Discovery of novel BTK inhibitors with carboxylic acids

Accepted Manuscript Discovery of novel BTK inhibitors with carboxylic acids Xiaolei Gao, James Wang, Jian Liu, Deodial Guiadeen, Arto Krikorian, Sobhana Babu Boga, Abdul-Basit Alhassan, Oleg Selyutin, Wensheng Yu, Younong Yu, Rajan Anand, Shilan Liu, Chundao Yang, Hao Wu, Jiaqiang Cai, Alan Cooper, Hugh Zhu, Kevin Maloney, Ying-Duo Gao, Thierry O. Fischmann, Jeremy Presland, My Mansueto, Zangwei Xu, Erica Leccese, Jie Zhang-Hoover, Ian Knemeyer, Charles G. Garlisi, Nathan Bays, Peter Stivers, Philip E. Brandish, Alexandra Hicks, Ronald Kim, Joeseph A. Kozlowski PII: DOI: Reference:

S0960-894X(16)31244-6 http://dx.doi.org/10.1016/j.bmcl.2016.11.079 BMCL 24474

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

8 November 2016 23 November 2016 24 November 2016

Please cite this article as: Gao, X., Wang, J., Liu, J., Guiadeen, D., Krikorian, A., Babu Boga, S., Alhassan, A-B., Selyutin, O., Yu, W., Yu, Y., Anand, R., Liu, S., Yang, C., Wu, H., Cai, J., Cooper, A., Zhu, H., Maloney, K., Gao, Y-D., Fischmann, T.O., Presland, J., Mansueto, M., Xu, Z., Leccese, E., Zhang-Hoover, J., Knemeyer, I., Garlisi, C.G., Bays, N., Stivers, P., Brandish, P.E., Hicks, A., Kim, R., Kozlowski, J.A., Discovery of novel BTK inhibitors with carboxylic acids, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl. 2016.11.079

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Xiaolei Gao*, James Wang, Jian Liu*, Deodial Guiadeen, Arto Krikorian, Sobhana Babu Boga, Abdul-Basit Alhassan, Oleg Selyutin, Wensheng Yu, Younong Yu, Rajan Anand, Shilan Liu, Chundao Yang, Hao Wu, Jiaqiang Cai, Alan Cooper, Hugh Zhu, Kevin Maloney, Ying-Duo Gao, Thierry O. Fischmann, Jeremy Presland, My Mansueto, Zangwei Xu, Erica Leccese, Jie Zhang-Hoover, Ian Knemeyer, Charles G. Garlisi, Nathan Bays, Peter Stivers, Philip E. Brandish, Alexandra Hicks, Ronald Kim, and Joeseph A. Kozlowski

Bioorganic & Medicinal Chemistry Letters

Discovery of novel BTK inhibitors with carboxylic acids Xiaolei Gao a,*, James Wang a, Jian Liua,* , Deodial Guiadeen a, Arto Krikorian a, Sobhana Babu Boga a, Abdul-Basit Alhassan a, Oleg Selyutina, Wensheng Yua, Younong Yu a, Rajan Anand a, Shilan Liu b, Chundao Yang b, Hao Wu b, Jiaqiang Caib, Alan Cooper a, Hugh Zhu a, Kevin Maloney a, Ying-Duo Gao a, Thierry O. Fischmann a, Jeremy Presland a, My Mansueto a, Zangwei Xu a, Erica Leccese a, Jie ZhangHoover a, Ian Knemeyer a, Charles G. Garlisi a, Nathan Bays a, Peter Stivers a, Philip E. Brandish a, Alexandra Hicks a, Ronald Kim a, and Joeseph A. Kozlowski a a b

Department of Early Development and Discovery Sciences, MRL, 126 East Lincoln Avenue, Rahway, NJ 07065 USA WuXi PharmaTech Co. Ltd, 288 FuTe Zhong Road, No. 1 Building, WaiGaoQiao Free Trade Zone, Shanghai 200131, P. R. China

A RT I C L E I N F O

A BS T RA C T

Article history: Received Revised Accepted Available online

We report the design and synthesis of a series of novel Bruton’s Tyrosine Kinase (BTK) inhibitors with a carboxylic acid moiety in the ribose pocket. This series of compounds has demonstrated much improved off-target selectivities including adenosine uptake (AdU) inhibition compared to the piperidine amide series. Optimization of the initial lead compound 4 based on BTK enzyme inhibition, and human peripheral blood mononuclear cell (hPBMC) and human whole blood (hWB) activity led to the discovery of compound 40, with potent BTK inhibition, reduced off target activities, as well as favorable pharmacokinetic profile in both rat and dog.

Keywords: BTK inhibitor Carboxylic acid ribose pocket Adenosine uptake activity Off target selectivities hPBMC and hWB

Bruton’s tyrosine kinase (BTK) is a Tec family kinase expressed in immune cells including B cells, mast cells, and macrophages.1,2 BTK plays a crucial role in B cell development, proliferation, and signaling, and is also involved in cellular signaling in myeloid cells.3 B cells have been suggested to contribute to the pathology of autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematous (SLE) and multiple sclerosis,4 and pharmacological modulation of B cell function with therapeutic antibodies such as rituximab and belimumab have demonstrated clinical efficacy in autoimmune diseases.5,6 Recently several groups reported the use of small molecule BTK selective inhibitors as cancer therapies or for the treatment of rheumatoid arthritis (RA).7 The covalent BTK inhibitor Ibrutinib is currently approved for mantle cell lymphoma and chronic lymphocytic leukemia.8 It is efficacious in the rat collagen induced arthritis model (CIA), indicating its potential use for the treatment of RA.8 Our group has recently reported the discovery of potent and selective reversible noncovalent BTK inhibitors based on 8-amino-imidazo[1,5a]pyrazines, as exemplified by compounds 1−3 (Figure 1).10 These inhibitors display high enzymatic potency, selectivity over other kinases, favorable PK properties and are orally efficacious in reducing paw thickness in a rat model of collagen induced arthritis.

2009 Elsevier Ltd. All rights reserved .

Figure 1. Structures of Ibrutinib and compounds 1-3.

However, while further profiling compound 3, we found that it is a potent inhibitor of adenosine uptake (AdU) activity, which caused concern due to the potential for unfavorable CV effects since the cardiovascular actions of adenosine are well documented.11 As a result, we undertook efforts to reduce AdU activity while retaining the favorable overall profile of the series. X-ray crystallography of the 8-amino-imidazo[1,5-a]pyrazines bound to BTK show that the piperidine amide moiety occupies the ribose binding pocket of the enzyme. 10 Extensive SAR studies were carried out to reduce the AdU activity and progress was made through exploration of the ribose pocket SAR. We discovered that replacing the amide with the carboxylic acid moiety led to superior off-target selectivity. The initial lead cyclohexylcarboxylic acid 4 (Figure 2) displayed significantly improved AdU selectivity as well as improved hERG and Cav1.2 selectivities compared to the previous lead compound 3. Zhu et al. has reported the inhibitory effect of a carboxylic acid group on hERG binding and suggested that the negatively charged

carboxylate group causes unfavorable interaction within the hERG channel binding cavity by electrostatic interaction.12

summarized in Table 1. Though several acid bioisosteres (13-15) maintain improved AdU selectivity, they showed no improvement in cellular potency; while others (16-20) showed improved cellular potency but lost the superior AdU selectivity. It is very likely that the improved AdU selectivities observed in compound 13 to 15 are related to the low permeability of those compounds, which also explains their poor cellular potency observed in the hPBMC assay. Table 1. Carboxylic acid bioisosteres Moiety in ribose pocket

Figure 2. Ribose pocket modification from piperidine amide to cyclohexylcarboxylic acid

Preparation of this series of BTK inhibitors is depicted in Scheme 1 and 2. (3-Chloropyrazin-2-yl)methanamine 5 is first coupled with cis-3-(methoxycarbonyl)cyclohexane-1-carboxylic acid 6 using HATU as coupling reagent and diisopropylethylamine as the base to provide compound 7 in 77% yield, which underwent a cyclization to form the imidazole by treatment with phosphorus oxychloride in acetonitrile to provide intermediate 8. Crude 8 was utilized in the next step without purification. Bromination of intermediate 8 was carried out using N-bromosuccinimide in DMF at room temperature for 1 h, producing intermediate 9 in 84% yield from compound 7. The chloro intermediate 9 was converted to 10 in high yield with 2,4dimethoxybenzylamine in dioxane at room temperature for overnight. LiOH hydrolysis followed by TFA deprotection provided intermediate 11 in quantitative yield. SAR studies with other carboxylic moieties in the ribose pocket were achieved with various carboxylic acids esters to replace 6, which were either commercially available or readily prepared. Bioisosteres of acid are readily prepared from the ester 10.

BTK IC50 a (nM)

hPBMC IC50a (nM)

AdU (nM)

Papp (10-6 cm/s)

0.5

98

7851

7.0

1.2

648

5600

2.0

0.5

1587

>10000

3.0

1.4

979

9086

NT

0.3

17

168

20

0.6

5

150

28

0.3

21

155

26

19

0.5

37

44

NT

20

0.2

17

45

NT

Cpd # 4 13 14 15 16 17 18

a

Scheme 1. Reagents. i. HATU, DIPEA, DMF, rt, 77%; ii. POCl3, CH3CN, 0 o C-rt; iii. NBS, DMF, rt, 84% for two steps; iv. 2,4-Dimethoxybenzylamine, dioxanes, rt, 93%; v. 1N LiOH, THF/MeOH, 40 °C; vi. TFA, triethysilane, 70 °C, 99% for two steps.

Boronic acid pinacol ester 12 was prepared as described in our previous report.10 Suzuki coupling (Scheme 2) of 11 and 12, using palladium catalyst such as PdCl2(dppf)CH2Cl2 and potassium phosphate tribasic in dioxane /water (4/1) at 85°C for 6 h generated 48% of compound 4.

Scheme 2. Reagents. i. PdCl2(dppf)CH2Cl2, K3PO4, dioxane/water (4/1), 85°C, 6 h, 48%.

All BTK inhibitors were evaluated in a BTK enzymatic assay, in a human peripheral blood mononuclear cell (hPBMC) and a whole blood (hWB) functional assays.10 Compound 4 had improved off target selectivities, but displayed weak hPBMC and hWB potency, and demonstrated low bioavailability in the rat. Initial optimization on 4 was carried out by exploring alternates to the carboxylic acid. Representative examples (13-20) are

R

The data are average of at least two repeated tests.

We explored different ring sizes and projections of the carboxylic acid in the ribose pocket with the representative examples shown in Table 2. We discovered that all (21-25) demonstrated enzymatic potency as well as good AdU selectivity; and some displayed good cellular potency as well (24, 25). The 4-transcyclohexyl carboxylic acid 21, and 3-cis-cyclopentyl carboxylic acid 23, showed similar potency to compound 4, while the 4-ciscyclohexyl carboxylic acid 22 showed a decrease in cellular potency. We also found that steric hindrance around the carboxylic acid seemed to improve the hPBMC and hWB potencies (compound 24 and 25). Compound 24 was later found to have poor rat PK profiles, while compound 25 showed poor off-target selectivity.

Table 2. Different unsubstituted carboxylic acids in ribose pocket Cpd #

26

Cpd#

R

BTK IC50a (nM) 0.4

4

hPBMC

IC50a

(nM) 98

hWBa (nM)

27 AdU

1.2

77

2307

23

24

25

a

0.5

1237

NT

>10000

0.8

132

NT

4435

0.1

11

232

1927

31

a

0.4

22

246

hPBMC IC50a (nM)

0.2

5

99

308

0.3

6

161

274

0.2

9

264

459

0.4

67

1909

6476

0.1

49

1267

812

0.3

24

67

9161

hWBa (nM)

AdU (nM)

>10000 30

22

R

BTK IC50a (nM)

7851 29

21

2

(nM) 28

2788

R

1

7263

The data are average of at least two repeated tests.

Unfortunately, the rat PK profile of compound 31 (Figure 3) was unacceptable, due to the very high clearance. Metabolic identification studies suggested the major metabolism was through glucuronidation of the carboxylic acid. We therefore increased steric hindrance around the acid moiety in an attempt to block or slow this pathway.

The data are average of at least two repeated tests.

This result encouraged us to further explore analogs with substituted ribose pocket ring. A series of bridgehead carboxylic acids were prepared and tested (Table 3). Despite the excellent hPBMC and hWB potencies, those bridgehead carboxylic acids (26-28) displayed poor AdU selectivity. Compounds 29 and 30 showed reduced hWB potency, but interestingly, when we introduced an ethoxy group onto the middle phenyl ring of compound 26, compound 29, we observed potent enzyme, hPBMC and hWB potency and reduced adenosine uptake activity. SAR studies on the middle phenyl ring have suggested that substitutions on middle phenyl rings have impact on AdU activity.13 Table 3. Bridgehead carboxylic acid moiety in ribose pocket

Figure 3. [2.2.2]-Bridgehead carboxylic acid 31

Several alpha-substituted carboxylic acids were then prepared and tested (Table 4). Several of these analogs showed improved hPBMC potency (compounds 32 and 35), though the hWB potency still needed improvement. There is no apparent correlation between hWB activity and protein binding (See the unbound fraction data in Table 4 and Table 5). Compounds 33, 34 and 36 show weaker cellular potency. Table 4. Alpha-Substituted carboxylic acids moiety in ribose pocket

Cpd #

BTK IC50a (nM)

R

hPBMC

IC50a

(nM)

hWB a

(nM)

AdU

(nM)

Table 6. Pharmacokinetic parameters and off target activities of compounds 37-40a

fub

37 0.9

32

33

26

3

34

128

1071

NT

2432

1938

NT

1.3

62

909

5396

NT

1.1

27

604

1711

0.8

4.5

36

546

NT

2492

39

40

0.002 rat

IV

Cl (mL/min/kg)

9

2

2.8

13

T1/2 (h)

3

2.4

2

5

PO

F (%)

NT

IV

PO

22

IV

dog

35

38

PO

IV

PO

39

IV

PO

IV

PO

44

IV

PO

75

IV

Cl (mL/min/kg)

13

T1/2 (h)

5.8

PO

F (%) a

77

The data are average of at least two repeated tests; b Unbound fraction

in rat

We explored the impact of added steric bulk to the benzylic position of the ribose pocket ring as well as adjacent to the carboxylic acid. These analogs (37 to 40) displayed good hPBMC and hWB potency while maintaining good AdU selectivity. This series of compounds also had improved offtarget selectivity as compared to our previous lead compound 3, and also displayed an excellent rat PK profile (Table 6). Compound 40 demonstrated an acceptable dog PK profile. Further profiling of compound 40 in in vivo studies is in progress. Table 5. alpha-substituted carboxylic acids moiety in ribose pocket with a tertiary methyl at benzylic position

hERG IC50 (M)

>60

54

>60

>60

Cav1.2 IC50 (M)

13

19

8

15

Nav1.5 IC50 (M)

>30

>30

>30

>30

a.

Formulations for both IV DMSO/PEG400/Water:20/60/20

and

PO

are

both

In summary, we described here a new carboxylic acid series of reversible BTK inhibitors based on the 8-amino-imidazo [1,5a]pyrazine core. This series of compounds are characterized by improved off-targets activities especially high AdU selectivity. Analogs with steric bulk added near the carboxylate, i.e. in the ribose pocket demonstrated potent BTK enzyme inhibition, hPBMC, human whole blood activity, kinase selectivity, and favorable pharmacokinetic profiles. Further investigation of the SAR of this series of BTK inhibitors will be the subject of another report. References and notes

Cpd #

37

R

BTK IC50a (nM)

hPBMC

0.1

18

IC50a

(nM)

hWBa (nM)

1. AdU

(nM)

fu

b

2. 210

6073

0.002 3.

38

0.2

8

145

1699

0.001

0.4

20

289

4010

0.003

4. 39

5. 40

a

1.0

17

272

1452

0.001

The data are average of at least two repeated tests. bUnbound fraction in rat

6.

7.

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