Novel pyrrolopyrimidines as Mps1/TTK kinase inhibitors for breast cancer

Article abstract of DOI:10.1016/j.bmc.2017.02.030

New targeted therapy approaches for certain subtypes of breast cancer, such as triple-negative breast cancers and other aggressive phenotypes, are desired. High levels of the mitotic checkpoint kinase Mps1/TTK have correlated with high histologic grade in breast cancer, suggesting a potential new therapeutic target for aggressive breast cancers (BC). Novel small molecules targeting Mps1 were designed by computer assisted docking analyses, and several candidate compounds were synthesized. These compounds were evaluated in anti-proliferative assays of a panel of 15 breast cancer cell lines and further examined for their ability to inhibit a variety of Mps1-dependent biological functions. The results indicate that the lead compounds have strong anti-proliferative potential through Mps1/TTK inhibition in both basal and luminal BC cell lines, exhibiting IC₅₀values ranging from 0.05 to 1.0?μM. In addition, the lead compounds 1 and 13 inhibit Mps1 kinase enzymatic activity with IC₅₀values from 0.356?μM to 0.809?μM, and inhibited Mps1-associated cellular functions such as centrosome duplication and the spindle checkpoint in triple negative breast cancer cells. The most promising analog, compound 13, significantly decreased tumor growth in nude mice containing Cal-51 triple negative breast cancer cell xenografts. Using drug discovery technologies, computational modeling, medicinal chemistry, cell culture and in vivo assays, novel small molecule Mps1/TTK inhibitors have been identified as potential targeted therapies for breast cancers.

Full text of DOI:10.1016/j.bmc.2017.02.030

Accepted Manuscript
Novel Pyrrolopyrimidines as Mps1/TTK Kinase Inhibitors for Breast Cancer
Yasuro Sugimoto, Dwitiya B. Sawant, Harold A. Fisk, Liguang Mao, Chenglong
Li, Somsundaram Chettiar, Pui-Kai Li, Michael V. Darby, Robert W.
Brueggemeier
PII:
S0968-0896(16)31130-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.02.030
BMC 13559
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 November 2016
10 February 2017
12 February 2017
Please cite this article as: Sugimoto, Y., Sawant, D.B., Fisk, H.A., Mao, L., Li, C., Chettiar, S., Li, P-K., Darby,
M.V., Brueggemeier, R.W., Novel Pyrrolopyrimidines as Mps1/TTK Kinase Inhibitors for Breast Cancer,
Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.02.030
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Novel Pyrrolopyrimidines as Mps1/TTK Kinase Inhibitors for
Breast Cancer
Yasuro Sugimoto,1 Dwitiya B. Sawant,2 Harold A. Fisk, 2 Liguang Mao,1 Chenglong Li, 1
Somsundaram Chettiar,1 Pui-Kai Li, 1 Michael V. Darby, 1 and Robert W. Brueggemeier1
1 Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy,
2 Department of Molecular Genetics, College of Arts & Sciences,
The Ohio State University, Columbus, OH 43210, U.S.A.
Corresponding Author: Robert W. Brueggemeier, Ph.D.; e-mail: Brueggemeier.1@osu.edu
Running Title: Mps1/TTK Kinase Inhibitors
Keywords: Mps-1, TTK, spindle checkpoint kinases, centrosome amplification,
pyrrolopyrimidine analogues, breast cancer
Conflict of interests: None

Abstract
New targeted therapy approaches for certain subtypes of breast cancer, such as triple-
negative breast cancers and other aggressive phenotypes, are desired. High levels of the
mitotic checkpoint kinase Mps1/TTK have correlated with high histologic grade in breast
cancer, suggesting a potential new therapeutic target for aggressive breast cancers (BC).
Novel small molecules targeting Mps1 were designed by computer assisted docking
analyses, and several candidate compounds were synthesized. These compounds were
evaluated in anti-proliferative assays of a panel of 15 breast cancer cell lines and further
examined for their ability to inhibit a variety of Mps1-dependent biological functions. The
results indicate that the lead compounds have strong anti-proliferative potential through
Mps1/TTK inhibition in both basal and luminal BC cell lines, exhibiting IC50 values ranging
from 0.05 to 1.0 M. In addition, the lead compounds 1 and 13 inhibit Mps1 kinase
enzymatic activity with IC50 values from 0.356 μM to 0.809 μM, and inhibited Mps1-
associated cellular functions such as centrosome duplication and the spindle checkpoint in
triple negative breast cancer cells. The most promising analog, compound 13, significantly
decreased tumor growth in nude mice containing Cal-51 triple negative breast cancer cell
xenografts. Using drug discovery technologies, computational modeling, medicinal
chemistry, cell culture and in vivo assays, novel small molecule Mps1/TTK inhibitors have
been identified as potential targeted therapies for breast cancers.
2

Graphical Abstract:
Site 1
Site 2
O
S
O
HO
HN
N
O
N
N
N
H
N
H
Compound 1
3

1. Introduction
Breast cancer (BC) is a heterogeneous group of tumors which can be subdivided on the
basis of histopathological features, genetic alterations, and gene-expression profiles.1, 2
Approximately 50-60% of all breast cancer patients and two-thirds of postmenopausal
breast cancer patients have estrogen receptor positive tumors (ER+). Adjuvant hormonal
therapy is the primary therapy for ER+ breast cancer. Triple-negative breast cancers
(TNBC) are defined by the absence of staining for estrogen receptors, progesterone
receptors, and HER2/neu. These tumors have poor clinical outcome and represent a
.
3
recognized prognostic group characterized by aggressiveness and resistance to available
systemic therapy. Approximately 10-25% of all breast cancers in the U.S. are TNBC.
A 2011 study reported a high-throughput RNAi screening of a series of pharmacologically
tractable genes followed by comprehensive functional viability assay profiles for a panel in
>30 commonly used breast tumor cell models to identify genes critical to the growth of
specific breast cancer subtypes.4 Analysis of these profiles identified a series of novel
genetic dependencies and suggested potential new therapeutic targets for aggressive
breast cancers. One group of genes identified was mitotic checkpoint kinases, including
Mps1/TTK. Other studies in 2011 and 2013 reported that high levels of Mps1 protein has
been correlated with high histologic grade in breast cancer, while reducing Mps1 levels in
BC cells by RNAi resulted in aberrant mitoses, induction of apoptosis, and decreased ability
of BC cells to grow as xenografts in nude mice.5, 6 These studies suggest Mps1 as a potential
therapeutic target in aggressive breast cancers.
4

While the Mps1 gene (monopolar spindle) was first identified in the budding
yeast, Saccharomyces cerevisiase,7 its human ortholog [phosphotyrosine-picked threonine
kinase/threonine and tyrosine kinase (PYT/TTK)] had been discovered as a dual specificity
protein kinase.8, 9 Mps1/TTK regulates cell cycle progression,10, 11 and both mRNA and
protein levels of Mps1 are readily detectable in proliferating breast cancer cells, while
markedly reduced or absent in breast cancer cells with a low proliferative index.
Mps1/TTK regulates several processes that are important for genomic integrity such as
centrosome duplication,12-14 DNA damage control,15 and the spindle assembly checkpoint
(SAC),12 and alterations to Mps1/TTK have been associated with cell transformation and
chromosome instability in different tumor models.16
As many as 80% of invasive breast tumors have extra centrosomes 17 that generate
aberrant mitotic spindles in situ,18 and the appearance of extra centrosomes is an early
occurrence in breast tumorigenesis 17 that correlates with aneuploidy in invasive tumors.17,
19 Preventing the degradation of Mps1 is sufficient to cause centrosome re-duplication,13, 14,
20 and defects in this control are correlated with centrosome amplification and
tumorigenesis.14, 21 Notably, breast cells are particularly sensitive to increases in
Mps1/TTK protein levels and, unlike in other cell types, overexpression of wild type
Mps1/TTK causes centrosome re-duplication in breast-derived cells.14 Several centrosome
defects have been observed in breast cancer, including centrosome amplification,
centrioles of unusual length, and accumulation of phosphorylated forms of Centrin 2
5

(Cetn2),19, 22, 23 an Mps1 substrate whose function and localization at the centrosome are
regulated by Mps1 phosphorylation.24-26
The role of Mps1 has been most widely studied in the SAC, which ensures proper
chromosome segregation by delaying progression through mitosis until chromosomes are
all properly aligned. Mps1 accumulates at unattached or misaligned kinetochores27 where
it plays a key role in recruitment of several other checkpoint proteins.28, 29 Also, reports
suggest that silencing Mps1/TTK, which has dual roles in checkpoint activation and
chromosome alignment, can sensitize cancer cells to sub-lethal doses of paclitaxel, whereas
non-tumorigenic cells cannot be sensitized.30, 31
These observations suggest that pharmacological inhibition of Mps1/TTK may be a
promising targeted cancer therapeutic. Several Mps1/TTK inhibitors have been developed
in recent years,32-38 such as Mps1-IN-1, Mps1-IN-3, reversine (originally identified as an
Aurora B inhibitor),39 AZ3146, Mps-BAY1, Mps1-BAY2a, BAY 1217389, MPI-0479605, and
CCT251455. In most cases, however, only the SAC function of Mps1 was assayed, or as for
Mps1-IN-1, Mps1-BAY1 and Mps1-BAY2a there was no inhibition of centrosome
duplication. Current therapies for certain subtypes of breast cancer, such as TNBC and
other aggressive phenotypes, rely on standard chemotherapy approaches with significant
side effects; therefore, newer targeted therapy approaches are needed. This research
focused on designing and developing novel small molecule Mps1/TTK inhibitors as
targeted therapies for TNBC and aggressive breast cancers. Such agents are envisioned to
replace standard cytotoxic chemotherapies as monotherapy or in combination with current
6

treatment regimens as safe, effective interventions that reduce life-threatening toxicities. In
this study we describe two novel small molecule inhibitors of Mps1, Compound 1 and
Compound 13, that inhibit both centrosome duplication and SAC function. Furthermore,
Compound 13 attenuates growth of triple negative breast cancer cells in mouse xenografts.
2. Results
2.1. Computational drug design: Using computer assisted docking studies, a series of
novel molecules were designed to interact at the Mps1-ATP competitive binding site. A
computational study of known chemical scaffolds led us to the design of lead compound 1
(Figure 1) to explore as Mps1/TTK inhibitors. In silico analysis of our unique lead scaffold
identified twin hydrogen bonds between our novel chemical scaffold, a pyrrolopyrimidine
ring, and the kinase hinge loop, which should impart strong inhibitor binding (Figure 1).
The hydrogen bonds are between carbonyl oxygen of Glu603 and pyrrole nitrogen and
between amide of Gly605 and pyrimidine nitrogen. The binding modes are very similar to
that of Mps1-IN-1,33 except that the twin hydrogen bonds are 0.9 Kcal/mol stronger due to
the substitution of pyridine ring of Mps1-IN-1 to pyrimidine ring, translating to a 5-fold
increase in potency at enzymatic level.
For lead optimization, research focuses on adding fragments into Site 1 to gain potency and
selectivity and on replacing fragments at Site 2 to gain potency and improve drug
properties (Figure 1). Site 1 is a particularly attractive site for modifications as this site is
7

not occupied by substituents in current inhibitors. Site 2 affords opportunities for fragment
substitutions that will enhance pharmacokinetic parameters. As a result of the
computational drug design, compound 1 (Figure 1) was synthesized as a lead compound
for investigation.
Figure 1. General chemical scaffold and lead Compound 1 binding to kinase ATP site.
2.2. Synthesis of the inhibitors: The synthesis of compounds 1, 7, 9 and 13 are shown in
Figure 2. The synthesis began with the known compound 2,4-dichloro-7H-pyrrolo[2,3-
d]pyrimidine 2.40 The N7 position of compound 2 was selectively protected using di-tert-
butyl dicarbonate to form the carboxylate 3.41 Crossing coupling of 3 with 2-
(isopropylsulfonyl)aniline afforded 4. Nucleophilic aromatic substitution with 2-
8

methoxyaniline and 1-(4-amino-3-methoxy)piperidin-4-ol formed compounds 5 and 6,
respectively. Boc-deprotection of 5 and 6 yielded the respective compounds 1 and 7.
Intermediate 4 was used for the synthesis of compound 9. Nucleophilic aromatic
substitution of 4 with 2-methoxy-4-morpholinoaniline obtained 8. Boc-deprotection of 8
yielded compound 9. Finally, cyclohexylamine was reacted with 2,6-
dichloropyrrolopyrimidine in presence of a base 42 to form 10. The N7 position of
compound 10 was then protected using di-tert-butyl dicarbonate to yield 11. Aromatic
substitution of 11 with 2-methoxy-4-morpholinoaniline followed by boc deprotection
afforded compound 13.
9

Figure 2: Syntheses of Compounds 1, 7, 9 and13. Reagents and conditions: a) di-tert-
Butyldicarbonate, DMAP, DIEA, DCM, reflux, 15 min. b) 2-(Isopropylsulfonyl)aniline,
Pd2(dba)3, XPhos, K2CO3, t-butanol, 100°C, 4 h. c) 1-(4-Amino-3-methoxyphenyl-
piperidin-4-ol, Pd2(dba)3, XPhos, K2CO3, t-butanol, 100°C, 6 h. d) 2-Methoxyaniline,
Pd2(dba)3, XPhos, K2CO3, t-butanol, 100°C, 4 h. c) 1-(4-amino-3-methoxyphenyl)-
piperidin-4-ol, Pd2(dba)3, XPhos, K2CO3, t-butanol, 100°C, 6 h. e) TFA, DCM, rt, 4 h.
f) 2-Methoxy-morpholinoaniline, Pd2(dba)3, XPhos, K2CO3, t-butanol, 100°C, 4 h.
g) Cyclohexylamine, TEA, EtOH, reflux, overnight. h) di-tert-butyldicarbonate, DMAP,
DIEA, DCM, reflux, 15 min. i) 2-methoxy-4-morpholinoaniline, Pd2(dba)3, XPhos, K2CO3,
t-butanol, 100°C, 6 h.
10

2.3. Drug validation: In vitro evaluation of synthesized molecules included the
determination of anti-proliferative effects on breast cancer cell lines, kinase inhibition
assays, and functional cell biology studies on centriole assembly and the spindle
checkpoint.
2.3.1 Anti-proliferative effects: The synthesized molecules were examined for anti-
proliferative effects on numerous breast cancer cell lines, including hormone-responsive
cell lines and triple-negative cell lines. The IC50 values of compounds 1, 7, 9 and 13 ranged
from 0.05 to 1.0 M in several breast cancer cell lines (Table 1). In general, the compounds
demonstrated anti-proliferative effects in TNBC basal cells and in luminal breast cancer cell
types. Compounds 1 and 13 were also tested in the primary cultures of normal breast
epithelial cells; both compounds exhibited no inhibition or incomplete dose-response
curves up to 20 M.
2.3.2 Mps1 kinase inhibition: To determine the efficacy of candidate compounds against
Mps1/TTK kinase activity, assays were performed as previously described 24 using
recombinant Mps1, Cetn2 (specific substrate), and varying concentrations of the inhibitors.
This enzyme assay using Cetn2 exhibits greater sensitivity than using a generic substrate
(e.g., MBP). In the enzyme assay, compound 1 inhibits in vitro kinase activity of Mps1 with
an IC50 value of 0.809 M and compound 13 with an IC50 value of 0.356 M. Compound 7
and compound 9 were less effective, with IC50 values of 3.4 and 13.1 M, respectively. On
the other hand, Mps1-IN-1 exhibits an IC50 value of 1.7 M in our assay system. Compound
13 was further evaluated in kinase profiling assays for twenty common kinases and
11

exhibited inhibition of only two kinases, FAK/PTK2 and JNK1, with IC50 values at 0.89 M
and 1.67 M, respectively (see Supplemental Materials). Thus, compound 13
demonstrated selectivity for Mps1/TTK kinase.
Table 1. IC50 determinations of compounds 1, 7, 9, 13, and known inhibitor, Mps1-IN-1,
in panel of breast cancer cell lines
IC50 ± SEM (M)
Cell line
BASAL
Cpd 1
Cpd 7
Cpd 9
Cpd 13
Mps1-IN-1
subtype
CAL-51
HCC1937
BT-20
TNBC basal
0.23 ± 0.04
0.73 ± 0.25
0.35 ± 0.38
0.81 ± 0.17
0.90 ± 0.28
0.19 ± 0.07
> 10
1.26 ± 0.24
0.84 ± 0.15
0.06 ± 0.01
2.19 ± 0.59
5.23 ± 3.94
1.57 ± 0.29
0.30 ± 0.01
1.10 ± 0.01
1.71 ± 0.21
1.30 ± 0.01
1.23 ± 1.01
0.32 ± 0.04
> 10
0.55 ± 0.10
1.02 ± 0.07
0.37 ± 0.20
0.06 ± 0.27
0.66 ± 0.35
0.35 ± 0.22
2.92 ± 1.08
> 10
TNBC basal
TNBC basal A
TNBC basal B
> 10
BT-549
1.73 ± 0.71
> 10
MDA-MB-436 TNBC basal B
MDA-MB-468 TNBC basal B
1.85 ± 0.51
> 10
Hs578T
TNBC basal B
> 10
> 10
> 10
0.60 ± 0.47
MDA-MB-231 TNBC basal B
> 10
0.40 ± 0.05
0.84 ± 0.71
0.75 ± 0.22
> 10
> 10
CAMA-1
Sk-Br-3
AU565
luminal
0.36 ± 0.02
> 10
0.65 ± 0.13
0.47 ± 0.01
> 10
> 10
luminal
0.40 ± 0.38
0.95 ± 0.15
> 10
luminal
> 10
> 10
0.33 ± 0.18
> 10
> 10
T47D
luminal
> 10
> 10
> 10
0.35 ± 0.06
> 10
0.05 ± 0.01
> 10
MDA-MB-453
MCF-7
luminal A
luminal A
luminal B
0.82 ± 0.10
0.44 ± 0.21
> 10
> 10
> 10
0.35 ± 0.01
0.47 ± 0.23
0.30 ± 0.09
0.29 ± 0.04
> 10
BT-474
> 10
IC50 values are averages of replicate independent assays, each determined by ten-point dosage treatments (n = 6 per dose).
2.4. Cell biology: To determine the functional cellular activity of candidate Mps1/TTK
inhibitors, we monitored their effects on centriole assembly and the spindle checkpoint.
For centriole assembly assays (Figure 3), Cal-51 and MDA-MB-468 cells were treated with
candidate compounds prior to a short pulse of BrdU. Because the centriole pair is
12

replicated at the G1/S transition, compounds that block centriole assembly will lead to an
increased number of BrdU-positive cells with two centrioles. In addition, like many breast
cancer cells Cal-51 and MDA-MB-468 are capable of centrosome amplification, and
compounds that block this centrosome amplification should reduce the number of BrdU-
positive cells with more than four centrioles. Roughly 80% of BrdU-positive DMSO-treated
Cal-51 cells have three or four centrioles, but treatment with compound 1 or compound 13
leads to a dose dependent increase in the percentage of BrdU positive cells that have failed
to complete centriole assembly and have just two centrioles (Figure 3A-C). Compounds 1
and 13 also blocked centrosome amplification in Cal-51 cells, as evidenced by a dose
dependent reduction in the percentage of cells with more than four centrioles. We also
observed a decrease in cell viability at higher concentrations (10 µM and above) of
compound 13 compared to the DMSO control and compound 1. At 25 µM concentration of
compound 13 there was a reduction in cell viability by 70% making it difficult to assay
BrdU positive cells for centriole numbers (Figure 3C). At 10 µM compounds 1 and 13
increased the percentage of BrdU positive Cal-51 cells with two centrioles compared to
other Mps1 inhibitors (Figure 3D). At 10 µM compounds 1 and 13 elicited similar effects in
MDA-MB-468 cells (Figure 3E) on centriole biogenesis during S-phase. On the other hand,
the effects of AZ3146 and Mps1-IN-1 at the same concentrations on centriole assembly and
centrosome amplification was similar to that of compound 5 that did not inhibit Mps1
kinase activity in vitro and was included as a negative control (Figure 3 D, E).
13

Figure 3: Compound 1 and compound 13 attenuate centriole duplication in breast cancer
cells. (A) Representative image of BrdU positive Cal-51 cells (top panel) and MDA-MB-
468 cells (bottom panel) treated with either DMSO or compound 1 (25µM). Cetn2
(green) was used as a centriolar marker and γ-tubulin (red) was used as a
pericentriolar matrix (PCM) marker. Scale bar: 5µM. Graphical representation of BrdU
positive Cal-51 cells showing inhibition of centriole duplication with different
concentrations of (B) compound 1 and (C) compound 13. Compounds 1 and 13 led to
an increase in the number of BrdU positive cells with 2 Cetn2 foci (off-white bar) in a
concentration dependent manner. (D) Graphical representation of BrdU positive Cal-51
cells comparing the effect of different Mps1 inhibitors on centriole numbers.
Compounds 1 and 13 increased the number of cells with 2 Cetn2 foci when compared
to other inhibitors and DMSO control. DMSO and compound 5 were used as negative
controls. (E) Graphical representation of BrdU positive MDA-MB-468 cells comparing
the effect of different compounds on centriole numbers. Similar to that seen in Cal-51
cells, compounds 1 and 13 led to an increase in the number of cells with 2 Cetn2 foci.
These experiments were done in triplicates and at least 75 cells were counted pre
replicate with the exception of 25µM compound 13 in 4C (#); due to high amount of cell
death in these samples, only 15 cells were counted in triplicate. Asterisks indicate P
values calculated using two-sided Student’s t-Test when comparing the percentage of
cells with 1 or 2 centrioles for each indicated treatment to that for DMSO as follows:
*, P<0.05<P>0.01; **, P<0.01<P>0.005; ***,P<0.005.
14

For SAC assays (Figure 4), S-phase arrested cells treated with candidate compounds were
released into medium containing the spindle poison nocodazole. Because the SAC prevents
the metaphase to anaphase transition in the presence of spindle damage, cells with an
intact SAC should arrest at metaphase with high levels of cyclin B in response to
nocodazole. However, compounds that block the SAC will prevent this arrest, leading to a
decreased number of mitotic cells and the loss of cyclin B. Roughly 40% of Cal-51 cells were
arrested in mitosis after being released from S-phase arrest into nocodazole, but compound
1 leads to a dose-dependent reduction in the ability of cells to arrest in response to
nocodazole (Figure 4 A, B). Compound 13 showed the maximal effect at 1 µM, the lowest
concentration tested (Figure 4 B), and both compounds 1 and 13 are more effective than
IN-1, AZ3146, or compound 5 that was included as a negative control (Figure 4 C).
Compounds 1 and 13 had similar effects in MDA-MB-468 cells (Figure 4 C). In addition,
compounds 1 and 13 lead to a loss of Cyclin B that required proteasome activity (Figure 4
D, E), and the loss was more pronounced than that observed in cells treated with either
IN-1 or AZ3146 (Figure 4 F).
15

Figure 4. Compound 1 and compound 13 attenuate the spindle assembly checkpoint in
breast cancer cells. (A) Representative image of Cal-51 cells stained with DAPI (blue)
and Cyclin B (red) showing a cell in S-phase (S), two cells in Mitosis (M) and a cell in G2.
(B) Graphical representation of the percentage of mitotic cells in the presence of
different concentrations of compound 1 (light grey) and compound 13 (dark grey)
compared to DMSO control in Cal-51 cells. (C) Graphical representation of the
percentage of Cal-51 (light grey) or MDA-MB-468 cells (dark grey) cells in mitosis in the
presence of various compounds. These experiments were done in triplicates and at
least 500 cells were counted per set. (D-F) Cyclin B immunoblots from nocodazole
treated Cal-51 cells in the presence of the indicated concentrations (in µM) of (D)
compound 1 and (E) compound 13 with or without MG115, (F), and in the presence of
increasing concentrations (in µM) of various compounds. Compound 13 has the
strongest effect on Cyclin B levels. Asterisks indicate P values calculated using two-
sided Student’s t-Test when comparing the mitotic index for each indicated treatment
to that for DMSO as follows: *, P<0.05<P>0.01; **, P<0.01<P>0.005; ***P<0.005.
16

2.5 Solubility and pharmacokinetics of compound 13:
Solubility of compound 13 in DMSO was determined to be 53.4 mg/ml. Its solubility in the
formulation of DMSO: Tween 80: PEG400: saline (10: 5: 10: 75) was 5 mg/ml. The
formulation was then used for the pilot plasma pharmacokinetic studies in mice using
intravascular (IV) and extravascular (IP) administrations (Supplemental Materials).
Systemic clearance following an IV dose was 17.5 L/hr/kg or roughly 3x mouse liver blood
flow, suggesting considerable extrahepatic clearance. The elimination half-life was 2.28
hours. The IP AUC was determined to be 20,031 hr*nmol/L or 8,463 hr*ng/mL following a
50 mg/kg dose. These data suggest that an IP administration of compound 13 at doses up
to 50 mg/kg could reasonably be expected to provide a level of drug sufficient for the
inhibition of Mps1 driven tumor growth.
2.6 In vivo evaluation in breast cancer xenograft model:
To explore the effects of compound 13 on tumor growth in vivo, athymic nude mice were
injected with the highly tumorigenic Cal 51 breast cancer cells. After the tumors reached a
certain size, mice were injected every day with either compound 13 (10mg/kg) or DMSO
(control). The growth rates of the xenografts in compound 13 administered mice were
significantly reduced compared to those in DMSO control mice (Figure 5A-C). The
reduction in growth rates was first observed after 3 weeks of initiation of drug
administration (Figure 5A). This reduced growth rate was observed until the mice were
sacrificed at week 7, when the most significant reduction was observed (p=0.002). There
was also a significant reduction in the tumor weight of the compound 13 treated mice
compared to the control mice (Figure 5B). Overall, the data suggest that compound 13
17

significantly inhibited growth of xenografted tumors in nude mice and had no toxic effect
on the mice, as there was no significant change in body weight over the course of the
experiment (Supplemental Materials).
A
300
200
100
0
**
*
Control
*
*
Compound 13
1
2
3
4
5
6
7
8
Weeks
C
B
400
300
200
100
0
*P= 0.013
1R 1L 2R 2L 3R 3L 4R 4L 5R 5L
No
Tumor
DMSO Cmpd 13
Figure 5. Compound 13 reduces the growth of breast cancer cell derived xenografts in
immunodeficient mice. Athymic nude mice carrying Cal-51 cells derived xenografts
were treated after 1 week with either DMSO control or compound 13 inhibitor i.p., once
daily and the tumor area was routinely measured using a caliper. (A) Graphical
representation of tumor volume data (mean) of compound 13 injected mice (black
dotted line) compared to control DMSO treated mice (black solid line) over a period of 7
weeks. The P values for the weeks are as follows; week 3 :0.08, week 4 :0.029, week 5
:0.042, week 6 :0.011, week 7 :0.002. Error bars indicate SEM and the P values were
calculated using two-sided Student’s t-test. *P<0.05, **P<0.01. (B) Box and whisker plot
showing the weight of tumors dissected from mice sacrificed at week 7 after
inoculation. The dots represent the outliers. (C) Representative image of tumors
dissected from DMSO treated mice compared to tumors from compound 13 treated
mice at week 7.
18

3. Discussion:
Current therapies for aggressive types of breast cancer such as TNBC rely heavily on
chemotherapy that can also affect non-cancer cells that divide quickly such as bone
marrow cells, lining of the intestines and hair follicles. Hence, there is an urgent need for
newer targeted therapies.
To facilitate the advancement of targeted therapies, studying the basic molecular
mechanisms that underlie the progression of a cell from normalcy to tumor onset and
metastasis is crucial. A high frequency of aneuploidy is commonly observed in cancer.
While normal mammalian cells are highly intolerant to aneuploidy, cancer cells develop the
capacity to tolerate and thrive with high frequency of aneuploidy. Expression of checkpoint
genes are frequently increased in cancers when compared to normal cells.43, 44 Also, high
levels of Mps1 are observed in breast cancer cells and reducing Mps1 levels in aneuploid
cells increases the frequency of aberrant mitoses and decreases survival.5 In addition, Mps1
inhibitor studies have shown SAC inactivation, which increased aneuploidy and aberrant
mitoses resulting in cell death in cancer cells.45, 46 These data suggest that Mps1 is a good
anticancer target.
Centrosome amplification is prevalent in breast cancer cells.23 This represents a potential
target unique to cancer cells, and a recent study reported that an inhibitor of the
centrosome regulator Plk4 kinase had potent antitumor activity.47 In our study, novel
small molecules targeting Mps1 were designed by computer assisted docking analyses, and
several candidate compounds were synthesized. These compounds were evaluated in anti-
19

proliferative assays of a panel of 15 breast cancer cell lines and further examined for their
ability to inhibit a variety of Mps1-dependent biological functions. The results indicate that
the lead compounds have strong anti-proliferative potential through Mps1/TTK inhibition
in both basal and luminal BC cell lines, exhibiting IC50 values ranging from 0.05 to 1.0 M.
In addition, lead compounds 1 and 13 inhibit Mps1 kinase enzymatic activity with IC50
values 0.809 M and 0.356 M, respectively. In functional cellular assays, compounds 1
and 13 produced a dose-dependent increase in the percentage of BrdU positive cells that
have failed to complete centriole assembly and also blocked centrosome amplification.
Furthermore, both compounds 1 and 13 were the most effective at abrogating the spindle
checkpoint.
Based on the computational study of the chemical scaffold (Figure 1), the addition of
fragments into Site 1 did enhance potency and selectivity, with both the cyclohexyl moiety
(compound 13) and the isopropylsulfonylphenyl moiety (compound 1) resulting in
excellent inhibition. In addition, the fragments at Site 2, a 4-morpholinophenyl (compound
13) and a 4-hydroxypiperidinyl (compound 1), led to increased activity and improved drug
properties such as solubility.
Unlike other Mps1 inhibitors, compounds 1 and 13 prevent centrosome amplification. It
will be interesting to determine in the future whether the primary efficacy of these
compounds is through their effects on centrosomes, on the SAC, or on other Mps1
functions. Because tumors consist of a heterogeneous population of cells with and without
20

centrosome amplification, it would also be interesting to test how depletion of a population
of cells with centrosome amplification affects the overall tumor progression.
The most promising analog, compound 13, was selected for in vivo evaluation. Compound
13 in the formulation of DMSO: Tween 80: PEG400: saline (10: 5: 10: 75) was used for the
pilot plasma pharmacokinetic studies in mice. The extravascular (IP) administrations
resulted in an AUC of 20,031 hr*nmol/L following a 50 mg/kg dose, suggesting excellent
bioavailability. A dose of 10 mg/kg of compound 13 was selected for evaluation of
antitumor activity in nude mice containing Cal 51 breast cancer cell xenografts. At this
dose, compound 13 significantly decreased tumor growth over the 7-week study.
In summary, novel small molecule Mps1/TTK inhibitors have been identified as potential
targeted therapies for basal and luminal types of breast cancers using drug discovery
technologies, computational modeling, medicinal chemistry, cell culture and in vivo assays.
Since Mps1 has many critical roles in cells, future in-depth investigations of both anticancer
activities and safety profiles of these Mps1 inhibitors are warranted.
4. Materials and Methods
Proton (1H) and Carbon (13C) NMR spectrums were obtained on Bruker 300 MHz FT-NMR
and Bruker 400 MHz FT-NMR instrument at College of Pharmacy, The Ohio State
University. High Resolution Mass Spectrums (HR-MS) were obtained on Micromass LCT
spectrometer at the Campus Chemical Instrumentation Center of The Ohio State University.
All chemicals and solvents were purchased from standard commercial suppliers.
21

4.1. Computational drug design:
AutoDock version 4.0.048, 49 was used for the docking simulation. The Lamarckian genetic
algorithm (LGA) was selected for ligand conformational searching because it has enhanced
performance relative to simulated annealing or the simple genetic algorithm. For all
compounds, all hydrogens were added and Gasteiger charges 50 were assigned, then non-
polar hydrogens were merged. 80 × 100 × 70 3-D affinity grids centered on the empty
binding site with 0.375 Å spacing were calculated for each of the following atom types: a)
protein: A (aromatic C), C, HD, N, NA, OA, SA; b) ligand: C, A, OA, HD, NA, SA, e (electrostatic)
and d (desolvation) using Autogrid4. The ligand’s translation, rotation and internal torsions
are defined as its state variables and each gene represents a state variable. LGA adds local
minimization to the genetic algorithm, enabling modification of the gene population. The
docking parameters were as follows: trials of 100 dockings, population size of 250, random
starting position and conformation, translation step ranges of 2.0 Å, rotation step ranges of
50º, elitism of 1, mutation rate of 0.02, crossover rate of 0.8, local search rate of 0.06, and
100 million energy evaluations. Final docked conformations were clustered using a
tolerance of 1.5 Å root-mean-square deviations (RMSD).
4.2. Chemistry:
4.2.1. tert-Butyl 2-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-7H-pyrrolo[2,3-
d]pyrimidine-7-carboxylate (4)
The synthesis of tert-butyl 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (3)
was achieved from compound 2 according to reported procedure.41 For the synthesis of
tert-butyl 2-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-
22

7-carboxylate (4), tert-butyl 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (3,
0.239 g, 0.83 mmol), 2-(isopropylsulfonyl)-aniline (0.165 g, 0.83 mmol) and K2CO3 ( 0.220
g, 1.6 mmol) was dissolved in 3 mL t-butanol. The reaction mixture was degassed for 15
minutes. Pd2(dba)3 (0.046 g, 0.05 mmol) and XPhos (0.036 g, 0.076 mmol) was added to
the reaction mixture under nitrogen atmosphere and stirred for 4 hours at 100C. The
reaction mixture was cooled to room temperature, filtered, and partitioned between ethyl
acetate and water. The aqueous layer was extracted three times with ethyl acetate. The
combined organic layer was washed with water and brine, dried over sodium sulfate and
concentrated under reduced pressure. Product 4 was purified using silica gel column
chromatography with 20-30 % ethyl acetate - hexane mixture as the solvent system (solid;
yield 56%). 1H NMR (300 MHz, CDCl3) δ 9.97 (s, 1H), 8.82 (d, J = 8.5 Hz, 1H), 7.87 (d, J = 7.7
Hz, 1H), 7.72 (t, J = 7.3 Hz, 1H), 7.53 (d, J = 4.0 Hz, 1H), 7.25 (t, J = 7.7 Hz, 1H), 6.59 (d, J = 3.9
Hz, 1H), 3.23 (m, 1H), 1.68 (s, 9H), 1.30 (d, J = 6.8 Hz, 6H).
4.2.2. tert-Butyl 2-((4-(4-hydroxypiperidin-1-yl)-2-methoxyphenyl)amino)-4-((2-
(isopropylsulfonyl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (5)
tert-Butyl 2-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-
7-carboxylate (4, 0.211 g, 0.47 mmol), 1-(4-amino-3-methoxyphenyl)piperidin-4-ol (0.125
g, 0.56 mmol) and K2CO3 ( 0.195 g, 1.41 mmol) was dissolved in 2 mL t-butanol. The
reaction mixture was degassed for 15 minutes. Pd2(dba)3 (0.025 g, 0.028 mmol) and XPhos
(0.020 g, 0.042 mmol) was added to the reaction mixture under nitrogen atmosphere and
stirred for 6 hours at 100°C. The reaction mixture was processed in the same manner as
Compound 4. Product 5 was purified using silica gel column chromatography with 80-90
23

% ethyl acetate - hexane mixture as the solvent system (solid, yield 59%). 1H NMR (300
MHz, CDCl3) δ 8.37 (d, J = 5.1 Hz, 1H), 7.99 (d, J = 5.2 Hz, 1H), 7.89 (s, 1H), 7.55 (m, 2H), 7.38
– 7.25 (m, 1H), 7.24 – 7.03 (m, 1H), 6.84 (s, 1H), 6.59 (s, 1H), 6.53 (d, J = 4.2 Hz, 1H), 6.30 (d,
J = 4.0 Hz, 1H), 4.24 – 4.05 (m, 1H), 3.87 (m, 4H), 3.50 (m, 2H), 3.29 (m, 1H), 2.91 (m, 2H),
2.06 (m, 2H), 1.78 (m, 2H), 1.69 (s, 9H), 1.29 (d, J = 3.6 Hz, 6H).
4.2.3. 1-(4-((4-((2-(Isopropylsulfonyl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-2-
yl)amino)-3-methoxyphenyl)piperidin-4-ol (Compound 1)
tert-Butyl 2-((4-(4-hydroxypiperidin-1-yl)-2-methoxyphenyl)amino)-4-((2-
(isopropylsulfonyl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (5, 0.063 g,
0.10 mmol) was dissolved in 5 mL DCM. 1 mL TFA was added to the reaction mixture and
stirred for 4 hour at room temperature. The reaction was neutralized with saturated
NaHCO3 solution and extracted three times with ethyl acetate. The combined organic layer
was washed with water and brine, dried over sodium sulfate and was concentrated under
reduced pressure. Compound 1 was purified using preparative thin layer chromatography
with ethyl acetate and few drops of methanol as the solvent system (solid, yield 35%).
1H NMR (300 MHz, DMSO-d6) δ 11.40 (s, 1H), 9.48 (s, 1H), 8.81 (d, J = 8.4 Hz, 1H), 7.81 (m,
2H), 7.74 – 7.66 (m, 1H), 7.50 (s, 1H), 7.36 – 7.17 (m, 1H), 6.99 (s, 1H), 6.64 (s, 1H), 6.48 (d,
J = 9.9 Hz, 1H), 6.22 (s, 1H), 4.69 (d, J = 3.2 Hz, 1H), 3.81 (s, 3H), 3.72 – 3.55 (m, 1H), 3.58 –
3.39 (m, 3H), 2.80 (t, J= 11.2 Hz, 2H), 1.93 – 1.77 (m, 2H), 1.65 – 1.41 (m, 2H), 1.17 (d, J = 6.2
Hz, 6H). HR-MS (M + Na)+ calculated 559.2103; observed 559.2132.
24

4.2.4. tert-Butyl 4-((2-(isopropylsulfonyl)phenyl)amino)-2-((2-methoxy-
phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (6)
tert-Butyl 2-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-
7-carboxylate (4, 0.350 g, 0.78 mmol), 2-methoxyaniline (0.106 g, 0.86 mmol) and K2CO3
(0.317 g, 2.3 mmol) was dissolved in 4 mL t-butanol. The reaction mixture was degassed
for 15 minutes. Pd2(dba)3 ( 0.045 g, 0.049 mmol) and Xphos (0.040 g, 0.084 mmol) was
added to the reaction mixture under nitrogen atmosphere and stirred for 6 hours at 100°C.
The reaction mixture was processed in the same manner as Compound 4. Product 6 was
purified using silica gel column chromatography (solid, yield 49 %). 1H NMR (300 MHz,
DMSO-d6) δ 9.56 (s, 1H), 8.47 (d, J = 8.8 Hz, 1H), 8.40 (d, J = 7.2 Hz, 1H), 7.88 (d, J = 8.6 Hz,
1H), 7.78 (m, 2H), 7.42 (m, 2H), 7.02 (d, J = 7.8 Hz, 1H), 6.96 (t, J = 6.5 Hz, 1H), 6.84 (t, J = 8.1
Hz, 1H), 6.54 (d, J = 3.8 Hz, 1H), 3.87 (s, 3H), 3.50 – 3.33 (m, 1H), 1.63 (s, 9H), 1.13 (d, J = 6.8
Hz, 6H).
4.2.5. N4-(2-(isopropylsulfonyl)phenyl)-N2-(2-methoxyphenyl)-7H-pyrrolo-
[2,3-d]pyrimidine-2,4-diamine (7)
tert-Butyl 4-((2-(isopropylsulfonyl)phenyl)amino)-2-((2-methoxyphenyl)amino)-
7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (6, 0.100 g, 0.19 mmol) was deprotected in a
manner similar as Compound 5. Compound 7 was purified using silica gel column
chromatography (solid, yield 48 %). 1H NMR (300 MHz, DMSO-d6) δ 11.51 (s, 1H), 9.48 (s,
1H), 8.71 (d, J = 8.5 Hz, 1H), 8.29 (d, J = 6.6 Hz, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.75 (t, J = 6.7
Hz, 1H), 7.63 (s, 1H), 7.32 (t, J = 6.8 Hz, 1H), 7.13 – 7.00 (m, 2H), 6.98 – 6.78 (m, 2H), 6.27 (s,
1H), 3.86 (s, 3H), 3.52 – 3.27 (m, 1H), 1.16 (d, J = 6.9 Hz, 6H). 13C NMR (DMSO-d6, 75 MHz):
25

δ 155.24, 152.69, 152.65, 148.31, 139.49, 135.06, 130.85, 129.58, 123.47, 123.34, 122.54,
121.35, 121.01, 120.22, 119.35, 110.47, 98.88, 97.15, 55.65, 55.04, 14.80. HR-MS (M + Na)+
calculated 460.1419; observed 460.1312.
4.2.6. tert-Butyl 4-((2-(isopropylsulfonyl)phenyl)amino)-2-((2-methoxy-4-
morpholinophenyl) amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (8)
tert-Butyl 2-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-
7-carboxylate (4, 0.350 g, 0.78 mmol), 2-methoxy-4-morpholinoaniline (0.162 g, 0.78
mmol) and K2CO3 ( 0.317 g, 2.3 mmol), was dissolved in 4 mL t-butanol. The reaction
mixture was degassed for 15 minutes. Pd2(dba)3 ( 0.045 g, 0.049 mmol) and Xphos (0.040
g, 0.084 mmol) was added to the reaction mixture under nitrogen atmosphere and stirred
for 4 hours at 100°C. The reaction mixture was processed in the same manner as
Compound 4. Product 8 was purified using silica gel column chromatography (solid, yield
42 %). 1H NMR (300 MHz, CDCl3) δ 9.58 (s, 1H), 8.85 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 9.1 Hz,
1H), 7.91 (d, J = 7.4 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.55 (s, 1H), 7.33 (d, J = 4.0 Hz, 1H), 7.23
(t, J = 7.8 Hz, 1H), 6.58 (d, J = 10.2 Hz, 2H), 6.51 (d, J = 4.0 Hz, 1H), 3.91 (m, 7H), 3.37 – 3.22
(m, 1H), 3.21 – 3.13 (m, 4H), 1.74 (s, 9H), 1.34 (d, J = 6.8 Hz, 6H).
4.2.7. N4-(2-(Isopropylsulfonyl)phenyl)-N2-(2-methoxy-4-morpholinophenyl)-7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine (9)
tert-Butyl 2-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-
7-carboxylate (8, 0.100 g, 0.16 mmol) was deprotected in a manner similar as Compound 5.
Compound 9 was purified using silica gel column chromatography (solid, yield 36 %).
26

1H NMR (300 MHz, CDCl3) δ 9.52 (s, 1H), 9.28 (s, 1H), 8.88 (d, J = 8.2 Hz, 1H), 8.27 (d, J = 8.6
Hz, 1H), 7.89 (dd, J = 8.1, 1.3 Hz, 1H), 7.65 (t, J = 7.2 Hz, 1H), 7.19 (dd, J = 9.1, 5.8 Hz, 2H),
6.80 – 6.73 (m, 1H), 6.60 (d, J = 2.4 Hz, 1H), 6.55 (dd, J = 9.0, 1.7 Hz, 1H), 6.47 – 6.37 (m,
1H), 3.90 (m, 7H), 3.29 (dt, J = 13.9, 6.9 Hz, 1H), 3.21 – 3.10 (m, 4H), 1.32 (d, J = 6.9 Hz, 6H).
HR-MS (M + Na)+ calculated 545.1947; observed 545.1882.
4.2.8. 2-Chloro-N-cyclohexyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (10)
2,4-Dichloro-7H-pyrrolo[2,3-d]pyrimidine 2 (0.695 g, 3.75 mmol), cyclohexyl amine (0.406
g, 4.1 mmol) and TEA ( 0.758 g, 7.5 mmol) were dissolved in 18 mL ethanol and refluxed
overnight. The solvent was reduced and the residue was dissolved in ethyl acetate. The
organic layer was washed with water and brine, dried over sodium sulfate and
concentrated under reduced pressure. Compound 10 was purified by silica gel column
chromatography with 30-40 % EA-Hex mixture (solid, yield 78 %). 1H NMR (300 MHz,
DMSO-d6) δ 11.60 (s, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.40 (d, J = 3.8 Hz, 1H), 6.60 (d, J = 3.3 Hz,
1H), 3.95 (m, 1H), 1.94 – 1.90 (m, 2H), 1.77 – 1.74 (m, 2H), 1.65 – 1.61 (m, 1H), 1.42 –1.20
(m, 4H), 1.18 – 1.13 (m, 1H).
4.2.9. tert-Butyl 2-chloro-4-(cyclohexylamino)-7H-pyrrolo[2,3-d]pyrimidine-7-
carboxylate (11)
2-Chloro-N-cyclohexyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (10, 0.400 g, 1.6 mmol) was
dissolved in 6 mL of DCM . Di-tert-butyl dicarbonate ( 0.419 g, 1.92 mmol), DIEA (0.248 g,
1.92 mmol) and DMAP (0.010 g, 0.08 mmol) was added to the reaction mixture and
refluxed for 10 minutes. The reaction mixture was cooled, diluted with water and the
27

aqueous layer was extracted three times with DCM. The combined organic layer was
washed with water and brine, dried over sodium sulfate and concentrated under reduced
pressure. Product 11 was purified using silica gel column chromatography with 5-10 %
ethyl acetate - hexane mixture as the solvent system (solid, yield 85%). 1H NMR (300 MHz,
DMSO-d6) δ 7.90 (d, J = 7.6 Hz, 1H), 7.40 (d, J = 3.8 Hz, 1H), 6.86 (d, J = 3.3 Hz, 1H), 3.94 (m,
1H), 1.93 (m, 2H), 1.73 (m, 2H), 1.57 (s, 9H), 1.32 (m, 4H), 1.17 (m, 2H).
4.2.10. tert-Butyl 4-(cyclohexylamino)-2-((2-methoxy-4-morpholinophenyl)amino)-
7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (12)
tert-Butyl 2-chloro-4-(cyclohexylamino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (11,
0.518 g, 1.48 mmol), 2-methoxy-4-morpholinoaniline(0.323 g, 1.55 mmol) and K2CO3
(0.290 g, 2.1 mmol) was dissolved in 4 mL t-butanol. The reaction mixture was degassed
for 15 minutes. Pd2(dba)3 (0.068 g, 0.075 mmol) and XPhos (0.048 g, 0.102 mmol) was
added to the reaction mixture under nitrogen atmosphere and stirred for 6 hours at 100°C.
The reaction mixture was processed in the same manner as Compound 4. Product 12 was
purified using silica gel column chromatography with 60-70 % ethyl acetate - hexane
1
mixture as the solvent system (solid, yield 45%). H NMR (300 MHz, DMSO-d6) δ 8.48 (d, J
= 8.8 Hz, 1H), 7.27 (d, J = 7.4 Hz, 1H), 7.17 (s, 1H), 7.14 (d, J = 4.0 Hz, 1H), 6.71 (d, J = 4.0 Hz,
1H), 6.66 (d, J = 2.3 Hz, 1H), 6.43 (dd, J = 8.9, 2.1 Hz, 1H), 3.98 (m, 1H), 3.89 (s, 3H), 3.81 –
3.70 (m, 4H), 3.15 – 3.01 (m, 4H), 1.96 (m, 1H), 1.78 (d, J = 9.8 Hz, 2H), 1.73 – 1.67 (m, 1H),
1.60 (s, 9H), 1.44 – 1.21 (m, 6H).
28

4.2.11. N4-Cyclohexyl-N2-(2-methoxy-4-morpholinophenyl)-7H-pyrrolo[2,3-
d]pyrimidine-2,4-diamine (13)
tert-Butyl 4-(cyclohexylamino)-2-((2-methoxy-4-morpholinophenyl)amino)-7H-
pyrrolo[2,3-d]pyrimidine-7-carboxylate (12, 0.100 g, 0.19 mmol) was deprotected in a
manner similar as Compound 5. The product, Compound 13, was purified using silica gel
column chromatography with 90% ethyl acetate-hexane as the solvent system (solid, yield
33%). 1H NMR (300 MHz, DMSO-d6) δ 11.22 (s, 1H), 8.10 (s, 1H), 7.72 (s, 2H), 6.83 (s, 1H),
6.67 (d, J = 2.1 Hz, 1H), 6.55 (s, 1H), 6.49 (dd, J = 8.8, 2.1 Hz, 1H), 3.96 (d, J = 5.6 Hz, 1H),
3.86 (s, 3H), 3.80 – 3.69 (m, 4H), 3.16 – 3.02 (m, 4H), 1.97 - 1.80 (d, J = 10.2 Hz, 2H), 1.78 (d,
J = 10.2 Hz, 2H), 1.67 (m, 1H), 1.35 -1.25 (m, 5H). 13C NMR (DMSO-d6, 75 MHz): δ 171.96,
155.27, 154.55, 149.81, 148.83, 146.37, 122.48, 119.61, 118.01, 106.77, 99.94, 99.59, 96.90,
66.20, 55.64, 49.54, 49.10, 32.70, 25.39, 25.03, 21.00. HR-MS (M + Na)+ calculated
445.2328; observed 445.2277.
4.3 Breast cancer cell culture:
The human breast cancer cell lines MCF-7, MDA-MB-231, MDA-MB-453, MDA-MB-468, BT-
20, Sk-Br-3, AU565, T47D CAMA-1 and BT-474 were purchased from American Type
Culture Collection (ATCC, VA). Cal-51, HCC-1937, BT-549 cell lines were gift from Dr. Ching-
Shih Chen (The Ohio State University). Hs578T and MDA-MB-436 cell lines were provided
by Dr. Majumder Sarmila (The Ohio State University). The cells, except Sk-Br-3, AU565 and
BT-474, were maintained in a mixture of Dulbecco's modified Eagle's medium and Ham's
F12 medium (1 : 1) (DMEM/F12) without phenol red (Sigma-Aldrich, MO) supplemented
with 5% fetal bovine serum (FBS, Sigma-Aldrich, MO) and 1x antibiotic–antimycotic
29