Improved Synthesis of Substituted 6 , 7-Dihydroxy-4-quinazolineamines : Tandutinib , Erlotinib and Gefitinib

The synthesis of three substituted 6,7-dihydroxy-4-quinazolineamines: tandutinib (1), erlotinib (2) and gefitinib (3) in improved yields is reported. The intermediates were characterized by NMR and the purities determined by HPLC.


Introduction
Many signal transduction pathways are governed by protein kinases regulating various aspects of cell functions.Mutations which deregulate their expression, their function or both can result in cancers.Protein kinase inhibitors, particularly phosphorylation inhibitors, have become important targets for selective cancer therapies.The genome project has identified more than 500 protein kinases and 16 protein tyrosine kinases (PTKs) are possible therapeutic targets.The epidermal growth factor receptor (EGFR), a cell membrane receptor, plays a key role in cancer development and progression [1].Tyrosine kinase inhibitors erlotinib (2, Tarceva ® , OSI-774) and gefitinib (3, Iressa ® ) have been launched for the treatment of non-small-cell lung cancer (NSCLC) and tandutinib (1, MLN-518, CT-53518) is in phase II clinical trials for myeloid leukemia (ML) or advanced myelodysplasia (MDS).Several substances that inhibit platelet-derived growth factor receptor (PDGFR) phosphorylation via competitive binding of adenosine 5´-triphosphate [2,3] have been reported previously.Furthermore gefitinib (3) and erlotinib (2) inhibit tyrosine kinase activity and restrict the receptor's catalytic activity, autophosphorylation, and its engagement with signal transducers [4].Gefitinib (3) is the first EGFRtargeting agent to be registered as an anticancer drug in Japan, in Australia and in the US for the third-line treatment of chemo-resistant NSCLC patients [1].Tandutinib (1) selectively inhibits FLT3 and PDGFR, while other tyrosine or serine/threonine kinases are not significantly inhibited [5].Here we report at the improved synthesis of these three PTK inhibitors: tandutinib (1), erlotinib (2) and gefitinib (3) (Figure 1).

Results and Discussion
For tandutinib (1) the synthesis of the key intermediate 8 starts from 4-hydroxy-3-methoxy-benzoic acid, which is reacted with 1-bromo-3-chloro-propane followed by nitration, yielding the desired regioisomer in 75% yield.These steps, as well as the following substitution with piperidine and subsequent catalytic hydrogenation were performed according to the published procedures [6], however all intermediates were purified and fully characterized (Scheme 1).
In our hands the reported cyclization of 7 to 8 using either formamide or formamide with addition of ammonium formiate resulted in yields of only 10-20 %.Using formamidine acetate and 2-methoxyethanol as a solvent, we obtained 8 in 85 % yields and >98 % HPLC purity.For the chlorination step we found that the use of thionyl chloride and DMF [6] gave less reliable results than our method using phosphoryl chloride and N,N-diethylaniline [7] where we obtained 70-86 %.The preparation of 9 is not mentioned in the literature.We prepared it by reaction of 4-isopropoxyphenylamine first with trichloromethyl chloroformate followed by reaction with excess of piperazine.A close example starting from benzylpiperazine and 4-methoxyphenylisocyanate is described in literature [5].The final convergent coupling of 9 with 10 gave 95 % of 1 in >90 % HPLC purity.Tandutinib (1) was obtained after recrystallization in >99 % HPLC purity.The synthesis of tandutinib 1 was also described by Scarborough et al. but without experimental details [2].
The synthesis of erlotinib (2, Scheme 2) starts with the O-alkylation of methyl 3,4-dihydroxybenzoate using either 1-chloro-2-methoxyethane or 1-bromo-2-methoxyethane.In both cases the yield was >90 %, with the chlorine compound requiring longer reaction times.Nitration of 11 gave the desired regioisomer on 92% yield and was followed by catalytic hydrogenation and cyclization with formamide performed according to the patent literature [8].Again, our preferred reagent for the chlorination was phosphoryl chloride and N,N-diethylaniline [7], giving 15 in 89 % yield and 96 % HPLC purity.The final product was obtained as the hydrochloride salt 2•HCl and converted to 2. Erlotinib (2) was thus obtained in 56 % overall yield, each step being improved substantially.All intermediates were fully characterized.The synthesis of gefitinib (3, Scheme 3) starts with regioselective demethylation of 6,7-dimethoxy-3H-quinazolin-4-one followed by O-protection.Attempts to prepare the desired chloro compound using thionyl chloride and DMF [9] gave only unsatisfactory yields.Similar to the syntheses described above, phosphoryl chloride and N, N-diethylaniline [7] proved to be advantageous.Each of the following steps could be improved substantially and the overall yield from 16 to 3 was 33 % as compared to 20 % previously [9].

General
Melting points were measured on a Büchi melting point apparatus B-545. 1 H-and 13 C-NMR spectra were recorded on a Bruker AC-200 (200 MHz) pulse Fourier-transform NMR spectrometer in CDCl 3 or DMSO-d 6 .Thin layer chromatography (TLC) was performed on Merck TLC aluminum sheets silica 60 F 254 .Visualization was by UV light at 254 and 366 nm or spray reagents (molybdophosphoric acid and heating).Column chromatography was performed using silica gel (Baker 40-60 µm).MPLC (medium pressure liquid chromatography) was performed using a LC-8A pump (Shimadzu), a SPD-6AV UV-detector (Shimadzu) and Büchi preparative columns.HPLC was performed using a Waters 2695 instrument and Merck Chromolith RP 18 columns and a gradient of 3 % to 60 % acetonitrile/water (0.1 % TFA) at a flow of 3.0 ml/min.The HPLC purity reported is the number generated for the peak area as calculated using the Waters Millennium Software with the Maxplot option for the UV maximum of the corresponding peak.

N-(4-Isopropoxyphenyl)piperazine-1-carboxamide (9)
A solution of 4-isopropoxyphenylamine (1.0 g, 6.61 mmol) in dry toluene (10 mL) was added drop-wise to a solution of trichloromethyl chloroformate (13.0 g, 9.94 mmol) in dry toluene (10 mL) at 0-5 °C and the mixture was stirred at the same temperature for 20 min.The reaction mixture was then stirred and heated to 100 °C over 1h and kept at this temperature for 1 h.Reaction progress was monitored by HPLC and found to be complete by this time.The reaction mixture was cooled to room temperature and extracted with saturated solution of sodium bicarbonate (1 x 10 mL).The toluene solution was separated and aqueous phase extracted with toluene (5 mL).The combined toluene layers were added dropwise to a solution of piperazine (2.80 g, 32.5 mmol) in toluene (10 mL) and this mixture was heated at 90 °C for 3 h, cooled and washed with water (2 x 15).The toluene layer was dried (Na 2 SO 4 ) and evaporated to afford the product (0.91 g, 52%, 95% HPLC purity). 1  A suspension of ethyl 3,4-dihydroxybenzoate (40.0 g, 220 mmol), potassium carbonate (70.0 g, 507 mmol) and tetrabutylammonium iodide (2.80 g, 7.6 mmol) in acetone (300 mL) was stirred for 20 min at room temperature.1-Chloro-2-methoxyethane (158.0 g, 1.671 mol) was added and the solution stirred and heated to reflux for 5 days.Reaction progress was monitored by HPLC and the reaction was found to be complete after this time.The reaction mixture was cooled to room temperature and diethyl ether (250 mL) was added.Inorganic salts were filtered off and washed with diethyl ether (2 x 250 mL).The combined organic layers were dried (Na 2 SO 4 ), decolorized (charcoal) and volatiles evaporated under reduced pressure to afford the product as light oil (72.5 g) which was dissolved in petrol ether (250 mL) and cooled to 0-5 °C.The resulting white precipitate was collected by filtration and dried on the air (m.p.: 55-58 °C, 61.8 g, yield: 94 %, 97% HPLC purity). 1

6-Hydroxy-7-methoxyquinazolin-4(3H)-one (16)
A mixture of 6,7-dimethoxyquinazolin-4(3H)-one (64.7 g, 0.31 mol) and L-methionine (53.7 g, 0.34 mol) was dissolved in methanesulfonic acid (425 mL) and heated to reflux for 12 h.Reaction was monitored by HPLC and no starting material was indicated after this time.Crushed ice/water mixture (200 mL) was added and the solution was cooled to 0 °C.Then NaOH (40% water solution) was added slowly (pH~7) resulting in precipitation of a white deposit.This product was collected by filtration using a sintered glass funnel, washed with water and dried at 50 °C and 23 mbar to afford the product (58.3g, 98%, 90% HPLC purity) which was used without further purification.