Bioorganic & Medicinal Chemistry Letters 25 (2015) 1597–1602
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Identification and optimization of pyridazinones as potent and selective c-Met kinase inhibitors Dieter Dorsch a,⇑, Oliver Schadt a, Frank Stieber a, Michael Meyring b, Ulrich Grädler a, Friedhelm Bladt a, Manja Friese-Hamim a, Christine Knühl a, Ulrich Pehl a, Andree Blaukat a a b
Merck Serono Research & Development, Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany Merck Serono Research & Development, Merck KGaA, Am Feld 32, 85567 Grafing, Germany
a r t i c l e
i n f o
Article history: Received 18 December 2014 Revised 30 January 2015 Accepted 2 February 2015 Available online 16 February 2015 Keywords: c-Met Tyrosine kinase Pyridazinone Structure-based design MSC2156119
a b s t r a c t In a high-throughput screening campaign for c-Met kinase inhibitors, a thiadiazinone derivative with a carbamate group was identified as a potent in vitro inhibitor. Subsequent optimization guided by cMet-inhibitor X-ray structures furnished new compound classes with excellent in vitro and in vivo profiles. The thiadiazinone ring of the HTS hit was first replaced by a pyridazinone followed by an exchange of the carbamate hinge binder with a 1,5-disubstituted pyrimidine. Finally an optimized compound, 22 (MSC2156119), with excellent in vitro potency, high kinase selectivity, long half-life after oral administration and in vivo anti-tumor efficacy at low doses, was selected as a candidate for clinical development. Ó 2015 Elsevier Ltd. All rights reserved.
The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase, which is activated upon binding of its high-affinity ligand hepatocyte growth factor (HGF). While the expression pattern of both c-Met and HGF is widely spread to a variety of tissues, the c-Met receptor is typically expressed in tissues of epithelial origin, and HGF expression is predominantly detected on tissues of mesenchymal origin.1 Binding of HGF to c-Met triggers multiple downstream signaling pathways, which result in cell proliferation, motility, migration, angiogenesis, morphogenic differentiation and cell survival during normal development and tissue repair.2 The activity of c-Met is strictly regulated during normal mammalian development and tissue homeostasis. Dysregulation of cMet function, as a result of overexpression, mutational activation or amplification, has been observed in many types of cancer, including liver and lung cancer.3 Furthermore, c-Met activation in these tumors has been associated with a malignant phenotype, characterized by rapid tumor growth, metastasis, invasiveness and induction of angiogenesis. Indeed, retrospective studies indicate a correlation between poor patient prognosis and overexpression of c-Met or HGF.4 Preclinical studies show that blockade of c-Met activation inhibits tumor growth and metastasis which points toward a pivotal
⇑ Corresponding author. Tel.: +49 6151 72 7569. E-mail address: [email protected] (D. Dorsch). http://dx.doi.org/10.1016/j.bmcl.2015.02.002 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
role of c-Met in cancer. A number of ongoing studies are currently evaluating the safety and efficacy profile of different agents inhibiting c-Met activity, either by blocking ligand-dependent activation of c-Met (monoclonal antibodies binding to HGF or c-Met) or by inhibiting ligand-dependent and -independent c-Met activation by small-molecule tyrosine kinase inhibitors.5 During an internal high-throughput screening campaign the thiadiazinone 1 (Fig. 1) which originated from a PDE inhibitor project was identified as a potent c-Met kinase inhibitor (in vitro IC50 = 400 nM, cellular IC50 = 1100 nM, Table 1). For further structure-based optimization, the kinase domain of human c-Met was co-crystallized with 1 and the structure was solved at 2.0 Å resolution.6,7 The X-ray structure analysis of 1 revealed a U-shaped conformation within the ATP binding pocket (Fig. 2). The carbamate carbonyl oxygen of 1 forms one hydrogen bond to the backbone nitrogen atom of Met1160 within the hinge region, while the carbamate NH-group is in water-mediated contact with Ile1084 of the GC-loop. The thiadiazinone ring is sandwiched between the side chains of Met1211 and Tyr1230 and forms an H-bond to the main chain nitrogen of Asp1222 of the DFG motif via its carbonyl oxygen. Finally, one of the methoxy groups is involved in an H-bond to Asn1167. This U-turn like binding mode is seen or anticipated for many known c-Met kinase inhibitors like JNJ-38877605,8 PF04217903,9 or AZD6094 (Volitinib) (Fig. 3).10 These structural insights guided the hit optimization process.
1598
D. Dorsch et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1597–1602 S O
N
O
O
N
N H
O
O
N
1 Figure 1. Thiadiazinone 1.
The enzymatic c-Met activity of 1 was acceptable for an HTS hit. In addition to the necessity to improve the biochemical and cellular potency of the structural class, it turned out that 1 also had poor pharmacokinetic properties (see Table 1) and an unfavorable residual PDE profile (PDE3 1500 nM, PDE4 60 nM).11 Due to the fact that PDE activity can be attributed to the 3,4-dimethoxyphenyl substituent, one of the first optimization cycles was dedicated to the identification of alternative substituents without significant PDE activity.12 This goal was achieved by replacing the 3,4dimethoxyphenyl substituent by a 3,5-difluorophenyl residue which resulted in cpd. 2 with increased biochemical and cellular potency and with improved iv clearance and oral bioavailability in mice. By replacing the thiadiazinone core with the generally chemically more stable and synthetically easier accessible bioisosteric pyridazinone core,13 potency and pharmacokinetic properties could be maintained at a fully satisfactory level (compd 3). The general synthesis of carbamates 1–5 is shown in Scheme 1. In order to avoid the potential liabilities connected with a carbamate unit like carcinogenicity,14 efforts were undertaken to replace the carbamate unit by alternative groups that could provide a hydrogen bond acceptor for hinge binding. Guided by availability of building blocks, several five-membered heterocycles were virtually combined with the pyridazinone template and docked into the X-ray structure (Supplementary data). One of the best-fitting groups, a 5-methyl-[1,2,4]oxadiazol-3-yl group, was introduced to give compound 6 (Table 2) with an IC50 already in the same range as comparable carbamates (compounds 4 and 5).15 As expected, the isomeric oxadiazole 7 was less active due
Figure 2. The X-ray structure of thiadiazinone 1 with c-Met kinase at 2.0 Å resolution (PDB-ID: 4R1Y) revealed H-bonds (dashed black lines) to Met1160 of the hinge region, to Asp1222 of the DFG motif, to Asn1167 and water-mediated contacts to Ile1084 of the GC-loop.
to the non-optimal position of the donor nitrogen. Several fivemembered heterocyclic derivatives with a donor nitrogen in the optimal position were then prepared. Imidazole 8 was only weakly active, whereas oxazole 9 and thiazole 10 were more active than oxadiazole 6. This activity gain may be due to a CH–p interaction between the oxazole/thiazole H-atom pointing toward the p-sys-
Table 1 Properties of carbamates O X
Compd
X
R
S O
1
N
N H
O
c-Met biochemIC50 (nM)a
N
R
R
c-Met cell IC50(nM)b
Mouse PKc CL (L/h/kg)
F (%)
6
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Identification and optimization of pyridazinones as potent and selective c-Met kinase inhibitors Dieter Dorsch a,⇑, Oliver Schadt a, Frank Stieber a, Michael Meyring b, Ulrich Grädler a, Friedhelm Bladt a, Manja Friese-Hamim a, Christine Knühl a, Ulrich Pehl a, Andree Blaukat a a b
Merck Serono Research & Development, Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany Merck Serono Research & Development, Merck KGaA, Am Feld 32, 85567 Grafing, Germany
a r t i c l e
i n f o
Article history: Received 18 December 2014 Revised 30 January 2015 Accepted 2 February 2015 Available online 16 February 2015 Keywords: c-Met Tyrosine kinase Pyridazinone Structure-based design MSC2156119
a b s t r a c t In a high-throughput screening campaign for c-Met kinase inhibitors, a thiadiazinone derivative with a carbamate group was identified as a potent in vitro inhibitor. Subsequent optimization guided by cMet-inhibitor X-ray structures furnished new compound classes with excellent in vitro and in vivo profiles. The thiadiazinone ring of the HTS hit was first replaced by a pyridazinone followed by an exchange of the carbamate hinge binder with a 1,5-disubstituted pyrimidine. Finally an optimized compound, 22 (MSC2156119), with excellent in vitro potency, high kinase selectivity, long half-life after oral administration and in vivo anti-tumor efficacy at low doses, was selected as a candidate for clinical development. Ó 2015 Elsevier Ltd. All rights reserved.
The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase, which is activated upon binding of its high-affinity ligand hepatocyte growth factor (HGF). While the expression pattern of both c-Met and HGF is widely spread to a variety of tissues, the c-Met receptor is typically expressed in tissues of epithelial origin, and HGF expression is predominantly detected on tissues of mesenchymal origin.1 Binding of HGF to c-Met triggers multiple downstream signaling pathways, which result in cell proliferation, motility, migration, angiogenesis, morphogenic differentiation and cell survival during normal development and tissue repair.2 The activity of c-Met is strictly regulated during normal mammalian development and tissue homeostasis. Dysregulation of cMet function, as a result of overexpression, mutational activation or amplification, has been observed in many types of cancer, including liver and lung cancer.3 Furthermore, c-Met activation in these tumors has been associated with a malignant phenotype, characterized by rapid tumor growth, metastasis, invasiveness and induction of angiogenesis. Indeed, retrospective studies indicate a correlation between poor patient prognosis and overexpression of c-Met or HGF.4 Preclinical studies show that blockade of c-Met activation inhibits tumor growth and metastasis which points toward a pivotal
⇑ Corresponding author. Tel.: +49 6151 72 7569. E-mail address: [email protected] (D. Dorsch). http://dx.doi.org/10.1016/j.bmcl.2015.02.002 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
role of c-Met in cancer. A number of ongoing studies are currently evaluating the safety and efficacy profile of different agents inhibiting c-Met activity, either by blocking ligand-dependent activation of c-Met (monoclonal antibodies binding to HGF or c-Met) or by inhibiting ligand-dependent and -independent c-Met activation by small-molecule tyrosine kinase inhibitors.5 During an internal high-throughput screening campaign the thiadiazinone 1 (Fig. 1) which originated from a PDE inhibitor project was identified as a potent c-Met kinase inhibitor (in vitro IC50 = 400 nM, cellular IC50 = 1100 nM, Table 1). For further structure-based optimization, the kinase domain of human c-Met was co-crystallized with 1 and the structure was solved at 2.0 Å resolution.6,7 The X-ray structure analysis of 1 revealed a U-shaped conformation within the ATP binding pocket (Fig. 2). The carbamate carbonyl oxygen of 1 forms one hydrogen bond to the backbone nitrogen atom of Met1160 within the hinge region, while the carbamate NH-group is in water-mediated contact with Ile1084 of the GC-loop. The thiadiazinone ring is sandwiched between the side chains of Met1211 and Tyr1230 and forms an H-bond to the main chain nitrogen of Asp1222 of the DFG motif via its carbonyl oxygen. Finally, one of the methoxy groups is involved in an H-bond to Asn1167. This U-turn like binding mode is seen or anticipated for many known c-Met kinase inhibitors like JNJ-38877605,8 PF04217903,9 or AZD6094 (Volitinib) (Fig. 3).10 These structural insights guided the hit optimization process.
1598
D. Dorsch et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1597–1602 S O
N
O
O
N
N H
O
O
N
1 Figure 1. Thiadiazinone 1.
The enzymatic c-Met activity of 1 was acceptable for an HTS hit. In addition to the necessity to improve the biochemical and cellular potency of the structural class, it turned out that 1 also had poor pharmacokinetic properties (see Table 1) and an unfavorable residual PDE profile (PDE3 1500 nM, PDE4 60 nM).11 Due to the fact that PDE activity can be attributed to the 3,4-dimethoxyphenyl substituent, one of the first optimization cycles was dedicated to the identification of alternative substituents without significant PDE activity.12 This goal was achieved by replacing the 3,4dimethoxyphenyl substituent by a 3,5-difluorophenyl residue which resulted in cpd. 2 with increased biochemical and cellular potency and with improved iv clearance and oral bioavailability in mice. By replacing the thiadiazinone core with the generally chemically more stable and synthetically easier accessible bioisosteric pyridazinone core,13 potency and pharmacokinetic properties could be maintained at a fully satisfactory level (compd 3). The general synthesis of carbamates 1–5 is shown in Scheme 1. In order to avoid the potential liabilities connected with a carbamate unit like carcinogenicity,14 efforts were undertaken to replace the carbamate unit by alternative groups that could provide a hydrogen bond acceptor for hinge binding. Guided by availability of building blocks, several five-membered heterocycles were virtually combined with the pyridazinone template and docked into the X-ray structure (Supplementary data). One of the best-fitting groups, a 5-methyl-[1,2,4]oxadiazol-3-yl group, was introduced to give compound 6 (Table 2) with an IC50 already in the same range as comparable carbamates (compounds 4 and 5).15 As expected, the isomeric oxadiazole 7 was less active due
Figure 2. The X-ray structure of thiadiazinone 1 with c-Met kinase at 2.0 Å resolution (PDB-ID: 4R1Y) revealed H-bonds (dashed black lines) to Met1160 of the hinge region, to Asp1222 of the DFG motif, to Asn1167 and water-mediated contacts to Ile1084 of the GC-loop.
to the non-optimal position of the donor nitrogen. Several fivemembered heterocyclic derivatives with a donor nitrogen in the optimal position were then prepared. Imidazole 8 was only weakly active, whereas oxazole 9 and thiazole 10 were more active than oxadiazole 6. This activity gain may be due to a CH–p interaction between the oxazole/thiazole H-atom pointing toward the p-sys-
Table 1 Properties of carbamates O X
Compd
X
R
S O
1
N
N H
O
c-Met biochemIC50 (nM)a
N
R
R
c-Met cell IC50(nM)b
Mouse PKc CL (L/h/kg)
F (%)
6
