Bioorganic & Medicinal Chemistry Letters 25 (2015) 2169–2173
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of potent, selective phenylimidazole-based FVIIa inhibitors Peter W. Glunz ⇑, Xuhong Cheng, Daniel L. Cheney, Carolyn A. Weigelt, Anzhi Wei, Joseph M. Luettgen, Pancras C. Wong, Ruth R. Wexler, E. Scott Priestley Bristol-Myers Squibb R&D, 311 Pennington-Rocky Hill Rd, Pennington, NJ 08534, United States
a r t i c l e
i n f o
Article history: Received 26 February 2015 Revised 19 March 2015 Accepted 23 March 2015 Available online 30 March 2015 Keywords: Factor VIIa Tissue factor TF–FVIIa inhibitor Serine protease inhibitors Amide isosteres
a b s t r a c t Heterocyclic amide isosteres were incorporated into a phenylglycine-based tissue factor/factor VIIa (TF–FVIIa) inhibitor chemotype, providing potent inhibitors. An X-ray co-crystal structure of phenylimidazole 19 suggested that an imidazole nitrogen atom effectively mimics an amide carbonyl, while the phenyl ring forms key hydrophobic interactions with the S10 pocket. Exploration of phenylimidazole substitution led to the discovery of potent, selective and efficacious inhibitors of TF–FVIIa. Ó 2015 Elsevier Ltd. All rights reserved.
Arterial thrombosis is a leading cause of mortality and morbidity in the developed world.1 Comparisons of antithrombotic mechanisms have identified the tissue factor–factor VIIa proteolytic complex (TF–FVIIa) as a therapeutic target that offers strong antithrombotic efficacy and a low bleeding liability.2 This serine protease initiates the coagulation cascade by activating factors IX and X to IXa and Xa, respectively, leading to thrombin generation. Thrombin cleaves fibrinogen to fibrin and activates platelets, resulting in clot formation. FVIIa has attracted considerable attention in the pharmaceutical industry as a promising anticoagulant target.3 We chose phenylglycine-based inhibitors 1–44 (Fig. 1) as a starting point for inhibitor design for their potency and well-defined SAR. We sought to make progress toward oral bioavailability by modifying their peptide-like structure and zwitterionic character. We have reported replacement of the basic amidine P1 group within related chemotypes resulting in oral bioavailability.5,6 Herein, we sought to replace the amide or acylsulfonamide group. Heterocycles have been utilized extensively as replacements of amides to impart chemical and enzymatic stability, and therefore improved pharmacokinetic properties, into peptide ligands.7 Our strategy was to mimic the interactions of the amide or acylsulfonamide moiety of inhibitors 2–4 with His 57 and Ser 195 ⇑ Corresponding author. Tel.: +1 609 818 5287; fax: +1 609 818 3550. E-mail address: [email protected] (P.W. Glunz). http://dx.doi.org/10.1016/j.bmcl.2015.03.062 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
OEt NH
EtO
OEt
H 2N
H 2N OH
N H
NH
OEt
NH
N H
O 1 FVIIa Ki = 190 nM Oi-Pr OEt
H2 N N H
H N O
S O O
3 FVIIa Ki = 1 nM
H N
2 O FVIIa Ki = 40 nM
NH
EtO
OEt
H2 N HO
N H
NH2
F H N
S O O O G17905, 4 FVIIa Ki = 0.35 nM
Figure 1. Phenylglycine-based FVIIa inhibitors.
(Fig. 2a) using a heterocyclic replacement (Fig. 2b). This change removes a potential site for proteolytic cleavage or a negative charge. We then undertook a structure-based approach to identify additional binding interactions, exploiting the critical hydrophobic S10 pocket.5 The general synthesis of inhibitors 9–22 (Scheme 1) involves addition of a lithiated heterocycle to 4-(benzylideneamino)benzonitrile 7 to generate amine 8. Lithiation of 4-phenyloxazole, en route to analog 22, required pre-complexation with BF3 to avoid
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P. W. Glunz et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2169–2173
(a)
Oi-Pr
H N
N H
(b)
S2
OEt
Oi-Pr
N H
S1'
N
S1'
R
25
H O
H N
Leu 41
H N
Ser 195
N H
N H
S
Cys 58
S
Cys 42
His 57
His 57
Figure 2. (a) Binding interactions of acylsulfonamide (X = SO2) and amide (X = CH2) inhibitors. (b) Design of FVIIa inhibitors to mimic carbonyl interactions and reach the S10 pocket.
OHC
OEt
NC
NC a NH2
R1
5
R3
OEt
N
R2 6a,b
7a,b
R1
R3 R2 R3
R3 EtO b
Het-H(Br)
R2
NC
R1
NH
EtO
R2
c-f H 2N
R1
Het
N H
N H 9-22
8a,b a: R1, R2 = H, R3 = Oi-Pr b: R1 = F, R2 = Oi-Pr, R3 = H
Het
Scheme 1. General synthetic strategy for heterocyclic FVIIa inhibitors. Reagents and conditions: (a) 4 Å mol sieves, toluene, reflux (7a, 87%; 7b, 99%); (b) BuLi, THF; then 7a or 7b (8a, 20–72%; 8b, 24–78%); (c) 3 M NH2OH, DMSO, 60 °C, 2 h; (d) Ac2O, CH2Cl2, rt; (e) H2 (balloon), Pd–C, MeOH; (f) 90% aq AcOH, 60 °C (24–79%).
EtO Tr N
7b Br 23
Oi-Pr
a NC
N
Oi-Pr F Tr N
N
H
Ser 195
EtO
N H X
O
O
O N
S2
OEt
F Tr N
N H
c,d,g
O N
EtO
F Tr N
N N H
N 24
Oi-Pr
N 25
Br
Br
Method B b,h,f
b-f Method A NH
EtO
H 2N N H N via Method A: 26-32 via Method B: 33-35
Oi-Pr F H N
Ar
Scheme 2. General synthetic routes for preparation of substituted phenylimidazole inhibitors. Reagents and conditions: (a) BuLi, THF, 0 °C, then 7b, 71%; (b) ArB(OH)2, Pd(PPh3)4, Na2CO3, DME/H2O, 150 °C, 5 min or 80 °C, 2 h; (c) NH2OH, DMSO, 60 °C; (d) Ac2O, CH2Cl2, rt; (e) H2, 10% Pd–C, MeOH; (f) 90% aq AcOH, 60 °C; (g) TBAF, THF, rt, 88% for 3 steps to 25; (h) H2 (1 atm), 10% Pd–C, 8:1 MeOH/TEA or MeOH + 1 drop AcOH. (Method A: 9–41%, 5 steps. Method B: 7–21%, 3 steps).
oxazole ring opening.8 The nitrile functionality was converted to the amidine, and the target was deprotected as necessary. Substituted phenylimidazoles were prepared as shown in Scheme 2. 4-Bromo-1-trityl-1H-imidazole (23) was lithiated at the 2-position,9 then was reacted with aldimine 7b, affording amine 24. Suzuki–Miyaura coupling, amidine formation and
N Br
NH
EtO
Oi-Pr F H N
H 2N a-d
N H
N
O R
36, R = NHMe 37, R = NMe 2,
Scheme 3. Synthesis of amide analogs. Reagents and conditions: (a) 2-(CO2Et)PhB(OH)2, Na2CO3, P(o-Tol)3, Pd(OAc)2, DME/H2O, 80 °C, 2 h, 92%; (b) aq LiOH, THF/ MeOH, 64%; (c) MeNH2HCl (R = NHMe), TEA or Me2NH (R = NMe2), BOP, DMF, (R = NHMe, 94%; R = NMe2, 92%); (d) H2, 10% Pd–C, MeOH/THF (1:1), AcOH (1 drop); 90% aq AcOH, 60 °C (36, 47%; 37, 43%).
deprotection produced the targeted inhibitors (Method A). Alternatively, nitrile 24 is converted to 1,2,4-oxadiazole 25,10 which serves as a protected amidine and allows a single deprotection step, following Suzuki coupling, to afford analogs 33–35, thus reducing the steps per analog from five to three (Method B). Amide derivatives were prepared as shown in Scheme 3. Suzuki coupling of 25 with (2-(ethoxycarbonyl)phenyl)boronic acid afforded the ester, en route to amides 36 and 37. FVIIa inhibitory potencies for an initial set of heterocycles is listed in Table 1. Neutral and basic monocyclic heterocycles 9–12 are weakly potent inhibitors, whereas the carboxylic acid isostere11 tetrazole 13 is 4-fold more potent than its acid counterpart 1. Fused heterocycles 14–17 generally display improved potency relative to their monocyclic counterparts; quinoline 16 is 15-fold more potent than 2-pyridyl analog 11. A phenyl substituent on the heterocycle had a dramatic effect. 4-Phenylimidazol-2-yl analog 19 is a potent inhibitor of FVIIa, Ki = 11 nM versus 520 nM for imidazole analog 12. 19 is 4-fold more potent than unsubstituted benzylamide 2 and 10-fold less potent than acylsulfonamide 3. Methylation of each imidazole nitrogen (20 and 21) provided evidence of the tautomeric binding mode of the phenylimidazole, suggesting interaction between the nitrogen lone pair adjacent to the phenyl ring and the enzyme active site. This binding orientation of 19 was confirmed by Xray crystallography in FVIIa, see below. Phenyloxazole 22 is similar in potency to phenylimidazole 19, suggesting similar binding interactions for the two heterocycles. Phenylimidazole 18 proved unstable under acidic HPLC conditions (MeOH/H2O + 0.1% TFA), precluding isolation of the pure compound and leading us to investigate acid stability. Compounds 9, 12 and 14 were found prone to hydrolysis, affording biarylmethanol and 4-aminobenzamidine degradants upon hydrolysis. We hypothesized that this instability is due to electron donation by the para-O-i-Pr group and could be circumvented by moving the 4-alkoxy group out of conjugation with the a-carbon of the core. Replacing the 3,4-dialkoxy P2 group with a 2-F-3,5-dialkoxy P2 had the intended effect of improving stability (Table 1, A vs B), and thus this P2 group was employed for subsequent SAR exploration. The X-ray crystal structure of compound 19 in FVIIa is shown in Figure 3a.12 The inhibitor adopts a similar binding mode to reported phenylglycine-based FVIIa inhibitors such as acylsulfonamide 4 (PDB ID: 1YGC)4e with superposition of the P1 and P2 groups (Fig. 3b) as shown in Figure 3b. Closely related analogs of acid 1 and benzylamide 2 (PDB ID: 2BZ64b and PDB ID: 1W7X,4a respectively) bind similarly (data not shown). The imidazole moiety aligns with the carbonyl as an isostere forming close contacts with His 57 and Ser 195. Owing to the planarity of the imidazole ring, the 4-phenyl substituent of 19 is directed into the hydrophobic S10 pocket, with the meta and para positions of the ring interacting with the Cys 42–Cys 58 disulfide bond and
P. W. Glunz et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2169–2173
2171
Table 1 FVIIa inhibition13 and chemical stability of heterocyclic isosteres
Oi-Pr
NH
OEt H2 N
P2 N H
i-PrO F
P1' A P10
Het-H/Br
S
S
N
N
9
10 11
Br N
N
Tr N
H N
N
N N N N N H
12
N N N N
13
14 15
16
17
BOM S
S
N
N
Br N
N
BOM N
H N
N
Ph
Ph
N
A
2100
A B
1100 750
A
520
B
49
>24a
A B
80 420
1 >24a
B
49
>24a
B
140
>24a
A B
— 11
B
15
22
B
1800
104
B
20
pH 1 stab. (t1/2, h) 0.4 83 >24a
12.5
Unstableb 42
Ph
Ph
N Ph
N Me O
Ph
Ph
N
Ph
O 22
N
N
N N Me
FVIIa Ki (nM)
Me N
20
21
P2
N
Me N N
B
H N
N Tr N
18 19
OEt
P2 :
>24c
Ki determination, n P 2. a 100% remaining at 24 h. b Unstable in preparative HPLC solvent (MeOH/H2O + 0.1% TFA). c 96% remaining at 21 h.
the Leu 41 side chain, respectively. We have exploited this critical hydrophobic S10 interaction via substitution of related acylsulfonamide5 and benzylamide6 chemotypes. The phenylimidazole forms this interaction directly. This crystal structure is consistent with the N-methylation results (20 and 21). Subsequent to our patent filing,14 a related triazolone chemotype was reported,15 which presumably makes similar interactions with the FVIIa binding site. A double-prodrug strategy was employed to attain oral bioavailability with these compounds.16 Unsubstituted phenylimidazole 19 shows good selectivity versus thrombin, FXa and trypsin (110-, 70- and 60-fold, respectively), but only weak efficacy in a modified-PT clotting assay17 (Table 2). Our efforts next focused on introducing substitution to enhance inhibitory potency and clotting efficacy, while maintaining or improving selectivity.
Figure 3. (a) X-ray crystal structure of 19 bound in FVIIa, solved at 2.07 Å, and depicting the initial Fo–Fc map of electron density (at 3 sigma) within 1.6 Å of the ligand. (b) Overlay with bound acylsulfonamide 4 (PDB ID: 1YGC).4e Arrows highlight different directionality of phenyl groups in acylsulfonamide and imidazole-based inhibitors.
The ortho and meta positions are more tolerant of substitution than the para position (28 and 31), consistent with the observed binding mode of 19 in FVIIa. We focused our SAR exploration on the ortho position, which based upon the X-ray structure of 19 either occupies a polar, exposed region (2-position) or is directed towards the catalytic triad (6-position). Hydrogen bond acceptors, including ketone and ester moieties, are accommodated in the ortho position. While carboxylic acid 34 is 2-fold more potent than 19, it is 6-fold more potent in the clotting assay, likely due to decreased protein binding. Efforts to identify other neutral, polar substituents led to primary amide 35. It is 6- and 8-fold more potent than 19 in the inhibition and clotting assays, respectively, and shows greatly improved selectivity versus other serine proteases due to enhanced activity against FVIIa. Compound 35 was inactive in the activated partial thromboplastin time assay (aPTT IC2x > 40 lM), consistent with selective FVIIa inhibition.2b Primary amide 35 is similar in
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Table 2 Substituted phenylimidazole SAR—FVIIa inhibitory potency,13 selectivity and clotting activity17
NH
EtO
Oi-Pr F H N
H2 N N H
N 2 R 3
22, 30-52 6 5
19 26 27 28 29 30 31 32 33 34 35 36 37
inhibitor chemotype, replacing an amide moiety with good inhibitory activity. Phenylimidazole 19 was identified as a potent inhibitor that mimics the binding interactions of the parent inhibitors’ carbonyl with the catalytic triad. The phenyl ring contributes significantly to binding through hydrophobic interactions with the S10 disulfide. Exploration of phenylimidazole substitution led to the discovery of primary amide 35, a potent, selective and efficacious inhibitor of FVIIa. Acknowledgments
4
R
FVIIa Ki (nM)
FIIa Ki (nM)
FXa Ki (nM)
Trypsin Ki (nM)
Mod. PT IC2x (lM)
H 2-Me 3-Me 4-Me 2-OMe 3-OMe 4-OMe 2-C(O)Me 2-CO2Me 2-CO2H 2-CONH2 2-CONHMe 2-CONMe2
11 19 37 207 10 48 1020 5.0 13 6.1 1.7 68 79
1200 710 750 760 570 790 640 1670 4470 190 1190 — —
760 680 450 430 460 560 540 430 4070 2190 2330 — —
690 400 500 610 430 580 490 560 1430 2410 1050 — —
37 48 63 ND 31 49 ND 26 >80 6.6 4.5 43 45
Ki determination, n P 2.
Figure 4. X-ray crystal structure of 35 bound in FVIIa, solved at 2.3 Å, and depicting the initial Fo–Fc map of electron density (at 3 sigma) within 1.6 Å of the ligand.
potency to acylsulfonamide 3 without the anionic moiety that renders the latter zwitterionic. The X-ray crystal structure of 35 in FVIIa13 was obtained (Fig. 4) and reveals a binding mode similar to that observed for 19. The primary amide is directed towards the partially solvent-exposed edge of the S10 binding pocket, forming hydrogen bonds to the His 57 backbone carbonyl and to the Asp 60 side chain via a water molecule. The amide substituent occupies the general hydrophilic region of the primary aniline of FVIIa inhibitor 4. Secondary and tertiary amides 36 and 37 are considerably less potent, consistent with the importance of these interactions present in the X-ray structure. In summary, we have developed an efficient and general method to synthesize heterocyclic amide-bond isosteres via addition of a lithiated heterocycle to an N-aryl imine. In this way, we have incorporated heterocycles into a phenylglycine-based FVIIa
The authors thank Frank Barbera, Jeffrey Bozarth, Randi Brown, Karen Hartl, Diane Normandin, Tara Peterson and Ge Zhang for conducting biological assays. Gerry Everlof is acknowledged for determining chemical stability. We would also like to thank Steven Sheriff for helping to prepare X-ray coordinates and data for PDB deposition. References and notes 1. Global Status Report on Noncommunicable Diseases 2010; WHO Press: Geneva, 2011. 2. (a) Szalony, J. A.; Taite, B. B.; Girard, T. J.; Nicholson, N. S.; LaChance, R. M. J. Thromb. Thrombolysis 2002, 14, 113–121; (b) Suleymanov, O. D.; Szalony, J. A.; Salyers, A. K.; Lachance, R. M.; Parlow, J. J.; South, M. S.; Wood, R. S.; Nicholson, N. S. J. Pharmacol. Exp. Ther. 2003, 306, 1115; (c) Salyers, A. K.; Szalony, J. A.; Suleymanov, O. D.; Parlow, J. J.; Wood, R. S.; South, M. S.; Nicholson, N. S. Pharmacology 2004, 70, 100; (d) Wong, P.; Crain, E.; Watson, C.; Staus, A.; Luettgen, J.; Rendina, A.; Abell, L.; Knabb, R.; Baomin, X.; Zhou, J.; DeLucca, I.; Priestley, E. S. J. Thromb. Haemost. 2005, 3, P0162. 3. (a) Shirk, R. A.; Vlasuk, G. P. Arterioscler Thromb. Vasc. Biol. 2007, 27, 1895; (b) Priestley, E. S. Drug Discovery Today 2014, 19, 1440. 4. (a) Zbinden, K. G.; Obst-Sander, U.; Hilpert, K.; Kuehne, H.; Banner, D. W.; Boehm, H.-J.; Stahl, M.; Ackermann, J.; Alig, L.; Weber, L.; Wessel, H. P.; Riederer, M. A.; Tschopp, T. B.; Lave, T. Bioorg. Med. Chem. Lett. 2005, 15, 5344– 5352; (b) Zbinden, K. G.; Banner, D. W.; Hilpert, K.; Himber, J.; Lavé, T.; Riederer, M. A.; Stahl, M.; Tschopp, T. B.; Obst-Sander, U. Bioorg. Med. Chem. 2006, 14, 5357; (c) Zbinden, K. G.; Banner, D. W.; Ackermann, J.; D’Arcy, A.; Kirchhofer, D.; Ji, Y.-H.; Tschopp, T. B.; Wallbaum, S.; Weber, L. Bioorg. Med. Chem. Lett. 2005, 15, 817–822; (d) Aliagas-Martin, I.; Artis, D. R.; Dina, M. S.; Flygare, J. A.; Goldsmith, R. A.; Munroe, R. A.; Olivero, A. G.; Pastor, R.; Rawson, T. E.; Robarge, K. D.; Sutherlin, D. P.; Weese, K. J.; Zhou, Z.; Zhu, Y. (Genentech, Inc., USA). U.S. Patent WO2000041531, 2000.; (e) Olivero, A. G.; Eigenbrot, C.; Goldsmith, R.; Robarge, K.; Artis, D. R.; Flygare, J.; Rawson, T.; Sutherlin, D. P.; Kadkhodayan, S.; Beresini, M.; Elliott, L. O.; DeGuzman, G. G.; Banner, D. W.; Ultsch, M.; Marzec, U.; Hanson, S. R.; Refino, C.; Bunting, S.; Kirchhofer, D. J. Biol. Chem. 2005, 280, 9160. 5. Glunz, P. W.; Zhang, X.; Zou, Y.; Delucca, I.; Nirschl, A. H.; Cheng, X.; Weigelt, C. A.; Cheney, D. L.; Wei, A.; Anumula, R.; Luettgen, J. M.; Rendina, A. R.; Harpel, M.; Luo, G.; Knabb, R.; Wong, P. C.; Wexler, R. R.; Priestley, E. S. Bioorg. Med. Chem. Lett. 2013, 23, 5244. 6. Zhang, X.; Jiang, W.; Jacutin-Porte, S.; Glunz, P. W.; Zou, Y.; Cheng, X.; Nirschl, A. H.; Wurtz, N. R.; Luettgen, J. M.; Rendina, A. R.; Luo, G.; Harper, T. M.; Wei, A.; Anumula, R.; Cheney, D. L.; Knabb, R. M.; Wong, P. C.; Wexler, R. R.; Priestley, E. S. ACS Med. Chem. Lett. 2014, 5, 188. 7. (a) Gordon, T.; Hansen, P.; Morgan, B.; Singh, J.; Baizman, E.; Ward, S. Bioorg. Med. Chem. Lett. 1993, 3, 915; (b) Hamada, Y.; Kiso, Y. Expert Opin. Drug Discov. 2012, 7, 903. 8. Vedejs, E.; Monahan, S. D. J. Org. Chem. 1996, 61, 5192. 9. Palmer, B. D.; Denny, W. A. J. Chem. Soc., Perkin Trans. I 1989, 95. 10. Gangloff, A. R.; Litvak, J.; Shelton, E. J.; Sperandio, D.; Wang, V. R.; Rice, K. D. Tetrahedron Lett. 2001, 42, 1441. 11. Herr, R. J. Bioorg. Med. Chem. 2002, 10, 3379. 12. Compounds 19 and 35 were diffused into pre-formed FVIIa/benzamidine crystals by soaking. The X-ray diffraction data were collected from these FVIIa inhibitor complex crystals extending to 2.07 Å, and 2.3 Å resolution with Rsym = 0.074, and 0.098, and completeness = 90.0%, and 100%, respectively. The complex structures were refined to R-work (R-free) of 0.177 (0.202) and 0.173 (0.198), respectively. The detailed X-ray diffraction data and refinement statistics are listed under PDB codes 4YT6 and 4YT7, respectively, in the Protein Data Bank. 13. Factor VIIa determinations were made in 0.005 M calcium chloride, 0.15 M sodium chloride, 0.05 M HEPES buffer containing 0.1% PEG 8000 at a pH of 7.5. Determinations were made using purified human Factor VIIa (Haematologic Technologies) or recombinant human Factor VIIa (Novo Nordisk) at a final assay concentration of 1–5 nM, recombinant soluble tissue factor at a concentration of 10–40 nM and the synthetic substrate H-D-Ile-Pro-Arg-pNA (-2288; Chromogenix or BMPM-2; AnaSpec) at a concentration of 0.001– 0.0075 M.
P. W. Glunz et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2169–2173 14. Glunz, P. W.; Wurtz, N.; Cheng, X. Heterocyclic Compounds as Inhibitors of Factor VIIa. US 7576098; Bristol-Myers Squibb Company, priority date: 8 December 2004. 15. (a) Clark, R.; Matsuura, F.; Kira, K.; Hirota, S.; Azuma, H.; Nagakura, T.; Horizoe, T.; Tabata, K.; Kusano, K.; Omae, T.; Inoue, A. Preparation of Triazolone Derivatives as Blood Coagulation Factor VIIa Inhibitors. WO2007111212; Eisai R&D Management Co., Ltd, priority date: 24 March 2006.; (b) Clark, R.;
2173
Matsuura, F.; Shinoda, M.; Hirota, S.; Kira, K.; Azuma, H.; Takemura, A.; Yasui, S.; Kusano, K.; Mikamoto, M.; Omae, T. Prodrug of Triazolone Compound. WO 2011145747; Eisai R&D Management Co., Ltd, priority date: 20 May 2010. 16. Nagakura, T.; Tabata, K.; Kira, K.; Hirota, S.; Clark, R.; Matsuura, F.; Hiyoshi, H. Thrombosis Res. 2013, 132, 271. 17. Wong, P. C.; Luettgen, J. M.; Rendina, A. R.; Kettner, C. A.; Xin, B.; Knabb, R. M.; Wexler, R.; Priestley, E. S. Thromb. Haemost. 2010, 104, 261.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of potent, selective phenylimidazole-based FVIIa inhibitors Peter W. Glunz ⇑, Xuhong Cheng, Daniel L. Cheney, Carolyn A. Weigelt, Anzhi Wei, Joseph M. Luettgen, Pancras C. Wong, Ruth R. Wexler, E. Scott Priestley Bristol-Myers Squibb R&D, 311 Pennington-Rocky Hill Rd, Pennington, NJ 08534, United States
a r t i c l e
i n f o
Article history: Received 26 February 2015 Revised 19 March 2015 Accepted 23 March 2015 Available online 30 March 2015 Keywords: Factor VIIa Tissue factor TF–FVIIa inhibitor Serine protease inhibitors Amide isosteres
a b s t r a c t Heterocyclic amide isosteres were incorporated into a phenylglycine-based tissue factor/factor VIIa (TF–FVIIa) inhibitor chemotype, providing potent inhibitors. An X-ray co-crystal structure of phenylimidazole 19 suggested that an imidazole nitrogen atom effectively mimics an amide carbonyl, while the phenyl ring forms key hydrophobic interactions with the S10 pocket. Exploration of phenylimidazole substitution led to the discovery of potent, selective and efficacious inhibitors of TF–FVIIa. Ó 2015 Elsevier Ltd. All rights reserved.
Arterial thrombosis is a leading cause of mortality and morbidity in the developed world.1 Comparisons of antithrombotic mechanisms have identified the tissue factor–factor VIIa proteolytic complex (TF–FVIIa) as a therapeutic target that offers strong antithrombotic efficacy and a low bleeding liability.2 This serine protease initiates the coagulation cascade by activating factors IX and X to IXa and Xa, respectively, leading to thrombin generation. Thrombin cleaves fibrinogen to fibrin and activates platelets, resulting in clot formation. FVIIa has attracted considerable attention in the pharmaceutical industry as a promising anticoagulant target.3 We chose phenylglycine-based inhibitors 1–44 (Fig. 1) as a starting point for inhibitor design for their potency and well-defined SAR. We sought to make progress toward oral bioavailability by modifying their peptide-like structure and zwitterionic character. We have reported replacement of the basic amidine P1 group within related chemotypes resulting in oral bioavailability.5,6 Herein, we sought to replace the amide or acylsulfonamide group. Heterocycles have been utilized extensively as replacements of amides to impart chemical and enzymatic stability, and therefore improved pharmacokinetic properties, into peptide ligands.7 Our strategy was to mimic the interactions of the amide or acylsulfonamide moiety of inhibitors 2–4 with His 57 and Ser 195 ⇑ Corresponding author. Tel.: +1 609 818 5287; fax: +1 609 818 3550. E-mail address: [email protected] (P.W. Glunz). http://dx.doi.org/10.1016/j.bmcl.2015.03.062 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
OEt NH
EtO
OEt
H 2N
H 2N OH
N H
NH
OEt
NH
N H
O 1 FVIIa Ki = 190 nM Oi-Pr OEt
H2 N N H
H N O
S O O
3 FVIIa Ki = 1 nM
H N
2 O FVIIa Ki = 40 nM
NH
EtO
OEt
H2 N HO
N H
NH2
F H N
S O O O G17905, 4 FVIIa Ki = 0.35 nM
Figure 1. Phenylglycine-based FVIIa inhibitors.
(Fig. 2a) using a heterocyclic replacement (Fig. 2b). This change removes a potential site for proteolytic cleavage or a negative charge. We then undertook a structure-based approach to identify additional binding interactions, exploiting the critical hydrophobic S10 pocket.5 The general synthesis of inhibitors 9–22 (Scheme 1) involves addition of a lithiated heterocycle to 4-(benzylideneamino)benzonitrile 7 to generate amine 8. Lithiation of 4-phenyloxazole, en route to analog 22, required pre-complexation with BF3 to avoid
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P. W. Glunz et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2169–2173
(a)
Oi-Pr
H N
N H
(b)
S2
OEt
Oi-Pr
N H
S1'
N
S1'
R
25
H O
H N
Leu 41
H N
Ser 195
N H
N H
S
Cys 58
S
Cys 42
His 57
His 57
Figure 2. (a) Binding interactions of acylsulfonamide (X = SO2) and amide (X = CH2) inhibitors. (b) Design of FVIIa inhibitors to mimic carbonyl interactions and reach the S10 pocket.
OHC
OEt
NC
NC a NH2
R1
5
R3
OEt
N
R2 6a,b
7a,b
R1
R3 R2 R3
R3 EtO b
Het-H(Br)
R2
NC
R1
NH
EtO
R2
c-f H 2N
R1
Het
N H
N H 9-22
8a,b a: R1, R2 = H, R3 = Oi-Pr b: R1 = F, R2 = Oi-Pr, R3 = H
Het
Scheme 1. General synthetic strategy for heterocyclic FVIIa inhibitors. Reagents and conditions: (a) 4 Å mol sieves, toluene, reflux (7a, 87%; 7b, 99%); (b) BuLi, THF; then 7a or 7b (8a, 20–72%; 8b, 24–78%); (c) 3 M NH2OH, DMSO, 60 °C, 2 h; (d) Ac2O, CH2Cl2, rt; (e) H2 (balloon), Pd–C, MeOH; (f) 90% aq AcOH, 60 °C (24–79%).
EtO Tr N
7b Br 23
Oi-Pr
a NC
N
Oi-Pr F Tr N
N
H
Ser 195
EtO
N H X
O
O
O N
S2
OEt
F Tr N
N H
c,d,g
O N
EtO
F Tr N
N N H
N 24
Oi-Pr
N 25
Br
Br
Method B b,h,f
b-f Method A NH
EtO
H 2N N H N via Method A: 26-32 via Method B: 33-35
Oi-Pr F H N
Ar
Scheme 2. General synthetic routes for preparation of substituted phenylimidazole inhibitors. Reagents and conditions: (a) BuLi, THF, 0 °C, then 7b, 71%; (b) ArB(OH)2, Pd(PPh3)4, Na2CO3, DME/H2O, 150 °C, 5 min or 80 °C, 2 h; (c) NH2OH, DMSO, 60 °C; (d) Ac2O, CH2Cl2, rt; (e) H2, 10% Pd–C, MeOH; (f) 90% aq AcOH, 60 °C; (g) TBAF, THF, rt, 88% for 3 steps to 25; (h) H2 (1 atm), 10% Pd–C, 8:1 MeOH/TEA or MeOH + 1 drop AcOH. (Method A: 9–41%, 5 steps. Method B: 7–21%, 3 steps).
oxazole ring opening.8 The nitrile functionality was converted to the amidine, and the target was deprotected as necessary. Substituted phenylimidazoles were prepared as shown in Scheme 2. 4-Bromo-1-trityl-1H-imidazole (23) was lithiated at the 2-position,9 then was reacted with aldimine 7b, affording amine 24. Suzuki–Miyaura coupling, amidine formation and
N Br
NH
EtO
Oi-Pr F H N
H 2N a-d
N H
N
O R
36, R = NHMe 37, R = NMe 2,
Scheme 3. Synthesis of amide analogs. Reagents and conditions: (a) 2-(CO2Et)PhB(OH)2, Na2CO3, P(o-Tol)3, Pd(OAc)2, DME/H2O, 80 °C, 2 h, 92%; (b) aq LiOH, THF/ MeOH, 64%; (c) MeNH2HCl (R = NHMe), TEA or Me2NH (R = NMe2), BOP, DMF, (R = NHMe, 94%; R = NMe2, 92%); (d) H2, 10% Pd–C, MeOH/THF (1:1), AcOH (1 drop); 90% aq AcOH, 60 °C (36, 47%; 37, 43%).
deprotection produced the targeted inhibitors (Method A). Alternatively, nitrile 24 is converted to 1,2,4-oxadiazole 25,10 which serves as a protected amidine and allows a single deprotection step, following Suzuki coupling, to afford analogs 33–35, thus reducing the steps per analog from five to three (Method B). Amide derivatives were prepared as shown in Scheme 3. Suzuki coupling of 25 with (2-(ethoxycarbonyl)phenyl)boronic acid afforded the ester, en route to amides 36 and 37. FVIIa inhibitory potencies for an initial set of heterocycles is listed in Table 1. Neutral and basic monocyclic heterocycles 9–12 are weakly potent inhibitors, whereas the carboxylic acid isostere11 tetrazole 13 is 4-fold more potent than its acid counterpart 1. Fused heterocycles 14–17 generally display improved potency relative to their monocyclic counterparts; quinoline 16 is 15-fold more potent than 2-pyridyl analog 11. A phenyl substituent on the heterocycle had a dramatic effect. 4-Phenylimidazol-2-yl analog 19 is a potent inhibitor of FVIIa, Ki = 11 nM versus 520 nM for imidazole analog 12. 19 is 4-fold more potent than unsubstituted benzylamide 2 and 10-fold less potent than acylsulfonamide 3. Methylation of each imidazole nitrogen (20 and 21) provided evidence of the tautomeric binding mode of the phenylimidazole, suggesting interaction between the nitrogen lone pair adjacent to the phenyl ring and the enzyme active site. This binding orientation of 19 was confirmed by Xray crystallography in FVIIa, see below. Phenyloxazole 22 is similar in potency to phenylimidazole 19, suggesting similar binding interactions for the two heterocycles. Phenylimidazole 18 proved unstable under acidic HPLC conditions (MeOH/H2O + 0.1% TFA), precluding isolation of the pure compound and leading us to investigate acid stability. Compounds 9, 12 and 14 were found prone to hydrolysis, affording biarylmethanol and 4-aminobenzamidine degradants upon hydrolysis. We hypothesized that this instability is due to electron donation by the para-O-i-Pr group and could be circumvented by moving the 4-alkoxy group out of conjugation with the a-carbon of the core. Replacing the 3,4-dialkoxy P2 group with a 2-F-3,5-dialkoxy P2 had the intended effect of improving stability (Table 1, A vs B), and thus this P2 group was employed for subsequent SAR exploration. The X-ray crystal structure of compound 19 in FVIIa is shown in Figure 3a.12 The inhibitor adopts a similar binding mode to reported phenylglycine-based FVIIa inhibitors such as acylsulfonamide 4 (PDB ID: 1YGC)4e with superposition of the P1 and P2 groups (Fig. 3b) as shown in Figure 3b. Closely related analogs of acid 1 and benzylamide 2 (PDB ID: 2BZ64b and PDB ID: 1W7X,4a respectively) bind similarly (data not shown). The imidazole moiety aligns with the carbonyl as an isostere forming close contacts with His 57 and Ser 195. Owing to the planarity of the imidazole ring, the 4-phenyl substituent of 19 is directed into the hydrophobic S10 pocket, with the meta and para positions of the ring interacting with the Cys 42–Cys 58 disulfide bond and
P. W. Glunz et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2169–2173
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Table 1 FVIIa inhibition13 and chemical stability of heterocyclic isosteres
Oi-Pr
NH
OEt H2 N
P2 N H
i-PrO F
P1' A P10
Het-H/Br
S
S
N
N
9
10 11
Br N
N
Tr N
H N
N
N N N N N H
12
N N N N
13
14 15
16
17
BOM S
S
N
N
Br N
N
BOM N
H N
N
Ph
Ph
N
A
2100
A B
1100 750
A
520
B
49
>24a
A B
80 420
1 >24a
B
49
>24a
B
140
>24a
A B
— 11
B
15
22
B
1800
104
B
20
pH 1 stab. (t1/2, h) 0.4 83 >24a
12.5
Unstableb 42
Ph
Ph
N Ph
N Me O
Ph
Ph
N
Ph
O 22
N
N
N N Me
FVIIa Ki (nM)
Me N
20
21
P2
N
Me N N
B
H N
N Tr N
18 19
OEt
P2 :
>24c
Ki determination, n P 2. a 100% remaining at 24 h. b Unstable in preparative HPLC solvent (MeOH/H2O + 0.1% TFA). c 96% remaining at 21 h.
the Leu 41 side chain, respectively. We have exploited this critical hydrophobic S10 interaction via substitution of related acylsulfonamide5 and benzylamide6 chemotypes. The phenylimidazole forms this interaction directly. This crystal structure is consistent with the N-methylation results (20 and 21). Subsequent to our patent filing,14 a related triazolone chemotype was reported,15 which presumably makes similar interactions with the FVIIa binding site. A double-prodrug strategy was employed to attain oral bioavailability with these compounds.16 Unsubstituted phenylimidazole 19 shows good selectivity versus thrombin, FXa and trypsin (110-, 70- and 60-fold, respectively), but only weak efficacy in a modified-PT clotting assay17 (Table 2). Our efforts next focused on introducing substitution to enhance inhibitory potency and clotting efficacy, while maintaining or improving selectivity.
Figure 3. (a) X-ray crystal structure of 19 bound in FVIIa, solved at 2.07 Å, and depicting the initial Fo–Fc map of electron density (at 3 sigma) within 1.6 Å of the ligand. (b) Overlay with bound acylsulfonamide 4 (PDB ID: 1YGC).4e Arrows highlight different directionality of phenyl groups in acylsulfonamide and imidazole-based inhibitors.
The ortho and meta positions are more tolerant of substitution than the para position (28 and 31), consistent with the observed binding mode of 19 in FVIIa. We focused our SAR exploration on the ortho position, which based upon the X-ray structure of 19 either occupies a polar, exposed region (2-position) or is directed towards the catalytic triad (6-position). Hydrogen bond acceptors, including ketone and ester moieties, are accommodated in the ortho position. While carboxylic acid 34 is 2-fold more potent than 19, it is 6-fold more potent in the clotting assay, likely due to decreased protein binding. Efforts to identify other neutral, polar substituents led to primary amide 35. It is 6- and 8-fold more potent than 19 in the inhibition and clotting assays, respectively, and shows greatly improved selectivity versus other serine proteases due to enhanced activity against FVIIa. Compound 35 was inactive in the activated partial thromboplastin time assay (aPTT IC2x > 40 lM), consistent with selective FVIIa inhibition.2b Primary amide 35 is similar in
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Table 2 Substituted phenylimidazole SAR—FVIIa inhibitory potency,13 selectivity and clotting activity17
NH
EtO
Oi-Pr F H N
H2 N N H
N 2 R 3
22, 30-52 6 5
19 26 27 28 29 30 31 32 33 34 35 36 37
inhibitor chemotype, replacing an amide moiety with good inhibitory activity. Phenylimidazole 19 was identified as a potent inhibitor that mimics the binding interactions of the parent inhibitors’ carbonyl with the catalytic triad. The phenyl ring contributes significantly to binding through hydrophobic interactions with the S10 disulfide. Exploration of phenylimidazole substitution led to the discovery of primary amide 35, a potent, selective and efficacious inhibitor of FVIIa. Acknowledgments
4
R
FVIIa Ki (nM)
FIIa Ki (nM)
FXa Ki (nM)
Trypsin Ki (nM)
Mod. PT IC2x (lM)
H 2-Me 3-Me 4-Me 2-OMe 3-OMe 4-OMe 2-C(O)Me 2-CO2Me 2-CO2H 2-CONH2 2-CONHMe 2-CONMe2
11 19 37 207 10 48 1020 5.0 13 6.1 1.7 68 79
1200 710 750 760 570 790 640 1670 4470 190 1190 — —
760 680 450 430 460 560 540 430 4070 2190 2330 — —
690 400 500 610 430 580 490 560 1430 2410 1050 — —
37 48 63 ND 31 49 ND 26 >80 6.6 4.5 43 45
Ki determination, n P 2.
Figure 4. X-ray crystal structure of 35 bound in FVIIa, solved at 2.3 Å, and depicting the initial Fo–Fc map of electron density (at 3 sigma) within 1.6 Å of the ligand.
potency to acylsulfonamide 3 without the anionic moiety that renders the latter zwitterionic. The X-ray crystal structure of 35 in FVIIa13 was obtained (Fig. 4) and reveals a binding mode similar to that observed for 19. The primary amide is directed towards the partially solvent-exposed edge of the S10 binding pocket, forming hydrogen bonds to the His 57 backbone carbonyl and to the Asp 60 side chain via a water molecule. The amide substituent occupies the general hydrophilic region of the primary aniline of FVIIa inhibitor 4. Secondary and tertiary amides 36 and 37 are considerably less potent, consistent with the importance of these interactions present in the X-ray structure. In summary, we have developed an efficient and general method to synthesize heterocyclic amide-bond isosteres via addition of a lithiated heterocycle to an N-aryl imine. In this way, we have incorporated heterocycles into a phenylglycine-based FVIIa
The authors thank Frank Barbera, Jeffrey Bozarth, Randi Brown, Karen Hartl, Diane Normandin, Tara Peterson and Ge Zhang for conducting biological assays. Gerry Everlof is acknowledged for determining chemical stability. We would also like to thank Steven Sheriff for helping to prepare X-ray coordinates and data for PDB deposition. References and notes 1. Global Status Report on Noncommunicable Diseases 2010; WHO Press: Geneva, 2011. 2. (a) Szalony, J. A.; Taite, B. B.; Girard, T. J.; Nicholson, N. S.; LaChance, R. M. J. Thromb. Thrombolysis 2002, 14, 113–121; (b) Suleymanov, O. D.; Szalony, J. A.; Salyers, A. K.; Lachance, R. M.; Parlow, J. J.; South, M. S.; Wood, R. S.; Nicholson, N. S. J. Pharmacol. Exp. Ther. 2003, 306, 1115; (c) Salyers, A. K.; Szalony, J. A.; Suleymanov, O. D.; Parlow, J. J.; Wood, R. S.; South, M. S.; Nicholson, N. S. Pharmacology 2004, 70, 100; (d) Wong, P.; Crain, E.; Watson, C.; Staus, A.; Luettgen, J.; Rendina, A.; Abell, L.; Knabb, R.; Baomin, X.; Zhou, J.; DeLucca, I.; Priestley, E. S. J. Thromb. Haemost. 2005, 3, P0162. 3. (a) Shirk, R. A.; Vlasuk, G. P. Arterioscler Thromb. Vasc. Biol. 2007, 27, 1895; (b) Priestley, E. S. Drug Discovery Today 2014, 19, 1440. 4. (a) Zbinden, K. G.; Obst-Sander, U.; Hilpert, K.; Kuehne, H.; Banner, D. W.; Boehm, H.-J.; Stahl, M.; Ackermann, J.; Alig, L.; Weber, L.; Wessel, H. P.; Riederer, M. A.; Tschopp, T. B.; Lave, T. Bioorg. Med. Chem. Lett. 2005, 15, 5344– 5352; (b) Zbinden, K. G.; Banner, D. W.; Hilpert, K.; Himber, J.; Lavé, T.; Riederer, M. A.; Stahl, M.; Tschopp, T. B.; Obst-Sander, U. Bioorg. Med. Chem. 2006, 14, 5357; (c) Zbinden, K. G.; Banner, D. W.; Ackermann, J.; D’Arcy, A.; Kirchhofer, D.; Ji, Y.-H.; Tschopp, T. B.; Wallbaum, S.; Weber, L. Bioorg. Med. Chem. Lett. 2005, 15, 817–822; (d) Aliagas-Martin, I.; Artis, D. R.; Dina, M. S.; Flygare, J. A.; Goldsmith, R. A.; Munroe, R. A.; Olivero, A. G.; Pastor, R.; Rawson, T. E.; Robarge, K. D.; Sutherlin, D. P.; Weese, K. J.; Zhou, Z.; Zhu, Y. (Genentech, Inc., USA). U.S. Patent WO2000041531, 2000.; (e) Olivero, A. G.; Eigenbrot, C.; Goldsmith, R.; Robarge, K.; Artis, D. R.; Flygare, J.; Rawson, T.; Sutherlin, D. P.; Kadkhodayan, S.; Beresini, M.; Elliott, L. O.; DeGuzman, G. G.; Banner, D. W.; Ultsch, M.; Marzec, U.; Hanson, S. R.; Refino, C.; Bunting, S.; Kirchhofer, D. J. Biol. Chem. 2005, 280, 9160. 5. Glunz, P. W.; Zhang, X.; Zou, Y.; Delucca, I.; Nirschl, A. H.; Cheng, X.; Weigelt, C. A.; Cheney, D. L.; Wei, A.; Anumula, R.; Luettgen, J. M.; Rendina, A. R.; Harpel, M.; Luo, G.; Knabb, R.; Wong, P. C.; Wexler, R. R.; Priestley, E. S. Bioorg. Med. Chem. Lett. 2013, 23, 5244. 6. Zhang, X.; Jiang, W.; Jacutin-Porte, S.; Glunz, P. W.; Zou, Y.; Cheng, X.; Nirschl, A. H.; Wurtz, N. R.; Luettgen, J. M.; Rendina, A. R.; Luo, G.; Harper, T. M.; Wei, A.; Anumula, R.; Cheney, D. L.; Knabb, R. M.; Wong, P. C.; Wexler, R. R.; Priestley, E. S. ACS Med. Chem. Lett. 2014, 5, 188. 7. (a) Gordon, T.; Hansen, P.; Morgan, B.; Singh, J.; Baizman, E.; Ward, S. Bioorg. Med. Chem. Lett. 1993, 3, 915; (b) Hamada, Y.; Kiso, Y. Expert Opin. Drug Discov. 2012, 7, 903. 8. Vedejs, E.; Monahan, S. D. J. Org. Chem. 1996, 61, 5192. 9. Palmer, B. D.; Denny, W. A. J. Chem. Soc., Perkin Trans. I 1989, 95. 10. Gangloff, A. R.; Litvak, J.; Shelton, E. J.; Sperandio, D.; Wang, V. R.; Rice, K. D. Tetrahedron Lett. 2001, 42, 1441. 11. Herr, R. J. Bioorg. Med. Chem. 2002, 10, 3379. 12. Compounds 19 and 35 were diffused into pre-formed FVIIa/benzamidine crystals by soaking. The X-ray diffraction data were collected from these FVIIa inhibitor complex crystals extending to 2.07 Å, and 2.3 Å resolution with Rsym = 0.074, and 0.098, and completeness = 90.0%, and 100%, respectively. The complex structures were refined to R-work (R-free) of 0.177 (0.202) and 0.173 (0.198), respectively. The detailed X-ray diffraction data and refinement statistics are listed under PDB codes 4YT6 and 4YT7, respectively, in the Protein Data Bank. 13. Factor VIIa determinations were made in 0.005 M calcium chloride, 0.15 M sodium chloride, 0.05 M HEPES buffer containing 0.1% PEG 8000 at a pH of 7.5. Determinations were made using purified human Factor VIIa (Haematologic Technologies) or recombinant human Factor VIIa (Novo Nordisk) at a final assay concentration of 1–5 nM, recombinant soluble tissue factor at a concentration of 10–40 nM and the synthetic substrate H-D-Ile-Pro-Arg-pNA (-2288; Chromogenix or BMPM-2; AnaSpec) at a concentration of 0.001– 0.0075 M.
P. W. Glunz et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2169–2173 14. Glunz, P. W.; Wurtz, N.; Cheng, X. Heterocyclic Compounds as Inhibitors of Factor VIIa. US 7576098; Bristol-Myers Squibb Company, priority date: 8 December 2004. 15. (a) Clark, R.; Matsuura, F.; Kira, K.; Hirota, S.; Azuma, H.; Nagakura, T.; Horizoe, T.; Tabata, K.; Kusano, K.; Omae, T.; Inoue, A. Preparation of Triazolone Derivatives as Blood Coagulation Factor VIIa Inhibitors. WO2007111212; Eisai R&D Management Co., Ltd, priority date: 24 March 2006.; (b) Clark, R.;
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Matsuura, F.; Shinoda, M.; Hirota, S.; Kira, K.; Azuma, H.; Takemura, A.; Yasui, S.; Kusano, K.; Mikamoto, M.; Omae, T. Prodrug of Triazolone Compound. WO 2011145747; Eisai R&D Management Co., Ltd, priority date: 20 May 2010. 16. Nagakura, T.; Tabata, K.; Kira, K.; Hirota, S.; Clark, R.; Matsuura, F.; Hiyoshi, H. Thrombosis Res. 2013, 132, 271. 17. Wong, P. C.; Luettgen, J. M.; Rendina, A. R.; Kettner, C. A.; Xin, B.; Knabb, R. M.; Wexler, R.; Priestley, E. S. Thromb. Haemost. 2010, 104, 261.
