Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx

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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl

Development of a novel class of potent and selective FIXa inhibitors Ting Zhang a,⇑, Patrick Andre b, Thomas J. Bateman c, Yi-Heng Chen a, Kunal Desai b, Kenneth Ellsworth d, Wayne M. Geissler d, Liangqin Guo a, Alan Hruza e, Tianying Jian a, Dongfang Meng a, Dann L. Parker Jr. a, Xiaoxia Qian a, Paul Reichert e, Edward C. Sherer f, Min Shu a, Cameron J. Smith a, Lisa M. Sonatore d, Richard Tschirret-Guth c, Andrew F. Nolting g, Robert Orr g, Louis-Charles Campeau g, Kazuto Araki h, Teruyuki Nishimura h, Isao Sakurada h, Harold B. Wood a a

Department of Medicinal Chemistry, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA Department of Cardiometabolic Disease, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA c Department of Pharmacokinetics, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA d Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA e Department of Structural Chemistry, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA f Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA g Department of Process Chemistry, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA h Discovery Research, Mochida Pharmaceutical Co., LTD, 7, Yotsuya 1-Chome, Shinjuku-ku, Tokyo 160-8515, Japan b

a r t i c l e

i n f o

Article history: Received 21 March 2015 Revised 14 April 2015 Accepted 17 April 2015 Available online xxxx Keywords: Benzimidazole FIXa inhibitor TGA Structure based drug design

a b s t r a c t Using structure based drug design (SBDD), a novel class of potent coagulation Factor IXa (FIXa) inhibitors was designed and synthesized. High selectivity over FXa inhibition was achieved. Selected compounds demonstrated oral bioavailability in rat IV/PO pharmacokinetic (PK) studies. Finally, the pharmacodynamics (PD) of this class of molecules was evaluated in Thrombin Generation Assay (TGA) in Corn Trypsin Inhibitor (CTI) citrated human plasma and demonstrated characteristics of a FIXa inhibitor. Ó 2015 Elsevier Ltd. All rights reserved.

Factor IXa is a plasma serine protease involved in the regulation of blood coagulation. While blood coagulation is a necessary and important part of the regulation of an organism’s homeostasis, abnormal blood coagulation can also have deleterious effects. For instance, thrombi formed in a blood vessel or cavity of the heart can become pathological if they obstruct blood flow at the site of vessel wall injury or downstream (i.e., upon embolization), leading to heart attack, stroke, pulmonary embolism or cardiogenic stroke, respectively. These thromboembolic disorders remain the largest cause of mortality and disability in the industrialized world. Defects in Factor IXa lead to hemophilia B, a debilitating, potentially life threatening hemorrhagic disease. Interestingly, it has been observed that FIXa-deficient patients, independent of the severity (mild, moderate and severe), as well as female carriers have a reduced incidence of thrombotic events while increased levels of Factor IXa in the blood lead to a significantly increased risk of thrombosis.1,2 These observations have been confirmed in

preclinical species as the regulation of Factor IXa activity can reduce thrombus formation in animal models.3 Unlike thrombin or FXa inhibitors which are inhibitors of the common pathway, FIXa inhibitors mostly target the ‘intrinsic’ pathway of the coagulation propagation cascade. Such ‘selective’ clotting mechanisms may lead to better safety margins clinically. Several groups have published their research toward finding potent and selective FIXa inhibitors.4 Previously, we have reported our early hit-to-lead efforts leading to an acyclic lead class of FIXa inhibitors with moderate potency and selectivity over FXa (compound 1).5,6

H N N

Cl NH O

N

N N

1

⇑ Corresponding author. Tel.: +1 732 594 3031. E-mail address: [email protected] (T. Zhang). http://dx.doi.org/10.1016/j.bmcl.2015.04.057 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Zhang, T.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.04.057

2

T. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx O O

Cl O

OEt 2

+

NH 2

N

HO

OEt Cl NH

a

N N

3

N N

N

O 4

O OH b

Cl

O

H N

c-e

NH N

N N

N

NH

5

1

Cl N N

N

O

Scheme 1. Reagents and conditions: (a) EDC, HOAt, pyridine, DMF, rt, 75%; (b) LiOH, dioxane, water, rt, 81%; (c) HATU, 4,5-dimethyl-1,2-phenylenediamine, Hunig’s base, DMF, rt; (d) AcOH, 80 °C, 3 h, then rt, overnight; (e) chiral separation on OJ column, 27% 3 steps.

Table 1 SAR of the S2–S4 binding moiety

Table 2 SAR of the tertiary phenyl binding moiety N

N

N

N

N O

Entry

X

Compd

X

Entry

hFIXa Ki (nM)

hFXa Ki (nM)

Ratio

15.7

394

25

N

N N

Compd

hFIXa Ki (nM)

hFXa Ki (nM)

Ratio

1

15.7

394

12

19

19.3

412

21

20

5.7

1010

177

21

8.4

1507

179

22

15.4

1601

104

F N

N N

N

N N

1

2 F

3 13

3.6

105

29 F

4

Cl

Cl

3

Ar

1

Cl

2

Cl N O

Cl

1

Ar

5

N

N N

N

N N

15

8.0

195

24

N

N N

16

21.8

340

16

N

N N

17

14.8

245

16

18

20.8

2473

119

14

15.2

544

N

36

F Cl

4 Me Cl

5 F3C Cl

6 MeO

Cl N

7

N N

Cl

Herein, we continue the discussion on further SAR studies covering the acyclic series, leading to potent and selective FIXa inhibitors which achieved oral bioavailability in PK studies and demonstrated efficacy in PD assay. The general syntheses of analogs of compound 1 are described in Scheme 1. Starting from compound 2, amide coupling with tail piece 3 afforded amide 4, which underwent ester hydrolysis to give acid 5. Condensing acid 5 with diamine piece yielded final compound, which was subjected to chiral separation to give

enantiomerically pure FIXa inhibitor 1. Analogs shown in Table 1 and Table 2, as well as entries 1–4 and entry 7 in Table 3 were synthesized using similar approaches described above. Alternatively, some P1 moieties were synthesized as shown in Scheme 2. Starting from heterocyclic halide 6, Pd-catalyzed a-arylation with compound 7 led to nitrile 8.7 Compound 8 was then reduced using literature methods to give primary amine 9.8 Converting acid piece 10 to acid chloride 11, followed by coupling with amine 9 gave the final compound, which was subjected to chiral resolution and gave FIXa inhibitor 12. Compounds in Table 3, entries 5–6 and entries 8–9 were synthesized using similar methods. Enzymatic activity of Factor IXa and Factor Xa was determined measuring the hydrolytic release of an amide-linked fluorophore from a fluorescently labeled tri-peptide substrate. Inhibitory activity (IC50) of test compounds was determined from a dose titration using a four parameter logistic fit, and subsequently converted to Ki using the Cheng–Prusoff equation for competitive inhibition.9 Selectivity of the compounds for Factor IXa is represented as a ratio of Ki values. According to crystal structures of lead compounds soaked into Factor IXa shown in our previous publication, we postulated that the 90s loop in the S4 region of human FIXa and FXa may allow for differentiation/selectivity gains.5,6 Overlays of FIXa and FXa indicate the S3/S4 region of FIXa is occupied by a significant portion of the FXa 90s loop (residues 95–99) providing a handle for selectivity. Therefore, we set out to explore the SAR of this region

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T. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx

3

Table 3 SAR of the S1 binding moiety F

Ar

NH

Cl N

O

Ar

N N

Cl

Compd

hFIXa Ki (nM)

hFXa Ki (nM)

Ratio

23

13.6

15160

1118

24

2.9

1932

677

N

1

N H

Cl O

2

N N H

Cl

N

3

25

9.8

20720

2119

26

6.2

>28890

>4679

12

9.1

2798

308

27

3.7

>28890

>7817

N

28

1.0

>28890

>28890

NH

29

3.3

>28890

>8684

N

30

10.4

>28890

>2775

N H

Cl

N

4

N H

Cl

5

HN N N

6

HN

O

NH

7 F O

8 F

9 F

by changing the S2–S4 binding moiety (Table 1). Adding one extra Cl on the chlorophenyl ring improved FIXa potency by 4–5 fold (Table 1, entries 1–2). Substitutions other than Cl were also investigated, but led to inferior FIXa potencies (Table 1, entries 3–6).

Figure 1. X-ray crystal structure (PDB code 4ZAE) of compound 13 (peach) bound to FIXa protein. A hydrophobic pocket is formed by the orange residues. Crystal waters are shown as red spheres.

Changing the 1,3,4-triazole to 1,2,4-methyltriazole had significant effects on FIXa/FXa selectivity (Table 1, entry 7) likely induced by a close steric interaction (50000 >50000 >50000 >50000

>50000 30400 >50000 31650 >50000

9089 19820 22380 24850 42760

456 5280 4043 1573 24400

10930 5351 3051 19900 >60000

13. TGA references: Kravtsov, D. V.; Matafonov, A.; Tucker, E. I.; Sun, M. F.; Walsh, P. N.; Gruber, A.; Gailani, D. Blood 2009, 114, 452; Hemker, H. C.; Giesen, P. L.; Ramjee, M.; Wagenvoord, R.; Béguin, S. Thromb. Haemost. 2000, 83, 589; De Smedt, E.; Al Dieri, R.; Spronk, H. M.; Hamulyak, K.; ten Cate, H.; Hemker, H. C. Thromb. Haemost. 2008, 100, 343. See the Supporting information for TGA assay protocol. 14. References for apixaban: Wong, P. C.; Pinto, D. J. P.; Zhang, D. J. Thromb. Thrombolysis 2011, 31, 478; Pinto, D. J. P.; Orwat, M. J.; Koch, S.; Rossi, K. A.; Alexander, R. S.; Smallwood, A.; Wong, P. C.; Rendina, A. R.; Luettgen, J. M.; Knabb, R. M.; He, K.; Xin, B.; Wexler, R. R.; Lam, P. Y. S. J. Med. Chem. 2007, 50, 5339.

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Development of a novel class of potent and selective FIXa inhibitors.

Using structure based drug design (SBDD), a novel class of potent coagulation Factor IXa (FIXa) inhibitors was designed and synthesized. High selectiv...
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