Design and Optimization Leading to an Orally Active TTK Protein

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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

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Journal of Medicinal Chemistry

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 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

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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 ivdosed TTK/CLK2 inhibitor to an orally bioavailable TTK inhibitor is described.


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Introduction. 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. Cellular mechanism of action studies confirmed inhibition of both TTK and CLK2 by CC671.2 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 CC671 (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.


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N O O

O

N H

N

O

N H

N

N H

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. SEMprotection 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


4

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Cl

Cl

C4

Cl C2 N

N

a

N N H

Cl

b N

R1

R1 N SEM

2

c

N Cl

N

N SEM

N

R2

3a-3h

N SEM

N

4-20 SEM

d, e or f

R1 N

R2

N

N 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.


5


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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.

Table 1. Modification of 1 leads to improved physicochemical properties N O O

O

O

N H

N

N

O

N H

O

N

N H

H

N

O

N H

N

N H

4

1

CAL-51 potency Analog

amean

IC50

(M)a

Mol Wt

PSAb

cLogPb

1

0.028

513

127

5.0

4

0.497

395

92

3.9

reported, SEM available in Supporting information, Table 2,

bpipeline

pilot calculation


6

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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.

Table 2. C-4 position SAR

C4

O N

O

N O

N H

N

N H


7


TTK enzyme Analog

C4

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CAL-51 potency

# kinases Mol

Wtb

PSAb

IC50 (M)a

IC50 (M)a

0.0013

0.497

395

92

15/46

0.0072

1.959

383

101

12/46

6

0.0032

1.265

369

92

11/46

7

0.0013

0.190

409

92

10/46

0.001

0.198d

411

101

12/46

4 5

O

>80% inhc

8

O

9

OH

0.0006

0.069

440

101

25/46

10

OH

0.0007

0.044

440

112

21/46

0.0010

0.131

440

101

8/46

11

O amean


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 solventexposed region.1 The oral exposure and selectivity of 11 were high, serving as a starting point for


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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.

Table 3. C-2 position SAR O O

R3

N O

N H

N

N H

CAL-51 Analog

R3

potency IC50

Mol Wt

Rat PO PK

# kinases

AUC (Mh)c

>80% inhd

30e

8/46

PSAb

(M)a

O

11

N

0.131

440

101


9


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O

12

N

0.406

481

101

ND

10/46

0.053

454

93

14.7f

7/46

0.071

482

104

11.0f

8/46

0.037

511

125

9.5e

10/46

0.084

509

97

24f

7/46

O

N

13

O

N

14 HO

15

N N

HO O

N

16

N O amean


bpipeline

pilot

calculation, cdose 10 mg/kg, dsingle point assessment at 3 M, d n=1, eformulated in CMCT (0.5% CMC/0.25%tween-80), fformulated 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 orthosubstituents 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


10

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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.

Table 4. C-2 and C-5 position SAR

HO O

O

N

C5

N

R4

O

N H

N

N H

CAL-51 Analog

R4

C5

potency IC50

Mol

Wtb

Rat PO PK

# kinases

AUC (Mh)c

>80% inhd

PSAb

(M)a

10

Me

H

0.044

440

112

21.8e

21/46

17

Et

H

0.026

453

112

ND

15/46

H

0.015

508

112

16.1f

11/46

H

0.024

521

112

30e

8/46

Cl

0.010

556

112

26e

18 19

F3C

F3C

10/46 20

F3C

amean

10/258 reported, SEM available in Supporting information, Table 2,

bpipeline

pilot

calculation, cdose 10 mg/kg, dsingle point assessment at 3 M, eformulated in CMCT


11


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(0.5% CMC/0.25%tween-80), fformulated in 5%TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate), 10% captisol, 50 mM citrate buffer

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 Analog

CAL-51 p-TTK CAL-51 potency

# kinases

IC50 (M)a

IC50 (M)a

IC50 (M)a

>80% inhb

15

0.0013

0.016

0.037

10/46

16

0.0025

0.044

0.084

7/46 10/46

20

0.0007

0.009

0.010 10/258

amean

reported, SEM available in Supporting information, Table 2, bsingle point

assessment at 3 M


12

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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.


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Figure 3. TTK substrate phosphorylation inhibition post treatment with compound 20 as measured by Western blot

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.


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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).


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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).

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

0.001

0.032

0.059

0.104

0.107

0.217

0.261

0.567

G2019S 0.0007

0.001

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


16

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Human S9 Analog Met.

Cmax

Cmax

Stabilitya (M)

AUC (M·h)

(M)

AUC (M·h)

15

79

2.53

9.52

9.86

17.6

16

85

4.43f

24f

11.8

35.3

20

65

4.35

26.3

5.2

22.5

a%

2,

remaining at 60 min, bmean reported, SEM available in Supporting information, Table cformulated

edose

as a solution in CMCT (0.5% CMC/0.25%tween-80), ddose 10 mg/kg,

30 mg/kg in SCID mice, fformulated in 5%TPGS (D-α-tocopheryl polyethylene

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 dosedependent 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.


17


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.

1750 1500

Tumor Volume (mm3)

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

Page 18 of 41

1250

Vehicle (QW) 120 mg/kg, QW 90 mg/kg, QW 60 mg/kg, QW 30 mg/kg, QW 30 mg/kg, BID-QW Docetaxel (5 mg/kg, IV, Q4D)

1000 750 500 250 0 10

Docetaxel (Q4D)

12

14

16

18

20

22

24

QW

26

28

30

32

Days After Tumor Cell Inoculation


18

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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 95%. 1H 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,Ndimethylbenzamide (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), 7.057.08 (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[2,3d]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-2yl)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,


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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, 3H). MS (ESI) m/z 440.2 [M+1]+. 4-((4-(((1s,4s)-4-Hydroxy-4-methylcyclohexyl)oxy)-7H-pyrrolo[2,3-d]pyrimidin-2yl)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]+. (S)-4-(4-(2,2-Dimethyltetrahydro-2H-pyran-4-yloxy)-7H-pyrrolo[2,3-d]pyrimidin-2ylamino)-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-2yl)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)-3methoxybenzoic 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 semipreparative HPLC to obtain the title compound (0.073 g, 0.152 mmol, 63% yield, HPLC purity >


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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]+. (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-2yl)amino)-3-methoxyphenyl)-4-methylpiperidin-4-ol (14). Yield 0.075 g, 0.16 mmol, 43% over two steps, HPLC purity >95%. 1H 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-2yl)amino)-3-methoxyphenyl)piperazin-1-yl)-2-hydroxyethanone (15). To a solution of 2hydroxyacetic acid (0.672 g, 8.84 mmol) in DMF (20 mL) was added HATU (3.7 g, 9.72 mmol),


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DIEA (4.63 mL, 26.5 mmol) and (S)-4-((2,2-dimethyltetrahydro-2H-pyran-4-yl)oxy)-N-(2methoxy-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 semipreparative 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-3yl)piperazin-1-yl]phenyl]-7H-pyrrolo[2,3-d]pyrimidin-2-amine (16). Yield 3.78 g, 7.4 mmol, 74%, HPLC purity > 99%. 1H 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-2yl)amino)-N,N-dimethylbenzamide (17). Yield 0.126 g, 0.28 mmol, 34%, HPLC purity >99%. 1H NMR (400 MHz, DMSO-d ) δ 11.46 - 11.58 (m, 1 H), 8.48 - 8.61 (m, 1 H), 7.54 - 7.62 (m, 1 6 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 -


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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 H), 1.33 - 1.45 (m, 3 H), 1.09 - 1.21 (m, 3 H). MS (ESI) m/z 454.3 [M+1]+. 4-((4-(((1s,4s)-4-Hydroxy-4-methylcyclohexyl)oxy)-7H-pyrrolo[2,3-d]pyrimidin-2yl)amino)-N,N-dimethyl-3-(2,2,2-trifluoroethoxy)benzamide (18). Yield 0.076 g, 0.15 mmol, 52%, HPLC purity >99%. 1H NMR (500 MHz, DMSO-d6) δ 11.42 - 11.63 (m, 1 H), 8.47 - 8.57 (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-2yl)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-2yl]amino]-N,N-dimethyl-3-[(1R)-2,2,2-trifluoro-1-methyl-ethoxy]benzamide

(20).

Yield 3.44 g, 6.2 mmol, 47%, HPLC purity >99%. 1H 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),


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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® 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® 647labeled, 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 412% 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 antimouse 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 (LICOR 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


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Envision Multilabel (Perkin Elmer) plate reader. The percent inhibition at each compound concentration was determined by normalizing data to the DMSO control values for each set of triplicate wells. All data were analyzed using XLfit from IDBS. The formula used for determining IC50 in XLfit was model number 205, which utilizes a 4-parameter logistic model or sigmoidal dose-response model to calculate IC50 values. Full experimental details have been previously described.25 Crystallization. –The TTK kinase domain with a thrombin-cleavable, N-terminal 6XHis tag was expressed in E.coli strain BL21(DE3) (Life Technologies). Cells were grown in LB media, and induced for 4 hours at 20°C. Initial capture was performed via nickel affinity chromatography using nickel-NTA resin. The 6XHis tag was cleaved by addition of thrombin, and the protein passed back over nickel-NTA resin. Cleaved TTK was purified by S-75 size exclusion chromatography (GE Healthcare), and concentrated to 11mg/mL. TTK was crystallized by sitting drop vapor diffusion at 4°C in the presence of 1mM of compound 20. The TTK:20 complex was mixed 1:1 with, and subsequently equilibrated against, a solution of 100mM Tris pH 8.5, 300mM Sodium acetate, 8% PEG 20K, 8% PEG 500 MME. Crystals were cryoprotected by addition of 20% ethylene glycol, and flash cooled under liquid nitrogen. Diffraction data was collected at the Canadian Light Source, beamline CMCF-08ID. Data were indexed, integrated and scaled using HKL2000.26 The structure was solved using PHASER molecular replacement.27 Manual model building was performed using COOT, and refined using CCP4.28-29 Coordinates and structure factors have been deposited in the Protein Data Bank with the accession code 6N6O.


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Computational Methods and Modeling. The Schrodinger Small-Molecule Drug Discovery Suite was used for small molecule-protein and crystal structure visualization and analysis. Maestro: Schrödinger Release 2017-1: MS Jaguar, S., LLC, New York, NY, 2017. The Supporting Information contains additional modeling studies. In Vivo Studies. All animal studies were performed under protocols approved by the Celgene Institutional Animal Care and Use Committee (IACUC). Animals were acclimatized to the animal housing facility for a period of 7 days prior to the beginning of the experiment. Female 6-8 weeks old CB17 SCID (severe combined immunodeficiency) (Charles River Laboratories) mice were housed in a barrier facility in micro-isolator cages at 10 animals per cage. Mice were fed with Harlan-Teklad LM- 485 Mouse/Rat Sterilizable Diet and autoclaved water ad libitum and maintained on a 12 h light/dark cycle. SCID mice were inoculated subcutaneously with 2×106 CAL-51 cells. Tumor volumes were determined prior to the initiation of treatment and considered as the starting volumes. Tumors were measured twice a week for the duration of the study. The long and short axes of each tumor were measured using a digital caliper in millimeters. The tumor volumes were calculated using the formula: width2 × length/2. The tumor volumes were expressed in cubic millimeters (mm3). Mechanism of Action Studies were done with mice bearing CAL-51 tumors ranging from 300-500 mm3. At predetermined time points after compound administration, the mice were euthanized, the tumors were dissected and snap frozen in liquid nitrogen MSD assays. MSD electrochemiluminescent immunoassay kits and protocol were used for measuring the pHH3 Ser10 and cleaved caspase. Xenograft and in vivo biomarker data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism. A one-way analysis of


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variance (ANOVA) was performed for tumor volume and PD marker measurements. Post-hoc analysis was performed using Dunnett’s test where all treatment groups are compared with the vehicle control.

Author Information *Corresponding Author: Tel: 858-795-4854, [email protected] Present Addresses: Current address information for K. Fultz, A. Kulkarni, K. Condroski, and K. Leftheris available from corresponding author upon request. Notes: The authors declare no competing financial interest. All authors are employees of Celgene, except K. Fultz, A. Kulkarni, K. Condroski, and K. Leftheris, who were Celgene employees at the time of their contribution to this work.

Acknowledgements The authors thank the Celgene San Diego DMPK department for analysis, the Celgene San Diego analytical and compound management groups for project support, Deborah Mortensen for editorial comments and helpful discussions, and Lawrence Hamann and Jorge DiMartino for project guidance. Crystal structure determination was performed at the Canadian Light Source using beamline 08ID-1. The Canadian Light Source is supported by the Canada Foundation for Innovation, Natural Sciences and Engineering Research Council of Canada, Western Economic


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Diversification Canada, the National Research Council Canada, the Canadian Institutes of Health Research, the University of Saskatchewan, and the Government of Saskatchewan.

Abbreviations: TNBC, triple negative breast cancer; Mps1, monopolar spindle 1; CLK2, CDC2like kinase; SAC, spindle assembly checkpoint; KNL1, kinetochore scaffold1; LRRK2, Leucinerich repeat kinase 2; JNK, c-Jun N-terminal kinase; DYRK3, dual specificity tyrosinephosphorylation-regulated kinase 3; TSSK, testis-specific serine/threonine-protein kinase 2; Ltk, Leukocyte receptor tyrosine kinase; SEM, standard error of the mean; TGI, tumor growth inhibition; BWL, body weight loss.

Supporting Information Rat PO PK for compound 1, averages and SEM values for potency and PK data, in vitro kinase selectivity data for compound 20, additional TTK crystal structure views, BWL graph for xenograft study, experimental procedures for intermediates, and molecular formula strings of final compounds are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

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R.; Pauls, H. W., Discovery of pyrazolo[1,5-a]pyrimidine TTK inhibitors: CFI-402257 is a potent, selective, bioavailable anticancer agent. ACS Med. Chem. Lett. 2016, 7 (7), 671-675. 11.

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I.; Bray, M. R.; Mak, T. W., Functional characterization of CFI-402257, a potent and selective Mps1/TTK kinase inhibitor, for the treatment of cancer. Proc. Natl. Acad. Sci. U S A 2017, 114 (12), 3127-3132. 12.

Faisal, A.; Mak, G. W. Y.; Gurden, M. D.; Xavier, C. P. R.; Anderhub, S. J.; Innocenti,

P.; Westwood, I. M.; Naud, S.; Hayes, A.; Box, G.; Valenti, M. R.; De Haven Brandon, A. K.; O'Fee, L.; Schmitt, J.; Woodward, H. L.; Burke, R.; vanMontfort, R. L. M.; Blagg, J.; Raynaud, F. I.; Eccles, S. A.; Hoelder, S.; Linardopoulos, S., Characterisation of CCT271850, a selective, oral and potent MPS1 inhibitor, used to directly measure in vivo MPS1 inhibition vs therapeutic efficacy. Br. J. Cancer 2017, 116 (9), 1166-1176. 13.

Innocenti, P.; Woodward, H. L.; Solanki, S.; Naud, S.; Westwood, I. M.; Cronin, N.;

Hayes, A.; Roberts, J.; Henley, A. T.; Baker, R.; Faisal, A.; Mak, G. W.; Box, G.; Valenti, M.; De Haven Brandon, A.; O'Fee, L.; Saville, H.; Schmitt, J.; Matijssen, B.; Burke, R.; van Montfort, R. L.; Raynaud, F. I.; Eccles, S. A.; Linardopoulos, S.; Blagg, J.; Hoelder, S., Rapid discovery of pyrido[3,4-d]pyrimidine inhibitors of monopolar spindle kinase 1 (MPS1) using a structure-based hybridization approach. J. Med. Chem. 2016, 59 (8), 3671-3688. 14.

Kusakabe, K.; Ide, N.; Daigo, Y.; Itoh, T.; Yamamoto, T.; Hashizume, H.; Nozu, K.;

Yoshida, H.; Tadano, G.; Tagashira, S.; Higashino, K.; Okano, Y.; Sato, Y.; Inoue, M.; Iguchi, M.; Kanazawa, T.; Ishioka, Y.; Dohi, K.; Kido, Y.; Sakamoto, S.; Ando, S.; Maeda, M.; Higaki, M.; Baba, Y.; Nakamura, Y., Discovery of imidazo[1,2-b]pyridazine derivatives: selective and


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orally available Mps1 (TTK) kinase inhibitors exhibiting remarkable antiproliferative activity. J. Med. Chem. 2015, 58 (4), 1760-1775. 15.

Uitdehaag, J. C. M.; de Man, J.; Willemsen-Seegers, N.; Prinsen, M. B. W.; Libouban,

M. A. A.; Sterrenburg, J. G.; de Wit, J. J. P.; de Vetter, J. R. F.; de Roos, J.; Buijsman, R. C.; Zaman, G. J. R., Target residence time-guided optimization on TTK kinase results in inhibitors with potent anti-proliferative activity. J. Mol. Biol. 2017, 429 (14), 2211-2230. 16.

Jemaa, M.; Galluzzi, L.; Kepp, O.; Senovilla, L.; Brands, M.; Boemer, U.; Koppitz, M.;

Lienau, P.; Prechtl, S.; Schulze, V.; Siemeister, G.; Wengner, A. M.; Mumberg, D.; Ziegelbauer, K.; Abrieu, A.; Castedo, M.; Vitale, I.; Kroemer, G., Characterization of novel MPS1 inhibitors with preclinical anticancer activity. Cell Death Differ. 2013, 20 (11), 1532-1545. 17.

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Woodward, H. L.; Innocenti, P.; Cheung, K. J.; Hayes, A.; Roberts, J.; Henley, A. T.;

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37


19.

Page 38 of 41

Compound 20 binds comparably to related 2, 5-trisubstituted-7H-pyrrolo-[2,3-

d]pyrimidine analogs in the TTK ATP binding pocket (see PDB Code 6B4W). Similar observed G-loop induced fit and binding site interactions are described in reference 1. 20.

The Schrodinger Small-Molecule Drug Discovery Suite 2017-1, S., LLC, New York,

NY, 2017 was used for all crystal structure and small molecule-protein visualization and analysis. Schrödinger Release 2017-1: Maestro, Schrödinger, LLC, New York, NY, 2017. Schrödinger Release 2017-1: Schrödinger Suite 2017-1 Protein Preparation Wizard; Epik, Schrödinger, LLC, New York, NY, 2016; Impact, Schrödinger, LLC, New York, NY, 2016; Prime, Schrödinger, LLC, New York, NY, 2017. Schrödinger Release 2017-1: SiteMap, Schrödinger, LLC, New York, NY, 2017. Halgren, T., "Identifying and characterizing binding sites and assessing druggability," J. Chem. Inf. Model., 2009, 49, 377–389. Halgren, T., "New method for fast and accurate binding-site identification and analysis," Chem. Biol. Drug Des., 2007, 69, 146–148. 21.

In the crystal structure, a molecule of pentaethylene glycol binds adjacent to the ligand

and interacts directly with catalytic residue Lys553. The pentaenthylene glycol wraps around residue Lys553 and may influence the conformation of proximal residues. Also, the activation loop is not depicted in the structure, as it was not resolved. The ligand may also form additional interactions with the activation loop residues which are not present in the crystal structure. 22.

The residue Cys604 is small and fairly unique residue that creates a larger pocket in the

hinge region of TTK relative to other kinases. For details refer to J. Med. Chem. 2017, 60, 89899002 and references therein. 23.

SelectScreen® Kinase Profiling Services: Thermo Fisher Scientific, M., WI, USA.


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24.

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K.; Honigberg, L.; Jubb, A. M.; Katavolos, P.; lee, D. W.; Lewin-Koh, S.; Lin, T.; Liu, X.; Liu, S.; Lyssikatos, J. P.; O'Mahony, J.; Reichelt, M.; Roose-Girma, M.; Sheng, Z.; Sherer, T.; Smith, A.; Solon, M.; Sweeney, Z. K.; Tarrant, J.; Urkowitz, A.; Warming, S.; Yaylaoglu, M.; Zhang, S.; Zhu, H.; Estrada, A. A.; Watts, R. J., Effect of selective LRRK2 kinase inhibition on nonhuman primate lung. Sci. Transl. Med. 2015, 7 (273), 1-12. 25.

Shultz, M. D., Two decades under the influence of the rule of five and the changing

properties of approved oral drugs. J. Med. Chem. [Online early access]. DOI: 10.1021/acs.jmedchem.8b00686. Published Online: Sept 13, 2018. https://pubs.acs.org/doi/10.1021/acs.jmedchem.8b00686 (accessed Sept 13, 2018). 26.

Otwinowski, Z.; Minor, W., Processing of X-ray diffraction data collected in oscillation

mode. Methods Enzymol. 1997, 276, 307-326. 27.

McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.;

Read, R. J., Phaser crystallographic software. J. Appl. Crystallogr. 2007, 40 (Pt 4), 658-674. 28.

Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K., Features and development of Coot.

Acta crystallographica. Section D, Biological crystallography 2010, 66 (Pt 4), 486-501. 29.

Murshudov, G. N.; Skubak, P.; Lebedev, A. A.; Pannu, N. S.; Steiner, R. A.; Nicholls, R.

A.; Winn, M. D.; Long, F.; Vagin, A. A., REFMAC5 for the refinement of macromolecular crystal structures. Acta crystallographica. Section D, Biological crystallography 2011, 67 (Pt 4), 355-367.


39


Table of Contents Graphic

HO

N O O

O

N H

O

O

N

O

N

N H

TTK IC50 = 0.005 M CAL-51 IC50 = 0.060 M %F = 0.9

O

N H

N

1500

Cl

N

N N H

TNBC CAL-51 Xenograft Data 1750

N H

CF3

TTK IC50 = 0.0007 M CAL-51 IC50 = 0.010 M %F = 60

Tumor Volume (mm3)

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

Page 40 of 41

1250

Vehicle (QW) 120 mg/kg, QW 90 mg/kg, QW 60 mg/kg, QW 30 mg/kg, QW 30 mg/kg, BID-QW Docetaxel (5 mg/kg, IV, Q4D)

1000 750 500 250 0 10

Docetaxel (Q4D)

12

14

16

18

20

22

24

QW

26

28

30

32

Days After Tumor Cell Inoculation


40

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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

Design and Optimization Leading to an Orally Active TTK Protein

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