. Cal-51 tumor xenograft studies with q3d (a) and q7d (b) dosing schedules of 23
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
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5
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8
9
0
1
2
3
4
5
6
7
8
9
0
(
*p<0.001, one way ANNOVA).
In addition to the data presented above, higher species PK, in vitro and in vivo
toxicological assessments, and additional in vivo models were used to select compound 23 (CC-
71) for IND enabling studies. Compound 23 has a favorable CEREP receptor profile and a
6
hERG (human ether-a-go-go-related gene) ion channel IC value of >33 ꢀM. Full biological
50
characterization and additional in vivo TNBC patient derived xenograft (PDX) data where
compound 23 causes regression at well tolerated doses will be published in due course.
Conclusion
In summary, we have described the SAR and optimization of the 2,4,5-trisubstituted-7H-
pyrrolo[2,3-d]pyrimidine series emanating from a TNBC phenotypic screen. Interrogation of the
biology of these compounds identified two inhibitory targets, TTK and CLK2. This unique dual
kinase profile of accelerated mitotic progression and mRNA splicing has been shown to be
efficacious in TNBC cell lines in vitro and in xenograft models in vivo. Multiple compounds in
this series were generated with the desired in vitro profile and good iv PK properties. This work
ultimately led to the identification and nomination of compound 23 for IND-enabling studies.
Compound 23 has excellent kinase selectivity, favorable iv pharmacokinetic properties in all
species tested, superior in vivo efficacy and tolerability as compared to Taxotere, and an
acceptable in vitro and in vivo toxicology profile. Additional biological characterization of
compound 23 will be disclosed elsewhere.
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Materials and Methods
Chemistry – All materials were obtained from commercial sources and used without further
purification unless otherwise noted. Chromatography solvents were HPLC grade and used as
purchased. All air-sensitive reactions were carried out under a positive pressure of an inert
1
nitrogen atmosphere. H 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 (δ) are reported in ppm and coupling constants (J) are
given in Hz. Thin Layer Chromatography (TLC) analysis was performed on Whatman thin layer
plates. 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 equipped with 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). Compounds were analyzed for purity by one of two methods: (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
final tested compounds was determined to be >95% by HPLC conducted on an Agilent 1100
system using a reverse phase C18 column and diode array detector (compounds 8, 9, 10, 11, 20,
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1, and 44 are >90% pure). Elemental analysis was performed at Robertson Microlit
Laboratories, Ledgewood, New Jersey. Reported yields are unoptimized. Compounds were
named using ChemDraw Ultra.
0
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2
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7
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9
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Synthetic procedures for all intermediates 2, 3a-3u, 4a-4u, 6a-6g and 7-44 SEM are
included in the supplemental material.
General procedure for the synthesis of compounds 1, and 7 – 23, 25 – 29, 31 - 44. The
appropriate SEM protected intermediate (1 equiv) was diluted with dichloromethane (0.2 M) and
treated with trifluoroacetic acid (25 – 200 equiv). The solution was stirred at rt for 1 – 18 h. The
mixture was concentrated under reduced pressure to afford an oil. The residue was treated with
2
M ammonia in methanol (50 – 500 equiv) for 3 to 18 h. The mixture was concentrated under
reduced pressure. The residue was diluted with either acetonitrile, dioxane, or methanol (0.1 M),
treated with a concentrated aqueous solution of ammonium hydroxide (50 – 500 equiv), and
stirred for 2 – 24 h at temperatures of rt to 50 °C. The mixture was concentrated under reduced
pressure and purified by reverse phase preparative HPLC. Fractions containing pure product
were loaded onto a Phenomenex Strata-XC strong cation ion exchange column. The column was
washed successively with water, methanol, and the product eluted with 2 M ammonia in
methanol. The combined product fractions were concentrated under reduced pressure to afford
the title compound.
4-((4-(Cyclopentyloxy)-5-(4-hydroxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-
methoxy-N-methylbenzamide (1). (0.120 g, 0.253 mmol, 12% yield over 2 steps, HPLC purity
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=
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99%). H NMR (400 MHz, DMSO-d ) δ 11.62 (br s, 1H), 9.27 (s, 1H), 8.65 – 8.63 (d, 1 H, J =
6
Hz), 8.30 – 3.28 (m, 1 H), 7.66 (s, 1H), 7.52 – 7.46 (m, 4 H), 7.12 (s, 1 H), 6.76 -6.73 (d, 2 H, J
9 Hz), 5.67 – 5.65 (br. m., 1 H), 3.97 (s, 3 H), 2.80 – 2.79 (d, J = 4.4 Hz, 3 H), 1.99 – 1.94 (m,
10
11
12
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+
H), 1.82 – 1.62 (m, 6 H). MS (ESI) m/z 474.2 [M+1] .
4-((4-(Cyclopentyloxy)-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-
1
methylbenzamide (7). (0.155 g, 0.339 mmol, 64% yield, HPLC purity >99%). H NMR (499
MHz, DMSO-d ) δ 11.78 (s, 1 H) 8.63 (d, J=8.86 Hz, 1 H) 8.25 - 8.34 (m, 1 H) 7.63 - 7.75 (m, 3
6
H) 7.46 - 7.55 (m, 2 H) 7.29 (d, J=2.46 Hz, 1 H) 7.18 (t, J=8.86 Hz, 2 H) 5.58 - 5.70 (m, J=5.78,
5.78, 2.71, 2.46 Hz, 1 H) 3.96 (s, 3 H) 2.79 (d, J=4.43 Hz, 3 H) 1.90 - 2.03 (m, 2 H) 1.74 - 1.85
+
(
m, 2 H) 1.56 - 1.73 (m, 4 H). MS (ESI) m/z 458.1 [M+1] .
4-((4-(Cyclopentyloxy)-5-(3-hydroxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-
1
methoxy-N-methylbenzamide (8). (0.014 g, 0.030 mmol, 9% yield, HPLC purity >93%). H
NMR (400 MHz, DMSO-d ) δ 11.69-11.75 (m, 1H), 9.17-9.25 (m, 1H), 8.59-8.68 (m, 1H), 8.27-
6
8.33 (m, 1H), 7.66-7.71 (m, 1H), 7.47-7.56 (m, 2H), 7.20-7.24 (m, 1H), 7.05-7.16 (m, 3H), 6.62-
6.67 (m, 1H), 5.62-5.69 (m, 1H), 3.91-4.07 (m, 3H), 2.74-2.84 (m, 3H), 1.92-2.02 (m, 2H), 1.55-
+
1
.87 (m, 6H). MS (ESI) m/z 474 [M+1] .
4-((4-(Cyclopentyloxy)-5-(pyridin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-
1
methoxy-N-methylbenzamide (9). (0.067 g, 0.15 mmol, 28% yield, HPLC purity >93%). H
NMR (400 MHz, DMSO-d ) δ 12.03-12.08 (m, 1H), 8.58-8.62 (m, 1H), 8.48-8.52 (m, 2H), 8.28-
6
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.34 (m, 1H), 7.76-7.78 (m, 1H), 7.70-7.74 (m, 2H), 7.64 (d, J=2.45 Hz, 1H), 7.49-7.54 (m, 2H),
.68-5.74 (m, 1H), 3.96 (s, 3H), 2.79 (d, J=4.65 Hz, 3H), 1.96-2.06 (m, 2H), 1.81-1.90 (m, 2H),
+
.63-1.79 (m, 4H). MS (ESI) m/z 459 [M+1] .
0
1
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3
4
5
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7
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9
0
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4-((4-(Cyclopentyloxy)-5-(pyridin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-
methoxy-N-methylbenzamide (10). (0.033 g, 0.071 mmol, 19% yield, HPLC purity >93%). MS
+
(ESI) m/z 459 [M+1] .
4
-((4-(Cyclopentyloxy)-5-(1H-pyrazol-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-
1
methoxy-N-methylbenzamide (11). (0.018 g, 0.040 mmol, 16% yield, HPLC purity >91%). H
NMR (400 MHz, DMSO-d ) δ 12.71-12.76 (m, 1H), 11.87-11.94 (m, 1H), 8.58-8.67 (m, 1H),
6
8
4
1
.34-8.44 (m, 1H), 7.66-7.75 (m, 1H), 7.44-7.57 (m, 3H), 6.63-6.74 (m, 1H), 5.65-5.73 (m, 1H),
.08-4.17 (m, 1H), 3.92-4.03 (m, 3H), 2.74-2.84 (m, 3H), 1.98-2.09 (m, 2H), 1.82-1.93 (m, 2H),
+
.58-1.82 (m, 4H). MS (ESI) m/z 448 [M+1] .
4
-((4-(Cyclopentyloxy)-5-(1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-
1
methoxy-N-methylbenzamide (12). (0.012 g, 0.027 mmol, 8% yield, HPLC purity >97%). H
NMR (400 MHz, DMSO-d ) δ 12.67-12.77 (m, 1H), 11.53-11.65 (m, 1H), 8.61-8.69 (m, 1H),
6
8.28-8.37 (m, 1H), 7.94-8.02 (m, 1H), 7.83-7.91 (m, 1H), 7.64-7.69 (m, 1H), 7.49-7.58 (m, 2H),
7.25-7.33 (m, 1H), 5.64-5.76 (m, 1H), 3.97 (s, 3H), 2.77-2.85 (m, 3H), 2.00-2.14 (m, 2H), 1.84-
+
1.94 (m, 2H), 1.64-1.81 (m, 4H). MS (ESI) m/z 448 [M+1] .
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-((4-(Cyclopentyloxy)-5-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-
1
methoxy-N-methylbenzamide (13). (0.033 g, 0.067 mmol, 17% yield, HPLC purity >95%). H
NMR (400 MHz, DMSO-d ) δ 11.66-11.70 (m, 1H), 8.61-8.67 (m, 1H), 8.27-8.33 (m, 1H), 7.66-
6
10
11
12
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.70 (m, 1H), 7.57-7.63 (m, 2H), 7.48-7.53 (m, 2H), 7.18-7.22 (m, 1H), 6.88-6.95 (m, 2H), 5.63-
5.69 (m, 1H), 3.96 (s, 3H), 3.77 (s, 3H), 2.76-2.84 (m, 3H), 1.92-2.03 (m, 2H), 1.76-1.86 (m,
+
2H), 1.61-1.76 (m, 4H). MS (ESI) m/z 488 [M+1] .
4-((4-(Cyclopentyloxy)-5-(4-(hydroxymethyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-
yl)amino)-3-methoxy-N-methylbenzamide (14). (0.020 g, 0.041 mmol, 60% yield, HPLC
1
purity >97%). H NMR (400 MHz, DMSO-d ) δ 11.73-11.79 (m, 1H), 8.62-8.68 (m, 1H), 8.26-
6
8
5
1
.36 (m, 1H), 7.69-7.71 (m, 1H), 7.62-7.68 (m, 2H), 7.49-7.54 (m, 2H), 7.27-7.32 (m, 3H), 5.65-
.71 (m, 1H), 5.16 (t, J=5.75 Hz, 1H), 4.52 (d, J=5.87 Hz, 2H), 3.97 (s, 3H), 2.78-2.83 (m, 3H),
+
.94-2.06 (m, 2H), 1.78-1.87 (m, 2H), 1.62-1.77 (m, 4H). MS (ESI) m/z 488.3 [M+1] .
4-[4-Cyclopentyloxy-5-(3-hydroxymethyl-phenyl)-7-(2-trimethylsilanyl-ethoxymethyl)-7H-
pyrrolo[2,3-d]pyrimidin-2-ylamino]-3-methoxy-N-methyl-benzamide (15). (0.008 g, 0. 016
1
mmol, 5% yield, HPLC purity >95%). H NMR (400 MHz, DMSO-d ) δ 8.70 (d, J = 8.4 Hz,
6
1H), 7.58 (s, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.41 (d, J = 7.6 Hz, 2H), 7.27 (t, J = 7.6 Hz, 1H),
7.20 (d, J = 7.6 Hz, 1H), 7.03 (s, 1H), 5.62 (s, 1H), 4.60 (s, 2H), 3.95 (s, 3H), 2.87 (s, 3H), 2.01-
+
1
.91 (m, 2H), 1.86-1.83 (m, 2H), 1.69-1.60 (m, 4H). MS (ESI) m/z 488.2 [M+H] .
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