Discovery of novel dual inhibitors of receptor tyrosine kinases EGFR and IGF-1R.

Novel 4-benzylamino benzo-anellated pyrrolo[2,3-b]pyridines have been synthesized with varied substitution patterns both at the molecular scaffold of the benzo-anellated ring and at the 4-benzylamino residue. With a structural similarity to substituted thieno[2,3-d]pyrimidines as epidermal growth factor receptor (EGFR) inhibitors, we characterized the inhibition of EGFR for our novel compounds. As receptor heterodimerization gained certain interest as mechanism of cancer cells to become resistant against novel protein kinase inhibitors, we additionally measured the inhibition of insulin-like growth factor receptor IGF-1R which is a prominent receptor for such heterodimerizations with EGFR. Structure-activity relationships are discussed for both kinase inhibitions depending on the varied substitution patterns. We discovered novel dual inhibitors of both receptor tyrosine kinases with interest for further studies to reduce inhibitor resistance developments in cancer treatment.


Introduction
Protein kinase inhibitors have been established as effective tool to combat cancer in case of a deregulation of such protein kinases that are being either overexpressed or overactivated in cancer cells to cause cell proliferation 1 . Early protein kinase inhibitor developments concentrated on single kinases to avoid expected side effects during anticancer therapies with such inhibitors 2 . However, resistance developments against such novel inhibitors occurred due to amino acid substitutions in the inhibitor-binding region of the respective kinase 1,2 . Monoclonal antibodies have been therapeutic alternatives that bind to the extracellular domain of the protein kinase receptor 3,4 . Resistance developments against those antibodies have also been described, and the costs of such therapies are high so that small molecule inhibitors, which act at the intracellular receptor site are attractive target compounds for anticancer drug developments, also to address receptor mutants 5,6,7 .
The epidermal growth factor receptor (EGFR) tyrosine kinase is one major target structure in cancer therapies. EGFR is found overexpressed in many epidermal tumors 8,9,10 . With additional contributions to angiogenesis and invasive tumor growth, EGFR contributes to cell proliferation and the formation of metastases 11,12 . Developed EGFR inhibitors have a 4-amino quinazoline molecular scaffold A that binds to the protein kinase target structure via the N-1 of the pyrimidine partial structure (Figure 1) as shown for erlotinib in Figure 2 13 .
In recent studies, the phenyl ring of the quinazoline has been replaced by a thiophene ring leading to novel thienopyrimidines B with a 4-benzylamino substitution and various substituted phenyl residues attached to the anellated thiophene ring 14 . Those thienopyrimidines reached activities to inhibit EGFR partially similar to the reported quinazolines.
We developed novel pyrrolopyridines C with a 4-benzylamino residues and a benzo-anellation to the pyrrole residue. The compounds have a structural similarity to the substituted thienopyrimidines, and we investigated their potential to inhibit EGFR. We additionally proved their ability to inhibit the insulin-like growth factor receptor (IGF-1R) that contributes to an EGFR resistance mechanism via a receptor heterodimerization as will be discussed later. So we discovered novel dual inhibitors of cancer-relevant tyrosine kinases EGFR und IGF-1R.

Experimental
General Commercial reagents were used without further purification. The bromo-substituted benzylamines have been synthesized via N-benzylamine phthalimides that underwent a following reaction with hydrazine to release the corresponding amines following literature 15 . The 1 H-NMR spectra (400 MHz) were measured using tetramethylsilane as internal standard. Thin-layer chromatography (TLC) was performed on E. Merck 5554 silica gel plates. The electrospray ionization (ESI) spectra were recorded on a Finnigan LCQ Classic mass spectrometer. Infrared (IR) spectra were recorded on a Fourier transform infrared (FT-IR) spectrometer. Elemental analysis indicated by the symbols of the elements was within ±0.4% of the theoretical values and was performed using a Leco CHNS-932 apparatus.
were added. The mixture was heated under reflux for 23 h and then poured into 1 L of ethylacetate. The precipitate was solved under addition of 100 mL of a potassium hydroxide solution (10%). The resulting phases were separated and the organic layer was washed with 300 mL of the potassium hydroxide solution for two times, dried over sodium sulfate and filtered. After evaporation of the solvent, the resulting product was used without further purification. Yield 80 g (97%); yellow-white needles; mp 110-112 C; 1   Polyphosphoric acid (29.4 g) was heated in a round flask to 170 C. Then, 11.4 g (58 mmol) of compound 1 were added under stirring for 3 h at the maintained temperature. Then, 50 mL of water was added and the solution was alkalized with a 10 M potassium hydroxide solution to a pH of 10. After stirring overnight, the suspension was poured into 250 mL of water, cooled down to 0 C in an ice bath and filtered. Precipitated disodium hydrogen phosphate was washed out with portions of water and the remaining residue was kept under vacuum. Yield 3.6 g (36%); beige solid; mp 201-212 C; 1 Formation of the 4-chloro-9-H-pyrido[2,3-b]indole 3 About 3.6 g (21 mmol) of compound 2 were dissolved in acetic acid and 4.4 g (45 mmol) of a 35% solution of hydrogen peroxide in water were added dropwise. The solution was heated under reflux for 5 h. Then, the solution volume was reduced in vacuum and the remaining oily product was treated with a saturated potassium carbonate solution to reach a pH of 8. After stirring overnight, the resulting precipitate was filtered off and dried in vacuum. After that 3.6 g (19.5 mmol) were dissolved in dimethylformamide (DMF) under stirring and argon atmosphere. The solution was cooled down to 0 C on an ice bath, and then, 4.2 mL (7.1 g, 46.9 mmol) of phosphoryl oxychloride was added. The whole mixture was stirred for 24 h and poured into 50 mL of water. The pH value was adjusted to 12 using a 12% solution of potassium hydroxide. After stirring for 30 min, the solution was filtered and the remaining solid was dried and purified by column chromatography using silica gel and a mixture of cyclohexane and ethylacetate (80/20) to wash out the impurities, and then, with the eluent mixture of 50/50 to isolate the poduct 3.   Formation of the 6-bromo-4-chloro-9-H-pyrido[2,3-b]indole 4 About 1 g (4.9 mmol) of compound 3 was dissolved in 30 mL of acetic acid, and 0.94 g (5.9 mmol) of bromine was added dropwise under stirring. The resulting sticky mixture was diluted with 20 mL of acetic acid and stirring continued for 24 h at room temperature. Then, 50 mL of a 1 M sodium thiosulfate solution was added. The solution was cooled on an ice bath and the pH value was adjusted to 10 with concentrated ammonia. Then extraction followed for three times with each 50 mL chloroform first and then with each 50 mL of ethylacetate. The unified organic layers were dried over sodium sulfate. Then, it was filtered and the layer was removed in vacuum. Yield  General procedure for the formation of the 4-benzylamino substituted 6-bromo-9-H-pyrido[2,3-b]indoles 5a-h One equivalent of compound 4 and 15 equivalents of the respective benzylamine were heated under stirring at 140 C for 48 h. After cooling 10 mL of chloroform were added and the mixture was stirred over night. The resulting precipitate was washed with tetrahydrofurane and filtered (Scheme 1).   General procedure for the formation of the 6-cyano substituted 9-H-pyrido[2,3-b]indoles 6a-c One equivalent of the respective 6-bromo 4-benzylamine compound 5 was dissolved in 3 mL NMP and 2.7 equivalents of copper(I) cyanid were added. Then, the mixture was heated under argon atmosphere and reflux for 7 h at 200 C and after that poured into 20 mL of ethylacetate. Washing with 10 mL of a 20% solution of ammonia in water followed and a saturated solution of sodium chloride in water was added to clear the suspension formation. The extraction of the water phase with 20 mL ethylacetate followed and the unified organic layers were dried over sodium sulfate, filtered and, finally, the solution volume was reduced in vacuum. Then, 20 mL of water was added so that the product 6 precipitated (Scheme 2).

Receptor tyrosine kinase inhibition
The protein kinases were expressed by means of the baculovirus expression system in Sf9 insect cells as human recombinant GST fusion proteins and purified by affinity chromatography using GSH-agarose. The kinase identity was confirmed by mass spectrometry using LC-ESI-MS/MS technique.
The measuring of protein kinase activity was performed in 96well FlashPlates TM from Perkin Elmer in a 50 mL reaction volume. The reaction mixture consisted of 20 mL of assay buffer solution, 5 mL of ATP solution in water, 5 mL of used test compound in a 10% DMSO solution and finally a premixture of each 10 mL of used substrate and enzyme solutions. The assay buffer solution contained 70 mM of HEPES-NAOH pH 7.5, each 3 mM of magnesium chloride and manganese(II) chloride, 3 mM of sodium orthovanadate, 1.2 mM of DTT, 50 mg/mL of PEG 20000 and finally 15 mM of [c-33 P]-ATP making approximately 7 Â 10 5 cpm per well.
The final kinase concentration has been 10 ng/50 mL for EGFR and IGF-1R. The used substrate was Poly(Glu,Tyr) 4:1 in a concentration of 125 ng/50 mL.
The reaction mixtures were incubated at 30 C for 60 min. The reaction was stopped with 50 mL of a 2% (v/v) solution of  (Table 1).

Chemistry
The benzo-anellated pyrrolo[2,3-b]pyridine was yielded from the primary reaction of benzotriazole and 2-bromopyridine in toluene under reflux to give the (pyridine-2-yl) benzotriazole that underwent a following polyphosphoric acid-catalyzed reaction to the tricyclic molecular scaffold. Next, a chlorination in the 4-position of the pyridine partial structure took place with phosphoryl chloride after the benzo-anellated pyrrolo[2,3-b]pyridine had been activated with hydrogen peroxide in acetic acid to the N-oxide that directed the chloro substituent preferably into the desired 4-position. Then, the bromo substituent was introduced into the preferred 6-position of the molecular scaffold using bromine in acetic acid. Finally, the varying benzylamine residues were introduced in the 4-position by heating under solvent-free conditions with the benzylamines. The bromo substituent exchange with the cyano function was managed with copper(I) cyanide by heating with NMP (N-methylpyrrolidone). The 6-carboxylic compound resulted from the 6-cyano substituted compound in a copper(I) iodide catalyzed reaction in strong alkaline medium.

Receptor tyrosine kinase inhibition
Insight in deregulated chemical pathways of cellular signal transduction in cancer cells offered the possibility to develop inhibitors of the responsible protein kinases 1 . Receptor tyrosine kinases are transmembrane receptor proteins that are regulated by extracellular ligands 9,18 . Being activated after ligand binding, the receptor undergoes a dimerization reaction after autophosphorylation 19,20 . The dimerized receptor activates following signal pathways by phosphorylation of respective substrates 19,21 . In the case of EGFR, which is found deregulated in many epidermal tumors, small molecule inhibitors have been developed to bind to the ATP-binding site of the receptor. Their binding is specific to single amino acid residues of the protein backbone. Mutations that cause exchanges of such amino acids led to resistance developments against established inhibitors 22 . Another recently discovered resistance mechanism of cancer cells is a possible heterodimerization of the EGFR receptor with IGF-1R as another activated tyrosine kinase 23,24 . Such described heterodimerizations made void the EGFR-specific inhibitory activity of an established EGFR inhibitor. That loss of inhibitory activity via receptor heterodimerization led to a proceeding of an aggressive tumor growth as described 24 . So there have been intense efforts to develop novel inhibitors of EGFR and IGF-1R. We investigated the inhibitory activity towards both kinases EGFR and IGF-1R for our novel benzo-anellated pyrrolo [2,3-b]pyridines that show structural relationship to reported thienopyrimidines as EGFR inhibitors. The varied 3-benzylamine substituted compounds 5a-d have been investigated first. The 3-methoxybenzylamine compound 5a showed submicromolar affinities towards EGFR. The 3-chlorobenzylamine derivative 5b showed similar EGFR affinities and the micromolar activity to inhibit IGF-1R was improved if compared to that of compound 5a. Slight improvements of the inhibitory activity toward both kinases were found for the 3-bromobenzylamine substituted derivative 5c. The 3amino function in compound 5d led to a further increased affinity toward EGFR with a K i value of 0.101 mM and to a submicromolar affinity towards IGF-1R with 0.537 mM. So compound 5d is a first dual inhibitor of both kinases in similar ranges. When the 3methoxy function of compound 5a moved to the 4-position of the benzylamine residue in derivative 5e, the affinity towards EGFR was reduced; however, the affinity towards IGF-1R increased. If the 3-chloro function of compound 5b moved to the 4-position of the benzylamine residue in derivative 5f, the affinity towards EGFR was lost, while the affinity towards IGF-1R remained in the range of the 4-methoxybenzylamine compound 5e. Finally, the movement of the 3-bromo substituent to the 4-position in the benzylamine residue of compound 5g reduced the EGFR affinity, but increased the affinity towards IGF-1R to give a second dual inhibitor of both kinases in the similar activity range. If the 4-bromo function was replaced with a 4-methyl function in the 4-methyl benzylamino derivative 5h both affinities increased. So we can state that a methyl function in the 4-position of the benzylamino residue is most favorable for both EGFR and IGF-1R affinities, whereas the 3-amino function is most favorable in the 3-benzylamine residue to inhibit both EGFR and IGF-1R.
We then investigated the affinity of our synthesized 5-cyano derivatives 6a-c towards our target kinases. The 3-methoxybenzylamine compound 6a showed significantly increased affinities towards EGFR with a determined K i value of 72 nM. Thus, nanomolar ranges were reached similar to the EGFR inhibitor erlotinib for which a K i value of 17.5 nM has been reported 25 . Moreover, the affinity towards IGF-1R in the submicromolar range was more than thirtyfold higher than that of the corresponding 6-bromo compound 5a. Erlotinib for comparison showed no activity toward IGF-1R 26 . The 4-methoxybenzylamine function of compound 6b was less favorable than the 3-methoxybenzylamine function of derivative 6a concerning the EGFR affinity, whereas the IGF-1R affinity slightly improved. If compared to the 6-bromo compounds 5a and 5e, we found similar tendencies in the affinities towards EGFR and IGF-1R with the methoxy substituent in the 3-position of the benzylamine residue being more favorable towards IGF-1R, but less favorable towards EGFR. However, the 6-cyano substitution was again more favorable if compared to the 6-bromo substitution of the molecular scaffold. Finally, we determined the affinities of the 4-methyl benzylamino derivative 6c. Both affinities towards EGFR and IGF-1R were found increased if compared to the 6-bromo substituted compound 5h. So we can state an allover better activity for the 6-cyano substituted compounds if compared to the 6-bromo substituted derivatives. We finally determined the affinity of the 6-carboxylic acid substituted compound 7. The affinity towards EGFR was less favorable than that of the corresponding derivative 6a. However, with a determined K i value of 2.36 mM, the affinity towards IGF-1R was almost tenfold lower than that of the corresponding 6-cyano compound 6a.
It can be summarized that we identified novel dual inhibitors of the receptor tyrosine kinases EGFR and IGF-1R. Both the benzylamine and the molecular scaffold substitutions were sensitive to influence the kinase affinities. Most favorable substitutions were the 6-cyano function of the molecular scaffold and the 3-amino and the 4-methly benzylamino residues as far as investigated. Our novel dual inhibitors may be promising lead structures to combat cancer resistance developments via receptor heterodimerization of the respective kinases by inhibiting both relevant kinases.