Optimization of Pan-Pim Kinase Activity and Oral Bioavailability

Subscriber access provided by EKU Libraries

Article

Optimization of Pan-Pim Kinase Activity and Oral Bioavailability Leading to Diaminopyrazole (GDC-0339) for the Treatment of Multiple Myeloma Xiaojing Wang, Wesley Peter Blackaby, Vivienne Allen, Grace Ka Yan Chan, Jae H Chang, PoChang Chiang, Coura Diene, Jason Drummond, Steven Do, Eric Fan, Eric Harstad, Alastair Hodges, Huiyong Hu, Wei Jia, William Kofie, Aleksandr Kolesnikov, Joseph p Lyssikatos, Justin Q. Ly, Mizio Matteucci, John G. Moffat, Veerendra Munugalavadla, Jeremy M. Murray, David Nash, Cameron Noland, Geoffrey Del Rosario, Leanne Ross, Craig Rouse, Andrew Sharpe, Dionysos Slaga, Minghua Sun, Vickie Tsui, Heidi J. A. Wallweber, Shang-Fan Yu, and Allen Ebens J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01857 • Publication Date (Web): 04 Feb 2019 Downloaded from http://pubs.acs.org on February 5, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 50

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Optimization of Pan-Pim Kinase Activity and Oral Bioavailability Leading to Diaminopyrazole (GDC-0339) for the Treatment of Multiple Myeloma Xiaojing Wanga, *, Wesley Blackabyb, Vivienne Allenb, Grace Ka Yan Chana, Jae H. Changa, Po-Chang Chianga, Coura Dièneb, Jason Drummonda, Steven Doa, Eric Fana, Eric Harstada, Alastair Hodgesb, Huiyong Hua, Wei Jiaa, William Kofieb, Aleksandr Kolesnikova, Joseph P. Lyssikatosa, Justin Lya, Mizio Matteuccib, John G. Moffata, Veerendra Munugalavadlaa, Jeremy Murraya, David Nashb, Cameron Nolanda, Geoff Del Rosarioa, Leanne Rossa, Craig Rouseb, Andrew Sharpeb, Dionysos Slagaa, Minghua Suna, Vickie Tsuia, Heidi Wallwebera, Shang-Fan Yua, Allen J. Ebensa aGenentech,

bCharles

Inc., 1 DNA Way, South San Francisco, California 94080, United States.

River Discovery Research Services UK Limited, Chesterford Research Park, Saffron

Walden, Essex, CB10 1XL, United Kingdom.


Page 2 of 50

Page 3 of 50 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


ABSTRACT Pim kinases have been targets of interest for a number of therapeutic areas. Evidence of durable single-agent efficacy in human clinical trials validated Pim kinase inhibition as a promising therapeutic approach for multiple myeloma patients. Here, we report the compound optimization leading to GDC-0339 (16), a potent, orally bioavailable, and well tolerated panPim kinase inhibitor that proved efficacious in RPMI8226 and MM.1S human multiple myeloma xenograft mouse models and has been evaluated as an early development candidate.

Keywords: Pim kinases; Kinase inhibitor; Oral bioavailability; Lead optimization; GDC-0339; multiple myeloma



INTRODUCTION Multiple myeloma is an incurable B-cell neoplasm characterized by the accumulation of malignant plasma cells. Upon accumulation in the bone marrow, cancerous plasma cells crowd out the healthy blood cells and cause bone lesions, increased calcium level, anemia, and renal insufficiency.1 Multiple myeloma accounts for 10% of all hematological cancers, and although rare, represents the second most common hematological cancer.2 Standard of care therapies for the treatment of multiple myeloma, including DNA alkylators, glycocorticoids, IMiDs and proteasome inhibitors3, have evolved over the past half of a century and significantly improved the 5-year survival from ~10% to 66% in 2014.4 Despite effective therapies that can prolong patients’ lives, the quality of life for elderly and fragile patients (median age at diagnosis is 70 years5) may be compromised due to the undesirable side effects of current therapies. In addition, as health care improvements allow more people to live into their seventies and beyond, higher incidence of multiple myeloma would be expected, requiring safer and effective novel therapies. The Proviral Integration site in Moloney murine leukemia virus (Pim) kinases are frequently overexpressed in multiple myeloma, and inhibition of Pim kinase activity was found to be beneficial for the treatment of multiple myeloma.6-9 The Pim proteins are a family of constitutively active serine/threonine kinases (Pim-1, Pim-2 and Pim-3) that are highly homologous. Besides multiple myeloma, Pim kinase inhibitors are potentially useful in targeting other malignancies, including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), non-Hodgkin lymphoma (NHL) and triple-negative breast cancer.10-14 Pim kinase inhibitors are therefore highly sought after as a new cancer therapy. Numerous groups have reported the discovery of Pim kinase inhibitors,15-32 including previous


Page 4 of 50

Page 5 of 50 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


work conducted in our laboratory (46 in Figure 1).33-35 To date, three Pim inhibitors have entered Phase I clinical trials but none has been approved, i.e. SGI-1776,36-38 AZD120839,40 and LGH447 (Figure 1).15 LGH447 provided evidence of durable single-agent efficacy in human clinical trials, validating Pim kinase inhibition as a promising therapeutic rationale for multiple myeloma patients.7 In this article, we report the discovery and characterization of GDC-0339, a pan-Pim kinase inhibitor clinical candidate for the treatment of multiple myeloma. Pim inhibitors tested in clinical trials

NH2 NH2

N N

N

NH

F O F F

N

O N

HN

N S

F

NH N

O

O 1 (SGI-1776)

F 2 (AZD1208)

F

3 (LGH447)

Our previous work N N N O

HN

F N H

NH

NH

F HN N

H2N

N N

N N

4

5

N

N N

N

N

N H

O

N

N N

N 6

Figure 1. Selected Pim kinase inhibitors disclosed in the literature. RESULTS AND DISCUSSION Burger et al first disclosed pan-Pim kinase inhibitors such as 7 in Table 1, containing a diaminopyridyl moiety (highlighted in red) that forms a hydrogen-bond with a catalytic Lys67 residue of Pim-1.16 Inspired by this work, we prepared compound 8 in which the 3aminopyridine (in black) was replaced by a bioisosteric 5-aminothiazole group. Compound 8 showed excellent biochemical potency (Ki: 0.01–0.05 nM), improved permeability (Papp A-B:



Page 6 of 50

3 vs. 0.5  10-6 cm/s), and cellular potency (IC50: 0.05 vs. 0.16 M), but decreased solubility (28 vs. 934 M) and oral bioavailability (23% vs. 54%) compared to 7. To mitigate potential liabilities such as hERG inhibition, diaminopyrazole moiety was used in place of the diaminopyridine. This change, combined with expansion of the piperidine to the homopiperidine for optimal placement of the primary amine provided a prototype molecule 9. Compound 9 displayed a nice combination of the best properties of 7 and 8 in terms of biochemical potency, cellular potency, and solubility. In addition, the potency of 9 against the hERG potassium channel is 100-fold lower than that of 8 (10.1 and 0.11 M respectively, Table S2 in supporting information). However, 9 suffered from low permeability and high transporter efflux ratio in MDCK epithelial cell lines, resulting in low oral bioavailability in rats. Table 1. Identifying hits using literature aided drug design. ID Structure

N

7

N

NH2

NH N

O

F

H 2N

N

8

In vitro Ki (nM)a Pim-1 Pim-2 Pim-3

N

NH2

NH

F

O

N

H2N

S F

MM.1S IC50 (M)b

In vivo Sol. Pappd Rat (M)c A-B CLe B-A F%f ER

0.16

934

0.5 6 13

15 54%

8.4 1.5 110

0.01 0.05 0.02

0.05

28

3 9 3

16 23%

8.4 1.6 110

0.02 0.1 0.007

0.08

2523

0.6 11 18

12 8%

9.3 1.2 115

0.03 0.2 0.06

In silico c_pKag cgLogDh tPSAi

NH2 N

N

9

N NH

F

O

N

H2N

S F


Page 7 of 50 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


aBiochemical

assays against three Pim isoforms; geometric mean values are shown here; 95%

confidence intervals where n > 3 are in Table S1. bCellular proliferation assay; geometric mean values are shown here; 95% confidence intervals where n > 3 are in Table S1. cMeasured

thermodynamic solubility at pH 6.5. dPapp: measured permeability using Madin-

Darby Canine Kidney (MDCK) epithelial cell lines, A to B or B to A (10-6 cm/s); ER: efflux ratio calculated as Papp (B to A) divided by Papp (A to B). eTotal clearance in rat in vivo (mL/min/kg); compound was dosed at 1 mg/kg in a solution of DMSO/Cremphor/water (5/5/90) for 7, 8, and 9. fRat bioavailability was calculated using (AUCoral÷AUCiv) x (Doseiv÷Doseoral) x 100; compound was dosed at 5 mg/kg in a solution of PEG400/water for 7 and 8, a suspension of MCT in water for 9; red denotes decreased bioavailability compared to 9. gCalculated logarithm of acid dissociation constant of the most basic amine. hCalculated Genentech logarithm of distribution-coefficient at pH 7.4. iCalculated topological polar surface area.

A co-crystal structure of compound 9 bound to Pim-1 at 2.39 Å resolution provided further insight into the pharmacophore (Figure 2). Besides van der Waals interactions, several key hydrogen-bond interactions were identified and represented by dashed lines in Figure 2. Consistent with our initial design hypothesis, the pyrazole nitrogen forms a hydrogen-bond with Lys67. The carbonyl oxygen of the amide was engaged in a network of hydrogen-bonds with Glu89 and Asp186 through stable water molecules. The aminothiazole is within hydrogen-bond distance to the backbone carbonyl oxygen of Glu121. Due to the resolution, the 4-aminoazepane moiety was not explicitly defined. We speculated that the primary amine



would make salt bridges with the acidic residues (Asp128 and Glu171) and the surrounding water molecules.

Figure 2. 2.39 Å crystal structure of compound 9 (PDB code: 5V80, authors will release the atomic coordinates and experimental data upon article publication) bound to Pim-1. Hydrogen bond interactions are depicted as black dashed line.

In rat pharmacokinetics (PK) studies, compound 9 suffered from low oral bioavailability. At pH 7.4, a total of six hydrogen bond donors (HBD) are displayed in 9 which may contribute to the low permeability and high efflux in the MDCK cell lines.41 Therefore, we set out to reduce the total number of HBD to improve the oral bioavailability. Compound 10, with a 1,4diazepane replacing the 4-aminoazepane (Table 2), suffered from a drastic loss in both biochemical (10–50-fold) and cellular potency (163-fold). While the basic primary amine of 9 is well positioned to form multiple salt-bridge interactions to the acidic patch formed by Asp128 and Glu171 residues, the secondary amine of 10 may not be close enough to maintain similar beneficial interactions, resulting in the observed potency loss for 10. However, with one less HBD and lower basicity of the amine (c_pKa: 7.5 for 10 vs. 9.3 for 9), 10


Page 8 of 50

Page 9 of 50 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


demonstrated significantly improved oral bioavailability (F%: 67% vs. 8%) commensurate with its higher kinetic solubility (127 vs. 55 M) and permeability (Papp A-B: 5.9 vs. 0.6  106

cm/s) relative to 9. Compound 11 with an alcohol in place of the primary amine in 9, also

suffered a loss of potency (4–30-fold in biochemical and 26-fold in cellular assays). The modest increase in permeability (Papp A-B: 4.6 vs. 0.6  10-6 cm/s) was offset by the loss of solubility (< 1 vs. 55 M), which ultimately resulted in a modest gain in oral bioavailability (F%: 38% vs. 8%) compared to 9. Based on the co-crystal structure of 9 with Pim-1, we observed that the sulfur atom of the aminothiazole moiety is in close proximity (3.98 Å) to the backbone carbonyl oxygen of Glu121, providing a potential opportunity for an OS -hole interaction.42 We therefore made 12 without a NH2 group on the thiazole, hoping that the thiazole sulfur would be closer to the carbonyl oxygen of Glu121. Unfortunately, the modest gain in permeability (Papp A-B: 1.6 vs. 0.6  10-6 cm/s) and oral bioavailability (F%: 32% vs. 8%) were not adequate to offset the marked potency loss comparing 12 to 9 (30–80-fold in biochemical and > 313-fold in cellular assays). While decreasing the number of HBD resulted in the loss of potency, the enhanced permeability and oral bioavailability encouraged us to continue the strategy of optimizing physiochemical properties to improve PK profile. Table 2. Reducing the number of hydrogen-bond donors to improve permeability and oral bioavailability. R2

N N

NH

R1

ID

R1

R2

In vitro Ki (nM)a

F N

O

S

MM.1S IC50 (M)b

F

Kin. Sol.

In vivo Pappd Rat


In silico NH+OHg c_pKah


Pim-1 (M)c A-B CLe Pim-2 B-A F%f Pim-3 ER NH 0.02 0.6 9 -NH2 12 6 0.1 0.08 55 11 N 8% 9.3 0.007 18 0.2 5.9 NH 10 -NH2 9 5 5 13 127 21 N 67% 7.5 0.2 3.6 OH 0.09 4.6 11i -NH2 7 4 2 4 2.1 < 1 N 38% 3.2 0.2 0.9 NH 0.6 1.6 12 -H 40 4 8.1 17.6 >25 50 N 32% 9.3 0.4 11 a,b,c,dsee footnotes from Table 1; red denotes significant erosion on potency compared to 9; 2

2

green denotes improvement compared to 9. eTotal clearance in rat in vivo (mL/min/kg); compound was dosed at 1 mg/kg in a solution of DMSO/PEG400/water for 10, 11, and PEG400/water (40/60) for 12. fRat bioavailability calculated using (AUCoral÷AUCiv) x (Doseiv÷Doseoral) x 100; compound was dosed at 5 mg/kg in a suspension of MCT in water for 10, 11, and 12; green denotes improvement over 9. gCalculated total number of active protons at pH 7.4. hCalculated logarithm of acid dissociation constant of the most basic amine. iCompound

11 is a eutomer with unknown stereochemistry.

In parallel, we investigated the effects of reduced basicity of the primary amine on permeability and oral bioavailability. Direct substitution of a trifluoroethyl (13) or difluoroethyl (14) group on the primary amine significantly reduced the c_pKa, but unfortunately was not well tolerated in terms of biochemical potency (Table 3). Difluoro substitutions at the -position of the primary amine as exemplified by 15 maintained biochemical potency but had weak cellular potency, most likely a result of the increased lipophilicity and plasma protein binding (Table S2). Monofluoro substitution at the -position


Page 10 of 50

Page 11 of 50 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


of the primary amine in 16 or 17 was well tolerated with a concurrent improvement in permeability and oral bioavailability. Given these encouraging results, all other stereoisomers of 16 and 17 were characterized but overall, none of them was superior to 16 and 17. When compared to 17, 16 showed better Pim-2 binding potency (4-fold), better oral bioavailability, and was easier to synthesize. Difluoro substitution at the -position of the primary amine (18 and 19) was also well tolerated in the biochemical assay. Compound 19 displayed a larger cellular shift than 18, again potentially due to the higher lipophilicity and plasma protein binding of 19 (Table S2). Analog 18 had improved permeability (Papp A-B: 3.4 vs. 0.6  10-6 cm/s) and oral bioavailability (F%: 71% vs. 8%) compared to 9. Mono-substitution on either  or position of the primary amine such as in 20 – 23 maintained excellent potency, however only modestly reduced the calculated c_pKa (< 0.7) of the most basic amine and consequently had poor permeability, high efflux and low oral bioavailability compared to 9. In summary, there seemed to be a trend that permeability and oral bioavailability can be improved by adjusting the basicity of the most basic primary amino group in the azepane subseries. Lastly, compounds such as 24 and 25, with a heptane ring attached to the central pyrazole ring instead of an azepane, maintained good potency and had a modest improvement in permeability or oral bioavailability, however both required complex synthesis. Table 3. Reducing the basicity of the primary amine to improve permeability and oral bioavailability. R

N N

NH

ID R

F

O

N

H2N

S F

In vitro

In vivo


In silico


NH2

9 N

HN

13

CF3 N

HN

14

CF2H N

NH2

15

F F

N

16

NH2 F

N

17 NH2

18 N

F F NH2

19 N

F

20

F

NH2 O

N

21

NH2 OH

N

22 23

NH2 N O

24

Abs

NH2 F

Ki (nM)a Pim-1 Pim-2 Pim-3 0.02 0.1 0.007 0.2 9 0.02 0.08 6 0.005 0.04 0.3 0.1 0.03 0.1 0.02 0.08 0.4 0.009 0.005 0.04 0.009 0.003 ND 0.01 0.05 0.2 0.19 0.03 0.2 0.01 0.01 0.05 0.01 0.007 0.04 0.02 0.03 0.3 0.01

MM.1S IC50 (M)b

Kin. Sol. (M)c

0.08

55

ND

1

8

1

0.5

2.8

0.07

67

0.1

108

0.04

81

0.9

164

0.05

52

0.03

100

0.07

112

0.008

62

0.2

88

Page 12 of 50

Pappd Rat A-B B-A ER 0.6 11 18

c_pKah

CLe F%f 12 8%

9.3

ND

ND

4.4

ND

ND

6.0

27 91%

6.2

8 33%

7.8

12 24%

7.8

26 71%

8.4

ND

8.5

5 16%

8.6

15 3%

8.7

25 5%

8.7

2 3%

8.8

ND

9.1

0.4 28.1 11 7 14.0 2 6.5 13.1 2 3.4 12.6 3.8 14.5 15.2 1.1 0.9 12.1 13 0.1 4.3 32 1.4 10.6 7.7 n/a 9.4 3.4 n/a -


Page 13 of 50 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


0.006 2.8 3 O ND 0.07 16 5.4 24% 9.1 0.005 3.2 a,b,c,dSee footnotes from Table 1; ND: not determined, n/a: not available due to low mass 25

NH2

recovery. eTotal clearance in rat in vivo (mL/min/kg); compound was dosed at 1 mg/kg in a solution of DMSO/PEG400/water for 1518, 2123, and 25, PEG400/water (40/60) for 20. fRat

bioavailability calculated using (AUCoral÷AUCiv) x (Doseiv÷Doseoral) x 100; compound

was dosed at 5 mg/kg in a suspension of MCT in water for 1518, 20–23, and 25; green/red denotes better/worse than 9. hCalculated logarithm of acid dissociation constant of the most basic amine.

Based on their overall profiles balancing potency, PK and ease of synthesis, we considered compounds 16 and 18 for additional characterization. One differentiating property between 16 and 18 was time dependent inhibition (TDI) activity of CYP3A4. Since Pim inhibitors have a high potential to be combined with other therapeutic agents for improved efficacy, drug-drug interaction (DDI) as a result of TDI is highly undesired.43 As shown in Table 4, both compounds exhibited reversible inhibition of CYP3A4 (IC50: 3.8–7.5 M) using either midazolam (M) or testosterone (T) as the probe substrate. While 16 exhibited a moderate TDI liability with low to moderate DDI potential, the TDI risk associated with 18 was much higher and similar to the positive control mifepristone (kinact/KI ratio: 64). We previously reported our efforts in removing the TDI liability of 18 by methyl substitution at the -position of the basic amine as in 19.44 Unfortunately, cellular activity of 19 was poor, likely due to high plasma protein binding. In addition, compound 16 compared favorably over 18 with higher selectivity in the kinase panel (Table S7). Based on all these, 16 was selected for further profiling. Table 4. CYP3A4 reversible and time dependent inhibition (TDI) of 16 and 18.



16

18

Rev. M 7.5±2.2 3.8±2.5 IC50±SD T 6.3±3.3 4.9±2.9 (M) a TDIb 1.12 1.16 KI (M) -1 kinact (min ) 0.027 0.061 Ratio 24 52 aCYP3A4 reversible inhibition assay using either midazolam (M) or testosterone (T) as the probe substrate; IC50 was reported as a mean +/- standard deviation with n=10 for 16 and n=7 for 18. bCYP3A4 time dependent inhibition using testosterone as a probe; KI is the inhibitor concentration that supports half the maximal rate of inactivation; kinact is the maximal rate of enzyme inactivation; ratio calculated using 1000 x kinact/KI (mL/min/mol).

A co-crystal structure of 16 and Pim-1 was obtained to confirm the binding mode. Similar to 9, compound 16 was well positioned in the Pim-1 adenosine triphosphate (ATP) binding pocket with multiple van der Waals and polar-polar interactions. By overlaying both co-crystal structures, it was clear that the aromatic rings were perfectly aligned. In the co-crystal structure with 16, the aminofluoroazepane moiety was well resolved with clear salt bridge interactions from the primary amine to Asp128 and Glu171.


Page 14 of 50

Page 15 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. 1.71 Å crystal structure of compound 16 (PDB code: 6NO9, authors will release the atomic coordinates and experimental data upon article publication) bound to Pim-1. Hydrogen bond interactions are depicted as black dashed line.

Kinase selectivity of 16 was obtained against a panel of 277 kinases (Table S7). When tested at 0.1 M (500–5000-fold above Pim Ki), 16 showed higher than 50% binding to only 12 kinases. Ki values for these 12 kinases were determined and only 6 kinases had a lower than 500-fold Ki ratio over Pim-2 (Table 5). Among these, we ran a dose-response in the cellular assays for mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) and glycogen synthase kinase 3 (GSK3. The IC50 of 16 in each cellular assay were in the micromolar range and were at least 57-fold higher than that in MM.1S assay. We also tested 16 against a non-kinase selectivity panel, including G-protein coupled receptors (GPCRs), ion channels, and transporters. When 16 was tested at a concentration of 10 M, 6 out of 39 targets showed higher than 50% binding. In functional cell-based assays for these 6 targets, only 3 of them yielded a measurable IC50 and these were in the micromolar range. The IC50 against dopamine (DOP) receptor was 0.5 M; however, this may not have any physiological consequence since 16 is not brain penetrant. Compound 16 inhibited hERG potassium channel in a patch-clamp (PC) study with an IC50 of 2.7 M, which provided a 70-fold window to the unbound maximum concentration (0.039 M) at the projected human efficacious dose equivalent to a tumor growth inhibition of 90% in the RPMI8226 xenograft model (Figure 6).45 In limited 7day dose ranging studies in rats and monkeys, 16 was well tolerated at doses up to 100 mg/kg/day, which was at or above the projected human therapeutic plasma exposure. In all,



these studies illustrated a favorable safety profile that supported more comprehensive safety evaluation to enable clinical evaluation of 16. Table 5. Selectivity and in vitro safety profile of 16. Assay a Kinase selectivity

Result Binding% > 50% @ 0.1 M

12 out of 277 kinases DMPK (24x), PASK (43x), MAP4K4 Ki ratio < 500 (kinase/Pim-2) (48x), GSK3 (52x), GSK3 (133x), MNK1 (152x) MAP4K4: 5.7 [57x] Cellular IC50 (uM) [fold over IC50 in MM.1S] GSK3: 23 [230x] Non6 out of 39 enzymes Binding% > 50% @ 10 M kinase alpha 1a: 2.5b selectivity Functional IC50 (M) Na PCc tonic/phasic: 11.5/8.8 DOPd: 0.5; 5HT1a: 4.9b/2.5e f 2.7 hERG PC IC50 (M) aSee the supporting information for assay methods and details. bAntagonist assay for alpha 1a adrenergic receptor (alpha 1a) or serotonin 1a receptor (5HT1a). cSodium channel patch clamp assay. dDOP, dopamine receptor. eAgonist assay for 5HT1a. fhERG potassium channel patch clamp assay. The Absorption, Distribution, Metabolism, and Excretion (ADME) properties of 16 across species are listed in Table 6. Predicated hepatic stability (assuming the same unbound fractions in blood and microsomes) in liver microsomes and hepatocytes was low to moderate and relatively similar between the two assays, suggesting that the main route of metabolism was phase 1. The in vivo clearance varied and was the lowest in rat and highest in mouse. Oral bioavailability was moderate in rat and cyno but low in mouse. Based on these preclinical data, both the clearance and oral bioavailability were anticipated to be moderate in human.

Table 6. Preclinical ADME profiling of 16. Species

In vitro

In vivo


Page 16 of 50

Page 17 of 50 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


LM CLhepa mouse 76 rat 39 dog 21 cyno 34 human 11 aProjected hepatic clearance using

Hep CLc F%d Vssc T1/2c CLhepb 54 135 11e – 34f 6.8 0.9 37 8 33e – 28f 0.9 1.8 --21 --24 34 35g – 60f 5.0 1.8 10 ----liver microsomes (mL/min/kg). bProjected hepatic

clearance using hepatocytes (mL/min/kg). cTotal clearance in vivo (CL, mL/min/kg); volume of distribution at steady state (Vss, L/kg); half life (T1/2, hr); compound was dosed at a 1 mg/kg dose in mouse (n = 2) or rat (n = 2) in a solution of DMSO/PEG400/water and at a 0.5 mg/kg dose in cynomolgus (cyno) monkey (n = 2) in a solution of PEG400/water (40/60). dF% is oral bioavailability with 16 dosed in a suspension of MCT. eAmorphous 16 in a 5 mg/kg dose (n = 2). fCrystalline 16 in a 100 mg/kg dose (n = 3). gAmorphous 16 in a 1 mg/kg dose (n = 2).

The physiological downstream signaling of Pim kinases is mediated through the phosphorylation of a large number of substrates (Figure 4a), including regulators of protein translation (4EBP1, S6 ribosomal protein) and a regulator of cellular apoptosis (BAD). Western blot analysis of MM.1S myeloma cell line treated with a dose range of compound 16 revealed a constellation of Pim down-stream signaling events consistent with inhibition of Pim kinases (Figure 4b). These data demonstrated the dose-dependent modulation of phospho-BAD (pBAD), phospho-4EBP1 (p4EBP1) and phospho-S6 ribosomal protein (pS6).



Figure 4. Compound 16 Modulates known Pim substrates. a)Protein level by western blot analysis after 4 hour treatment with DMSO or increasing concentrations of 16 in MM.1S cells. b)Schematic

illustration of Pim-kinase signaling pathway.

Given that Pim kinases share common substrates with the PI3K-AKT-mTOR signaling pathway,46,47 a dose-response experiment combining both Pim inhibitor 16 and phosphoinositide 3 kinase (PI3K) inhibitor GDC-0941 was conducted.48 Compound 16 inhibited cell viability at a maximum of -60% (the bottom of the dose-response curve) in contrast to GDC-0941 at -100%. This was consistent with 16 being cytostatic while GDC0941 being cytotoxic. The relative IC50 of the combination treatment improved by 3-fold compared to each single agent, indicating a potential synergistic combination.


Page 18 of 50

Page 19 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Compound 16 is active in multiple myeloma cancer cells both as a single agent and in combination with PI3K inhibitor GDC-0941.

Compound 16 was then evaluated in vivo using multiple myeloma xenograft mouse models. In the RPMI8226 mouse model, 16 was orally administered with doses ranging from 1 to 300 mg/kg once per day for 21 days. Mice were well tolerated with body weight change less than 10%. Both clear dose-response and tumor regression were observed with 16 dosed as a single agent (Figure 6a). In a separate experiment, pharmacodynamic (PD) markers pS6 and pBAD were also shown to decrease with increasing doses of 16 (Figure 6b,c), consistent with the western blot data.



Figure 6. Single agent activity of 16 was observed in a RPMI8226 human multiple myeloma xenograft mouse model. a) Effect of 16 on tumor growth. Female C.B-17 SCID mice bearing subcutaneous tumors were administered with escalating doses of 16 or vehicle (0.5% methylcellulose/0.2% Tween-80) orally once daily for 21 days. Changes in tumor volumes over time for each treatment group are depicted as cubic spline fits generated via linear mixed effects analysis of log-transformed volumes. b,c) Effect of 16 on Pharmacodynamic (PD) markers. Tumors (n = 5/group) were excised from animals at indicated time after a single dose of 16 and processed for analysis of Pim kinase pathway markers as described in the supplemental method section. Levels of pS6 (Ser240/244) and pBAD (Ser112) were normalized to the matched total protein and shown as mean (±SEM) for each treatment group.


Page 20 of 50

Page 21 of 50 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


A second multiple myeloma model, MM.1S, was also evaluated dosing mice with 16 orally once per day. Similar to the RPMI8226 model, dose-dependent tumor growth inhibition was observed albeit with higher doses required for the same extent of inhibition. All mice were well tolerated with body weight change less than 15%. Tumor regression was observed at 450 mg/kg. However, given the significant weight loss at 450 mg/kg, data were not reported here.

Figure 7. Single agent activity of 16 was observed in a MM.1S human multiple myeloma xenograft mouse model. Female C.B-17 SCID mice bearing subcutaneous tumors were administered escalating doses of 16 or vehicle (0.5% methylcellulose/0.2% Tween-80) orally once daily for 21 days. Changes in tumor volumes over time for each treatment group are depicted as cubic spline fits generated via linear mixed effects analysis of log-transformed volumes.



To study the combination potential of Pim and PI3K inhibitors in vivo, we tested prototype compound 8 with GDC-0941 in xenograft mouse models early on before 16 was discovered. In the RPMI8226 xenograft mouse model, 8 and GDC-0941 combined well rendering a better tumor growth inhibition than each single agent alone (Figure 8a). Such combination effect was also evidenced by the tumor growth inhibition at the end of the study across 4 different multiple myeloma xenograft mouse models (Figure 8b). When used as single agents, Pim inhibitor 8 achieved slightly better tumor growth inhibition than PI3K inhibitor GDC-0941 in all models except NCI-H929. However, the combo group achieved better tumor growth inhibition than each single agent alone in all models.

Figure 8. Combination effect of 8 with a PI3K inhibitor (GDC-0941) was observed in human multiple myeloma xenograft models. Female C.B-17 SCID or SCID-beige mice bearing subcutaneous tumors were administered daily oral doses of vehicle (60% PEG400/40% water), 8 in 50 mg/kg, GDC-0941 in 75 mg/kg or the combination of 8 and GDC-0941 for 21 days. a)Changes

in tumor volumes over time for each treatment group in a RPMI8226 model are


Page 22 of 50

Page 23 of 50 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


depicted as cubic spline fits generated via linear mixed effects analysis of log-transformed volumes. b)Tumor growth inhibition (TGI) at the end of the study is shown for each treatment group in a RPMI8226 (Fig. 8a), MM.1S, NCI-H929 and OPM-2 model. TGI was calculated as percent area under the fitted tumor volume-time curve (AUC) per day in relation to the vehicle, using the formula: %TGI = 100 x [1 – (AUCtreatment/day ÷ AUCvehicle/day)].

The synthesis of building block 30 was accomplished in 4 steps and 16% overall yield (Scheme 1). Homoallyl bromide and ammonium in methanol were heated to yield precursor 27. Homoallyl bromide and 27 were then heated under weakly basic condition to yield the secondary amine which was then protected with a tert-butyloxycarbonyl (Boc) group to provide 28. Cyclization of 28 using Hoveyda-Grubbs catalyst yielded the Boc protected azepine 29. Subsequent deprotection under acidic condition yielded the HCl salt of building block 30. This reaction sequence was carried out on a multi-gram scale. Scheme 1. Synthesis of building block 30. Br 26

i

ii

NH2

N 28 Boc

27

iii iv H

N 30

H

Cl

N Boc 29

Reagents and conditions: (i) 12 M NH3 in MeOH, 90 C, 16 hr, 88%; (ii) (a) 26 in THF, NaHCO3, 60 C, 2 d; (b) Boc2O, (Et)3N, THF, 0 C to rt, 16 hr, 35% for two steps; (iii) Hoveyda-Grubbs catalyst, DCM, rt, 2 d, 53%. (iv) 4 N HCl in dioxane, MeOH, rt, 4 hr, 95%.



Page 24 of 50

With azepine 30 in hand, the building block 38 was synthesized in 7 steps and 44% yield prior to the SFC chiral separation (Scheme 2). We could not find examples in the literature of straightforward way to synthesize 4,5-diaminopyrazole substituted at the N1 position so we developed a facile regioselective synthesis. The readily commercially available 1-methyl-3nitropyrazole

31

was

treated

with

lithium

hexamethyldisilazane

(LiHMDS)

and

hexachloroethane at low temperature to yield the desired 5-chloro-1-methyl-4-nitropyrazole 32. Subsequent nucleophilic aromatic substitution (SNAr) reaction of 32 and 30, epoxidation of 33, and azide addition of 34 were all accomplished in excellent yields to generate 35. Deoxyfluorination using Deoxo-Fluor® led to compound 36 in a good yield, with the fluorine group in a trans orientation with respect to the azide group. Isolation of the trans diastereomer was surprising since typically, deoxyfluorination leads to an inversion of the alcohol stereocenter via a SN2 reaction mechanism. However, in our case, we believe azide group participated and intervened with the transition state and consequently led to the double inversion product 36. A similar neighboring group participation has been reported previously.49,50 Reduction of the azide group by Staudinger reaction, followed by Boc protection, and supercritical fluid chromatography (SFC) chiral separation yielded building block 38 as a single stereoisomer. Scheme 2. Synthesis of building block 38.


Page 25 of 50 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


N N

NO2

31 i N N

O Cl

ii

N N

NO2 32

iii

N NO2 33

34

OH

iv

N

N N

N3

Rel N

N N

NO2

NO2

35 v

NHBoc

Abs

F N

N N 38

NH2

Rel

N3

Rel

F

F vii

N

N N

NO2

37

vi N N

NO2

N

36

NO2

Reagents and conditions: (i) Cl3CCCl3, LiHMDS, THF, -70 C, 4 hr, 68%; (ii) 30, KF, DMSO, 70 C, 16 hr, 98%; (iii) mCPBA, DCM, rt, 3 hr, 98%. (iv) NaN3, NH4Cl, MeOH/H2O, 70 C, 16 hr, 100%; (v) Deoxo-Fluor®, DCM, rt, 16 hr, 82%; (vi) Ph3P, THF/H2O, 60 C, 3 hr, 88%; (vii) (a) (Boc)2O, DIPEA, DCM, rt, 16 hr, 94%; (b) SFC chiral separation.

The thiazole-containing building block 44 was prepared in 5 steps and 23% yield (Scheme 3). Ethyl (hydroxyimino)cyanoacetate 39 is readily commercially available and was reduced to aminocyanoacetate which was coupled with 2,5-difluorobenzoyl chloride in situ to yield intermediate 41. Treatment of 41 with Lawesson’s reagent, followed by Boc protection, and hydrolysis of the ester furnished building block 44. Scheme 3. Synthesis of building block 44.



O N

EtO

O

i OH

NH2

EtO

CN 39

F

O

ii

H N

EtO

CN 40

Page 26 of 50

CN

41

F

O iii

O

O

F N

HO

R

S

BocHN

F N

EtO

v F

44

S

N H

F

42 R = H 43 R = Boc

iv

Reagents and conditions: (i) aq. NaHCO3, Na2S2O4, H2O, 4 C to rt, 16 hr; (ii) 2,5difluorobenzoyl chloride, NaHCO3, DCM, 0 C, 50 min, 59% for two steps; (iii) Lawesson’s reagent, pyridine, reflux, 16 hr, 98%. (iv) (Boc)2O, DMAP, CH3CN, rt, 12 hr, 41%; (v) (a) LiOH•H2O, MeOH/H2O, 50 C, 14 hr; (b) 2 M HCl, 0 C, 96%.

Compound 16 was made in 3 steps and 32% yield starting from the two key building blocks 38 and 44 (Scheme 4). Iron-mediated reduction of the nitro group in 38 yielded aminopyrazole 45 which was then coupled with carboxylic acid 44 to yield 46. Acidpromoted deprotection of the Boc group in 46, afforded 16 in excellent yield. It is worth noting that a single crystal structure of 16 was obtained, confirming the stereochemistry (Figure S1 and Table S6). Scheme 4. Synthesis of final compound 16. Abs NHBoc

Abs

N N

F N NO2 38

NHBoc

Abs

N

N

H N

N

F

F i N N

N

ii

NH

NH2 45

R

F N

O N H

S

46 R = Boc 16 R = H


F iii

R

Page 27 of 50 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


Reagents and conditions: (i) NH4Cl, Fe powder, EtOH/H2O, 99 C, 2.75 hr, 91%; (ii) 44, DIPEA, PyBOP, DCM, rt, 48 hr, 39.8%; (iii) 4 N HCl in 1,4-dioxane, MeOH, rt, 20 hr, 89%.

CONCLUSIONS In summary, a novel scaffold containing a diaminopyrazole moiety was described as pan-Pim inhibitors. The development of a convenient and practical synthesis enabled the SAR development and scale-up synthesis for both efficacy and safety studies. Through lead optimization of the PK properties balanced with potency, selectivity, and ease of synthesis, 16 (GDC-0339) was identified as an early development candidate. Compound 16 is a potent and efficacious pan-Pim inhibitor, orally bioavailable, and well tolerated in safety studies. Compound 16 combined well with PI3K inhibition in vitro by decreasing IC50 and likely also in vivo by enhancing tumor growth inhibition. Looking forward, Pim inhibition may be also useful in combination with other pathway targets such as USP7,51 or applied to other disease indications such as T-cell acute lymphoblastic leukemia subset12 and triple-negative breast cancer. 13,14,52

EXPERIMENTAL SECTION All chemicals (including 31 and 39) and solvents were used directly as received from commercial suppliers. Syntheses of compounds 1 – 716,17,34-36,40,53 and 18 – 1944 were reported previously. Syntheses and characterizations of compounds 8 – 15, 17, and 20 – 25 are in supporting information. 1H NMR spectra were recorded on Bruker Avance 400 or 500 spectrometers. Chemical shifts are expressed in δ ppm referenced to an internal standard,



tetramethylsilane (δ = 0 ppm). Abbreviations used in describing peak signals are: br = broad signal, s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, m = multiplet. All final compounds were purified to have purity higher than 95% by reverse phase high performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC) or normal phase silica gel chromatography flash chromatography. The purity was assessed by reverse phase HPLC with an isocratic gradient of 5-95% acetonitrile in water (with either acid or base modifier) and monitored by ultraviolet diode array detection at wavelength 254 nm. Optical rotation was recorded using Autopol VI automatic polarimeter from Rudolph Research Analytical. Low-resolution mass spectra (LCMS) were recorded in a liquid chromatography-mass spectrometer in electrospray positive (ES+) mode. High-resolution mass spectra (HRMS) experiments were performed on a Dionex LC Ultimate 3000 coupled with a Thermo Scientific Q Exactive Orbitrap mass spectrometer using ESI as the ionization source and a Phenomenex XB-C18, 1.7 mm, 50 mm x 2.1 mm column with a 0.7 mL/min flow rate at 40 C for liquid chromatography (LC) separation. Solvent A was 0.1% formic acid in water and solvent B was 0.1% formic acid in acetonitrile. The gradient consisted of 298% solvent B over 7 min and held at 98% B for 1.5 min following equilibration for 1.0 min. The LC was monitored by UV absorbance at 220 and 254 nm. MS full scans with 35000 resolution were applied to all experiments.

5-Amino-N-(5-((4R,5R)-4-amino-5-fluoroazepan-1-yl)-1-methyl-1H-pyrazol-4-yl)-2-(2,6difluorophenyl)thiazole-4-carboxamide (16, GDC-0339). tert-butyl (4R,5R)-1-(4-(5-tertbutoxycarbonylamino-2-(2,6-difluorophenyl)thiazole-4-carboxamido)-1-methyl-1H-pyrazol5-yl)-5-fluoroazepan-4-ylcarbamate (10.11 g, 15.2 mmol) in 4 N HCl in dioxane (200 mL)


Page 28 of 50

Page 29 of 50 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


and methanol (200 mL) was stirred at room temperature for 20 hr. Evaporation under reduced pressure afforded a pale brown solid which was dissolved in 50% methanol in dichloromethane and added to an SCX cartridge (strong cation exchange chromatography). After washing with dichloromethane and methanol, elution with 1 N ammonia in methanol and evaporation of the eluent under reduced pressure afforded 5-amino-N-(5-((4R,5R)-4amino-5-fluoroazepan-1-yl)-1-methyl-1H-pyrazol-4-yl)-2-(2,6-difluorophenyl)thiazole-4carboxamide as the free base (6.3 g, 89%). Purity = 100%; m.p.: 185.73 °C; [α]D20 (deg cm3 g−1 dm−1) = +6.312 (c = 0.00301 g cm−3 in methanol); 1H NMR (400 MHz, DMSO-d6) δ 8.84 (s, 1H), 7.57 – 7.45 (m, 1H), 7.53 (s, 1H), 7.49 (s, 2H), 7.32 – 7.22 (m, 2H), 4.42 (m, 1H), 3.64 (s, 3H), 3.24 – 3.02 (m, 5H), 2.18 – 2.01 (m, 1H), 2.01 – 1.73 (m, 4H), 1.65 – 1.52 (m, 1H); 13C NMR (101 MHz, DMSO-d6) δ 162.52, 159.86 (dd, J = 6.2, 251.9 Hz), 158.68 (t, J = 2.6 Hz), 142.04, 133.91 (t, J = 4.0 Hz), 132.56, 131.56 (t, J = 10.8 Hz), 122.71, 113.10 – 112.85 (m), 112.67, 111.36 (t, J = 15.9 Hz), 98.39 (d, J = 167.4 Hz), 55.52 (d, J = 20.4 Hz), 48.86, 46.41 (d, J = 11.2 Hz), 35.93, 33.58 (d, J = 21.8 Hz), 32.73 (d, J = 7.6 Hz); 19F NMR (376 MHz, DMSO-d6) δ -111.24, -171.78; HRMS (m/z): [M+1]+ calcd for C20H23F3N7OS, 466.1559; found, 466.1626; elemental analysis (% calcd, % found for C20H22F3N7OS): C (51.60, 51.23), H (4.76, 4.5), F (12.24, 11.89), N (21.06, 20.81), O (3.44, oxygen could not be analyzed in the presence of fluorine) , S (6.89, 6.67). But-3-en-1-amine (27). 4-bromobut-1-ene (660 g, 4.9 mol) was added to 18.0 L of 12 M ammonia in MeOH. Then the mixture was stirred at 90 C in an autoclave overnight. The mixture was cooled to room temperature and the solvent was removed by concentration. The crude product was obtained as white solid (660 g, yield 88%).



tert-Butyl di(but-3-en-1-yl)carbamate (28). The mixture of but-3-en-1-amine (660 g, 4.41 mol), NaHCO3 (801 g, 9.57 mol) and 4-bromobut-1-ene (420 g, 3.12 mol) in 7.5 L THF was stirred at 60 C for two days. Then the mixture was cooled to 0 C, TEA (1098 g, 10.86 mol) was added, then (Boc)2O (633 g, 2.25 mol) in 1.5 L THF was added in dropwise. The result mixture was stirred at room temperature overnight. After the reaction was complete, which was quenched by 7.5 L water, extracted with EtOAc, the solvent was removed and the residue was purified by silica gel column chromatography (hexanes:ethyl acetate = 30:1) to afford the product as oil (350 g, yield 35%). 1H-NMR (CDCl3, 400 MHz) δ 5.78 – 5.72 (m, 2H), 5.07 – 4.99 (m, 4H), 3.22 (br, 4H), 2.31 – 2.23 (m, 4H), 1.44 (s, 9H). tert-Butyl 2,3,6,7-tetrahydro-1H-azepine-1-carboxylate (29). Hoveyda-Grubbs catalyst (10.0 g, 15.9 mmol) was added to a solution of tert-butyl di(but-3-en-1-yl)carbamate (224 g, 994 mmol) in degassed DCM (20 L). After the reaction mixture was stirred for two days at room temperature, the mixture was flowed through a silica column and washed with 5% EtOAc in PE in order to remove the catalyst. The crude product was obtained as two parts: 70.0 g (80% purity) and 60 g (unpurified). The unpurified 60 g product was re-dissolved in degassed DCM (6 L) and Hoveyda-Grubbs catalyst (0.6 g, 0.95 mmol) was added, after stirred for two days at room temperature, the reaction mixture was also flowed through a silica column and washed with 5% EtOAc in PE. Then 55.2 g (purified) product was obtained. The 125.2 g combined product was purified via silica gel column chromatography (0 – 3% EtOAc in hexanes) to give the product (104 g, 53% yield) as light yellow oil. 1H-NMR (CDCl3, 400 MHz) δ 5.73 (t, J = 2.7 Hz, 2H), 3.44 (br, 4H), 2.29 (br, 4H), 1.47 (s, 9H). (Z)-2,3,6,7-Tetrahydro-1H-azepine hydrochloride (30). 4 N Hydrogen chloride in 1,4dioxane (250 mL; 1 mol) was added over 5 minutes to a stirred, ice cooled solution of (Z)-


Page 30 of 50

Page 31 of 50 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


tert-butyl 2,3,6,7-tetrahydro-1H-azepine-1-carboxylate (50 g; 0.254 mol) in methanol (250 mL). On complete addition, the ice bath was removed and stirring continued at room temperature for 3.75 h. Volatiles were removed under reduced pressure and the residue triturated twice with diethyl ether (300 mL) to afford (Z)-2,3,6,7-tetrahydro-1H-azepine hydrochloride as a pale pink solid (32.3 g; 95%). 1H-NMR (DMSO-d6, 400 MHz) δ 9.54 (br, 2H), 6.40 – 6.25 (m, 2H), 3.15 – 3.05 (m, 4H), 2.55 – 2.40 (m, 4H). 5-Chloro-1-methyl-4-nitro-1H-pyrazole (32). To a solution of 1-methyl-4-nitro-1H-pyrazole (100 g, 0.78 mol) in THF (500 mL) was added lithium hexamethyldisilazane (LiHMDS) in THF (1 M, 1000 mL, 1.0 mol) dropwise via an addition funnel at -70 C for over 2 hr under nitrogen. The resultant dark mixture was stirred for another 1 hr at -70 C. A solution of hexachloroethane (236.7 g, 1.0 mol) in THF (500 mL) was added dropwise via an addition funnel over 75 min. The mixture was stirred for another 1.5 hr and quenched with a solution of saturated NH4Cl (1000 mL), followed by the addition of ethyl acetate (600 mL). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (1000 mL x 3). The combined organic layers were washed with saturated brine (800 mL), dried over Na2SO4 and the organic solvent was evaporated under reduced pressure. The crude product was purified on silica gel chromatography column (15% EtOAc in hexanes) to yield a white solid (86 g, 68%) as the desired product. 1H-NMR (CDCl3, 400 MHz) δ 8.14 (s, 1H), 3.92 (s, 3H). (Z)-1-(1-Methyl-4-nitro-1H-pyrazol-5-yl)-2,3,6,7-tetrahydro-1H-azepine (33). A mixture of (Z)-2,3,6,7-tetrahydro-1H-azepine hydrochloride (32.3 g; 0.24 mol), 5-chloro-1-methyl-4nitro-1H-pyrazole (37.2 g; 0.23 mol), potassium fluoride (56.24 g; 0.96 mol) and diisopropylethylamine (64 mL; 0.362 mol) in anhydrous DMSO (650 mL) was heated at 75



°C for 21 hr. On cooling, the mixture was poured into water (1500 mL), extracted with ethyl acetate (4 x 500 mL) and the combined organics washed with water (2 x 400 mL), brine (300 mL) and dried (MgSO4). The solvent was removed under reduced pressure to afford (Z)-1-(1methyl-4-nitro-1H-pyrazol-5-yl)-2,3,6,7-tetrahydro-1H-azepine as a light brown solid (50.74 g; 99%). 1H-NMR (CDCl3, 400 MHz) δ 8.00 (s, 1H), 5.95 – 5.85 (m, 2H), 3.80 (s, 3H), 3.30 – 3.20 (m, 4H), 2.45 – 2.35 (m, 4H). MS calcd for C10H15N4O2 [M+1]+: m/z 223.1, observed: 223.1. 4-(1-Methyl-4-nitro-1H-pyrazol-5-yl)-8-oxa-4-azabicyclo[5.1.0]octane (34). 77% metaChloroperbenzoic acid (77 g; 0.343 mol) was added portion-wise over 10 minutes to a stirred, ice cooled solution of (Z)-1-(1-methyl-4-nitro-1H-pyrazol-5-yl)-2,3,6,7-tetrahydro-1Hazepine (50.74 g; 0.23 mol) in dichloromethane (1000 mL). Ice bath used to control minor exotherm observed during a smaller scale reaction. On complete addition, the ice bath was removed and stirring continued at room temperature for 18 hr. The reaction mixture was washed with saturated sodium hydrogen carbonate (750 mL), 1 N sodium hydroxide (2 x 500 mL) and brine (350 mL). The organics were dried (MgSO4) and the solvent removed under reduced pressure to afford 4-(1-methyl-4-nitro-1H-pyrazol-5-yl)-8-oxa-4azabicyclo[5.1.0]octane as a pale yellow solid (55.6 g, 100% yield). 1H-NMR (CDCl3, 400 MHz) δ 7.98 (s, 1H), 3.75 (s, 3H), 3.50 – 3.35 (m, 2H), 3.30 – 3.20 (m, 2H), 2.95 – 2.80 (m, 2H), 2.35 – 2.15 (m, 4H). MS calcd for C10H15N4O3 [M+1]+: m/z 239.1, observed: 239.2. rel-(4R,5R)-5-Azido-1-(1-methyl-4-nitro-1H-pyrazol-5-yl)azepan-4-ol (35): To a stirred solution of 4-(1-methyl-4-nitro-1H-pyrazol-5-yl)-8-oxa-4-azabicyclo[5.1.0]octane (29.28 g) in methanol (350 mL) and water (90 mL) was added ammonium chloride (16.5 g; 0.308 mol) followed by sodium azide (20 g; 0.307 mol). The mixture was heated behind a blast screen at


Page 32 of 50

Page 33 of 50 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


70 °C for 22 hr, cooled then concentrated to 100 mL under reduced pressure at 40 °C. The concentrated solution was poured into water (1300 mL), extracted with dichloromethane (4 x 400 mL) and the combined organics dried (MgSO4). Evaporation under reduced pressure at 35 °C gave rel-(4R,5R)-5-azido-1-(1-methyl-4-nitro-1H-pyrazol-5-yl)azepan-4-ol (anti-isomer and a racemic mixture) as a pale yellow oil (34.5 g; 96% over 2 steps). 1H-NMR (CDCl3, 400 MHz) δ 8.03 (s, 1H), 3.85 – 3.77 (m, 1H), 3.77 (s, 3H), 3.65 – 3.55 (m, 1H), 3.45 – 3.15 (m, 4H), 2.85 – 2.70 (m, 1H), 2.25 – 2.10 (m, 2H), 2.05 – 1.85 (m, 2H). MS calcd for C10H16N7O3 [M+1]+: m/z 282.1, observed: 282.1. rel-(4R,5R)-4-Azido-5-fluoro-1-(1-methyl-4-nitro-1H-pyrazol-5-yl)azepane (36). 50% Deoxofluor in THF (111 mL; 0.307 mol) was added slowly over 20 minutes to a stirred, ice cooled solution of rel-(4R,5R)-5-azido-1-(1-methyl-4-nitro-1H-pyrazol-5-yl)azepan-4-ol (32.5 g; 0.115 mol) in dichloromethane (500 mL). On complete addition, the ice bath was removed and stirring continued at room temperature for 20 hr. The reaction mixture was re-cooled in an ice bath and saturated sodium hydrogen carbonate (400 mL) added dropwise (effervescence!). After stirring for 30 minutes the layers were separated and the aqueous layer extracted with dichloromethane (2 x 500 mL). Pooled organics were dried (MgSO4) and the solvent removed under reduced pressure. Flash column chromatography on silica eluting with 0–100% ethyl acetate in isohexane gradient afforded rel-(4R,5R)-4-azido-5-fluoro-1-(1methyl-4-nitro-1H-pyrazol-5-yl)azepane as a pale orange oil (26 g; 80%). 1H-NMR (CDCl3, 400 MHz) δ 8.03 (s, 1H), 4.90 – 4.65 (m, 1H), 4.00 – 3.85 (m, 1H), 3.77 (s, 3H), 3.40 – 3.10 (m, 4H), 2.35 – 2.05 (m, 3H), 1.95 – 1.75 (m, 1H). MS calcd for C10H15FN7O2 [M+1]+: m/z 284.1, observed: 284.3.



rel-(4R,5R)-5-Fluoro-1-(1-methyl-4-nitro-1H-pyrazol-5-yl)azepan-4-amine (37). A mixture of rel-(4R,5R)-4-azido-5-fluoro-1-(1-methyl-4-nitro-1H-pyrazol-5-yl)azepane (26 g; 91.8 mmol) and triphenylphosphine (24.1 g; 92 mmol) in tetrahydrofuran (400 mL) and water (80 mL) was heated at 60 °C for 20 hr, cooled and concentrated to approximate 80 mL under reduced pressure. Ethyl acetate (500 mL) was added and the mixture extracted with 1 N HCl (4 x 125 mL). Pooled acidic extracts were washed with ethyl acetate (500 mL), basified to pH 14 with 6 N NaOH and extracted with dichloromethane (3 x 400 mL). Combined extracts were dried (MgSO4) and the solvent removed under reduced pressure to give rel-(4R,5R)-5fluoro-1-(1-methyl-4-nitro-1H-pyrazol-5-yl)azepan-4-amine as a pale yellow oil (22 g; 93%). 1H-NMR

(CDCl3, 400 MHz) δ 8.03 (s, 1H), 4.60 – 4.40 (m, 1H), 3.77 (s, 3H), 3.45 – 3.10 (m,

5H), 2.35 – 1.90 (m, 3H), 1.80 – 1.65 (m, 1H), 1.60 (br s, 2H). MS calcd for C10H17FN5O2 [M+1]+: m/z 258.1, observed: 258.3. tert-Butyl (4R,5R)-5-fluoro-1-(1-methyl-4-nitro-1H-pyrazol-5-yl)azepan-4-ylcarbamate (38). Di-tert-butyl dicarbonate (28 g; 128.3 mmol) was added to a stirred, ice cooled solution of rel-(4R,5R)-5-fluoro-1-(1-methyl-4-nitro-1H-pyrazol-5-yl)azepan-4-amine (22 g; 85.6 mmol) and diisopropylethylamine (22.4 mL; 128.6 mmol) in dichloromethane (600 mL). On complete addition, the ice bath was removed and stirring continued at room temperature for 20 hr. The reaction mixture was washed with saturated sodium hydrogen carbonate (500 mL) and the aqueous layer re-extracted with dichloromethane (2 x 300 mL). Pooled organics were dried (MgSO4) and the solvent removed under reduced pressure. Flash column chromatography on silica eluting with 0–100% ethyl acetate in isohexane gradient afforded a pale yellow solid (29.7 g; 97%). Chiral separation (70 mL/min flow rate, 100 bar pressure, 40 °C, 6 min/cycle) of the racemic mixture by Berger MGII SFC using Chiralpak IA column


Page 34 of 50

Page 35 of 50 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


(250 x 21 mm, 5 m) with an isocratic mobile phase of 15% methanol (with 0.1% NH4OH) in carbon dioxide gave the desired product (second peak) as a single enantiomer. 1H-NMR (CDCl3, 400 MHz) δ 8.04 (s, 1H), 5.05 (m, 1H), 4.80 – 4.55 (m, 1H), 4.15 – 4.05 (m, 1H), 3.79 (s, 3H), 3.45 – 3.10 (m, 4H), 2.35 – 2.05 (m, 3H), 1.95 – 1.80 (m, 1H), 1.47 (s, 9H). MS calcd for C15H25FN5O4 [M+1]+: m/z 358.2, observed: 358.3. Ethyl 2-cyano-2-(2,6-difluorobenzamido)acetate (41). To a stirred solution of (E)-ethyl 2cyano-2-(hydroxyimino)acetate (200 g, 1.4 mol) in water (2.0 L) at 4 °C was added saturated NaHCO3 (200 ml) followed by Na2S2O4 (1200 g, 8.46 mol). After being stirred at room temperature overnight, the reaction mixture was basified with saturated NaHCO3 to adjusted PH to 8.09.0. To the resulting mixture was added DCM (2.0 L), followed by 2,5difluorobenzoyl chloride (102 g, 577 mmol) at 0 °C. The reaction mixture was stirred for 50 min and the organic layer was separated. The aqueous layer was extracted with DCM (1.0 L × 2). The combined organic layers were washed with brine (1.0 L), dried over Na2SO4, filtered, and concentrated under reduce pressure. The residue was purified by column chromatography on silica gel, eluting with a mixture of hexanes:ethyl acetate (5:1) to afford the desired compound (220 g, 59% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.42-7.46 (m, 1H), 6.95-7.00 (m, 3H), 5.67 (d, J = 7.6 Hz, 1H), 4.40 (q, J = 7.2 Hz, 2H), 1.38 (t, J = 4.0 Hz, 3H). MS calcd for C12H11F2N2O3 [M+1]+: m/z 269.1, observed: 269.2. Ethyl 5-amino-2-(2,6-difluorophenyl)thiazole-4-carboxylate (42). To a stirred solution of ethyl 2-cyano-2-(2,6-difluorobenzamido)acetate (110 g, 410 mmol) in pyridine (1500 ml) at room temperature was added Lawesson’s reagent (166 g, 410 mmol). The reaction mixture was heated under reflux for 16 hr, cooled to room temperature and concentrated under reduce pressure. The residue was partitioned between ethyl acetate (1.5 L) and water (1.0 L). The



organic layer was separated, washed with brine (1.0 L), dried over Na2SO4 and filtered. The solvent was removed under reduce pressure and the residue was purified by column chromatography on silica gel, eluting with a mixture of hexanes:ethyl acetate (10:1) to afford the desired compound (50 g, 43% yield) as a brown crude solid. Ethyl 5-((tert-butoxycarbonyl)amino)-2-(2,6-difluorophenyl)thiazole-4-carboxylate (43). To a solution of crude ethyl 5-amino-2-(2,6-difluorophenyl)thiazole-4-carboxylate (90 g, 317 mmol) in CH3CN (1.5 L) at room temperature was added 4-dimethylaminopyridine (DMAP, 2.0 g, 15.8 mmol) followed by (Boc)2O (83 g, 380 mmol). The reaction mixture was stirred at room temperature for 12 hr and concentrated under reduce pressure. The residue was dissolved in ethyl acetate (1.0 L), washed with saturated NaHCO3 (500 mL) and brine (500 mL). The organic solution was dried over Na2SO4, filtered and concentrated, and the crude residual was purified by column chromatography on silica gel, eluting with a mixture of hexanes:ethyl acetate (20:1) to give the desired compound (50 g, 41% yield) as a yellow solid. 1H

NMR (400 MHz, DMSO-d6) δ 9.94 (s, 1H), 7.55-7.59 (m, 1H), 7.24-7.29 (m, 2H), 4.34 (q,

J = 7.6 Hz, 2H), 1.47 (s, 9H), 1.30 (t, J = 7.6 Hz, 3H). MS calcd for C17H19F2N2O4S [M+1]+: m/z 385.1, observed: 385.2. 5-((tert-Butoxycarbonyl)amino)-2-(2,6-difluorophenyl)thiazole-4-carboxylic acid (44). To a solution of 43 (62 g, 161 mmol) in MeOH:H2O (2 L, 1:1) was added LiOH.H2O (34 g, 807 mmol). The reaction mixture was heated at 50 ºC for 14 hr, cooled to 0 ºC and acidified with 2 M HCl to pH 4. The mixture was stirred for 10 min, filtered and dried in vacuo to afford the desired compound (55 g, 96% yield) as a pale white solid: 1H NMR (400 MHz, DMSO-d6) δ 12.87 (bs, 1H), 7.53 – 7.51 (m, 1H), 7.30 – 7.26 (m, 2H), 1.49 (s, 9H). MS calcd for C15H15F2N2O4S [M+1]+: m/z 357.1, observed: 357.2.


Page 36 of 50

Page 37 of 50 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


tert-Butyl (4R,5R)-5-fluoro-1-(4-amino-1-methyl-1H-pyrazol-5-yl)azepan-4-ylcarbamate (45). To a stirred solution of tert-butyl (4R,5R)-5-fluoro-1-(1-methyl-4-nitro-1H-pyrazol-5yl)azepan-4-ylcarbamate (15.0 g; 42 mmol) in ethanol (1000 mL) and water (100 mL) was added ammonium chloride (11.34 g; 210 mmol) and iron powder (9.4 g; 168 mmol). The mixture was heated at 99 °C for 2.75 hr, cooled, filtered through Celite® and evaporated to approximately 100 mL. The concentrate was diluted with water (1000 mL) and extracted with ethyl acetate (2 x 500 mL). The pooled extracts were washed with water (200 mL), dried (MgSO4) and the solvent removed under reduced pressure to give tert-butyl (4R,5R)-5-fluoro1-(4-amino-1-methyl-1H-pyrazol-5-yl)azepan-4-ylcarbamate as a pale brown solid (12.6 g; 91%). 1H-NMR (CDCl3, 400MHz) δ 7.13 (s, 1H), 6.35 – 6.20 (m, 1H), 4.85 – 4.65 (m, 1H), 4.35 – 4.15 (m, 1H), 3.66 (s, 3H), 3.45 – 3.30 (m, 2H), 3.15 – 2.90 (m, 2H), 2.65 (s, 2H), 2.30 – 2.15 (m, 1H), 2.15 – 1.95 (m, 2H), 1.90 – 1.80 (m, 1H), 1.45 (s, 9H). MS calcd for C15H27FN5O2 [M+1]+: m/z 328.2, observed: 328.2. tert-Butyl (4R,5R)-1-(4-(5-tert-butoxycarbonylamino-2-(2,6-difluorophenyl)thiazole-4carboxamido)-1-methyl-1H-pyrazol-5-yl)-5-fluoroazepan-4-ylcarbamate (46). A mixture of tert-butyl (4R,5R)-5-fluoro-1-(4-amino-1-methyl-1H-pyrazol-5-yl)azepan-4-ylcarbamate (12.6 g, 38.5 mmol), 5-(tert-butoxycarbonylamino)-2-(2,6-difluorophenyl)thiazole-4carboxylic acid (14.4 g, 40.4 mmol), diisopropylethylamine (13.4 mL, 77 mmol) and PyBOP (26.1 g, 50 mmol) in dichloromethane (400 mL) was stirred at room temperature for 48 hr. Saturated sodium hydrogen carbonate (600 mL) was added and stirring continued for 0.5 hr. The mixture was filtered, the layers separated and the aqueous extracted with dichloromethane (500 mL). The pooled organics were dried (MgSO4) and the solvent removed under reduced pressure. Flash column chromatography on silica eluting with 0–100% ethyl acetate in



Page 38 of 50

isohexane gradient afforded tert-butyl (4R,5R)-1-(4-(5-tert-butoxycarbonylamino-2-(2,6difluorophenyl)thiazole-4-carboxamido)-1-methyl-1H-pyrazol-5-yl)-5-fluoroazepan-4ylcarbamate as a pale yellow solid (2.5 g, 9.8%). Further elution with 0-10% methanol in ethyl acetate, then 10% methanol in dichloromethane, evaporation of relevant fractions under reduced pressure and trituration of the residue with cold diethyl ether gave a further 7.61g (30%) of product. 1H-NMR (CDCl3, 400MHz) δ 10.34 (s, 1H), 8.75 (s, 1H), 7.89 (s, 1H), 7.40 – 7.25 (m, 1H), 7.15 – 7.00 (m, 2H), 4.95 – 4.85 (m, 1H),4.85 – 4.65 (m, 1H), 4.15 – 4.00 (m, 1H), 3.77 (s, 3H), 3.45 – 3.30 (m, 2H), 3.25 – 3.00 (m, 2H), 2.35 – 2.10 (m, 3H), 1.95 – 1.75 (m, 1H), 1.55 (s, 9H), 1.43 (s, 9H).

ANCILLARY INFORMATION Supporting

Information.

Potency,

ADME

properties,

physiochemical

properties,

experimental procedures, crystallographic data, compound characterization, assay protocols, and molecular formula strings are included. This material is available free of charge via the Internet at http://pubs.acs.org. PDB ID of New co-Crystal Structures: xxx (9) and xxx (16), authors will release the atomic coordinates upon article publication AUTHOR INFORMATION Corresponding Author *telephone number: 6504677959; e-mail: [email protected] (X. Wang).

ORCID


Page 39 of 50 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


Xiaojing Wang: 0000-0003-1575-3227

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources Thanks to Genentech, a member of the Roche group for the research funds.

Acknowledgment Special thanks to Emily Hanan for a synthetic method development, Steven Magnuson, Fabio Broccatelli, and Kim Huard for reviewing the manuscript, and Antonio DiPasquale for single crystal structure data gathering and analysis. We are grateful to analytical, purification chemistry and compound management group for compound characterization, purification and handling. This research used resources of the Advanced Light Source, which is a DOE Office of Science User Facility under contract no. DE-AC02-05CH11231. We are grateful for the assistance from beamline staff at beamline 5.0.1. Use of the IMCA-CAT beamline 17-ID (or 17-BM) at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman-Woodward Medical Research Institute. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC0206CH11357. We are grateful for the assistance from beamline staff at beamline 17-ID.



Abbreviations PIM, Proviral Integration site in Moloney murine leukemia virus; hERG, the human Ether-àgo-go-Related Gene; SAR, structural activity relationship; MCT, methylcellulose tween 80; PEG400, polyethylene glycol 400; ADME, absorption, distribution, metabolism and excretion; PK, pharmacokinetics; PD, pharmacodynamics; SNAr, nucleophilic aromatic substitution; TDI, time dependent inhibition; DMPK, myotonin-protein kinase; PASK, PAS domain-containing serine/threonine-protein kinase; MAP4K4, mitogen-activated protein kinase kinase kinase kinase 4; GSK3, glycogen synthase kinase-3 alpha; GSK3, glycogen synthase kinase-3 beta; MNK1, MAP kinase-interacting serine/threonine-protein kinase 1; alpha 1a, alpha 1a adrenergic receptor; 5HT1a, serotonin 1a receptor; DOP, dopamine receptor; Boc, tert-butyloxycarbonyl; SFC, supercritical fluid chromatography; DCM: dichloromethane; DIPEA: diisopropylethylamine; DMA: N,N-dimethylacetamide; DMSO: diemthylsulfoxide; LiHMDS, lithium hexamethyldisilazane; mCPBA, metachloroperoxybenzoic acid; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; TEA: trimethylamine; THF: tetrahydrofuran.

REFERENCES

(1)

Kyle, R. A.; Rajkumar, S. V. Multiple Myeloma. New Engl. J. Med. 2004, 351 (18), 1860–1873.

(2)

Kazandjian, D. Multiple Myeloma Epidemiology and Survival: a Unique Malignancy. Semin. Oncol. 2016, 43 (6), 676–681.


Page 40 of 50

Page 41 of 50 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


(3)

Yong, K.; Gonzalez-McQuire, S.; Szabo, Z.; Schoen, P.; Hajek, R. The Start of a New Wave: Developments in Proteasome Inhibition in Multiple Myeloma. Eur. J. Haematol. 2018, 9 (Suppl 1), 3.

(4)

Bergsagel, P. L. Where We Were, Where We Are, Where We Are Going: Progress in Multiple Myeloma. Am. Soc. Clin. Oncol. Educ. Book 2014, 34, 199–203.

(5)

Gozzetti, A.; Candi, V.; Papini, G.; Bocchia, M. Therapeutic Advancements in Multiple Myeloma. Front. Oncol. 2014, 4, 241.

(6)

Keane, N. A.; Reidy, M.; Natoni, A.; Raab, M. S.; O'Dwyer, M. Targeting the Pim Kinases in Multiple Myeloma. Blood Cancer J. 2015, 5 (7), e325.

(7)

Raab, M. S.; Ocio, E. M.; Thomas, S. K.; Günther, A.; Goh, Y.-T.; Lebovic, D.; Jakubowiak, A.; Song, D.; Xiang, F.; Patel, A.; Vanasse, K. G.; Kumar, S. Phase 1 Study Update of the Novel Pan-Pim Kinase Inhibitor LGH447 in Patients with Relapsed/ Refractory Multiple Myeloma. Blood 2014, 124 (21), 301–301.

(8)

Lu, J.; Zavorotinskaya, T.; Dai, Y.; Niu, X. H.; Castillo, J.; Sim, J.; Yu, J.; Wang, Y.; Langowski, J. L.; Holash, J.; Shannon, K.; Garcia, P. D. Pim2 Is Required for Maintaining Multiple Myeloma Cell Growth Through Modulating TSC2 Phosphorylation. Blood 2013, 122 (9), 1610–1620.

(9)

Neri, P.; Bahlis, N. J. Pinning Down Myeloma with Pim2 Inhibitors! Blood 2013, 122 (9), 1534–1536.

(10)

Garcia, P. D.; Langowski, J. L.; Wang, Y.; Chen, M.; Castillo, J.; Fanton, C.; Ison, M.; Zavorotinskaya, T.; Dai, Y.; Lu, J.; Niu, X.-H.; Basham, S.; Chan, J.; Yu, J.; Doyle, M.; Feucht, P.; Warne, R.; Narberes, J.; Tsang, T.; Fritsch, C.; Kauffmann, A.; Pfister, E.; Drueckes, P.; Trappe, J.; Wilson, C.; Han, W.; Lan, J.; Nishiguchi, G.; Lindvall,



M.; Bellamacina, C.; Aycinena, J. A.; Zang, R.; Holash, J.; Burger, M. T. Pan-PIM Kinase Inhibition Provides a Novel Therapy for Treating Hematologic Cancers. Clin. Cancer Res. 2014, 20 (7), 1834–1845. (11)

Jiménez-García, M.-P.; Lucena-Cacace, A.; Robles- Frías, M.-J.; Ferrer, I.; NarlikGrassow, M.; Blanco-Aparicio, C.; Carnero, A. Inflammation and Stem Markers Association to PIM1/PIM2 Kinase-Induced Tumors in Breast and Uterus. Oncotarget 2017, 8 (35), 58872–58886.

(12)

Padi, S. K. R.; Luevano, L. A.; An, N.; Pandey, R.; Singh, N.; Song, J. H.; Aster, J. C.; Yu, X.-Z.; Mehrotra, S.; Kraft, A. S. Targeting the PIM Protein Kinases for the Treatment of a T-Cell Acute Lymphoblastic Leukemia Subset. Oncotarget 2017, 8 (18), 30199–30216.

(13)

Horiuchi, D.; Camarda, R.; Zhou, A. Y.; Yau, C.; Momcilovic, O.; Balakrishnan, S.; Corella, A. N.; Eyob, H.; Kessenbrock, K.; Lawson, D. A.; Marsh, L. A.; Anderton, B. N.; Rohrberg, J.; Kunder, R.; Bazarov, A. V.; Yaswen, P.; McManus, M. T.; Rugo, H. S.; Werb, Z.; Goga, A. PIM1 Kinase Inhibition as a Targeted Therapy Against TripleNegative Breast Tumors with Elevated MYC Expression. Nat. Med. 2016, 22 (11), 1321–1329.

(14)

Brasó-Maristany, F.; Filosto, S.; Catchpole, S.; Marlow, R.; Quist, J.; FranceschDomenech, E.; Plumb, D. A.; Zakka, L.; Gazinska, P.; Liccardi, G.; Meier, P.; GrisOliver, A.; Cheang, M. C. U.; Perdrix-Rosell, A.; Shafat, M.; Noël, E.; Patel, N.; McEachern, K.; Scaltriti, M.; Castel, P.; Noor, F.; Buus, R.; Mathew, S.; Watkins, J.; Serra, V.; Marra, P.; Grigoriadis, A.; Tutt, A. N. PIM1 Kinase Regulates Cell Death,


Page 42 of 50

Page 43 of 50 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


Tumor Growth and Chemotherapy Response in Triple-Negative Breast Cancer. Nat. Med. 2016, 22 (11), 1303–1313. (15)

Burger, M. T.; Nishiguchi, G.; Han, W.; Lan, J.; Simmons, R.; Atallah, G.; Ding, Y.; Tamez, V.; Zhang, Y.; Mathur, M.; Muller, K.; Bellamacina, C.; Lindvall, M. K.; Zang, R.; Huh, K.; Feucht, P.; Zavorotinskaya, T.; Dai, Y.; Basham, S.; Chan, J.; Ginn, E.; Aycinena, A.; Holash, J.; Castillo, J.; Langowski, J. L.; Wang, Y.; Chen, M. Y.; Lambert, A.; Fritsch, C.; Kauffmann, A.; Pfister, E.; Vanasse, K. G.; Garcia, P. D. Identification of N-(4-((1R,3S,5S)-3-Amino-5-Methylcyclohexyl)Pyridin-3-Yl)-6(2,6-Difluorophenyl)-5-Fluoropicolinamide (PIM447), a Potent and Selective Proviral Insertion Site of Moloney Murine Leukemia (PIM) 1, 2, and 3 Kinase Inhibitor in Clinical Trials for Hematological Malignancies. J. Med. Chem. 2015, 58 (21), 8373– 8386.

(16)

Burger, M. T.; Han, W.; Lan, J.; Nishiguchi, G.; Bellamacina, C.; Lindval, M.; Atallah, G.; Ding, Y.; Mathur, M.; McBride, C.; Beans, E. L.; Muller, K.; Tamez, V.; Zhang, Y.; Huh, K.; Feucht, P.; Zavorotinskaya, T.; Dai, Y.; Holash, J.; Castillo, J.; Langowski, J.; Wang, Y.; Chen, M. Y.; Garcia, P. D. Structure Guided Optimization, in Vitro Activity, and in Vivo Activity of Pan-PIM Kinase Inhibitors. ACS Med. Chem. Lett. 2013, 4 (12), 1193–1197.

(17)

Nishiguchi, G. A.; Burger, M. T.; Han, W.; Lan, J.; Atallah, G.; Tamez, V.; Lindvall, M.; Bellamacina, C.; Garcia, P.; Feucht, P.; Zavorotinskaya, T.; Dai, Y.; Wong, K. Design, Synthesis and Structure Activity Relationship of Potent Pan-PIM Kinase Inhibitors Derived From the Pyridyl Carboxamide Scaffold. Bioorg. Med. Chem. Lett. 2016, 26 (9), 2328–2332.



(18)

Pettus, L. H.; Andrews, K. L.; Booker, S. K.; Chen, J.; Cee, V. J.; Chavez, F.; Chen, Y.; Eastwood, H.; Guerrero, N.; Herberich, B.; Hickman, D.; Lanman, B. A.; Laszlo, J.; Lee, M. R.; Lipford, J. R.; Mattson, B.; Mohr, C.; Nguyen, Y.; Norman, M. H.; Powers, D.; Reed, A. B.; Rex, K.; Sastri, C.; Tamayo, N.; Wang, P.; Winston, J. T.; Wu, B.; Wu, T.; Wurz, R. P.; Xu, Y.; Zhou, Y.; Tasker, A. S.; Wang, H.-L. Discovery and Optimization of Quinazolinone-Pyrrolopyrrolones as Potent and Orally Bioavailable Pan-Pim Kinase Inhibitors. J. Med. Chem. 2016, 59 (13), 6407–6430.

(19)

Cee, V. J.; Frank Chavez, J.; Herberich, B.; Lanman, B. A.; Pettus, L. H.; Reed, A. B.; Bin Wu; Wurz, R. P.; Andrews, K. L.; Chen, J.; Hickman, D.; Jimmy Laszlo, I.; Lee, M. R.; Guerrero, N.; Mattson, B. K.; Nguyen, Y.; Mohr, C.; Rex, K.; Sastri, C. E.; Wang, P.; Wu, Q.; Wu, T.; Xu, Y.; Zhou, Y.; Winston, J. T.; Lipford, J. R.; Tasker, A. S.; Wang, H.-L. Discovery and Optimization of Macrocyclic Quinoxaline-PyrroloDihydropiperidinones as Potent Pim-1/2 Kinase Inhibitors. ACS Med. Chem. Lett. 2016, 7 (4), 408–412.

(20)

Wu, B.; Wang, H.-L.; Cee, V. J.; Lanman, B. A.; Nixey, T.; Pettus, L.; Reed, A. B.; Wurz, R. P.; Guerrero, N.; Sastri, C.; Winston, J.; Lipford, J. R.; Lee, M. R.; Mohr, C.; Andrews, K. L.; Tasker, A. S. Discovery of 5-(1H-Indol-5-Yl)-1,3,4-Thiadiazol-2Amines as Potent PIM Inhibitors. Bioorg. Med. Chem. Lett. 2015, 25 (4), 775–780.

(21)

Wurz, R. P.; Pettus, L. H.; Jackson, C.; Wu, B.; Wang, H.-L.; Herberich, B.; Cee, V.; Lanman, B. A.; Reed, A. B.; Chavez, F.; Nixey, T.; Laszlo, J.; Wang, P.; Nguyen, Y.; Sastri, C.; Guerrero, N.; Winston, J.; Lipford, J. R.; Lee, M. R.; Andrews, K. L.; Mohr, C.; Xu, Y.; Zhou, Y.; Reid, D. L.; Tasker, A. S. The Discovery and


Page 44 of 50

Page 45 of 50 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


Optimization of Aminooxadiazoles as Potent Pim Kinase Inhibitors. Bioorg. Med. Chem. Lett. 2015, 25 (4), 847–855. (22)

Wan, X.; Zhang, W.; Li, L.; Xie, Y.; Li, W.; Huang, N. A New Target for an Old Drug: Identifying Mitoxantrone as a Nanomolar Inhibitor of PIM1 Kinase via Kinome-Wide Selectivity Modeling. J. Med. Chem. 2013, 56 (6), 2619–2629.

(23)

Nakano, H.; Hasegawa, T.; Kojima, H.; Okabe, T.; Nagano, T. Design and Synthesis of Potent and Selective PIM Kinase Inhibitors by Targeting Unique Structure of ATPBinding Pocket. ACS Med. Chem. Lett. 2017, 8 (5), 504–509.

(24)

Xu, Y.; Brenning, B. G.; Kultgen, S. G.; Foulks, J. M.; Clifford, A.; Lai, S.; Chan, A.; Merx, S.; McCullar, M. V.; Kanner, S. B.; Ho, K.-K. Synthesis and Biological Evaluation of Pyrazolo[1,5-a]Pyrimidine Compounds as Potent and Selective Pim-1 Inhibitors. ACS Med. Chem. Lett. 2015, 6 (1), 63–67.

(25)

Casuscelli, F.; Ardini, E.; Avanzi, N.; Casale, E.; Cervi, G.; D’Anello, M.; Donati, D.; Faiardi, D.; Ferguson, R. D.; Fogliatto, G.; Galvani, A.; Marsiglio, A.; Mirizzi, D. G.; Montemartini, M.; Orrenius, C.; Papeo, G.; Piutti, C.; Salom, B.; Felder, E. R. Discovery and Optimization of Pyrrolo[1,2-a]Pyrazinones Leads to Novel and Selective Inhibitors of PIM Kinases. Bioorg. Med. Chem. 2013, 21 (23), 7364–7380.

(26)

More, K. N.; Hong, V. S.; Lee, A.; Park, J.; Kim, S.; Lee, J. Discovery and Evaluation of 3,5-Disubstituted Indole Derivatives as Pim Kinase Inhibitors. Bioorg. Med. Chem. Lett. 2018, 28 (14), 2513–2517.

(27)

More, K. N.; Jang, H. W.; Hong, V. S.; Lee, J. Pim Kinase Inhibitory and Antiproliferative Activity of a Novel Series of Meridianin C Derivatives. Bioorg. Med. Chem. Lett. 2014, 24 (11), 2424–2428.



(28)

Schroeder, R. L.; Goyal, N.; Bratton, M.; Townley, I.; Pham, N. A.; Tram, P.; Stone, T.; Geathers, J.; Nguyen, K.; Sridhar, J. Identification of Quinones as Novel PIM1 Kinase Inhibitors. Bioorg. Med. Chem. Lett. 2016, 26 (13), 3187–3191.

(29)

Fan, Y.-B.; Li, K.; Huang, M.; Cao, Y.; Li, Y.; Jin, S.-Y.; Liu, W.-B.; Wen, J.-C.; Liu, D.; Zhao, L.-X. Design and Synthesis of Substituted Pyrido[3,2-D]-1,2,3-Triazines as Potential Pim-1 Inhibitors. Bioorg. Med. Chem. Lett. 2016, 26 (4), 1224–1228.

(30)

Dwyer, M. P.; Keertikar, K.; Paruch, K.; Alvarez, C.; Labroli, M.; Poker, C.; Fischmann, T. O.; Mayer-Ezell, R.; Bond, R.; Wang, Y.; Azevedo, R.; Guzi, T. J. Discovery of Pyrazolo[1,5-a]Pyrimidine-Based Pim Inhibitors: a Template-Based Approach. Bioorg. Med. Chem. Lett. 2013, 23 (22), 6178–6182.

(31)

Gavara, L.; Suchaud, V.; Nauton, L.; Théry, V.; Anizon, F.; Moreau, P. Identification of Pyrrolo[2,3-G]Indazoles as New Pim Kinase Inhibitors. Bioorg. Med. Chem. Lett. 2013, 23 (8), 2298–2301.

(32)

Ishchenko, A.; Zhang, L.; Le Brazidec, J.-Y.; Fan, J.; Chong, J. H.; Hingway, A.; Raditsis, A.; Singh, L.; Elenbaas, B.; Hong, V. S.; Marcotte, D.; Silvian, L.; Enyedy, I.; Chao, J. Structure-Based Design of Low-Nanomolar PIM Kinase Inhibitors. Bioorg. Med. Chem. Lett. 2014, 25 (3), 474–480.

(33)

Wang, X.; Kolesnikov, A.; Tay, S.; Chan, G.; Chao, Q.; Do, S.; Drummond, J.; Ebens, A. J.; Liu, N.; Ly, J.; Harstad, E.; Hu, H.; Moffat, J.; Munugalavadla, V.; Murray, J.; Slaga, D.; Tsui, V.; Volgraf, M.; Wallweber, H.; Chang, J. H. Discovery of 5Azaindazole (GNE-955) as a Potent Pan-Pim Inhibitor with Optimized Bioavailability. J. Med. Chem. 2017, 60 (10), 4458–4473.


Page 46 of 50

Page 47 of 50 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


(34)

Hu, H.; Wang, X.; Chan, G. K. Y.; Chang, J. H.; Do, S.; Drummond, J.; Ebens, A.; Lee, W.; Ly, J.; Lyssikatos, J. P.; Murray, J.; Moffat, J. G.; Chao, Q.; Tsui, V.; Wallweber, H.; Kolesnikov, A. Discovery of 3,5-Substituted 6-Azaindazoles as Potent Pan-Pim Inhibitors. Bioorg. Med. Chem. Lett. 2015, 25 (22), 5258–5264.

(35)

Wang, X.; Magnuson, S.; Pastor, R.; Fan, E.; Hu, H.; Tsui, V.; Deng, W.; Murray, J.; Steffek, M.; Wallweber, H.; Moffat, J.; Drummond, J.; Chan, G.; Harstad, E.; Ebens, A. J. Discovery of Novel Pyrazolo[1,5-a]Pyrimidines as Potent Pan-Pim Inhibitors by Structure- and Property-Based Drug Design. Bioorg. Med. Chem. Lett. 2013, 23 (11), 3149–3153.

(36)

Chen, L. S.; Redkar, S.; Pietro Taverna; Cortes, J. E.; Gandhi, V. Mechanisms of Cytotoxicity to Pim Kinase Inhibitor, SGI-1776, in Acute Myeloid Leukemia. Blood 2011, 118 (3), 693–702.

(37)

Natarajan, K.; Bhullar, J.; Shukla, S.; Burcu, M.; Chen, Z.-S.; Ambudkar, S. V.; Baer, M. R. The Pim Kinase Inhibitor SGI-1776 Decreases Cell Surface Expression of PGlycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2) and Drug Transport by Pim-1-Dependent and -Independent Mechanisms. Biochem. Pharmacol. 2013, 85 (4), 514–524.

(38)

Chen, L. S.; Redkar, S.; Bearss, D.; Wierda, W. G.; Gandhi, V. Pim Kinase Inhibitor, SGI-1776, Induces Apoptosis in Chronic Lymphocytic Leukemia Cells. Blood 2009, 114 (19), 4150–4157.

(39)

Dakin, L. A.; Block, M. H.; Chen, H.; Code, E.; Dowling, J. E.; Feng, X.; Ferguson, A. D.; Green, I.; Hird, A. W.; Howard, T.; Keeton, E. K.; Lamb, M. L.; Lyne, P. D.; Pollard, H.; Read, J.; Wu, A. J.; Zhang, T.; Zheng, X. Discovery of Novel



Benzylidene-1,3-Thiazolidine-2,4-Diones as Potent and Selective Inhibitors of the PIM-1, PIM-2, and PIM-3 Protein Kinases. Bioorg. Med. Chem. Lett. 2012, 22 (14), 4599–4604. (40)

Keeton, E. K.; McEachern, K.; Dillman, K. S.; Palakurthi, S.; Cao, Y.; Grondine, M. R.; Kaur, S.; Wang, S.; Chen, Y.; Wu, A.; Shen, M.; Gibbons, F. D.; Lamb, M. L.; Zheng, X.; Stone, R. M.; Deangelo, D. J.; Platanias, L. C.; Dakin, L. A.; Chen, H.; Lyne, P. D.; Huszar, D. AZD1208, a Potent and Selective Pan-Pim Kinase Inhibitor, Demonstrates Efficacy in Preclinical Models of Acute Myeloid Leukemia. Blood 2014, 123 (6), 905–913.

(41)

Desai, P. V.; Raub, T. J.; Blanco, M.-J. How Hydrogen Bonds Impact P-Glycoprotein Transport and Permeability. Bioorg. Med. Chem. Lett. 2012, 22 (21), 6540–6548.

(42)

Beno, B. R.; Yeung, K.-S.; Bartberger, M. D.; Pennington, L. D.; Meanwell, N. A. A Survey of the Role of Noncovalent Sulfur Interactions in Drug Design. J. Med. Chem. 2015, 58 (11), 4383–4438.

(43)

Bode, C. The Nasty Surprise of a Complex Drug-Drug Interaction. Drug Discovery Today 2010, 15 (9-10), 391–395.

(44)

Wang, X.; Sun, M.; New, C.; Nam, S.; Blackaby, W. P.; Hodges, A. J.; Nash, D.; Matteucci, M.; Lyssikatos, J. P.; Fan, P. W.; Tay, S.; Chang, J. H. Probing Mechanisms of CYP3A Time-Dependent Inhibition Using a Truncated Model System. ACS Med. Chem. Lett. 2015, 6 (8), 925–929.

(45)

Redfern, W. S.; Carlsson, L.; Davis, A. S.; Lynch, W. G.; Mackenzie, I.; Palethorpe, S.; Siegl, P. K. S.; Strang, I.; Sullivan, A. T.; Wallis, R.; Camm, A. J.; Hammond, T. G. Relationships Between Preclinical Cardiac Electrophysiology, Clinical QT Interval


Page 48 of 50

Page 49 of 50 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


Prolongation and Torsade De Pointes for a Broad Range of Drugs: Evidence for a Provisional Safety Margin in Drug Development. Cardiovasc. Res. 2003, 58 (1), 32– 45. (46)

Le, X.; Antony, R.; Razavi, P.; Treacy, D. J.; Luo, F.; Ghandi, M.; Castel, P.; Scaltriti, M.; Baselga, J.; Garraway, L. A. Systematic Functional Characterization of Resistance to PI3K Inhibition in Breast Cancer. Cancer Discov. 2016, 6 (10), 1134– 1147.

(47)

Ferrarelli, L. K. Inhibit PIM to Inhibit PI3K. Sci. Signal. 2016, 9 (449), ec237–ec237.

(48)

Folkes, A. J.; Ahmadi, K.; Alderton, W. K.; Alix, S.; Baker, S. J.; Box, G.; Chuckowree, I. S.; Clarke, P. A.; Depledge, P.; Eccles, S. A.; Friedman, L. S.; Hayes, A.; Hancox, T. C.; Kugendradas, A.; Lensun, L.; Moore, P.; Olivero, A. G.; Pang, J.; Patel, S.; Pergl-Wilson, G. H.; Raynaud, F. I.; Robson, A.; Saghir, N.; Salphati, L.; Sohal, S.; Ultsch, M. H.; Valenti, M.; Wallweber, H. J. A.; Wan, N. C.; Wiesmann, C.; Workman, P.; Zhyvoloup, A.; Zvelebil, M. J.; Shuttleworth, S. J. The Identification of 2-(1H-Indazol-4-Yl)-6-(4-Methanesulfonyl-Piperazin-1-Ylmethyl)-4-Morpholin-4Yl-Thieno[3,2-D]Pyrimidine (GDC-0941) as a Potent, Selective, Orally Bioavailable Inhibitor of Class I PI3 Kinase for the Treatment of Cancer. J. Med. Chem. 2008, 51 (18), 5522–5532.

(49)

Karban, J.; Sýkora, J.; Kroutil, J.; Císarová, I.; Padelková, Z.; Budesínský, M. Synthesis of All Configurational Isomers of 1,6-Anhydro-2,3,4-Trideoxy-2,3Epimino-4-Fluoro-Beta-D-Hexopyranoses. J. Org. Chem. 2010, 75 (10), 3443–3446.

(50)

Bouzard, D.; Di Cesare, P.; Essiz, M.; Jacquet, J. P.; Kiechel, J. R.; Remuzon, P.; Weber, A.; Oki, T.; Masuyoshi, M.; Kessler, R. E. Fluoronaphthyridines and



Quinolones as Antibacterial Agents. 2. Synthesis and Structure-Activity Relationships of New 1-Tert-Butyl 7-Substituted Derivatives. J. Med. Chem. 1990, 33 (5), 1344– 1352. (51)

Kategaya, L.; Di Lello, P.; Rougé, L.; Pastor, R.; Clark, K. R.; Drummond, J.; Kleinheinz, T.; Lin, E.; Upton, J.-P.; Prakash, S.; Heideker, J.; McCleland, M.; Ritorto, M. S.; Alessi, D. R.; Trost, M.; Bainbridge, T. W.; Kwok, M. C. M.; Ma, T. P.; Stiffler, Z.; Brasher, B.; Tang, Y.; Jaishankar, P.; Hearn, B. R.; Renslo, A. R.; Arkin, M. R.; COHEN, F.; Yu, K.; Peale, F.; Gnad, F.; Chang, M. T.; Klijn, C.; Blackwood, E.; Martin, S. E.; Forrest, W. F.; Ernst, J. A.; Ndubaku, C.; Wang, X.; Beresini, M. H.; Tsui, V.; Schwerdtfeger, C.; Blake, R. A.; Murray, J.; Maurer, T.; Wertz, I. E. USP7 Small-Molecule Inhibitors Interfere with Ubiquitin Binding. Nature 2017, 550 (7677), 534–538.

(52)

PIM1 May Be a Therapeutic Target in Triple-Negative Breast Cancer. Cancer Discov. 2016, 1303.

(53)

Pastor, J.; Oyarzabal, J.; Saluste, G.; Alvarez, R. M.; Rivero, V.; Ramos, F.; Cendón, E.; Blanco-Aparicio, C.; Ajenjo, N.; Cebriá, A.; Albarrán, M. I.; Cebrián, D.; Corrionero, A.; Fominaya, J.; Montoya, G.; Mazzorana, M. Hit to Lead Evaluation of 1,2,3-Triazolo[4,5-B]Pyridines as PIM Kinase Inhibitors. Bioorg. Med. Chem. Lett. 2012, 22 (4), 1591–1597.


Page 50 of 50

Page 51 of 50 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


TOC GRAPHIC


Optimization of Pan-Pim Kinase Activity and Oral Bioavailability

Recommend Documents