Fused bi-heteroaryl substituted hydantoin compounds as TACE inhibitors

Bioorganic & Medicinal Chemistry Letters 27 (2017) 3037–3042

Contents lists available at ScienceDirect

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

Fused bi-heteroaryl substituted hydantoin compounds as TACE inhibitors Ling Tong a,⇑, Seong Heon Kim a, Kristin Rosner a, Wensheng Yu a, Bandarpalle B. Shankar a, Lei Chen a, Dansu Li a, Chaoyang Dai a, Vinay Girijavallabhan a, Janeta Popovici-Muller a, Liping Yang a, Guowei Zhou a, Aneta Kosinski a, M. Arshad Siddiqui a, Neng-Yang Shih a, Zhuyan Guo b, Peter Orth a, Shiying Chen c, Daniel Lundell d, Xiaoda Niu d, Shelby Umland d, Joseph A. Kozlowski a,b,c,d a

Department of Discovery Chemistry, Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, NJ 07033, USA Department of Chemistry, Modeling and Informatics, Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, NJ 07033, USA c Department of Pharmacokinetics, Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, NJ 07033, USA d Department of Immunology, Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, NJ 07033, USA b

a r t i c l e

i n f o

Article history: Received 23 April 2017 Revised 16 May 2017 Accepted 20 May 2017 Available online 22 May 2017

a b s t r a c t We have identified a series of hydantoin-derived TNF-a converting enzyme (TACE) inhibitors containing a pendant fused bi-heteroaryl group, which demonstrate sub-nanomolar potency (Ki), excellent activity in human whole blood assay, and improved DMPK profiles over prior series. Ó 2017 Published by Elsevier Ltd.

Keywords: Tumor necrosis factor-alpha TACE inhibitors Hydantoin

The overexpression of tumor necrosis factor-a (TNF-a), a proinflammatory cytokine, has been associated with inflammatory diseases such as rheumatoid arthritis (RA) and Crohn’s disease.1,2 Current treatment for these diseases includes the use of injectable anti-TNF antibodies or soluble TNF receptors, such as RemicadeÒ and EnbrelÒ, which aim to control the levels of active TNF- a. While these agents have been successful, an orally active, selective small molecule with a similar function as the biologic agents could provide potential advantages such as ease of administration and cost reduction, while still maintaining adequate control of TNF-a levels. TNF- a is synthesized as a 26 kDa membrane bound form, which is cleaved by tumor necrosis factor-alpha converting enzyme (TACE) to generate the soluble 17 kDa form.2 TACE or ADAM17 is a member of the ‘‘A Disintegrin And Metalloprotease” or ADAM family, which includes an extracellular zinc-dependent protease domain. Both the membrane bound and soluble forms of TNF-a are biologically active, however soluble TNF- a is believed to be the major driver of TNF-mediated proinflammatory processes via interaction with tumor necrosis factor receptor 1 (TNFR1).3 Based on this hypothesis, a selective inhibitor of TACE is predicted

⇑ Corresponding author. E-mail address: [email protected] (L. Tong). http://dx.doi.org/10.1016/j.bmcl.2017.05.062 0960-894X/Ó 2017 Published by Elsevier Ltd.

to be an effective anti-inflammatory agent based on its ability to decrease soluble TNF-a levels.4 Our early research efforts on small molecule TACE inhibitors led to a series of hydantoin compounds which were designed based on the X-ray structure of the enzyme.5,6 Further SAR development identified hydantoins containing a pendant acetylene group that demonstrated improved affinity and activity in a human whole blood assay (hWBA), represented by 17 (Fig. 1). While the acetylene functionality was a structural feature that helped to produce potent compounds, the overall properties of these inhibitors were suboptimal since 1 exhibited low oral bioavailability in rat, monkey and dog.7 Therefore, efforts were devoted to developing analogs that could provide a combination of potent hWBA and acceptable pharmacokinetic (PK) profiles. Previous structural analysis revealed that the pendant acetylene group in 1 projects into solvent exposed S1 pocket.5 Therefore, it presented an opportunity to modify physicochemical properties which could improve PK parameters. One strategy was to utilize the existing acetylene as a synthetic handle to access other functionalities such as the fused bi-aryl scaffold 3 shown in Fig. 1. Synthesis of compound 4, as shown in Scheme 1, represents a general approach for accessing these analogs. Advanced intermediate 4a was obtained using published patent procedure8 which can be coupled to various aryl and heteroaryl hydroxy halides using

3038

L. Tong et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 3037–3042

Fig. 1. Development of fused biaryl hydantoin compounds.

Scheme 1. Reagents and conditions: a) CuI, Pd(PPh3)2Cl2, triethylamine, DMF, 80 °C, overnight, 42%.

standard Sonagashira reaction conditions to provide compounds of type 4.9 An example of accessing 5,5-bicyclic heteroaryl hydantoin compounds is shown in Scheme 2 with the synthesis of the representative hydantoin 11. The advanced intermediate 11b can be obtained through published procedures8 via the corresponding ketone 11a where 11a was prepared from intermediate 4a. Bromo keone 11b was treated with 2-aminothiazole to afford 11,10 via intermediate 11c, which was used in the next step without purification. Table 1 displays the TACE Ki and hWBA activities11 of various fused bi-cyclic aryl and heteroaryl derived hydantoin compounds. Their rapid rat AUC values12 were also included whenever available. Compounds 3–11 displayed potent TACE enzyme activity; however their hWBA activities varied significantly. The benzofuran 3, with sub-nanomolar TACE Ki, showed a marked right shift in hWBA (2.31 lM). Addition of a nitrogen atom in the benzofuran led to improved hWBA activity (to <300 nM range) as shown by 4 and 5 (Table 1). A second nitrogen atom, as in the aza benzofuran 7, did not further improve hWBA (0.77 lM) when compared with 4 and 5. The corresponding aza indole analog 8 showed weaker potency in both TACE Ki (3.6 nM) and hWBA (2.97 lM) when

compared with 5. Selected variations of 6,5 and 5,5 fused bi-cyclic aromatic ring systems were investigated (9–11). None of these analogs offered meaningful improvement over 4 and 5 in terms of either their hWBA activity or their rapid rat AUC values. Among the compounds in Table 1, 4 and 5 showed the best combination of hWBA and rapid rat AUC values. We were encouraged by the hWBA and rapid rat AUC that were displayed by aza benzofuran analogs such as 4 and 5 and we sought to further investigate this series by examining the impact of substitutions of the aza benzofuran portion of the molecule on these assays. Previous structural analysis revealed that this R group projected into solvent exposed S1 pocket,5 and we observed that incorporation of polar functionality in this position could potentially improve hWBA.7 Table 2 displays the data for substituted 5-aza benzofuran hydantoins. For R1 substitution, various polar groups were able to maintain hWBA similar to the parent compound 4 as demonstrated by 12, 14 and 15. However, polar substitutions seemed to be hurting PK properties as shown by poor AUC of 14. Interestingly, a cyano group at R1 was detrimental towards hWBA (16, hWBA = 1.24 lM). A lipophilic group such as CF3 at this position also decreased the hWBA as shown by 17. A similar trend

Scheme 2. Reagents and conditions: a) 2 N H2SO4, HgO, MeOH, 80 °C, 1 h, 76%; b) Bromine, rt to 50 °C, acetic acid, 3 h, crude; c) 2-aminothiazole, acetone, 65 °C, 1 h.; d) 2 N HCl, 80 °C, 1 h, 21% over 2 steps.

3039

L. Tong et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 3037–3042 Table 1 TACE Kia, hWBAb and rapid rat PK of selected fused bi-aryl/heteroaryl hydantoins.

TACE Ki (nM)

hWBA (lM)11

Rapid rat AUC (lM.h)12

3

0.3

2.31

0.20

4

0.7

0.27

13

5

0.5

0.29

27

6

0.7

0.59

34

7

1.6

0.77

2.2

8

3.6

2.97

NDc

9

1.9

0.71

3.2

10

1.4

1.48

NDc

11

1.3

0.54

2.2

Compound

R

a The compounds were tested in a FRET assay using the catalytic domain of TACE, each value is a determination of a minimum of three measurements, assay variation is up to 3-fold. b Human whole blood was diluted with 1:1 serum free medium; each value is a determination of a minimum of three measurements, assay variation is up to 3-fold. c ND = not determined.

was observed at R2 position. Polar substitution at this position, such as imidazolidin-2-one (18), demonstrated potent hWBA (0.21 lM) while a CF3 group displayed a loss of hWBA that was similar to the loss observed when R1 = CF3 (19, hWBA = 1.62 lM). Interestingly, a methyl group at both R1 and R2 position was well tolerated as demonstrated by 13 and 20. Compound 20 also demonstrated an improved rapid rat AUC of 19.1 lM.h. Further modification of this compound, with addition of a fluorine atom to the lactam phenyl, gave rise to 22, which resulted in a reasonably potent hWBA value and a high rapid rat AUC. When the methyl group was moved to R3 position (21), the hWBA (0.76 lM) was trending worse, although the rapid rat PK was

reasonable (8.7 lM.h). A polar substitution such as an NH2 was also well tolerated at R3 position as shown by 24. A similar investigation was carried out with 4-aza benzofuran hydantoins. Table 3 summarizes the result for this exercise. Addition of the NH2 group on R1, shown in 25, demonstrated reasonably potent hWBA (187 nM). However, this compound exhibited low rapid rat AUC (0.4 lM.h). The hWBA of the C-linked pyrazole analog 26 (99 nM) was similar or slightly better than 5, although the rapid rat AUC was low. Interestingly, when the pyrazole was linked to aza benzofuran through the nitrogen (27), the rapid rat AUC was significantly improved to 21.0 lM.h. However, this modification resulted in a loss of hWBA potency (1370 nM for 27 vs. 99 nM

3040

L. Tong et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 3037–3042

Table 2 TACE Ki/hWBAa,b activities of substituted 5-azabenzofuran hydantoins.

Compound

R1

R2

R3

X

Ki (nM)

hWBA (lM)11

Rapid rat AUC (lM.h)12

12 13 14

OH Me

H Me H

H H H

H H H

0.2 0.5 2.5

0.25 0.68 0.23

ND 4.2 0

H

H

H

0.2

0.15

NDc

H H

H H H

H H H

0.7 0.4 1.8

1.24 1.07 0.21

0.5 NDc 1.2

CF3 Me H Me H H

H H Me H F NH2

H H H F H H

0.5 0.4 0.5 0.4 2.0 0.4

1.62 0.35 0.76 0.21 1.09 0.31

19.3 19.1 8.7 26.3 2.7 0.1

15

16 17 18

CF3 H

19 20 21 22 23 24

H H H H H H

a The compounds were tested in a FRET assay using the catalytic domain of TACE, each value is a determination of a minimum of three measurements, assay variation is up to 3-fold. b Human whole blood was diluted with 1:1 serum free medium; each value is a determination of a minimum of three measurements, assay variation is up to 3-fold. c ND = not determined.

for 26). This trend seemed to show that a hydrogen bond donor in this region of the molecule, although solvent exposed, has a large positive impact on hWBA potency. Compound 28 also demonstrated hWBA similar to 26, and it also had the ability to be a hydrogen bond donor. Interestingly, 29 did not have a hydrogen bonding donor substitution, but contained a basic nitrogen, and also demonstrated potent hWBA (76 nM). This data demonstrated that basicity in this position could also have a positive impact on hWBA. The origin of this positive effect was unknown and appeared empirical. We then incorporated these two findings into a molecule which contained functional groups that provide both hydrogen bond donor ability and also a basic amine. Compound 31 has a tertiary nitrogen while 30 contains a secondary nitrogen. Although both compounds demonstrated potent hWBA values (93 nM for 30 and 187 nM for 31), they did not show additive effects derived from the two functionalities. The R2 position also tolerated polar substitutions as shown by 32 and 33. Although most of these substitutions demonstrated potent hWBA, the majority of them showed poor rapid rat AUC values. The lack of desirable PK properties with compounds bearing polar groups could be due low permeability. For example, in Caco-2 mono-layer permeability assay,13 30 and 31 displayed AP to BL low permeability values of 2.2 nm/s and 1 nm/s respectively. On the other hand, compounds bearing lipophilic substituent such as 20 demonstrated improved permeability value of 20.5 nm/s, and higher rat AUC value was observed. A more balanced profile was achieved when combining a polar substituent on R1 and a fluorine atom on R2, shown by 34 (hWBA = 101 nM, rapid rat AUC = 3.2 lM.h).

Due to their favorable overall in vitro profiles, compounds 4, 5, and 22 were selected for further characterization. Their PK profiles in rat, dog, and monkey are illustrated in Table 4. In comparison, 4 and 5 demonstrated significant improved PK properties over the acetylene series (1).7 Significant improvement of oral exposures in all three species were achieved with 4 and 5. Higher bioavailability was observed in both rat and dog for these compounds. Compound 5 also exhibited increased bioavailability in monkey. Compound 22 demonstrated higher oral exposure in rat than 1 with no improvement in bioavailability. The origin of the higher bioavailability obtained by 4 and 5 were not entirely clear. In Caco-2 mono-layer permeability assay, 4 and 5 displayed AP to BL permeability values of 15 nm/s and 12 nm/s respectively, comparable to 16 nm/s for 1. Table 5 displays clearance data for 1,4 and 5 in rat, dog and monkey. The higher bioavailability for 4 and 5 in dog could be attributed to their improved clearance values (3 mL/ min/kg for 4 and 3 mL/min/kg for 5) over 1 (19 mL/min/kg). In summary, we have discovered hydantoin compounds, which contain a pendant aza benzofuran group as a key moiety, which display potent hWBA and improved PK profiles. Substitutions on the aza benzofuran ring with polar functional groups, especially basic groups and hydrogen bonding donor groups, demonstrated positive effect on improving hWBA. In particular, we were able to improve hWBA to less than 100 nM with compounds such as 29. Most of these compounds, bearing polar groups, were limited by poor PK profiles. However, compounds 4,14 5 and 22 were profiled further and have shown overall improved PK profiles as well as good hWBA. Further optimization of the human whole blood

3041

L. Tong et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 3037–3042 Table 3 TACE Ki/hWBAa,b activates of substituted 4-azabenzofuran hydantoins.

R1

R2

X

Ki (nM)

hWBA (nM)11

Rapid rat AUC (lM.h)12

25

H

H

0.5

187

0.4

26

H

H

0.5

99

0.1

27

H

H

0.4

1370

21.0

28

H

F

0.6

95

0.1

29

H

F

NDc

76

0.04

30

H

H

0.4

93

NDc

31

H

H

1.0

187

0.06

Compound

32

H

H

0.9

351

0

33

Me

F

NDc

308

NDc

F

0.4

101

3.2

34

F

a The compounds were tested in a FRET assay using the catalytic domain of TACE, each value is a determination of a minimum of three measurements, assay variation is up to 3-fold. b Human whole blood was diluted with 1:1 serum free medium; each value is a determination of a minimum of three measurements, assay variation is up to 3-fold. c ND = not determined.

Table 4 In vivo pharmacokinetic profiles for 1 vs. 4, 5 and 22.a No.

1 (Na+ salt) 4 (HCl salt) 5 (Na+ salt) 22 (HCl salt) a b

Rat

Dog

Monkey

PO dose (mg/kg)

AUC (lM.h)

F%

PO dose (mg/kg)

AUC (lM.h)

F%

PO dose (mg/kg)

AUC (lM.h)

F%

10 10 10 10

4.0 11.0 10.0 13.4

15 58 35 11

5 3 3 2

1.1 52.0 41.0 13.5

9 98 82 NDb

3 3 10 3

2.6 10.0 22.0 1.5

23 19 46 NDb

Formulations: 0.4% HPMC. ND = not determined.

3042

L. Tong et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 3037–3042

Table 5 IV clearance for 1, 4 and 5 in rat, dog and monkey. No.

1 (Na+ salt) 4 (HCl salt) 5 (Na+ salt)

CL (mL/min/kg) Rat

Dog

Monkey

19 23 15

19 3 3

13 7 11

activity and PK profiles of our lead hydantoin TACE inhibitors will be reported in future communications. A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.05. 062. References 1. Cerretti DP. Biochem Soc Trans. 1999;27:219. 2. Vassalli P. Annu Rev Immunol. 1992;10:411. 3. Ruuls S, Hoek R, Ngo V, et al. Immunity. 2001;15:533.

4. (a) Bemelmans MH, van Tits LJ, Buurman WA. Crit Rev Immunol. 1996;16:1; (b) Aggarwal BB, Natarajan K. Eur Cytokine Netw. 1996;7:93. 5. Yu W, Guo Z, Orth P, et al. Bioorg Med Chem Lett. 2010;20:1877. 6. Yu W, Tong L, Kim SH, et al. Bioorg Med Chem Lett. 2010;20:5286. 7. Girijavallabhan V, Chen L, Dai C, et al. Bioorg Med Chem Lett. 2010;20:7283. 8. Yu W, Tong L, Chen L, et al., US2007/0219218 (A1); 2007. 9. NMR data for compound 4: 1H NMR (400 Hz, DMSO-d6) d:11.36 (s, 1H), 9.31 (s, 1H, NH), 9.10 (d, 1H, J = 1.4 Hz), 8.8 (bs, 1H), 8.23 (d, 1H, J = 6.2 Hz), 7.50 (m, 2H), 7.18 (m, 2H), 4.39 (m, 4H), 3.80 (s, 3H). MS m/z: calcd for C20H16N4O5, 392.11; found 393.1[M+H]+. 10. Tong L, Lavey BJ, Shankar BB, et al., PCT Int. Appl. (2010), WO 2010054278 A2 20100514. 11. Assay for inhibition of TNF-a production from human whole blood (hWBA): human whole bood was diluted 1:1 with serum free medium (RPMI, Lglutamine, Pen-Strep, HEPES) and incubated with a test compound in a final volumne of 360 lL for 1 h at 37 oC. Forty microlters of LPS (10 lL/mL) was then added. Supernatant was collected after 3.5 h incubation and the concentration of TNF-a was determined by ELISA (R&D Systems). The concentration of the test compound which inhibits 50% of the amount of TNF-a from the untreated control was determined. Assay variation is up to 3-fold. 12. For rapid rat AUC determination, see example 205 in Ref. 8. For detailed protocol, please see: Korfmacher W, Cox K, Ng K, Veals J, Hsieh Y, Wainhaus S, Broske L, Prelusky D, Nomeir A, White R. Rapid Commun Mass Spectrometry. 2001;15:335. 13. Chen K, Vibulbhan B, Liu T, et al. Drug Metab Lett. 2009;3:290. 14. Compound 4 is a selective TACE inhibitor with IC50 values of 200 to >40,000 nM versus a set of related metalloproteases. ADAM10 is the closest homolog of TACE. Compound 4 inhibits ADAM10 enzyme (Ki = 144 nM). For further detail, refer to Table S1 in the supplementary material.