Frank Himmelsbach

Target Binding Properties and Cellular Activity of Afatinib (BIBW 2992), an Irreversible ErbB Family Blocker

Deregulation of the ErbB (proto-oncogene B of the avian erythroblastosis virus AEV-H strain) receptor network is well recognized as an oncogenic driver in epithelial cancers. Several targeted drugs have been developed, including antibodies and small-molecule kinase inhibitors, each of them characterized by distinct patterns of ErbB receptor interactions. Understanding the precise pharmacological properties of these compounds is important for optimal use in clinical practice. Afatinib [BIBW 2992; N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2-butenamide] is an ATP-competitive anilinoquinazoline derivative harboring a reactive acrylamide group. It was designed to covalently bind and irreversibly block enzymatically active ErbB receptor family members. Here, we show by X-ray crystallography the covalent binding of afatinib to wild-type epidermal growth factor receptor (EGFR) and by mass spectrometry the covalent interaction with EGFR, EGFRL858R/T790M, human epidermal growth factor receptor 2 (HER2), and ErbB-4. Afatinib potently inhibits the enymatic activity of ErbB-4 (EC50 = 1 nM) and the proliferation of cancer cell lines driven by multiple ErbB receptor aberrations at concentrations below 100 nM. N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2-butanamide (BI 37781), a close analog of afatinib lacking the acrylamide group and thus incapable of covalent bond formation, had similar potency on cells driven by EGFR or EGFRL858R, but less or no detectable activity on cells expressing EGFRL858R/ T790M HER2 or ErbB-4. These results stress the importance of the acrylamide group and show that afatinib differs from approved ErbB targeting agents by irreversibly inhibiting the kinase activity of all ErbB family members. They provide a mechanistic rationale for the distinct pharmacological features of this compound and explain the clinical activity seen in some patients who are resistant to antibody or kinase inhibitor therapy because of secondary mutations or ErbB receptor “reprogramming.”

Empagliflozin, a novel selective sodium glucose cotransporter‐2 (SGLT‐2) inhibitor: characterisation and comparison with other SGLT‐2 inhibitors

Aims: Empagliflozin is a selective sodium glucose cotransporter‐2 (SGLT‐2) inhibitor in clinical development for the treatment of type 2 diabetes mellitus. This study assessed pharmacological properties of empagliflozin in vitro and pharmacokinetic properties in vivo and compared its potency and selectivity with other SGLT‐2 inhibitors.

(R)-8-(3-Amino-piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione (BI 1356), a Novel Xanthine-Based Dipeptidyl Peptidase 4 Inhibitor, Has a Superior Potency and Longer Duration of Action Compared with Other Dipeptidyl Peptidase-4 Inhibitors

BI 1356 [proposed trade name ONDERO; (R)-8-(3-amino-piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione] is a novel dipeptidyl peptidase (DPP)-4 inhibitor under clinical development for the treatment of type 2 diabetes. In this study, we investigated the potency, selectivity, mechanism, and duration of action of BI 1356 in vitro and in vivo and compared it with other DPP-4 inhibitors. BI 1356 inhibited DPP-4 activity in vitro with an IC50 of approximately 1 nM, compared with sitagliptin (19 nM), alogliptin (24 nM), saxagliptin (50 nM), and vildagliptin (62 nM). BI 1356 was a competitive inhibitor, with a Ki of 1 nM. The calculated koff rate for BI 1356 was 3.0 × 10–5/s (versus 2.1 × 10–4/s for vildagliptin). BI 1356 was ≥10,000-fold more selective for DPP-4 than DPP-8, DPP-9, amino-peptidases N and P, prolyloligopeptidase, trypsin, plasmin, and thrombin and was 90-fold more selective than for fibroblast activation protein in vitro. In HanWistar rats, the DPP-4 inhibition 24 h after administration of BI 1356 was more profound than with any of the other DPP-4 inhibitors. In C57BL/6J mice and Zucker fatty (fa/fa) rats, the duration of action on glucose tolerance decreased in the order BI 1356 > (sitagliptin/saxagliptin) > vildagliptin. These effects were mediated through control of glucagon-like peptide-1 and insulin. In conclusion, BI 1356 inhibited DPP-4 more effectively than vildagliptin, sitagliptin, saxagliptin, and alogliptin and has the potential to become the first truly once-a-day DPP-4 inhibitor for the treatment of type 2 diabetes.

3,5-Dihydro-imidazo[4,5-d]pyridazin-4-ones: a class of potent DPP-4 inhibitors.

Systematic variations of the xanthine scaffold in close analogs of development compound BI 1356 led to the class of 3,5-dihydro-imidazo[4,5-d]pyridazin-4-ones which provided, after substituent screening, a series of highly potent DPP-4 inhibitors.

Long-term treatment with empagliflozin, a novel, potent and selective SGLT-2 inhibitor, improves glycaemic control and features of metabolic syndrome in diabetic rats.

Empagliflozin is a potent, selective sodium glucose co‐transporter‐2 inhibitor that is in development for the treatment of type 2 diabetes. This series of studies was conducted to assess the in vivo pharmacological effects of single or multiple doses of empagliflozin in Zucker diabetic fatty rats. Single doses of empagliflozin resulted in dose‐dependent increases in urinary glucose excretion and reductions in blood glucose levels. After multiple doses (5 weeks), fasting blood glucose levels were reduced by 26 and 39% with 1 and 3 mg/kg empagliflozin, respectively, relative to vehicle. After 5 weeks, HbA1c levels were reduced (from a baseline of 7.9%) by 0.3 and 1.1% with 1 and 3 mg/kg empagliflozin, respectively, versus an increase of 1.1% with vehicle. Hyperinsulinaemic–euglycaemic clamp indicated improved insulin sensitivity with empagliflozin after multiple doses versus vehicle. These findings support the development of empagliflozin for the treatment of type 2 diabetes.

Bis-(p-nitrophenylethyl)phosphoromonochloridate, a new versatile phosphorylating agent

Abstract A new phosphorylating agent, bis-(p-nitrophenylethyl)phoshoromonochloridate, has been prepared and is used for 3′- and/or 5′-phosphorylations of nucleosides. The resulting bis-(p-nitrophenylethyl)phosphotriesters are versatile synthons in oligonucleotide syntheses leading finally to 3′- and/or 5′-terminated oligonucleotides in excellent yields.

Pharmacological characterization of the selective 11β-hydroxysteroid dehydrogenase 1 inhibitor, BI 135585, a clinical candidate for the treatment of type 2 diabetes.

To combat the increased morbidity and mortality associated with the developing diabetes epidemic new therapeutic interventions are desirable. Inhibition of intracellular cortisol generation from cortisone by blocking 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) has been shown to ameliorate the risk factors associated with the metabolic syndrome. A challenge in developing 11β-HSD1 inhibitors has been the species selectivity of small molecules, as many compounds are primate specific. Here we describe our strategy to identify potent selective 11β-HSD1 inhibitors while ensuring target engagement in key metabolic tissues, liver and fat. This strategy enabled the identification of the clinical candidate, BI 135585.

Antagonism of the GPIIb/IIIa receptor with the nonpeptidic molecule BIBU52: inhibition of platelet aggregation in vitro and antithrombotic efficacy in vivo.

The glycoprotein (GP) IIb/IIIa (the alphallb beta3 integrin) found on platelets binds fibrinogen or von Willebrand factor when the platelet is activated, thereby mediating the aggregation of platelets. Blockade of the GPIIb/IIIa should prevent platelet aggregation independent of the substance or substances responsible for activating the platelets. This comprehensive inhibition of platelet aggregation is thought to be an effective therapeutic approach to various clinical thromboembolic syndromes. This study investigated the platelet inhibition provided by blocking GPIIb/IIIa by using a new nonpeptidic molecule, BIBU52, in both in vitro and in vivo models. BIBU52 competes with [125I]fibrinogen for binding sites on human platelets in a Ca2+ and pH-dependent manner with a 50% inhibitory concentration (IC50) of 35 +/- 12 nM. BIBU52 inhibited the aggregation of human platelets in platelet-rich plasma induced by collagen (1-2 microg/ml), adenosine diphosphate (ADP; 2.5 microM), and a thrombin receptor-activating peptide (TRAP; SFLLRNPNDKYEPF-NH2; 25 microM) with IC50 values of 82, 83, and 200 nM, respectively. The inhibition of platelet aggregation by BIBU52 was found to be highly species dependent. BIBU52 inhibited aggregation in plasma from rhesus and marmoset monkeys with an IC50 of 150 nM but was totally ineffective in rat plasma. The selectivity of BIBU52 for inhibiting GPIIb/IIIa in comparison with other adhesion molecules was investigated in a human endothelial cell adhesion assay. The adhesion of human endothelial cells to matrices of vitronectin, fibronectin, collagen I, or laminin was not affected by concentrations as high as 100 microM BIBU52; thus BIBU52 demonstrates a high selectivity for the human GPIIb/IIIa. The antithrombotic effect of BIBU52 in vivo was investigated in three animal models of recurrent arterial thrombus formation. In the guinea pig aorta, BIBU52 inhibited thrombus formation dose dependently, with lack of thrombus formation for 1 h after a bolus dose of 1.0 mg/kg i.v.. Both acetylsalicylic acid and dazoxiben were less effective in this model. In pigs with recurrent thrombus formation in the carotid artery, 1.0 mg/kg i.v. also inhibited thrombus formation. Heparin was not effective in the pig, and acetylsalicylic acid was only partially effective. In the pig, the dose of 1.0 mg/kg i.v. BIBU52 also was associated with a 70% inhibition of collagen-induced platelet aggregation ex vivo but with only a transient prolongation of sublingual bleeding time to a maximum of 2.5-fold and without other hemodynamic effects. In the marmoset monkey, a dose of 10 microg/kg i.v. could abolish recurrent arterial thrombosis. Hemodynamic effects of BIBU52 in anesthetized pigs were not detected in doses < or = 10 mg/kg. These data demonstrate that BIBU52 is a potent and selective antagonist of the human GPIIb/IIIa receptor and capable of substantial inhibition of platelet aggregation in vitro and ex vivo as well as inhibition of arterial thrombus formation in vivo in animal models of thrombosis.

Nucleotides, Part LXIII, New 2‐(4‐Nitrophenyl)ethyl(Npe)‐ and 2‐(4‐Nitrophenyl)ethoxycarbonyl(Npeoc)‐Protected 2′‐Deoxyribonucleosides and Their 3′‐Phosphoramidites – Versatile Building Blocks for Oligonucleotide Synthesis

A series of new base-protected and 5′-O-(4-monomethoxytrityl)- or 5′-O-(4,4′-dimethoxytrityl)-substituted 3′-(2-cyanoethyl diisopropylphosphoramidites) and 3′-[2-(4-nitrophenyl)ethyl diisopropylphosphoramidites] 52 – 66 and 67 – 82, respectively, are prepared as potential building blocks for oligonucleotide synthesis (see Scheme). Thus, 3′,5′-di-O-acyl- and N 2,3′-O,5′-O-triacyl-2′-deoxyguanosines can easily be converted into the corresponding O6-alkyl derivatives 6, 8, 10, 12, 14, and 16 by a Mitsunobu reaction using the appropriate alcohol. Mild hydrolysis removes the acyl groups from the sugar moiety (→ 9, 11, 13, 15, and 19 (via18), resp.) which can then be tritylated (→ 38 – 42) and phosphitylated (→ 57 – 61) in the usual manner. N 2-[2-(4-nitrophenyl)ethoxycarbonyl]-substituted and N 2-[2-(4-nitrophenyl)ethoxycarbonyl]-O6-[2-(4-nitrophenyl)ethyl]-substituted 2′-deoxyguanosines 5 and 7, respectively, are synthesized as new starting materials for tritylation (→ 28, 35, and 37) and phosphitylation (→ 54, 56, 70, and 78). Various O4-alkylthymidines (see 20 – 24) are also converted to their 5′-O-dimethoxytrityl derivatives (see 43 – 47) and the corresponding phosphoramidites (see 62 – 66 and 79 – 82).

The use of the p-nitrophenylethoxycarbonyl group for amino protection in cytidine and adenosine chemistry

Abstract Amino protection in 2′-deoxycytidine ( 1 = ) and cytidine ( 2 = ) as well asin 2′-deoxyadenosine adenosine ( 8 = ) and adenosine ( 9 = ) respectively can be achieved by the new reagents 1-(p-nitrophenylethoxycarbonyl)-benzotriazole ( 3 = ) and 1-methyl-3-(p-nitrophenylethoxy-carbonyl)-imidazolium chloride ( 7 = ) to form the corresponding carbamates ( 4 = , 5 = , 1 = 3 = , 1 = 4 = ) in high yields.