Patent Application
20230357165
The present disclosure relates to the preparation of new compounds having Structure I in free form or in an acceptable salt form for modulation of Ubiquitin Ligase COP1 through its stabilization as a potential therapeutic target for Non-Alcoholic Fatty Liver Disease (NAFLD).
The present disclosure relates to a compound having Structure I, where R1, R2, R3, R4 and R5 are as defined in the description. Some of the synthesized molecules are capable of increasing the level of adipose triglyceride lipase (ATGL) through modulation of Ubiquitin Ligase COP1 through its stabilization as a potential therapeutic target for treatment of Non-Alcoholic Fatty Liver Disease (NAFLD).
Non Alcoholic Fatty Liver Disease (NAFLD) has garnered considerable attention due to the increasing worldwide prevalence of this disease spectrum. NAFLD is an umbrella term encompassing simple steatosis progressing to steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Steatosis is mostly a reversible condition whereby fat droplets, mostly in the form of triglycerides, accumulate in the liver without pronounced hepatocyte injury. Steatohepatitis (nonalcoholic steatohepatitis, NASH) denotes the stage wherein hepatocytes are significantly injured and is histologically characterized by the presence of ballooned hepatocytes, Mallory-Denk bodies, glycogenated nuclei and other distinguishing features. NASH may, in some cases, progress to fibrosis and cirrhosis which are more critical stages whereby extracellular matrix proteins, notably collagen fibres, accumulate in the liver encircling hepatocytes and forming scar tissue resulting in irreversible damage to the normal physiology of the liver. The prevalence of NAFLD is reported to be 20%-30% in Western countries and 5%-18% in Asia. While the incidence of NAFLD is rising at an alarming rate, with it being considered now as the second most common reason for liver transplantation, no robust therapies are available to reverse the advanced stages of this condition.
NAFLD is a complex multifactorial disorder involving the interplay of several molecules and their associated signaling pathways. A multitude of risk factors have been attributed to the development of NAFLD with type 2 diabetes and metabolic syndrome considered as the most important ones. As is evident, the most prominent feature of NAFLD is the deposition of excessive triacylglycerols (TAG) in hepatocytes and, therefore, deregulation of enzymes responsible for controlling intracellular lipid turnover and homeostasis may play an important role in NAFLD (Ong et al. Hepatology. 2011, 53, 116-126). A pivotal enzyme associated with the intracellular degradation of TAG is Adipose triglyceride lipase (ATGL) also known as patatin-like phospholipase domain-containing protein 2 (PNPLA2). It catalyses the initial and rate limiting step in the TAG lipolysis cascade. Indeed, studies have shown that ATGL levels are decreased in NAFLD patients and liver injury is aggravated in mice with liver specific ATGL depletion (Jha et al. Hepatology, 2014, 59, 858-869).
Ubiquitin-proteasome system is a pivotal pathway for regulation of protein turnover in cells. Ubiquitination of a protein requires the stepwise involvement of 3 enzymes: E1-ubiquitin-activating enzymes, E2-ubiquitin-conjugating enzymes, and E3 ubiquitin ligases. COP1 is one such evolutionary conserved ubiquitin ligase which plays a central role in a myriad of important cellular pathways like insulin secretion from pancreatic β cells, regulating the stability of p53, etc.
Our previous study has identified a novel interaction between COP1 and the VP motif of ATGL. This interaction targets ATGL for proteasomal degradation by K-48 linked polyubiquitination, predominantly at the lysine 100 residue. In NAFLD, increased degradation of ATGL by COP1 would cause more TAG to accumulate in the liver manifesting a more severe form of the disease (Ghosh et al. Diabetes, 2016, 65, 3561-3572). Therefore, curtailing the ubiquitin mediated degradation of ATGL by inhibiting COP1 can be a potential area for therapeutics. In the same study, it was validated that steatosis in mice liver could be ameliorated with adenovirus mediated depletion of COP1 in mice. In this context, if small molecules with the potential to target the interacting site of COP1 and ATGL can be developed to target COP1 and hinder its capability to ubiquitinate ATGL, ATGL would be able to hydrolyze the accumulated TAG in the liver and abort the progression of NAFLD. Therefore, if this increased lipolysis in liver in the context of NAFLD can be achieved, then a robust therapy is available at hand to combat the progression of steatosis to steatohepatitis ultimately restricting NAFLD at its very onset (Niyogi et al., Biochemical and Biophysical Research Communications, 2019, 512, 806-811).
At present, treatment strategies are mainly directed towards various targets that mediate hepatocyte dysregulation, inflammation, apoptosis and oxidative stress. Extrahepatic targets whose role are implicated in NASH like microbiome, gut liver axis, organs like muscle and adipose tissue are also being considered for designing therapeutic targets. Certain drugs are in clinical trials at various phases. Notably, elafibranor (PPAR-α/δ ligand), selonsertib (ASK-1 inhibitor), obeticholic acid (FXR agonist), cenicriviroc (CCR 2/5 inhibitor) are in Phase 3 trial. All these drugs aim at a much advanced stage of fibrosis in NASH. Few drugs like Aramchol (SCD-1 inhibitor), IMM-124E (Anti-LPS), MGL-3196 (THR-β agonist), NGM282 (FGF19 analog), and PF-05221304 (ACC inhibitor), which are in Phase 2 clinical trials aim at an improvement in liver fat and therefore, target mainly the steatotic stage. Targeting the fibrotic stage in NASH may not always prove to be beneficial since mostly the stage is irreversible and much damage has already been inflicted in liver with deposition of collagen fibres and beginning of scar tissue formation. Hence, if the progression of NASH can be curbed at the reversible stage of steatosis by curtailing the deposition of fat, a much effective therapy can be established.
The main objective of the present disclosure is to provide a compound having Structure I.
Another objective of the present disclosure is to provide a process for the preparation of compound having Structure I.
Still another objective of the present disclosure is to evaluate the efficacy of active compounds using screening methods including fluorescence microscopy and measurement of levels of ATGL protein.
Yet another objective of the present disclosure is to provide a method for testing the specificity of the compounds for targeting the interaction of ATGL-COP1.
Still another objective of the present disclosure is to increase the level of ATGL in hepatocytes that can decrease the level of cellular lipids.
Yet another objective of the present disclosure is to decrease the ubiquitination and proteasomal degradation of ATGL.
Still another objective of the present disclosure is to identify the specific E1 and E2 enzyme in ubiquitination process.
Yet another objective of the present disclosure is to decrease the level of triglycerides in hepatocytes.
Still another objective of the present disclosure is to test the efficacy of the compounds in vivo in preclinical models.
Yet another objective of the present disclosure is to provide a composition comprising compounds of Structure I for use in a number of clinical applications, including pharmaceutical agents and methods for treating conditions like Non-Alcoholic Fatty Liver Disease (NAFLD).
Still another objective of the present disclosure is to provide a composition and methods of using the compounds having general Structure I without considerable cytotoxicity in hepatocytes.
An embodiment of present disclosure provides compound having Structure I or a pharmaceutically acceptable salt thereof:
wherein
R5 is independently selected from the group consisting of:
Another embodiment of the present disclosure provides the compound having Structure I selected from the group consisting of:
Yet another embodiment of the present disclosure provides a process for the preparation of compounds having Structure I, wherein the steps comprising:
Still another embodiment of the present application provides a compound having Structure I or salts thereof for use in treating diseases and disorders related to modulation of COP1 enzyme through its stabilization or modulation of ATGL.
Another embodiment of the present disclosure provides a compound having Structure I or salts thereof for use in decreasing the level of triglycerides in hepatocytes.
Yet another embodiment of the present disclosure provides a compound having Structure I or salts thereof for use in treatment of disease selected from Non-Alcoholic Fatty Liver Disease (NAFLD) or Non-Alcoholic Steatohepatitis (NASH).
Another embodiment of the present disclosure provides a compound having Structure I or salts thereof along with pharmaceutically acceptable excipients.
Still another embodiment of the present disclosure provides a method of modulation COP1 enzyme through its stabilization by compound having Structure I.
Yet another aspect of the present disclosure provides a method of increasing the level of ATGL by compound having Structure I.
The objects and features of the present disclosure will become apparent from the following detailed description, when taken in conjunction with the accompanying drawings.
The present disclosure relates to a compound having Structure I or salts thereof:
wherein
All the compounds of this disclosure having Structure I are depicted in the Table 1:
The process for preparation of the compound having Structure I as given in Table 1 comprises the following steps:
Table 2 provides the structures of reactants and products obtained with reaction via chloroformate intermediates:
Table 3 provides the structures of reactants and products obtained with reaction with isocyanates:
Table 4 provides the structures of reactants and products obtained for Suzuki reaction:
Provided below are the schemes for preparing the compounds disclosed in the present application.
In an embodiment of the present disclosure, there is provided a compound having Structure I for use in treating diseases and disorders related to modulation of COP1 enzyme through its stabilization or modulation of ATGL.
In another embodiment of the present disclosure, there is provided a compound having Structure I for use in decreasing the level of triglycerides in hepatocytes.
In yet another embodiment of the present disclosure, there is provided a compound having Structure I for use in treatment of disease selected from Non-Alcoholic Fatty Liver Disease (NAFLD) or Non-Alcoholic Steatohepatitis (NASH).
In still another embodiment of the present disclosure, there is provided a composition comprising the compound having Structure I along with pharmaceutically acceptable excipients.
Another embodiment of the present disclosure provides a method of modulation COP1 enzyme through its stabilization by the compound having Structure I.
Yet another embodiment of the present disclosure provides a method of increasing the level of ATGL by the compound having Structure I.
Following examples are given by way of illustration and therefore should not be construed to limit the scope of the present disclosure.
Temperatures are given in degree Celsius. The structures of final products, intermediates and starting materials are confirmed by standard analytical methods, spectroscopic characterization e.g., MS, NMR. Abbreviations used are those conventional in the art.
All starting materials, reagents, catalysts, building blocks, acids, bases, dehydrating agents and solvents utilized to synthesize the compounds of the present disclosure are either commercially available or can be produced by known organic synthesis methods in the art.
Suitable carboxylic acid (1 mmol) was taken in DMF (1-2 mL) and HATU (1-1.2 equivalent) was added followed by stirring for 15 min-1 hour to obtain a reaction mixture. Suitable substituted aliphatic or aromatic amine was added dropwise (1-1.5 equivalent) to the reaction mixture followed by TEA (2.5-3 equivalent) and the contents of the reaction mixture were stirred for another 45 min. Reaction was monitored by checking TLC. Upon completion, the reaction mixture was washed thoroughly with ice cold water to remove DMF and extracted with EtOAc. Column chromatography was performed to get the pure product.
An amide compound (1 mmol) prepared by general procedure A provided in Example 1 was taken in trimethylorthoformate (TMOF) (5-10 equivalent) and heated at 110° C. for 12-18 hrs. Reaction was monitored by checking TLC. Upon completion, the reaction mixture was evaporated in vacuum to remove excess TMOF and washed with water followed by extraction with EtOAc. Column chromatography was performed to get the pure product.
A compound prepared by general procedure B (1 mmol) provided in example 2 was dissolved in methanol (2-5 mL) and a pinch of 10% wet Pd—C was added. The reaction mixture was degassed by passing nitrogen and H2 gas for 2-5 hours to get fully reduced compound. Reaction was thoroughly monitored by checking TLC. Upon completion of the reaction, Pd—C was filtered through celite bed and methanol was evaporated in vacuum to get the desired compound. Column chromatography was performed to get the pure product.
A compound prepared by general procedure C (1 mmol) provided in example 3 was dissolved in dry THF (5-10 mL). 4-nitrophenylchloroformate (1-1.5 equivalent) was added portion wise and reaction mixture was stirred for 15 min-3 hour till the amine got consumed. Reaction was monitored by checking TLC. Further, suitable amine (1-1.5 equivalent) was added to the reaction mixture followed by TEA (2-4 equivalent) and reaction mixture was stirred for another 2-8 hours. Upon completion of the reaction, reaction mass was evaporated in vacuum to remove THF and washed with satd. NaHCO3 solution and extracted with EtOAc. Column chromatography was performed to get the pure product.
A compound prepared by general procedure C (1 mmol) provided in example 3 was taken in dry THF (5-10 mL) and suitable aromatic substituted isocyanate (1-1.5 equivalent) was added followed by TEA (2-4 equivalent). The reaction mixture was stirred for 2-8 hours. Reaction was monitored by checking TLC. Upon completion of the reaction, reaction mass was evaporated in vacuum to remove THF and washed with satd. NaHCO3 solution and extracted with EtOAc. Column chromatography was performed to get the pure product.
Suzuki reaction was performed with suitable halo compound (1 mmol), an aliphatic or aromatic (substituted) boronic acid (1-2 equivalent) in presence of Cs2CO3 or 2M Na2CO3 or 2M K2CO3 (2-4 equivalent) solution taken in a pressure tube and dissolved in dioxane: H2O (9:1) (8 mL). The reaction mixture was purged with Ar-gas for 15 minutes. Pd2(dba)3 or Pd(PPh3)4 (10 mol %) and ligands such as X-phos (20 mol %) were added and the reaction mixture was stirred at 100° C.-110° C. for 10-16 hours. Reaction was monitored by checking TLC. After completion, reaction mass was washed with water and extracted with ethyl acetate and evaporated. Column chromatography was performed to purify the compound.
An uncyclized diamide compound (1 mmol) was taken in DMF (8-10 mL) and ZnCl2 (4-8 equivalent) was added followed by HMDS (8-10 equivalent) and the reaction mixture was heated at 100° C.-110° C. for 30 mins-16 hours. Reaction was monitored by checking TLC. After completion; the reaction mixture was washed with ice cold water and extracted with EtOAc. Column chromatography was performed to purify the compound.
An ester compound (1 mmol) was dissolved in toluene (5-7 mL) and anhydrous AlCl3 (4-8 equivalent) was added under N2 atmosphere to obtain a reaction mixture. Suitable aliphatic or aromatic or substituted aromatic amine (1-2 equivalent) was added to the reaction mixture followed by TEA (0.3 mL, 2.43 mmol) and the reaction mixture was stirred for 25 mins-10 hours at a temperature ranging from room temperature to 100° C.-110° C. The reaction was monitored by checking TLC. Upon completion of the reaction, the reaction mass was washed with water and 0.1 (N) NaOH solution and extracted with EtOAc. Compound was purified by column chromatography.
Synthesis of 2-amino-N-(2-methoxyethyl)-5-nitrobenzamide (2): The compound was prepared by general procedure A provided in example 1 using 2-amino-5-nitrobenzoic acid 1 (4 g, 21.97 mmol), DMF (12 mL), HATU (9.1 g, 24.17 mmol), 2-methoxyethylamine (2.1 mL, 24.17 mmol) and TEA (7.6 mL, 54.93 mmol). After evaporation, the crude mass was diluted with chloroform and pet ether was added to obtain the precipitation. The precipitate was washed with pet ether to afford compound 2 (4.2 g, 80%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.71 (br.s, —NH), 8.46 (d, J=2.8 Hz, 1H), 7.96 (dd, J=9.4 Hz, 2.4 Hz, 1H), 7.71 (br.s, 2H), 6.75 (d, J=9.2 Hz, 1H), 3.43-3.40 (m, 2H), 3.37-3.33 (m, 2H), 3.23 (s, 3H). ESI-HRMS m/z 240.0995 (M+H+). Melting Point: 168° C.
Synthesis of 3-(2-methoxyethyl)-6-nitroquinazolin-4 (3H)-one (3): The compound was prepared by general procedure B provided in example 2 using compound 2 (2 g, 8.63 mmol), (TMOF) (9 mL, 86.3 mmol) to obtain compound 3 (1.8 g, 87%) as pale yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.78 (d, J=2.4 Hz, 1H), 8.50 (dd, J=9 Hz, 2.8 Hz, 1H), 8.45 (s, 1H), 7.82 (d, J=9.2 Hz, 1H), 4.16 (t, J=5.2 Hz, 2H), 3.59 (t, J=4.8 Hz, 2H), 3.21 (s, 3H). ESI-HRMS m/z 250.0834 (M+H+). Melting Point: 152° C.
Synthesis of 6-amino-3-(2-methoxyethyl)quinazolin-4 (3H)-one (4): The compound was prepared by general procedure C provided in example 3 using compound 3 (1 g, 4.01 mmol), methanol (10 mL) to obtain compound 4 (0.8 g, 91%) as brown solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 7.94 (s, 1H), 7.37 (d, J=8.7 Hz, 1H), 7.21 (d, J=2.4 Hz, 1H), 7.07 (dd, J=8.5 Hz, 2.7 Hz, 1H), 5.66 (br.s, 2H), 4.09 (t, J=5.4 Hz, 2H), 3.58 (t, J=5.1 Hz, 2H), 3.24 (s, 3H). ESI-HRMS m/z 220.1095 (M+H+). Melting Point: 172° C.
Synthesis of 1-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-3-phenylurea (5): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.14 g, 0.63 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.16 g, 0.79 mmol), aniline (0.07 mL, 0.79 mmol), TEA (0.4 mL, 2.63 mmol) to obtain compound 5 (0.075 g, 35%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.09 (s, 1H), 8.78 (s, 1H), 8.35 (d, J=2.4 Hz, 1H), 8.17 (s, 1H), 7.79 (dd, J=8.7 Hz, J=2.4 Hz, 1H), 7.62 (d, J=8.7 Hz, 1H), 7.48 (d, J=7.8 Hz, 2H) 7.32-7.27 (m, 2H), 7.01-6.96 (m, 1H), 4.15 (t, J=5.7 Hz, 2H), 3.61 (t, J=5.1 Hz, 2H), 3.25 (s, 3H). ESI-MS m/z 339.0 (M+H+). Melting Point: 218° C.
Synthesis of 1-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-3-(3-methoxyphenyl)urea (6): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.12 g, 0.54 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.137 g, 0.68 mmol), m-anisidine (0.076 mL, 0.68 mmol), TEA (0.3 mL, 2.25 mmol) to obtain compound 6 (0.066 g, 33%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.01 (s, 1H), 8.71 (s, 1H), 8.30 (d, J=2.4 Hz, 1H), 8.10 (d, J=1.2 Hz, 1H), 7.73 (dd, J=8 Hz, J=2.8 Hz, 1H), 7.56 (d, J=8.4 Hz, 1H), 7.17-7.11 (m, 2H) 6.90 (d, J=8.1 Hz, 1H), 6.51 (d, J=10.8 Hz, 1H), 4.09 (t, J=4.8 Hz, 2H), 3.68 (s, 3H), 3.56 (t, J=5.2 Hz, 2H), 3.19 (s, 3H). ESI-MS m/z 369.0 (M+H+). Melting Point: 150° C.
Synthesis of 1-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-3-(3-(methylamino)phenyl)urea (7): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.12 g, 0.54 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.137 g, 0.68 mmol), N1-methylbenzene-1,3-diamine (0.083 g, 0.68 mmol), TEA (0.3 mL, 2.25 mmol) to obtain compound 7 (0.05 g, 25%) as light yellow solid.
Synthesis of 1-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-3-(3-nitrophenyl)urea (8): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.12 g, 0.54 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.137 g, 0.68 mmol), 3-nitroaniline (0.094 g, 0.68 mmol), TEA (0.3 mL, 2.25 mmol) to obtain compound 8 (0.07, 33%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.31 (d, J=16.2 Hz, 2H), 8.57 (s, 1H), 8.37 (d, J=2.1 Hz, 1H), 8.18 (s, 1H), 7.86-7.75 (m, 3H), 7.65-7.55 (m, 2H), 4.16 (t, J=5.1 Hz, 2H), 3.62 (t, J=4.8 Hz, 2H), 3.25 (s, 3H). ESI-MS m/z 384.07 (M+H+). Melting Point: 208° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (9): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.1 g, 0.45 mmol), dry THF (6 mL), 4-nitrophenylchloroformate (0.114 g, 0.57 mmol), 3′-aminoacetophenone (0.077 g, 0.57 mmol), TEA (0.3 mL, 1.88 mmol) to obtain compound 9 (0.08, 46%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.15 (s, 1H), 9.02 (s, 1H), 8.37 (d, J=1.5 Hz, 1H), 8.17 (s, 1H), 8.10 (s, 1H), 7.82 (dd, J=8.7 Hz, J=1.8 Hz, 1H), 7.70 (d, J=7.8 Hz, 1H), 7.64-7.59 (m, 2H), 7.48-7.43 (m, 1H), 4.15 (t, J=4.8 Hz, 2H), 3.61 (t, J=4.8 Hz, 2H), 3.25 (s, 3H), 2.57 (s, 3H). ESI-MS m/z 381.03 (M+H+). Melting Point: 210° C.
Synthesis of 1-(4-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (10): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.12 g, 0.54 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.137 g, 0.68 mmol), 4′-aminoacetophenone (0.092 g, 0.68 mmol), TEA (0.3 mL, 2.25 mmol) to obtain compound 10 (0.08, 46%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.26 (s, 1H), 9.24 (s, 1H), 8.37 (d, J=2.4 Hz, 1H), 8.17 (s, 1H), 7.92 (s, 1H), 7.89 (s, 1H), 7.81 (dd, J=8.8 Hz, 2.7 Hz, 1H), 7.64-7.60 (m, 3H), 4.15 (t, J=5.1 Hz, 2H), 3.61 (t, J=5.1 Hz, 2H), 3.24 (s, 3H), 2.51 (s, 3H). ESI-MS m/z 381.07 (M+H+). Melting Point: 202° C.
Synthesis of 1-(3-(1-hydroxyethyl)phenyl)-3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (11): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.12 g, 0.54 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.137 g, 0.68 mmol), 1-(3-aminophenyl)ethanol (0.093 g, 0.68 mmol), TEA (0.3 mL, 2.25 mmol) to obtain compound 11 (0.08 g, 38%) as light yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.02 (s, 1H), 8.73 (s, 1H), 8.34 (d, J=2.4 Hz, 1H), 8.14 (s, 1H), 7.77 (dd, J=9 Hz, 2.4 Hz, 1H), 7.59 (d, J=9 Hz, 1H), 7.44 (s, 1H), 7.32 (d, J=7.8 Hz, 1H), 7.23-7.17 (m, 1H), 6.94 (d, J=7.5 Hz, 1H), 5.14 (d, J=3.9 Hz, 1H), 4.70-4.62 (m, 1H), 4.13 (t, J=5.1 Hz, 2H), 3.59 (t, J=4.8 Hz, 2H), 3.23 (s, 3H), 1.29 (d, J=6.6 Hz, 3H). ESI-MS m/z 383.02 (M+H+). Melting Point: 168° C.
Synthesis of methyl 4-methoxy-3-(3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)ureido)benzoate (12): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.13 g, 0.593 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.15 mg, 0.74 mmol), methyl 3-amino-4-methoxybenzoate (0.134 g, 0.74 mmol), TEA (0.3 mL, 2.44 mmol) to obtain compound 12 (0.08 g, 32%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.75 (s, 1H), 8.84 (s, 1H), 8.43 (s, 1H), 8.39 (d, J=2.1 Hz, 1H), 8.17 (s, 1H), 7.77 (dd, J=8.7 Hz, 2.1 Hz, 1H), 7.66-7.61 (m, 2H), 7.15 (d, J=8.4 Hz, 1H), 4.16 (t, J=4.8 Hz, 2H), 3.98 (s, 3H), 3.83 (s, 3H) 3.62 (t, J=4.8 Hz, 2H), 3.25 (s, 3H). ESI-MS m/z 427.1 (M+H+). Melting Point: 224° C.
Synthesis of 1-(3-ethylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (13): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.12 g, 0.54 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.137 g, 0.68 mmol), 3-ethylaniline (0.085 g, 0.68 mmol), TEA (0.3 mL, 2.25 mmol) to obtain compound 13 (0.07 g, 35%) as off white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.00 (s, 1H), 8.63 (s, 1H), 8.30 (d, J=2.4 Hz, 1H), 8.10 (s, 1H), 7.73 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.56 (d, J=8.8 Hz, 1H), 7.30-7.29 (m, 1H), 7.21 (d, J=8.8 Hz, 1H), 7.15-7.12 (m, 1H), 6.78 (d, J=7.6 Hz, 1H), 4.09 (t, J=5.2 Hz, 2H), 3.56 (t, J=5.32 Hz, 2H), 3.19 (s, 3H), 2.52 (q, J=Hz, 2H), 1.12 (t, J=Hz, 3H). ESI-MS m/z 367.3 (M+H+). Melting Point: 182° C.
Synthesis of 1-(3-benzoylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (14): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.12 g, 0.54 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.137 g, 0.68 mmol), (3-aminophenyl)(phenyl)methanone (0.135 g, 0.68 mmol), TEA (0.3 mL, 2.25 mmol) to obtain compound 14 (0.065 g, 27%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.17 (s, 1H), 9.10 (s, 1H), 8.34 (d, J=2.4 Hz, 1H), 8.16 (s, 1H), 7.93 (s, 1H), 7.82-7.77 (m, 4H), 7.68 (d, J=7.2 Hz, 1H), 7.63-7.55 (m, 3H), 7.52-7.46 (m, 1H), 7.35 (d, J=7.8 Hz, 1H), 4.15 (t, J=5.1 Hz, 2H), 3.61 (t, J=5.1 Hz, 2H), 3.24 (s, 3H). ESI-MS m/z 443.2 (M+H+). Melting Point: 136° C.
Synthesis of N-cyclohexyl-3-(3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)ureido)benzamide (15): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.12 g, 0.54 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.137 g, 0.68 mmol), 3-amino-N-cyclohexylbenzamide (0.15 g, 0.68 mmol), TEA (0.3 mL, 2.25 mmol) to obtain compound 15 (0.085 g, 33%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.12 (s, 1H), 8.94 (s, 1H), 8.37 (d, J=2.4 Hz, 1H), 8.20 (s, 1H), 8.17 (s, 1H), 7.85 (s, 1H), 7.81 (dd, J=9 Hz, 2.7 Hz, 1H), 7.66-7.61 (m, 2H), 7.44 (d, J=7.8 Hz, 1H), 7.38-7.33 (m, 1H), 4.15 (t, J=5.1 Hz, 2H), 3.77-3.70 (m, 1H), 3.61 (t, J=5.1 Hz, 2H), 3.25 (s, 3H), 1.83-1.79 (m, 2H), 1.75-1.71 (m, 2H), 1.64-1.57 (m, 1H), 1.38-1.23 (m, 4H), 1.17-1.1 (m, 1H). ESI-MS m/z 464.2 (M+H+). Melting Point: 206° C.
Synthesis of methyl 2-(3-(3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)ureido)benzamido)-3-methylbutanoate (16): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.12 g, 0.54 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.137 g, 0.68 mmol), methyl 2-(3-aminobenzamido)-3-methylbutanoate (0.17 g, 0.68 mmol), TEA (0.3 mL, 2.25 mmol) to obtain compound 16 (0.08 g, 30%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.14 (s, 1H), 8.94 (s, 1H), 8.60 (d, J=7.8 Hz, 1H), 8.36 (d, J=2.1 Hz, 1H), 8.17 (s, 1H), 7.86 (s, 1H), 7.81 (dd, J=9 Hz, 2.1 Hz, 1H), 7.71 (d, J=8.1 Hz, 1H), 7.62 (d, J=8.7 Hz, 1H), 7.50 (d, J=7.8 Hz, 1H), 7.42-7.37 (m, 1H), 4.29 (t, J=7.5 Hz, 1H), 4.15 (t, J=4.8 Hz, 2H), 3.66 (s, 3H), 3.61 (t, J=5.1 Hz, 2H), 3.25 (s, 3H), 2.24-2.13 (m, 1H), 0.965 (q, J=6.9 Hz, 6H). ESI-HRMS m/z 496.2181 (M+H+). Melting Point: 198° C.
Synthesis of 3-(3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)ureido)-N,N-dimethylbenzamide (17): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.06 g, 0.27 mmol), dry THF (6 mL), 4-nitrophenylchloroformate (0.069 g, 0.34 mmol), 3-amino-N,N-dimethylbenzamide (0.056 g, 0.34 mmol), TEA (0.1 mL, 1.12 mmol) to obtain compound 17 (0.056 g, 50%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.32 (d, J=18.3 Hz, 1H), 9.02 (d, J=18.3 Hz, 1H), 8.36 (s, 1H), 8.17 (s, 1H), 7.86-7.79 (m, 1H), 7.65-7.57 (m, 2H), 7.47-7.30 (m, 2H), 7.00-6.96 (m, 1H), 4.15 (t, J=4.5 Hz, 2H), 3.61 (t, J=4.8 Hz, 2H), 3.25 (s, 3H), 2.98 (s, 3H), 2.92 (s, 3H). ESI-HRMS m/z 410.1837 (M+H+). Melting Point: 182° C.
Synthesis of 1-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-3-(3-(pyrrolidine-1-carbonyl)phenyl)urea (18): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.12 g, 0.54 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.137 mg, 0.68 mmol), (3-aminophenyl)(pyrrolidin-1-yl)methanone (0.13 g, 0.68 mmol), TEA (0.3 mL, 2.25 mmol) to obtain compound 18 (0.052 g, 22%) as light yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.19 (s, 1H), 8.95 (s, 1H), 8.36 (d, J=2.1 Hz, 1H), 8.17 (s, 1H), 7.80 (dd, J=9 Hz, 2.4 Hz, 1H), 7.70 (s, 1H), 7.62 (d, J=8.7 Hz, 1H), 7.47-7.41 (m, 1H), 7.37-7.33 (m, 1H), 7.12-7.08 (m, 1H), 4.15 (t, J=4.2 Hz, 2H), 3.61 (t, J=4.8 Hz, 2H), 3.46 (t, J=4.8 Hz, 4H), 3.25 (s, 3H), 1.89-1.79 (m, 4H). ESI-MS m/z 435.8 (M+H+). Melting Point: 198° C.
Synthesis of 1-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-3-(3-(morpholine-4-carbonyl)phenyl)urea (19): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.10 g, 0.45 mmol), dry THF (8 mL) and 4-nitrophenylchloroformate (0.137 g, 0.68 mmol), (3-aminophenyl)(morpholino)methanone (0.12 g, 0.54 mmol), TEA (0.3 mL, 1.88 mmol) to obtain compound 19 (0.05 g, 24%) as light yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.09 (s, 1H), 8.86 (s, 1H), 8.30 (s, 1H), 8.10 (s, 1H), 7.74 (d, J=8.8 Hz, 1H), 7.55-7.51 (m, 2H), 7.43-7.37 (m, 1H), 7.32-7.29 (m, 1H), 6.96-6.92 (m, 1H), 4.09 (t, J=4.4 Hz, 2H), 3.58-3.53 (m, 8H), 3.34-3.31 (m, 2H), 3.19 (s, 3H). ESI-MS m/z 452.3 (M+H+). Melting Point: 102° C.
Synthesis of 1-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-3-(4-(pyrrolidine-1-carbonyl)phenyl)urea (20): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.12 g, 0.54 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.137 g, 0.68 mmol), (4-aminophenyl)(pyrrolidin-1-yl)methanone (0.13 g, 0.68 mmol), TEA (0.3 mL, 2.25 mmol) to obtain compound 20 (0.075 g, 31%) as light yellow solid. H NMR (300 MHz, d6-DMSO) δ in ppm 9.21 (d, J=14.4 Hz, 1H), 8.99 (d, J=12.9 Hz, 1H), 8.37 (d, J=2.4 Hz, 1H), 8.17 (s, 1H), 7.80 (dd, J=9 Hz, 2.7 Hz, 1H), 7.62 (d, J=8.7 Hz, 1H), 7.55-7.47 (m, 4H), 4.15 (t, J=4.5 Hz, 2H), 3.61 (t, J=4.8 Hz, 2H), 3.47-3.42 (m, 4H) 3.25 (s, 3H), 1.88-1.79 (m, 4H). ESI-HRMS m/z 436.1987 (M+H+). Melting Point: 202° C.
Synthesis of 1-(3-(benzo[d]oxazol-2-yl)phenyl)-3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (21): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.08 g, 0.36 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.092 g, 0.45 mmol), 3-(benzo[d]oxazol-2-yl)aniline (0.096 g, 0.45 mmol), TEA (0.2 mL, 1.50 mmol) to obtain compound 21 (0.042 g, 25%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.06 (s, 1H), 8.81 (s, 1H), 8.33 (s, 1H), 8.16 (s, 1H), 7.76 (dd, J=8.8 Hz, 2.7 Hz, 1H), 7.60 (d, J=8.7 Hz, 1H), 7.46 (d, J=8.4 Hz, 1H), 7.33-7.29 (m, 4H), 7.26-7.20 (m, 2H), 6.87 (d, J=7.8 Hz, 1H), 4.15 (t, J=4.8 Hz, 2H), 3.61 (t, J=5.1 Hz, 2H), 3.25 (s, 3H). Melting Point: 176° C.
Synthesis of N-(3-(3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)ureido)phenyl)acetamide (22): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.13 g, 0.59 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.149 g, 0.74 mmol), N-(3-aminophenyl)acetamide (0.111 g, 0.74 mmol), TEA (0.4 mL, 2.44 mmol) to obtain compound 22 (0.05 g, 21%) as light yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.93 (s, 1H), 9.09 (s, 1H), 8.89 (s, 1H), 8.38 (d, J=2.4 Hz, 1H), 8.16 (s, 1H), 7.80-7.75 (m, 2H), 7.61 (d, J=8.7 Hz, 1H), 7.24-7.16 (m, 3H), 4.15 (t, J=5.1 Hz, 2H), 3.61 (t, J=5.1 Hz, 2H), 3.25 (s, 3H), 2.04 (s, 3H). ESI-HRMS m/z 396.1689 (M+H+). Melting Point: 162° C.
Synthesis of N-(3-(3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)ureido)phenyl)-N-methylacetamide (23): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.130 g, 0.59 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.149 g, 0.74 mmol), N-(3-aminophenyl)-N-methylacetamide (0.12 g, 0.74 mmol), TEA (0.4 mL, 2.44 mmol) to obtain compound 23 (0.052 g, 21%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.25 (s, 1H), 8.96 (s, 1H), 8.36 (s, 1H), 8.17 (s, 1H), 7.80 (d, J=9.3 Hz, 1H), 7.62 (d, J=8.4 Hz, 1H), 7.51 (s, 1H), 7.37-7.32 (m, 2H), 6.94 (s, 1H), 4.15 (t, J=4.5 Hz, 2H), 3.61 (t, J=4.8 Hz, 2H), 3.25 (s, 3H), 3.15 (s, 3H), 1.80 (s, 3H). ESI-MS m/z 410.4 (M+H+). Melting Point: 98° C.
Synthesis of N-benzyl-N-(3-(3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)ureido)phenyl)acetamide (24): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.15 g, 0.68 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.172 g, 0.85 mmol), N-(3-aminophenyl)-N-benzylacetamide (0.20 g, 0.85 mmol), TEA (0.4 mL, 2.82 mmol) to obtain compound 24 (0.08 g, 24%) as light yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.13 (s, 1H), 8.86 (s, 1H), 8.34 (s, 1H), 8.16 (s, 1H), 7.77 (dd, J=8.7 Hz, 1.5 Hz, 1H), 7.61 (d, J=8.7 Hz, 1H), 7.38-7.35 (m, 2H), 7.31-7.27 (m, 3H), 7.24-7.19 (m, 3H), 6.80 (d, J=7.5 Hz, 1H), 4.85 (s, 2H), 4.15 (t, J=4.8 Hz, 2H), 3.61 (t, J=4.8 Hz, 2H), 3.24 (s, 3H), 1.88 (s, 3H). ESI-MS m/z 486.2 (M+H+). Melting Point: 184° C.
Synthesis of N-(3-(3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)ureido)benzyl)acetamide (25): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.10 g, 0.45 mmol), dry THF (6 mL), 4-nitrophenylchloroformate (0.137 g, 0.68 mmol), N-(3-aminobenzyl)acetamide (0.089 g, 0.54 mmol), TEA (0.3 mL, 1.88 mmol) to obtain compound 25 (0.08 g, 43%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.08 (s, 1H), 8.81 (s, 1H), 8.37 (s, 1H), 8.36 (s, 1H), 8.16 (s, 1H), 7.78 (dd, J=8.7 Hz, J=1.2 Hz, 1H), 7.61 (d, J=8.7 Hz, 1H), 7.39-7.31 (m, 2H), 7.26-7.21 (m, 1H), 6.88 (d, J=7.5 Hz, 1H), 4.17 (t, J=4.8 Hz, 2H), 4.15 (t, J=4.8 Hz, 2H), 3.61 (t, J=4.8 Hz, 2H), 3.25 (s, 3H), 1.88 (s, 3H). ESI-HRMS m/z 410.1827 (M+H+). Melting Point: 202° C.
Synthesis of N-(3-(3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)ureido)benzyl)-N-methylacetamide (26): The compound was prepare by general procedure D provided in example 4 using compound 4 (0.10 g, 0.45 mmol), dry THF (6 mL) and 4-nitrophenylchloroformate (0.114 g, 0.68 mmol), N-(3-aminobenzyl)-N-methylacetamide (0.097 g, 0.54 mmol), TEA (0.3 mL, 1.88 mmol) to obtain compound 26 (0.075 g, 39%) as off white solid.
Synthesis of 3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-1,1-dimethylurea (27): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.10 g, 0.45 mmol), dry THF (6 mL), 4-nitrophenylchloroformate (0.114 g, 0.57 mmol), dimethylamine 2M in THF solution (1.1 mL, 0.57 mmol), TEA (0.3 mL, 1.88 mmol) to obtain compound 27 (0.047 g, 35%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.86 (s, 1H), 8.29 (d, J=2.1 Hz, 1H), 8.13 (s, 1H), 7.97 (dd, J=8.8 Hz, 2.1 Hz, 1H), 7.55 (d, J=9 Hz, 1H), 4.13 (t, J=5.1 Hz, 2H), 3.60 (t, J=5.1 Hz, 2H), 3.24 (s, 3H), 2.95 (s, 6H). ESI-MS m/z 291.2 (M+H+). Melting Point: 182° C.
Synthesis of N-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)piperidine-1-carboxamide (28): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.08 g, 0.36 mmol), dry THF (6 mL), 4-nitrophenylchloroformate (0.092 g, 0.45 mmol), piperidine (0.04 mL, 0.45 mmol), TEA (0.2 mL, 1.50 mmol) to obtain compound 28 (0.052 g, 43%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.84 (s, 1H), 8.27 (d, J=2.1 Hz, 1H), 8.13 (s, 1H), 7.95 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.55 (d, J=9 Hz, 1H), 4.13 (t, J=5.1 Hz, 2H), 3.60 (t, J=5.1 Hz, 2H), 3.44 (t, J=4.2 Hz, 4H), 3.24 (s, 3H), 1.61-1.56 (m, 2H), 1.51-1.48 (m, 4H). ESI-HRMS m/z 331.1769 (M+H+). Melting Point: 162° C.
Synthesis of 1-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-3-(1-methylpiperidin-4-yl)urea (29): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.10 g, 0.45 mmol), dry THF (6 mL), 4-nitrophenylchloroformate (0.114 g, 0.57 mmol), 4-amino-1-methylpiperidine (0.07 mL, 0.57 mmol), TEA (0.3 mL, 1.88 mmol) to obtain compound 29 (0.072, 44%)) as light yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.94 (s, 1H), 8.26 (d, J=2.1 Hz, 1H), 8.11 (s, 1H), 7.73 (dd, J=8.7 Hz, 2.1 Hz, 1H), 7.54 (d, J=8.7 Hz, 1H), 6.45 (d, J=7.5 Hz, 1H), 4.14-4.07 (m, 3H), 3.60 (t, J=5.1 Hz, 2H), 3.24 (s, 3H), 2.66-2.63 (m, 2H), 2.15 (s, 3H), 2.02-1.96 (m, 2H), 1.80-1.76 (m, 2H), 1.46-1.35 (m, 2H). ESI-HRMS m/z 360.2032 (M+H+). Melting Point: 142° C.
Synthesis of 4-benzyl-N-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)piperidine-1-carboxamide (30): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.10 g, 0.45 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.114 g, 0.57 mmol), 4-benzylpiperidine (0.1 mL, 0.57 mmol), TEA (0.3 mL, 1.88 mmol) to obtain compound 30 (0.066 g, 35%) as light yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.85 (s, 1H), 8.27 (d, J=1.2 Hz, 1H), 8.13 (s, 1H), 7.94 (dd, J=8.7 Hz, 1.5 Hz, 1H), 7.55 (d, J=8.7 Hz, 1H), 7.31-7.26 (m, 2H), 7.19-7.17 (m, 3H), 4.15-4.10 (m, 4H), 3.60 (t, J=4.5 Hz, 2H), 3.24 (s, 3H), 2.78-2.70 (m, 2H), 2.54-2.52 (m, 2H), 1.76-1.70 (m, 1H), 1.61-1.57 (m, 2H), 1.19-1.06 (m, 2H). ESI-HRMS m/z 421.2242 (M+H+). Melting Point: 146° C.
Synthesis of 1-(1-benzylpiperidin-4-yl)-3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (31): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.10 g, 0.45 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.114 g, 0.57 mmol), 4-amino-1-benzylpiperidine (0.1 mL, 0.57 mmol), TEA (0.3 mL, 1.88 mmol) to obtain compound 31 (0.059 g, 30%) as light brown solid. ESI-HRMS m/z 436.2340 (M+H+).
Synthesis of 1-(5-acetyl-2-hydroxyphenyl)-3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (32): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.1 g, 0.45 mmol), THF (6 mL), 4-nitrophenylchloroformate (0.114 g, 0.57 mmol), 1-(3-amino-4-hydroxyphenyl)ethanone (0.103 g, 0.68 mmol), TEA (0.3 mL, 1.88 mmol) to obtain compound 32 (0.0, 38%) as off white solid.
Synthesis of 1-(3-acetyl-5-chloro-2-hydroxyphenyl)-3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (33): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.1 g, 0.45 mmol), dry THF (6 mL), 4-nitrophenylchloroformate (0.114 g, 0.57 mmol), 1-(3-amino-5-chloro-2-hydroxyphenyl)ethanone (0.126 g, 0.68 mmol), TEA (0.3 mL, 1.88 mmol) to obtain compound 33 (0.065, 33%) as white solid.
Synthesis of 1-(3-acetyl-2-hydroxy-5-methylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (34): The compound was prepared by general procedure D provided in example 4 using compound 4 (0.1 g, 0.45 mmol), dry THF (6 mL), 4-nitrophenylchloroformate (0.114 g, 0.57 mmol), 1-(3-amino-2-hydroxy-5-methylphenyl)ethanone (0.113 g, 0.68 mmol), TEA (0.3 mL, 1.88 mmol) to obtain compound 34 (0.07, 37%) as yellow solid.
Synthesis of 1-(4-fluorophenyl)-3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (35): The compound was prepared by general procedure E provided in example 5 using compound 4 (0.085 g, 0.38 mmol), dry THF (5 mL), 4-fluorophenyl isocyanate (0.05 mL, 0.48 mmol), TEA (0.1 mL, 0.81 mmol) to obtain compound 35 (0.056 g, 43%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.04 (s, 1H), 8.75 (s, 1H), 8.29 (d, J=2.4 Hz, 1H), 8.11 (s, 1H), 7.75 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.56 (d, J=8.8 Hz, 1H), 7.46-7.42 (m, 2H), 7.11-7.06 (m, 2H), 4.10 (t, J=5.2 Hz, 2H), 3.56 (t, J=5.2 Hz, 2H), 3.20 (s, 3H). ESI-MS m/z 357.03 (M+H+). Melting Point: 206° C.
Synthesis of 1-(3-chloro-4-fluorophenyl)-3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (36): The compound was prepared by general procedure E provided in example 5 using compound 4 (0.10 g, 0.45 mmol), dry THF (5 mL), 3-chloro-4-fluorophenyl isocyanate (0.085 mL, 0.68 mmol), TEA (0.2 mL, 1.36 mmol) to obtain compound 36 (0.052 g, 29%) as white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.19 (s, 1H), 8.98 (s, 1H), 8.34 (d, J=2.4 Hz, 1H), 8.17 (s, 1H), 7.82-7.78 (m, 2H), 7.62 (d, J=9 Hz, 1H), 7.36-7.33 (m, 2H), 4.15 (t, J=5.1 Hz, 2H), 3.61 (t, J=4.8 Hz, 2H), 3.24 (s, 3H). ESI-MS m/z 390.99 (M+H+). Melting Point: 202° C.
Synthesis of 1-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-3-(4-(trifluoromethoxy)phenyl)urea (37): The compound was prepared by general procedure E provided in example 5 using compound 4 (0.10 g, 0.45 mmol), dry THF (6 mL), 4-(trifluoromethoxy)phenyl isocyanate (0.1 mL, 0.68 mmol), TEA (0.2 mL, 1.36 mmol) to obtain compound 37 (0.06 g, 31%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.31 (s, 1H), 9.15 (s, 1H), 8.35 (d, J=2.4 Hz, 1H), 8.17 (s, 1H), 7.81 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.63-7.57 (m, 3H), 7.31 (s, 1H), 7.29 (s, 1H), 4.15 (t, J=4.8 Hz, 2H), 3.61 (t, J=4.8 Hz, 2H), 3.24 (s, 3H). ESI-MS m/z 423.0 (M+H+). Melting Point: 182° C.
Synthesis of 1-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-3-(4-(trifluoromethyl)phenyl)urea (38): The compound was prepared by general procedure E provided in example 5 using compound 4 (0.10 g, 0.45 mmol), dry THF (5 mL), 4-(trifluoromethyl)phenyl isocyanate (0.1 mL, 0.68 mmol), TEA (0.2 mL, 1.36 mmol) to obtain compound 38 (0.07 g, 38%) as white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.25 (d, J=6.3 Hz, 2H), 8.37 (d, J=2.4 Hz, 1H), 8.18 (s, 1H), 7.82 (dd, J=9 Hz, 2.4 Hz, 1H), 7.71-7.62 (m, 5H), 4.15 (t, J=5.1 Hz, 2H), 3.61 (t, J=5.1 Hz, 2H), 3.25 (s, 3H). ESI-MS m/z 407.2 (M+H+). Melting Point: 196° C.
Synthesis of 1-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-3-(2-(trifluoromethyl)phenyl)urea (39): The compound was prepared by general procedure E provided in example 5 using compound 4 (0.10 g, 0.45 mmol), dry THF (5 mL), 2-(Trifluoromethyl)phenyl isocyanate (0.1 mL, 0.68 mmol), TEA (0.2 mL, 1.36 mmol) to obtain compound 39 (0.066 g, 37%) as white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.79 (s, 1H), 8.38 (d, J=2.4 Hz, 1H), 8.19 (s, 1H), 8.17 (s, 1H), 7.96 (d, J=8.1 Hz, 1H), 7.78 (dd, J=9 Hz, J=2.7 Hz, 1H), 7.71-7.62 (m, 3H), 7.33-7.28 (m, 1H), 4.15 (t, J=5.1 Hz, 2H), 3.61 (t, J=5.1 Hz, 2H), 3.25 (s, 3H). ESI-MS m/z 407.2 (M+H+). Melting Point: 220° C.
Synthesis of 1-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-3-(4-methoxyphenyl)urea (40): The compound was prepared by general procedure E provided in example 5 using compound 4 (0.10 g, 0.45 mmol), dry THF (5 mL), 4-methoxyphenylisocyanate (0.083 mL, 0.68 mmol), TEA (0.2 mL, 1.36 mmol) to obtain compound 40 (0.072 g, 43%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.02 (s, 1H), 8.58 (s, 1H), 8.33 (s, 1H), 8.15 (s, 1H), 7.79 (dd, J=8.7 Hz, J=1.2 Hz, 1H), 7.60 (d, J=9 Hz, 1H), 7.38 (d, J=8.7 Hz, 2H), 6.88 (d, J=8.7 Hz, 2H), 4.14 (t, J=5.1 Hz, 2H), 3.72 (s, 3H), 3.61 (t, J=5.1 Hz, 2H), 3.24 (s, 3H). ESI-HRMS m/z 369.1 (M+H+). Melting Point: 206° C.
Synthesis of 1-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-3-(2-methoxyphenyl)urea (41): The compound was prepared by general procedure E provided in example 5 using compound 4 (0.10 g, 0.45 mmol), dry THF (5 mL), 2-methoxyphenylisocyanate (0.083 mL, 0.68 mmol), TEA (0.2 mL, 1.36 mmol) to obtain compound 41 (0.070 g, 41%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.71 (s, 1H), 8.37 (d, J=2.4 Hz, 1H), 8.29 (s, 1H), 8.17 (s, 1H), 8.14 (d, J=1.8 Hz, 1H), 7.76 (dd, J=8.8 Hz, 2.7 Hz, 1H), 7.62 (d, J=8.7 Hz, 1H), 7.05-6.88 (m, 3H), 4.15 (t, J=4.8 Hz, 2H), 3.89 (s, 3H), 3.61 (t, J=5.1 Hz, 2H), 3.25 (s, 3H). ESI-MS m/z 369.3 (M+H+). Melting Point: 192° C.
Synthesis of ethyl 3-(3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)ureido)benzoate (42): The compound was prepared by general procedure E provided in example 5 using compound 4 (0.12 g, 0.54 mmol), dry THF (8 mL), 3-(Ethoxycarbonyl)phenyl isocyanate (0.1 mL, 0.65 mmol), TEA (0.2 mL, 1.36 mmol) to obtain compound 41 (0.080 g, 36%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.14 (s, 1H), 9.07 (s, 1H), 8.36 (s, 1H), 8.17 (s, 2H), 7.81 (d, J=7.2 Hz, 1H), 7.69-7.57 (m, 3H), 7.46-7.41 (m, 1H), 4.32 (q, J=4.5 Hz, 2H), 4.15 (t, J=4.8 Hz, 2H), 3.61 (t, J=5.1 Hz, 2H), 3.25 (s, 3H), 1.31 (t, J=7.2 Hz, 3H). ESI-MS m/z 411.2 (M+H+). Melting Point: 170° C.
Synthesis of 6-amino-5-bromo-3-(2-methoxyethyl) quinazolin-4 (3H)-one (43): Compound 4 (2 g, 6.73 mmol) was dissolved in acetic acid (15 mL). Liq. bromine (0.56 mL, 1.2 equiv.) was added in DCM (4 mL) and then the resultant solution was added dropwise at 0° C. to the reaction mixture over a period for 15 minutes. Then, the reaction mixture was allowed to stir at room temperature for 3 hrs. After completion of reaction, it was worked up with ethyl acetate and aqueous NaHCO3 soln. Then, it was purified by column chromatography (Silica gel, mesh size 100-200) eluting (70% EtOAc/Pet ether) to obtain compound 43 as light brown solid at 66% yield. 1H NMR (400 MHz, CDCl3) δ in ppm 7.90 (s, 1H), 7.48 (d, J=8.8 Hz, 1H), 7.15 (d, J=8.8 Hz, 1H), 4.56 (s, 2H), 4.11 (t, J=4.4 Hz, 2H), 3.65 (t, J=4.4 Hz, 2H), 3.30 (s, 3H). ESI-HRMS m/z 298.0184 (M+H+). Melting Point: 132° C.
Synthesis of 6-amino-3-(2-methoxyethyl)-5-phenylquinazolin-4 (3H)-one (44): The compound was prepared by general procedure F provided in example 6 using compound 43 (0.20 g, 0.67 mmol), benzene boronic acid (0.099 g, 0.80 mmol), cesium carbonate (0.44 g, 1.34 mmol), solution of dioxane, H2O (9:1) (10 mL), Pd2(dba)3 (0.061 g, 0.06 mmol), X-phos (0.064 g, 0.13 mmol) to obtain compound 44 (0.125 g, 63%) as light yellow solid. 1H NMR (400 MHz, CDCl3) δ in ppm 7.88 (s, 1H), 7.55 (d, J=8.8 Hz, 1H), 7.48-7.45 (m, 2H), 7.40-7.36 (m, 1H), 7.24-7.22 (m, 2H), 7.15 (d, J=8.4 Hz, 1H), 3.97 (t, J=4.4 Hz, 2H), 3.68 (br.s., 2H), 3.49 (t, J=4.4 Hz, 2H), 3.26 (s, 3H). ESI-HRMS m/z 296.1410 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-5-phenyl-3,4-dihydroquinazolin-6-yl)urea (45): The compound was prepared by general procedure D provided in example 4 using compound 44 (0.10 g, 0.33 mmol), dry THF (5 mL), 4-nitrophenylchloroformate (0.096 g, 0.47 mmol), 3′-aminoacetophenone (0.051 g, 0.37 mmol), TEA (0.19 mL, 1.35 mmol) to obtain compound 45 (0.07 g, 45%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.35 (s, 1H), 8.32 (d, J=8.8 Hz, 1H), 8.13 (s, 1H), 7.88 (s, 1H), 7.62 (d, J=8.8 Hz, 1H), 7.57 (d, J=8.4 Hz, 1H), 7.50 (d, J=7.6 Hz, 1H), 7.42-7.32 (m, 5H), 7.12 (s, 1H), 7.11 (s, 1H), 3.90 (t, J=5.2 Hz, 2H), 3.39 (t, J=5.2 Hz, 2H), 3.16 (s, 3H), 2.48 (s, 3H). ESI-HRMS m/z 457.1876 (M+H+).
Synthesis of 6-amino-5-(4-fluorophenyl)-3-(2-methoxyethyl)quinazolin-4 (3H)-one (46): The compound was prepared by general procedure F provided in example 6 using compound 43 (0.20 g, 0.67 mmol), 4-fluorophenylboronic acid (0.112 g, 0.80 mmol), cesium carbonate (0.437 g, 1.34 mmol), dioxane/H2O (9:1) (10 mL), Pd2(dba)3 (0.062 g, 0.06 mmol), X-phos (0.064 g, 0.13 mmol) to obtain compound 46 (0.094 g, 45%) as yellow solid. 1H NMR (600 MHz, d6-DMSO) δ in ppm 7.93 (s, 1H), 7.42 (d, J=9 Hz, 1H), 7.23 (d, J=9 Hz, 1H), 7.19-7.16 (m, 2H), 7.12-7.09 (m, 2H), 4.77 (s, 2H), 3.89 (t, J=4.8 Hz, 2H), 3.41 (t, J=2.7 Hz, 2H), 3.19 (s, 3H). ESI-MS m/z 314.1 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(5-(4-fluorophenyl)-3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (47): The compound was prepared by general procedure D provided in example 4 using compound 46 (0.08 g, 0.25 mmol), dry THF (4 mL), 4-nitrophenylchloroformate (0.072 g, 0.35 mmol), 3′-aminoacetophenone (0.037 g, 0.28 mmol), TEA (0.17 mL, 1.22 mmol) to obtain compound 47 (0.049 g, 41%) as yellow solid. 1H NMR (300 MHz, CDCl3) δ in ppm 8.66 (d, J=9 Hz, 1H), 8.02 (s, 1H), 7.77 (d, J=9 Hz, 1H), 7.71-7.67 (m, 2H), 7.46-7.43 (m, 1H), 7.37-7.32 (m, 1H), 7.13-7.09 (m, 2H), 7.05-6.99 (m, 2H), 6.86 (s, 1H), 6.43 (s, 1H), 3.99 (t, J=4.5 Hz, 2H), 3.50 (t, J=4.8 Hz, 2H), 3.28 (s, 3H), 2.58 (s, 3H). ESI-MS m/z 475.1 (M+H+).
Synthesis of 6-amino-3-(2-methoxyethyl)-5-(pyridin-2-yl)quinazolin-4 (3H)-one (48): The compound was prepared by general procedure F provided in example 6 using compound 43 (0.20 g, 0.67 mmol), 2-pyridineboronic acid (0.98 g, 0.80 mmol), cesium carbonate (0.437 g, 1.34 mmol) dioxane/H2O (9:1) (10 mL), Pd2(dba)3 (0.062 g, 0.06 mmol), X-phos (0.064 g, 0.13 mmol) to obtain compound 48 (0.103 g, 52%) as yellow solid.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-5-(pyridin-2-yl)-3,4-dihydroquinazolin-6-yl)urea (49): The compound was prepared by general procedure D provided in example 4 using compound 48 (0.08 g, 0.25 mmol), dry THF, 4-nitrophenylchloroformate (0.072 g, 0.35 mmol). 3′-aminoacetophenone (0.037 g, 0.28 mmol), TEA (0.17 mL, 1.22 mmol) to obtain compound 49 (0.046 g, 38%) as yellow solid.
Synthesis of 6-amino-3-(2-methoxyethyl)-5-(pyridin-3-yl)quinazolin-4 (3H)-one (50): The compound was prepared by general procedure F provided in example 6 using compound 43 (0.20 g, 0.67 mmol), 3-pyridineboronic acid (0.98 g, 0.80 mmol), cesium carbonate (0.437 g, 1.34 mmol), dioxane/H2O (9:1) (10 mL), Pd2(dba)3 (0.062 g, 0.06 mmol), (0.064 g, 0.13 mmol) to obtain compound 50 (0.115 g, 58%) as yellow solid. 1H NMR (600 MHz, d6-DMSO) δ in ppm 8.49 (dd, J=3.6 Hz, 1.2 Hz, 1H), 8.27 (d, J=1.8 Hz, 1H), 7.95 (s, 1H), 7.54-7.52 (m, 1H), 7.46 (d, J=9 Hz, 1H), 7.40-7.38 (m, 1H), 7.26 (d, J=9 Hz, 1H), 4.91 (s, 2H), 3.90 (t, J=4.8 Hz, 2H), 3.42 (t, J=5.4 Hz, 2H), 3.18 (s, 3H). ESI-MS m/z 297.2 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-5-(pyridin-3-yl)-3,4-dihydroquinazolin-6-yl)urea (51): The compound was prepared by general procedure D provided in example 4 using compound 50 (0.08 g, 0.25 mmol), dry THF (4 mL) and 4-nitrophenylchloroformate (0.072 g, 0.35 mmol), 3′-aminoacetophenone (0.037 g, 0.28 mmol), TEA (0.17 mL, 1.22 mmol) to obtain compound 51 (0.046 g, 38%) as yellow solid. ESI-MS m/z 458.3 (M+H+).
Synthesis of 6-amino-3-(2-methoxyethyl)-5-(pyridin-4-yl)quinazolin-4 (3H)-one (52): The compound was prepared by general procedure F provided in example 6 using compound 43 (0.20 g, 0.67 mmol), 4-pyridineboronic acid (0.98 g, 0.80 mmol), cesium carbonate (0.437 g, 1.34 mmol), dioxane/H2O (9:1) (10 mL), Pd2(dba)3 (0.062 g, 0.06 mmol), X-phos (0.064 g, 0.13 mmol) to obtain compound 52 (0.103 g, 52%) as yellow solid. 1H NMR (600 MHz, CDCl3) δ in ppm 8.73 (d, J=3.6 Hz, 2H), 7.92 (s, 1H), 7.62 (d, J=8.4 Hz, 1H), 7.23 (d, J=6 Hz, 2H), 7.18 (d, J=8.4 Hz, 1H), 4.00 (t, J=4.8 Hz, 2H), 3.68 (s, 2H), 3.53 (t, J=4.8 Hz, 2H), 3.29 (s, 3H). ESI-MS m/z 297.2 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-5-(pyridin-4-yl)-3,4-dihydroquinazolin-6-yl)urea (53): The compound was prepared by general procedure D provided in example 4 using compound 52 (0.08 g, 0.25 mmol), dry THF (4 mL), 4-nitrophenylchloroformate (0.072 g, 0.35 mmol), 3′-aminoacetophenone (0.037 g, 0.28 mmol), TEA (0.17 mL, 1.22 mmol) to obtain compound 53 (0.039 g, 32%) as yellow solid.
Synthesis of 6-amino-3-(2-methoxyethyl)-5-(6-methoxypyridin-3-yl)quinazolin-4 (3H)-one (54): The compound was prepared by general procedure F provided in example 6 using compound 43 (0.20 g, 0.67 mmol), 2-methoxypyridine-5-boronic acid pinacol ester (0.19 g, 0.80 mmol), cesium carbonate (0.437 g, 1.34 mmol), dioxane/H2O (9:1) (10 mL), Pd2(dba)3 (0.062 g, 0.06 mmol), X-phos (0.064 g, 0.13 mmol) to obtain compound 54 (0.1 g, 46%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 7.95 (s, 1H), 7.86 (d, J=2.1 Hz, 1H), 7.46-7.42 (m, 2H), 7.25 (d, J=8.7 Hz, 1H), 6.83 (d, J=8.4 Hz, 1H), 4.95 (s, 2H), 3.92 (t, J=5.1 Hz, 2H), 3.89 (s, 3H), 3.44 (t, J=5.1 Hz, 2H), 3.20 (s, 3H). ESI-HRMS m/z 327.1470 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-5-(6-methoxypyridin-3-yl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (55): The compound was prepared by general procedure D provided in example 4 using compound 54 (0.10 g, 0.30 mmol), dry THF (4 mL), 4-nitrophenylchloroformate (0.087 g, 0.42 mmol), 3′-aminoacetophenone (0.046 g, 0.33 mmol), TEA (0.17 mL, 1.22 mmol) to obtain compound 55 (0.075 g, 46%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.30 (s, 1H), 8.41 (d, J=8.8 Hz, 1H), 8.13 (s, 1H), 7.90-7.88 (m, 2H), 7.64 (d, J=9.2 Hz, 1H), 7.58 (d, J=6.0 Hz, 1H), 7.51 (d, J=8.0 Hz, 1H), 7.46 (dd, J=8.4 Hz, 1H), 7.39-7.36 (m, 2H), 6.85 (d, J=8.4 Hz, 1H), 3.92 (t, J=5.2 Hz, 2H), 3.88 (s, 3H), 3.40 (t, J=5.2 Hz, 2H), 3.16 (s, 3H), 2.49 (s, 3H). ESI-MS m/z 488.1 (M+H+).
Synthesis of 6-amino-3-(2-methoxyethyl)-5-(2-methoxypyridin-3-yl)quinazolin-4 (3H)-one (56): The compound was prepared by general procedure F provided in example 6 using compound 43 (0.20 g, 0.67 mmol), 2-methoxy-3-pyridinylboronic acid (0.123 g, 0.80 mmol), cesium carbonate (0.437 g, 1.34 mmol), dioxane/H2O (9:1) (10 mL), Pd2(dba)3 (0.062 g, 0.06 mmol), X-phos (0.064 g, 0.13 mmol) to obtain compound 56 (0.12 g, 58%) as yellow solid.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-5-(2-methoxypyridin-3-yl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (57): The compound was prepared by general procedure D provided in example 4 using compound 56 (0.10 g, 0.30 mmol), dry THF (4 mL), 4-nitrophenylchloroformate (0.087 g, 0.42 mmol), 3′-aminoacetophenone (0.046 g, 0.33 mmol), TEA (0.17 mL, 1.22 mmol) to obtain compound 57 (0.075 g, 46%) as yellow solid.
Synthesis of tert-butyl 4-(6-amino-3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-5,6-dihydropyridine-1 (2H)-carboxylate (58): The compound was prepared by general procedure F provided in example 6 using compound 43 (0.2 g, 0.67 mmol), tert-butyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1 (2H)-carboxylate (0.25 g, 0.80 mmol), cesium carbonate (0.437 g, 1.34 mmol), dioxane, H2O (9:1) (10 mL), Pd2(dba)3 (0.062 g, 0.06 mmol), X-phos (0.064 g, 0.13 mmol) to obtain compound 58 (0.12 g, 47%) as light brown solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 7.93 (s, 1H), 7.32 (d, J=8.7 Hz, 1H), 7.19 (d, J=8.7 Hz, 1H), 5.36 (s, 1H), 5.18 (s, 1H), 4.12-4.09 (m, 2H), 4.03 (t, J=4.8 Hz, 2H), 3.80-3.73 (m, 2H), 3.53 (t, J=5.1 Hz, 2H), 3.23 (s, 3H), 2.82-2.31 (m, 1H), 2.09-2.03 (m, 1H), 1.45 (s, 9H). ESI-MS m/z 401.2185 (M+H+).
Synthesis of tert-butyl 4-(6-(3-(3-acetylphenyl)ureido)-3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-5,6-dihydropyridine-1 (2H)-carboxylate (59): The compound was prepared by general procedure D provided in example 4 using compound 58 (0.1 g, 0.25 mmol), dry THF (3 mL), 4-nitrophenylchloroformate (0.071 g, 0.35 mmol), 3′-aminoacetophenone (0.037 g, 0.27 mmol), TEA (0.07 mL, 0.50 mmol) to obtain compound 59 (0.07 g, 53%) as off white solid. 1H NMR (400 MHz, CDCl3), δ in ppm 8.55 (d, J=9.2 Hz, 1H), 8.36 (brs, 1H), 8.07 (s, 1H), 8.00 (s, 1H), δ 7.71 (d, J=8.4 Hz, 1H), 7.67 (d, J=9.2 Hz, 1H), 7.62 (d, J=6.4 Hz, 2H), 7.38 (t, J=7.6 Hz, 1H), 5.55 (brs, 1H), 4.38 (d, J=11.4 Hz, 1H), 4.10 (t, J=4.8 Hz, 2H), 4.38 (d, J=15.4 Hz, 1H), 3.61 (t, J=5.2 Hz, 2H), 3.50-3.43 (m, 1H), 3.31 (s, 3H), 2.58 (s, 3H), 2.27 (brs, 3H), 1.45 (s, 9H). ESI-HRMS m/z 562.2667 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-5-(1,2,3,6-tetrahydropyridin-4-yl)-3,4-dihydroquinazolin-6-yl)urea (60): Compound 59 (0.10 g, 0.17 mmol) was dissolved in dry DCM (2 mL) and TFA (0.5 mL) was added dropwise at 0° C. and reaction mixture was stirred at room temperature for 2 hours. After completion of reaction, it was washed with ethyl acetate and aq. NaHCO3 solution and purified by column chromatography (Silica gel, mesh size 100-200, Merck) eluting (5% MeOH—CHCl3) to obtain compound 60 (0.05 g, 61%) as off white solid. 1H NMR (300 MHz, CDCl3), δ in ppm 8.71 (d, J=9.0 Hz, 1H), 8.10 (s, 1H), 7.94 (s, 1H), 7.68 (d, J=8.1 Hz, 1H), 7.58 (d, J=9.0 Hz, 1H), 7.51 (d, J=7.5 Hz, 1H), 7.31 (d, J=8.1 Hz, 1H), 5.52 (s, 1H), 4.02 (t, J=4.5 Hz, 2H), 3.68-3.62 (m, 2H), 3.54 (t, J=4.5 Hz, 2H), 3.37-3.30 (m, 2H), 3.24 (s, 3H), 2.73-2.66 (m, 1H), 2.52 (s, 3H), 2.38-2.33 (m, 1H). ESI-HRMS m/z 462.2142 (M+H+).
Synthesis of tert-butyl 4-(6-(3-(3-acetylphenyl)ureido)-3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-5-yl)piperidine-1-carboxylate (61): The compound was prepared by general procedure C provided in example 3 using compound 59 (0.10 g, 0.17 mmol), MeOH (5 mL), a pinch of Pd/C under hydrogen atmosphere to obtain compound 61 (0.032 g, 32%) as off white solid. 1H NMR (300 MHz, d6-DMSO), δ in ppm 9.41 (s, 1H), 8.38-8.32 (m, 1H), 8.17 (s, 1H), 7.77 (s, 1H), 7.57 (d, J=9.0 Hz, 1H), 7.38 (d, J=7.5 Hz, 2H), 7.22 (t, J=7.8 Hz, 1H), 6.94 (d, J=7.5 Hz, 1H), 5.47 (s, 1H), 5.14 (s, 1H), 4.69-4.67 (m, 1H), 4.17 (d, J=18.0 Hz, 1H), 4.08 (t, J=4.8 Hz, 2H), 3.86-3.80 (m, 2H), 3.56 (t, J=4.8 Hz, 2H), 3.25 (s, 3H), 2.24-2.15 (m, 3H), 1.45 (s, 9H), 1.30 (d, J=6.3 Hz, 3H). ESI-HRMS m/z 564.2819 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-5-(piperidin-4-yl)-3,4-dihydroquinazolin-6-yl)urea (62): The compound was prepared by general procedure C provided in example 3 using compound 60 (0.10 g, 0.21 mmol), MeOH (8 mL), pinch of Pd/C was added under nitrogen hydrogen atmosphere to obtain compound 62 (0.023 g, 23%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.74 (d, J=9.3 Hz, 1H), 7.96 (br.s, 2H), 7.62 (d, J=9.3 Hz, 1H), 7.44 (s, 1H), 7.36 (d, J=8.1 Hz, 1H), 7.16-7.10 (m, 1H), 6.79 (d, J=7.5 Hz, 1H), 5.55 (s, 1H), 4.05 (t, J=4.5 HZ, 2H), 3.91-3.86 (m, 1H), 3.72-3.66 (m, 1H), 3.57 (t, J=4.5 Hz, J=4.5 Hz, 2H), 3.43-3.40 (m, 2H), 3.28 (s, 3H), 3.08 (s, 3H), 3.01-2.95 (m, 3H), 2.60-2.43 (m, 3H). ESI-MS m/z 464.2 (M+H+).
Synthesis of 6-amino-3-(2-methoxyethyl)-5-(4-methoxyphenyl)quinazolin-4 (3H)-one (63): The compound was prepared by general procedure F provided in example 6 using compound 43 (0.150 g, 0.50 mmol), 4-methoxyphenylboronic acid (0.1 g, 0.60 mmol, dioxane/H2O (9:1) (5 mL) and K2CO3 (0.2 mL, 2 M) solution, Pd(PPh3)4 (0.058 g, 0.05 mmol) to obtain compound 63 (0.1 g, 61%) as brown solid. 1H NMR (600 MHz, d6-DMSO) δ in ppm 7.92 (s, 1H), 7.40 (d, J=9 Hz, 1H), 7.22 (d, J=9 Hz, 1H), 7.00 (d, J=9 Hz, 2H), 6.94 (d, J=9 Hz, 2H), 4.70 (t, J=5.4 Hz, 2H), 3.89 (t, J=5.4 Hz, 2H), 3.78 (s, 3H), 3.42 (t, J=5.4 Hz, 2H), 3.19 (s, 3H). ESI-MS m/z 326.1 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-5-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (64): The compound was prepared by general procedure D provided in example 4 using compound 63 (0.1 g, 0.30 mmol), dry THF (3 mL), 4-nitrophenylchloroformate (0.093 g, 0.46 mmol), 3′-Aminoacetophenone (0.06 g, 0.46 mmol), TEA (0.1 mL, 0.75 mmol) to obtain compound 64 (0.06 g, 40%) as off white solid. 1H NMR (300 MHz, CDCl3) δ in ppm 8.68 (d, J=9 Hz, 1H), 7.99 (s, 1H), 7.74 (d, J=9 Hz, 1H), 7.63 (d, J=7.8 Hz, 1H), 7.49 (d, J=7.8 Hz, 1H), 7.33-7.30 (m, 1H), 7.05 (d, J=8.1 Hz, 3H), 6.85 (d, J=8.4 Hz, 2H), 6.58 (s, 1H), 3.97 (t, J=4.8 Hz, 2H), 3.76 (s, 3H), 3.49 (t, J=4.8 Hz, 2H), 3.26 (s, 3H), 2.56 (s, 3H). ESI-MS m/z 487.2003 (M+H+).
Synthesis of 6-amino-3-(2-methoxyethyl)-5-(4-(trifluoromethyl)phenyl)quinazolin-4 (3H)-one (65): The compound was prepared by general procedure F provided in example 6 using compound 43 (0.20 g, 0.67 mmol), 4-(trifluoromethyl)phenylboronic acid (0.153 g, 0.80 mmol), cesium carbonate (0.437 g, 1.34 mmol), dioxane/H2O (9:1) (8 mL), Pd2(dba)3 (0.062 g, 0.06 mmol), X-phos (0.064 g, 0.13 mmol) to obtain compound 65 (0.146 g, 60%) as yellow solid. ESI-HRMS m/z 364.1281 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-5-(4-(trifluoromethyl)phenyl)-3,4-dihydroquinazolin-6-yl)urea (66): The compound was prepared by general procedure D provided in example 4 using compound 65 (0.1 g, 0.27 mmol), dry THF (3 mL), 4-nitrophenylchloroformate (0.081 g, 0.40 mmol), 3′-Aminoacetophenone (0.054 g, 0.40 mmol), TEA (0.1 mL, 0.65 mmol) to obtain compound 66 (0.054 g, 38%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.29 (s, 1H), 8.37 (d, J=9 Hz, 1H), 8.21 (s, 1H), 7.94 (brs, 1H), 7.80 (d, J=8.1 Hz, 2H), 7.72 (d, J=9 Hz, 1H), 7.62-7.55 (m, 3H), 7.43 (d, J=2.7 Hz, 1H), 7.41 (d, J=3 Hz, 1H), 7.27 (s, 1H), 3.96 (t, J=4.5 Hz, 2H), 3.44 (t, J=4.8 Hz, 2H), 3.21 (s, 3H), 2.54 (s, 3H). ESI-HRMS m/z 525.1769 (M+H+).
Synthesis of 6-amino-5-cyclohexyl-3-(2-methoxyethyl)quinazolin-4 (3H)-one (67): The compound was prepared by general procedure F provided in example 6 using compound 43 (0.20 g, 0.67 mmol), cyclohexylboronic acid (0.102 g, 0.80 mmol), cesium carbonate (0.437 g, 1.34 mmol), dioxane/H2O (9:1) (8 mL), Pd2(dba)3 (0.062 g, 0.06 mmol), X-phos (0.064 g, 0.13 mmol) to obtain compound 67 (0.121 g, 60%) as yellow solid.
Synthesis of 1-(3-acetylphenyl)-3-(5-cyclohexyl-3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (68): The compound was prepared by general procedure D provided in example 4 using compound 67 (0.1 g, 0.33 mmol), dry THF (3 mL), 4-nitrophenylchloroformate (0.100 g, 0.49 mmol), 3′-Aminoacetophenone (0.066 g, 0.49 mmol), TEA (0.11 mL, 0.82 mmol) to obtain compound 68 (0.053 g, 35%) as off white solid.
Synthesis of 6-amino-5-cyclopentyl-3-(2-methoxyethyl)quinazolin-4 (3H)-one (69): The compound was prepared by general procedure F provided in example 6 using compound 43 (0.20 g, 0.67 mmol), cyclopentylboronic acid (0.091 g, 0.80 mmol), cesium carbonate (0.437 g, 1.34 mmol), dioxane/H2O (9:1) (8 mL), Pd2(dba)3 (0.062 g, 0.06 mmol), X-phos (0.064 g, 0.13 mmol) to obtain compound 69 (0.116 g, 60%) as yellow solid.
Synthesis of 1-(3-acetylphenyl)-3-(5-cyclopentyl-3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (70): The compound was prepared by general procedure D provided in example 4 using compound 69 (0.1 g, 0.34 mmol), dry THF (3 mL), 4-nitrophenylchloroformate (0.102 g, 0.51 mmol), 3′-Aminoacetophenone (0.068 g, 0.51 mmol), TEA (0.10 mL, 0.85 mmol) to obtain compound 70 (0.065 g, 42%) as off white solid.
Synthesis of 6-amino-5-isopropyl-3-(2-methoxyethyl)quinazolin-4 (3H)-one (71): The compound was prepared by general procedure F provided in example 6 using compound 43 (0.20 g, 0.67 mmol), isopropylboronic acid (0.070 g, 0.80 mmol), cesium carbonate (0.437 g, 1.34 mmol), dioxane/H2O (9:1) (8 mL), Pd2(dba)3 (0.062 g, 0.06 mmol), X-phos (0.064 g, 0.13 mmol) to obtain compound 71 (0.126 g, 72%) as yellow solid.
Synthesis of 1-(3-acetylphenyl)-3-(5-isopropyl-3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (72): The compound was prepared by general procedure D provided in example 4 using compound 71 (0.1 g, 0.38 mmol), dry THF (3 mL) and 4-Nitrophenylchloroformate (0.114 g, 0.57 mmol), 3′-Aminoacetophenone (0.07 g, 0.57 mmol), TEA (0.13 mL, 0.85 mmol) to obtain compound 72 (0.072 g, 45%) as off white solid.
Synthesis of 1-(3-acetylphenyl)-3-(5-bromo-3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (73):
The compound was prepared by general procedure D provided in example 4 using compound 43 (0.1 g, 0.33 mmol), dry THF (6 mL) and 4-nitrophenylchloroformate (0.101 g, 0.50 mmol), 3′-aminoacetophenone (0.054 g, 0.40 mmol), TEA (0.2 mL, 1.38 mmol) to obtain compound 73 (0.071 g, 46%) as off white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.80 (s, 1H), 8.46 (s, 1H), 8.41 (d, J=9.2 Hz, 1H), 8.20 (s, 1H), 8.04-8.03 (m, 1H), 7.67-7.64 (m, 1H), 7.60 (d, J=8.8 Hz, 1H), 7.56 (d, J=7.6 Hz, 1H), 7.43-7.39 (m, 1H), 4.07 (t, J=5.2 Hz, 2H), 3.55 (t, J=5.2 Hz, 2H), 3.20 (s, 3H), 2.52 (s, 3H). ESI-MS m/z 459.2 (M+H+). Melting Point: 204° C.
Synthesis of tert-butyl (1-((3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (74): N-Boc Valine (1 g, 4.60 mmol) was taken in DMF (8 mL) and HATU (2 g, 5.06 mmol) was added followed by TEA (1.5 mL, 11.41 mmol) and compound 4 (1 g, 5.06 mmol) and reaction mixture was stirred at room temperature for 5 hours. After completion of reaction, reaction mixture was washed thoroughly with cold water and extracted with EtOAc to afford reddish coloured crude mass which was then purified by column chromatography (Silica gel, mesh size 100-200) eluting (50% EtOAc/Pet ether) to obtain compound 74 (0.8 g, 42%) as brown solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 10.36 (s, 1H), 8.52 (d, J=1.8 Hz, 1H), 8.19 (s, 1H), 7.95 (br.s, —NH), 7.64 (d, J=9 Hz, 1H), 6.98 (d, J=8.4 Hz, 1H), 4.15 (t, J=5.1 Hz, 2H), 3.93 (t, J=7.8 Hz, 1H), 3.60 (t, J=5.1 Hz, 2H), 3.24 (s, 3H), 2.04-1.96 (m, 1H), 1.38 (s, 9H), 0.90 (d, J=6.6 Hz, 6H). ESI-HRMS m/z 419.2296 (M+H+). Melting Point: 120° C.
Synthesis of 2-amino-N-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-3-methylbutanamide (75): Compound 74 (0.2 g, 0.47 mmol) was taken in DCM (3 mL) and TFA (0.8 mL) was added dropwise under cooling condition. After 2 hours, the reaction was completed and reaction mixture was neutralized by NaHCO3 solution and extracted with DCM; evaporated to obtain compound 75 (0.11 g, 72%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.85 (s, 1H), 8.26 (d, J=7.2 Hz, 1H), 8.20 (d, J=2 Hz, 1H), 7.98 (s, 1H), 7.65 (d, J=8.8 Hz, 1H), 4.14 (t, J=4.4 Hz, 2H), 3.64 (t, J=4.4 Hz, 2H), 3.44-3.41 (m, 1H), 3.28 (s, 3H), 2.46-2.40 (m, 1H), 1.02 (d, J=6.8 Hz, 3H), 0.86 (d, J=6.8 Hz, 3H). ESI-HRMS m/z 319.1772 (M+H+). Melting Point: 118° C.
Synthesis of 2-(3-(3-acetylphenyl)ureido)-N-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-3-methylbutanamide (76): The compound was prepared by general procedure E provided in example 5 using compound 75 (0.087 g, 0.27 mmol), dry THF (2 mL), 3-acetylphenyl isocyanate (0.05 mL, 0.32 mmol), TEA (0.1 mL, 0.60 mmol) to obtain compound 76 (0.062, 47%) as white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 10.55 (s, 1H), 8.95 (s, 1H), 8.53 (s, 1H), 8.20 (s, 1H), 8.00-7.95 (m, 2H), 7.65 (d, J=8.4 Hz, 1H), 7.59 (d, J=7.8 Hz, 1H), 7.52 (d, J=6.6 Hz, 1H), 7.41-7.36 (m, 1H), 6.57 (d, J=8.7 Hz, 1H), 4.32 (t, J=6.3 Hz, 1H), 4.14 (t, J=4.8 Hz, 2H), 3.60 (t, J=4.8 Hz, 2H), 3.24 (s, 3H), 2.53 (s, 3H), 2.13-2.02 (m, 1H), 0.95 (q, J=5.4 Hz, 6H). ESI-HRMS m/z 480.2266 (M+H+). Melting Point: 202° C.
Synthesis of N-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-3-nitrobenzamide (77): 3-Nitrobenzoic acid (0.365 g, 2.19 mmol) was taken in dry DCM and oxalyl chloride (0.3 mL, 3.27 mmol) was added dropwise under cooling conditions and reaction mixture was stirred at room temperature for 1 hour. Excess oxalyl chloride was evaporated in rotavapor and diluted with DCM (5 mL). To it, compound 4 (0.4 g, 1.82 mmol) diluted with (5 mL) was added dropwise over a period of 5 minutes and the reaction mixture was kept for another 5 hours. After completion of reaction, reaction mixture was evaporated to obtain a yellow coloured crude mass which was purified by column chromatography (Silica gel, mesh size 100-200) eluting (50% EtOAc/Pet ether) to obtain compound 77 as yellow solid (0.35 g, 83% yield). 1H NMR (400 MHz, d6-DMSO) δ in ppm 10.85 (s, 1H), 8.80 (brs, —NH), 8.62 (d, J=2.4 Hz, 1H), 8.42-8.39 (m, 2H), 8.20-8.17 (m, 2H), 7.83-7.79 (m, 1H), 7.66 (d, J=9.2 Hz, 1H), 4.13 (t, J=5.2 Hz, 2H), 3.58 (t, J=5.2 Hz, 2H), 3.21 (s, 3H). ESI-HRMS m/z 369.1214 (M+H+). Melting Point: 212° C.
Synthesis of 3-amino-N-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)benzamide (78): The compound was prepared by general procedure C provided in example 3 using compound 77 (0.25 g, 0.67 mmol), MeOH (8 mL) pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 78 as yellow solid (0.2 g, 87% yield). 1H NMR (400 MHz, d6-DMSO) δ in ppm 10.37 (s, 1H), 8.62 (d, J=2.4 Hz, 1H), 8.16 (s, 1H), 8.13 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.61 (d, J=9.2 Hz, 1H), 7.14-7.05 (m, 3H), 6.72 (d, J=9.6 Hz, 1H), 5.29 (brs, 2H), 4.12 (t, J=5.2 Hz, 2H), 3.58 (t, J=5.2 Hz, 2H), 3.21 (s, 3H). ESI-HRMS m/z 339.1214 (M+H+). Melting Point: 132° C.
Synthesis of 3-(3-(3-acetylphenyl)ureido)-N-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)benzamide (79): The compound was prepared by general procedure E provided in example 5 using compound 78 (0.11 g, 0.32 mmol), dry THF (8 mL), 3-acetylphenyl isocyanate (0.05 mL, 0.38 mmol), TEA (0.1 mL, 0.81 mmol) to obtain compound 79 (0.08 g, 49%) as light yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 10.62 (s 1H), 8.99 (s 1H), 8.98 (s 1H), 8.69 (d, J=2.4 Hz, 1H), 8.23-8.18 (m, 2H), 8.10-8.09 (m, 1H), 8.05-8.03 (m, 1H), 7.73-7.68 (m, 3H), 7.64-7.59 (m, 2H), 7.50-7.42 (m, 2H), 4.17 (t, J=5.1 Hz, 2H), 3.62 (t, J=5.1 Hz, 2H), 3.26 (s, 3H), 2.57 (s, 3H). ESI-HRMS m/z 500.1945 (M+H+). Melting Point: 140° C.
Synthesis of ethyl 2-(2-amino-5-nitrobenzamido)acetate (80): The compound was prepared by general procedure A provided in example 1 using compound 1 (2 g, 10.98 mmol), DMF (12 mL), HATU (4.5 g, 12.08 mmol), Glycine ethyl ester hydrochloride (1.7 g, 12.08 mmol), TEA (4.5 mL, 32.96 mmol) to obtain compound 80 (2.5 g, 85%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.11 (br.s, —NH), 8.52 (d, J=2.8 Hz, 1H), 7.99 (dd, J=9.2 Hz, 2.8 Hz, 1H), 7.73 (br.s, 2H), 6.77 (d, J=9.2 Hz, 1H), 4.08 (q, J=7.2 Hz, 2H), 3.92 (d, J=5.6 Hz, 2H), 1.16 (t, J=6.8 Hz, 3H). ESI-HRMS m/z 268.0941 (M+H+). Melting Point: 178° C.
Synthesis of ethyl 2-(6-nitro-4-oxoquinazolin-3 (4H)-yl)acetate (81): The compound was prepared by general procedure B provided in example 2 using compound 80 (2.5 g, 9.36 mmol), trimethylorthoformate (TMOF) (10 mL, 93.63 mmol) to obtain compound 81 (2.3 g, 89%) as pale yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.82 (d, J=2.8 Hz, 1H), 8.60-8.57 (m, 2H), 7.92 (d, J=8.8 Hz, 1H), 4.87 (s, 2H), 4.17 (q, J=7.2 Hz, 2H), 1.21 (t, J=7.2 Hz, 3H). ESI-HRMS m/z 278.0785 (M+H+). Melting Point: 166° C.
Synthesis of ethyl 2-(6-amino-4-oxoquinazolin-3 (4H)-yl)acetate (82): The compound was prepared by general procedure C provided in example 3 using compound 81 (1 g, 3.60 mmol), methanol (10 mL) pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 82 (0.8 g, 90%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.02 (s, 1H), 7.40 (d, J=8.7 Hz, 1H), 7.19 (d, J=2.4 Hz, 1H), 7.09 (dd, J=8.7 Hz, 2.7 Hz, 1H), 5.71 (s, 2H), 4.75 (s, 2H), 4.16 (q, J=7.2 Hz, 2H), 1.21 (t, J=7.2 Hz, 3H). ESI-HRMS m/z 248.1041 (M+H+). Melting Point: 138° C.
Synthesis of ethyl 2-(6-(3-(3-chloro-4-fluorophenyl)ureido)-4-oxoquinazolin-3 (4H)-yl)acetate (83): The compound was prepared by general procedure E provided in example 5 using compound 82 (0.1 g, 0.40 mmol), dry THF (6 mL), 3-chloro-4-fluorophenyl isocyanate (0.08 mL, 0.68 mmol), TEA (0.2 mL, 1.36 mmol) to obtain compound 83 (0.059 g, 35%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.23 (s, 1H), 9.00 (s, 1H), 8.33 (d, J=2.4 Hz, 1H), 8.25 (s, 1H), 7.86-7.80 (m, 2H), 7.66 (d, J=9 Hz, 1H), 7.35 (d, J=7.5 Hz, 2H), 4.81 (s, 2H), 4.17 (q, J=7.2 Hz, 2H), 1.22 (t, J=6.9 Hz, 3H). ESI-MS m/z 419.1 (M+H+). Melting Point: 220° C.
Synthesis of ethyl 2-(4-oxo-6-(3-(4-(trifluoromethoxy)phenyl)ureido)quinazolin-3 (4H)-yl)acetate (84): The compound was prepared by general procedure E provided in example 5 using compound 82 (0.1 g, 0.40 mmol), dry THF (5 mL), 4-(trifluoromethoxy)phenyl isocyanate (0.07 mL, 0.50 mmol), TEA (0.2 mL, 1.66 mmol) to obtain compound 84 (70 mg, 43%) as white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.18 (s, 1H), 9.00 (s, 1H), 8.34 (d, J=2.1 Hz, 1H), 8.25 (s, 1H), 7.83 (dd, J=8 Hz, 2.1 Hz, 1H), 7.67-7.56 (m, 3H), 7.30 (d, J=8.4 Hz, 2H), 4.81 (s, 2H), 4.17 (q, J=7.2 Hz, 2H), 1.22 (t, J=6.9 Hz, 3H). ESI-MS m/z 451.13 (M+H+). Melting Point: 230° C.
Synthesis of ethyl 2-(6-(3-(3-acetylphenyl)ureido)-4-oxoquinazolin-3 (4H)-yl)acetate (85): The compound was prepared by general procedure E provided in example 5 using compound 82 (60 mg, 0.24 mmol), dry THF (5 mL), 3-acetylphenyl isocyanate (0.04 mL, 0.34 mmol), TEA (0.1 mL, 0.68 mmol) to obtain compound 85 (80 mg, 49%) as white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.21 (s, 1H), 9.05 (s, 1H), 8.36 (d, J=2.4 Hz, 1H), 8.25 (s, 1H), 8.10 (s, 1H), 7.85 (dd, J=8.8 Hz, 2.7 Hz, 1H), 7.72-7.59 (m, 3H), 7.48-7.43 (m, 1H), 4.82 (s, 2H), 4.18 (q, J=7.2 Hz, 2H), 2.57 (s, 3H), 1.22 (t, J=6.9 Hz, 3H). ESI-MS m/z 409.4 (M+H+). Melting Point: 196° C.
Synthesis of 2-amino-N-(3-methoxypropyl)-5-nitrobenzamide (86): The compound was prepared by general procedure A provided in example 1 using compound 1 (0.8 g, 4.93 mmol), DMF (6 mL), HATU (1.8 g, 4.83 mmol), 3-methoxypropylamine (0.5 mL, 4.83 mmol), TEA (1.5 mL, 10.98 mmol) to obtain compound 86 (0.9 g, 90%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.70 (br.s., —NH), 8.49 (d, J=2.4 Hz, 1H), 8.01 (dd, J=9.1 Hz, 2.4 Hz, 1H), 7.76 (br.s, 2H), 6.79 (d, J=9 Hz, 1H), 3.37 (t, J=6.3 Hz, 2H), 3.28 (t, J=6 Hz, 2H), 3.24 (s, 3H), 1.80-1.71 (m, 2H). ESI-HRMS m/z 254.1133 (M+H+). Melting Point: 102° C.
Synthesis of 3-(3-methoxypropyl)-6-nitroquinazolin-4 (3H)-one (87): The compound was prepared by general procedure B provided in example 2 using compound 86 (0.6 g, 2.36 mmol), trimethylorthoformate (TMOF) (5 mL, 47.43 mmol) to obtain compound 87 (0.59 g, 95%) as pale yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.84 (d, J=2.4 Hz, 1H), 8.58-8.54 (m, 2H), 7.88 (d, J=9 Hz, 1H), 4.05 (t, J=7.2 Hz, 2H), 3.93 (t, J=6 Hz, 2H), 3.20 (s, 3H), 1.99-1.91 (m, 2H). ESI-MS m/z 264.2 (M+H+). Melting Point: 110° C.
Synthesis of 6-amino-3-(3-methoxypropyl)quinazolin-4 (3H)-one (88): The compound was prepared by general procedure C provided in example 3 using compound 87 (0.3 g, 1.13 mmol), methanol (6 mL), pinch of 10% wet Pd—C under hydrogen atmosphere was added to obtain compound 88 (0.25 g, 94%) as brown solid. ESI-HRMS m/z 234.1253 (M+H+). Melting Point: 98° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(3-methoxypropyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (89): The compound was prepared by general procedure D provided in example 4 using compound 88 (0.12 g, 0.51 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.124 g, 0.61 mmol), 3′-aminoacetophenone (0.084, 0.61 mmol), TEA (0.2 mL, 1.54 mmol) to obtain compound 89 (0.075 g, 37%) as off white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.08 (s, 1H), 8.95 (s, 1H), 8.30 (d, J=2.8 Hz, 1H), 8.16 (d, J=2 Hz, 1H), 8.05-8.04 (m, 1H), 7.77 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.67-7.64 (m, 1H), 7.58-7.54 (m, 2H), 7.42-7.38 (m, 1H), 3.97 (t, J=6.8 Hz, 2H), 3.31-3.30 (m, 2H) 3.17 (s, 3H), 2.52 (s, 3H), 1.91-1.85 (m, 2H). ESI-MS m/z 395.3 (M+H+). Melting Point: 162° C.
Synthesis of 2-amino-N-(2-ethoxyethyl)-5-nitrobenzamide (90): The compound was prepared by general procedure A provided in example 1 using compound 1 (0.8 g, 4.93 mmol), DMF (6 mL), HATU (1.8 g, 4.83 mmol), 2-ethoxyethylamine (0.5 mL, 4.83 mmol), TEA (1.5 mL, 10.98 mmol) to obtain compound 90 (1 g, 90%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.71 (br.s, —NH), 8.46 (d, J=2.8 Hz, 1H), 7.96 (dd, J=6.4 Hz, 2.8 Hz, 1H), 7.73 (s, 2H), 6.75 (d, J=9.2 Hz, 1H), 3.46-3.39 (m, 4H), 3.33 (q, J=6 Hz, 2H), 1.07 (t, J=6.8 Hz, 3H). ESI-HRMS m/z 254.1135 (M+H+). Melting Point: 104° C.
Synthesis of 3-(2-ethoxyethyl)-6-nitroquinazolin-4 (3H)-one (91): The compound was prepared by general procedure B provided in example 2 using compound 90 (0.85 g, 3.37 mmol), trimethylorthoformate (TMOF) (7 mL, 67.19 mmol) to obtain compound 91 (0.8 g, 96%) as pale yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.80 (d, J=2.4 Hz, 1H), 8.51 (dd, J=6.4 Hz, 2.4 Hz, 1H), 8.45 (s, 1H), 7.83 (d, J=8.4 Hz, 1H), 4.15 (t, J=5.2 Hz, 2H), 3.62 (t, J=5.2 Hz, 2H), 3.40 (q, J=7.2 Hz, 2H), 1.00 (t, J=6.8 Hz, 3H). ESI-HRMS m/z 264.0975 (M+H+). Melting Point: 138° C.
Synthesis of 6-amino-3-(2-ethoxyethyl)quinazolin-4 (3H)-one (92): The compound was prepared by general procedure C provided in example 3 using compound 91 (0.55 g, 2.09 mmol), methanol (6 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 92 (0.45 g, 92%) as light brown solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 7.94 (s, 1H), 7.37 (d, J=8.4 Hz, 1H), 7.21 (d, J=2.4 Hz, 1H), 7.07 (dd, J=8.7 Hz, 2.7 Hz, 1H), 5.66 (s, 2H), 4.08 (t, J=5.4 Hz, 2H), 3.61 (t, J=5.4 Hz, 2H), 3.42 (q, J=6.9 Hz, 2H), 1.04 (t, J=7.2 Hz, 3H). ESI-HRMS m/z 234.1229 (M+H+). Melting Point: 102° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-ethoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (93): The compound was prepared by general procedure E provided in example 5 using compound 92 (0.12 g, 0.51 mmol), THF (6 mL), 3-acetylphenyl isocyanate (0.09 mL, 0.61 mmol), TEA (0.2 mL, 1.28 mmol) to obtain compound 93 (0.06 g, 30%) as light yellow solid. 1H NMR (600 MHz, d6-DMSO) δ in ppm 9.12 (s, 1H), 8.99 (s, 1H), 8.35 (d, J=2.4 Hz, 1H), 8.16 (s, 1H), 8.08 (s, 1H), 7.80 (dd, J=9 Hz, 2.4 Hz, 1H), 7.70-7.68 (m, 1H), 7.62-7.58 (m, 2H), 7.45-7.42 (m, 1H), 4.12 (t, J=5.4 Hz, 2H), 3.63 (t, J=5.4 Hz, 2H), 3.42 (q, J=7.2 Hz, 2H), 2.56 (s, 3H), 1.03 (t, J=6.6 Hz, 3H). ESI-HRMS m/z 395.1728 (M+H+). Melting Point: 192° C.
Synthesis of 1-(4-acetylphenyl)-3-(3-(2-ethoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (94): The compound was prepared by general procedure E provided in example 5 using compound 92 (0.12 g, 0.51 mmol), dry THF (6 mL), 4-acetylphenyl isocyanate, (0.09 mL, 0.61 mmol), TEA (0.2 mL, 1.28 mmol) to obtain compound 94 (0.07 g, 34%) as light yellow solid. 1H NMR (600 MHz, d6-DMSO) δ in ppm 9.21 (s, 1H), 9.18 (s, 1H), 8.36 (d, J=2.4 Hz, 1H), 8.16 (s, 1H), 7.90 (d, J=9 Hz, 2H), 7.79 (dd, J=8.4 Hz, 2.4 Hz, 1H), 7.62-7.59 (m, 3H), 4.12 (t, J=4.8 Hz, 2H), 3.63 (t, J=5.4 Hz, 2H), 3.42 (q, J=6.6 Hz, 2H), 2.50 (s, 3H), 1.03 (t, J=7.2 Hz, 3H). ESI-HRMS m/z 395.1727 (M+H+). Melting Point: 102° C.
Synthesis of 2-amino-N-ethyl-5-nitrobenzamide (95): The compound was prepared by general procedure A provided in example 1 using compound 1 (1 g, 5.49 mmol), DMF (6 mL), HATU (2.2 g, 6.04 mmol), ethylamine 2 M in THF (2.5 mL), TEA (1.9 mL, 13.73 mmol) to obtain compound 95 (1.1 g, 96%) as yellow solid. 1H NMR (300 MHz, CDCl3) δ in ppm 8.33 (d, J=2.7 Hz, 1H), 8.07 (dd, J=9 Hz, 2.4 Hz, 1H), 6.65 (d, J=9 Hz, 1H), 6.28 (br.s, —NH), 5.05 (br.s, 2H), 3.52-3.43 (m, 2H), 1.28 (t, J=7.2 Hz, 3H). ESI-HRMS m/z 210.0881 (M+H+). Melting Point: 136° C.
Synthesis of 3-ethyl-6-nitroquinazolin-4 (3H)-one (96): The compound was prepared by general procedure B provided in example 2 using compound 95 (0.80 g, 3.82 mmol), trimethylorthoformate (TMOF) (8 mL, 76.55 mmol) to obtain compound 96 (0.8 g, 95%) as pale yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.82 (d, J=2.7 Hz, 1H), 8.61 (s, 1H), 8.53 (dd, J=9 Hz, 2.7 Hz, 1H), 7.86 (d, J=8.7 Hz, 1H), 4.03 (q, J=7.2 Hz, 2H), 1.29 (t, J=7.2 Hz, 3H). ESI-MS m/z 220.0728 (M+H+). Melting Point: 146° C.
Synthesis of 6-amino-3-ethylquinazolin-4 (3H)-one (97): The compound was prepared by general procedure C provided in example 3 using compound 96 (0.50 g, 2.28 mmol), methanol (6 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 97 (0.39 g, 90%) as light brown solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.06 (s, 1H), 7.37 (d, J=8.7 Hz, 1H), 7.21 (d, J=2.7 Hz, 1H), 7.06 (dd, J=8.7 Hz, 2.7 Hz, 1H), 5.64 (s, 2H), 3.95 (q, J=7.2 Hz, 2H), 1.25 (t, J=7.2 Hz, 3H). ESI-MS m/z 190.1 (M+H+). Melting Point: 164° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-ethyl-4-oxo-3,4-dihydroquinazolin-6-yl)urea (98): The compound was prepared by general procedure E provided in example 5 using compound 97 (0.12 g, 0.63 mmol), dry THF (6 mL), 3-acetylphenyl isocyanate (0.10 mL, 0.76 mmol), TEA (0.2 mL, 1.58 mmol) to obtain compound 98 (0.08 g, 36%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.07 (s, 1H), 8.93 (s, 1H), 8.30 (d, J=2.4 Hz, 1H), 8.23 (s, 1H), 8.05-8.04 (m, 1H), 7.76 (dd, J=9 Hz, 2.4 Hz, 1H), 7.66-7.63 (m, 1H), 7.57-7.53 (m, 2H), 7.41-7.37 (m, 1H), 3.95 (q, J=7.2 Hz, 2H), 2.52 (s, 3H), 1.23 (t, J=7.2 Hz, 3H). ESI-HRMS m/z 351.1465 (M+H+). Melting Point: 202° C.
Synthesis of 1-(4-acetylphenyl)-3-(3-ethyl-4-oxo-3,4-dihydroquinazolin-6-yl)urea (99): The compound was prepared by general procedure E provided in example 5 using compound 97 (0.12 g, 0.63 mmol), dry THF (6 mL), 4-acetylphenyl isocyanate (0.10 mL, 0.76 mmol), TEA (0.2 mL, 1.58 mmol) to obtain compound 99 (0.08 g, 36%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.13 (d, J=8.8 Hz, 2H), 8.31 (d, J=2.4 Hz, 1H), 8.24 (s, 1H), 7.86 (d, J=8.8 Hz, 2H), 7.75 (dd, J=6.4 Hz, 2.4 Hz, 1H), 7.58-7.55 (m, 3H), 3.95 (q, J=6.8 Hz, 2H), 2.96 (s, 3H), 1.23 (t, J=7.2 Hz, 3H). ESI-HRMS m/z 351.1464 (M+H+). Melting Point: 212° C.
Synthesis of 2-amino-N-(3-methoxyphenyl)-5-nitrobenzamide (100): The compound was prepared by general procedure A provided in example 1 using compound 1 (0.8 g, 4.93 mmol), DMF (6 mL), HATU (1.8 g, 4.83 mmol), m-anisidine (0.55 mL, 4.83 mmol), TEA (1.5 mL, 10.98 mmol) to obtain compound 100 (1.1 g, 90%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 10.34 (s, 1H), 8.55 (d, J=2.4 Hz, 1H), 8.03 (dd, J=6.4 Hz, 2.8 Hz, 1H), 7.59 (s, 2H), 7.34-7.33 (m, 1H), 7.27-7.19 (m, 2H), 6.81 (d, J=9.2 Hz, 1H), 6.67-6.64 (m, 1H), 3.71 (s, 3H). ESI-HRMS m/z 288.0990 (M+H+). Melting Point: 178° C.
Synthesis of 3-(3-methoxyphenyl)-6-nitroquinazolin-4 (3H)-one (101): The compound was prepared by general procedure B provided in example 2 using compound 100 (0.80 g, 2.78 mmol), trimethylorthoformate (TMOF) (6 mL, 55.74 mmol) to obtain compound 101 (0.79 g, 96%) as light yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.83 (d, J=2.8 Hz, 1H), 8.57 (dd, J=6 Hz, 2.8 Hz, 1H), 8.51 (s, 1H), 7.90 (d, J=9.2 Hz, 1H), 7.47-7.43 (m, 1H), 7.16-7.15 (m, 1H), 7.10-7.06 (m, 2H), 3.76 (s, 3H). ESI-MS m/z 298.0 (M+H+). Melting Point: 204° C.
Synthesis of 6-amino-3-(3-methoxyphenyl)quinazolin-4 (3H)-one (102): The compound was prepared by general procedure C provided in example 3 using compound 101 (0.7 g, 2.35 mmol), methanol (6 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 102 (0.59 g, 94%) as light brown solid. 1H NMR (300 MHz, d6-DMSO) 8.01 (s, 1H), 7.47-7.42 (m, 2H), 7.29 (d, J=2.7 Hz, 1H), 7.15-7.09 (m, 2H), 7.08-7.04 (m, 2H), 3.80 (s, 3H). ESI-MS m/z 268.1 (M+H+). Melting Point: 188° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(3-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (103): The compound was prepared by general procedure E provided in example 5 using compound 102 (0.12 g, 0.45 mmol), dry THF (7 mL), 3-acetylphenyl isocyanate (0.08 mL, 0.53 mmol), TEA (0.2 mL, 1.12 mmol) to obtain compound 103 (0.072 g, 37%) as light yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.13 (s, 1H), 8.95 (s, 1H), 8.34 (d, J=2.4 Hz, 1H), 8.16 (s, 1H), 8.04-8.03 (m, 1H), 7.84 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.67-7.63 (m, 2H), 7.55 (d, J=7.6 Hz, 1H), 7.43-7.38 (m, 2H), 7.10-7.09 (m, 1H), 7.05-7.01 (m, 2H), 3.75 (s, 3H), 2.52 (s, 3H). ESI-MS m/z 429.1 (M+H+). Melting Point: 194° C.
Synthesis of 2-acetamido-N-(2-methoxyethyl)-5-nitrobenzamide (104): Compound 2 (0.6 g, 2.50 mmol) was taken in dry DCM (10 mL) and TEA (0.77 mL, 5.52 mmol) was added. Acetyl chloride (0.21 mL, 3.01 mmol) was added dropwise at 0° C. and reaction mixture was allowed to stir at room temperature for 3 hours. Reaction was monitored by checking TLC. After completion of reaction, reaction mixture was washed with satd. NaHCO3 solution and extracted with DCM, evaporated to afford yellow coloured mass which was further diluted with chloroform and pet ether was added to get precipitation. The precipitate was filtered and washed with 20% (EtOAc/Pet ether) solution to obtain compound 104 (0.5 g, 71%) as light yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 11.57 (s, 1H), 9.18 (br.s, —NH), 8.60 (d, J=7.6 Hz, 1H), 8.58 (d, J=1.2 Hz, 1H), 8.32 (dd, J=10.8 Hz, 2 Hz, 1H), 3.46-3.39 (m, 4H), 3.24 (s, 3H), 2.12 (s, 3H). ESI-HRMS m/z 282.1089 (M+H+). Melting Point: 160° C.
Synthesis of 3-(2-methoxyethyl)-2-methyl-6-nitroquinazolin-4 (3H)-one (105): The compound was prepared by general procedure G provided in example 7 using compound 104 (0.4 g, 1.42 mmol), DMF (6 mL), Hexamethyldisilazane (3 mL, 14.22 mmol), ZnCl2 (0.96 g, 7.11 mmol) to obtain compound 105 (0.32 g, 86%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.75 (d, J=2.4 Hz, 1H), 8.48 (dd, J=6 Hz, 2.8 Hz, 1H), 7.72 (d, J=9.2 Hz, 1H), 4.23 (t, J=5.2 Hz, 2H), 3.60 (t, J=5.6 Hz, 2H), 3.20 (s, 3H), 2.65 (s, 3H). ESI-MS m/z 264.1 (M+H+). Melting Point: 154° C.
Synthesis of 6-amino-3-(2-methoxyethyl)-2-methylquinazolin-4 (3H)-one (106): The compound was prepared by general procedure C provided in example 3 using compound 105 (0.22 g, 0.83 mmol), methanol (6 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 106 (0.18 g, 93%) as light brown solid. 1H NMR (600 MHz, d6-DMSO) δ in ppm 7.26 (d, J=9 Hz, 1H), 7.13 (d, J=2.4 Hz, 1H), 7.02 (dd, J=8.4 Hz, 2.4 Hz, 1H), 5.52 (br.s, 2H), 4.16 (t, J=5.4 Hz, 2H), 3.56 (t, J=5.4 Hz, 2H), 3.21 (s, 3H), 2.51 (s, 3H). ESI-MS m/z 234.2 (M+H+). Melting Point: 196° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-2-methyl-4-oxo-3,4-dihydroquinazolin-6-yl)urea (107): The compound was prepared by general procedure D provided in example 4 using compound 106 (0.14 g, 0.61 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.15 g, 0.76 mmol), 3′-aminoacetophenone (0.10 g, 0.76 mmol), TEA (0.4 mL, 2.53 mmol) to obtain compound 107 (0.08 g, 33%) as light yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.06 (s, 1H), 8.99 (s, 1H), 8.30 (d, J=2.4 Hz, 1H), 8.09 (s, 1H), 7.77 (dd, J=8.7 Hz, J=2.4 Hz, 1H), 7.70 (d, J=9 Hz, 1H), 7.60 (d, J=7.5 Hz, 1H), 7.53 (d, J=9 Hz, 1H), 7.48-7.42 (m, 1H), 4.23 (t, J=5.4 Hz, 2H), 3.62 (t, J=5.4 Hz, 2H), 3.24 (s, 3H), 2.60 (s, 3H), 2.57 (s, 3H). ESI-MS m/z 395.3 (M+H+). Melting Point: 218° C.
Synthesis of 2-isobutyramido-N-(2-methoxyethyl)-5-nitrobenzamide (108): Compound 2 (0.6 g, 2.50 mmol) was taken in dry DCM (10 mL) and TEA (0.8 mL, 5.52 mmol) was added to it. Under cooling condition, isobutyryl chloride (0.3 mL, 3.01 mmol) was added dropwise at 0° C. and reaction was monitored by checking TLC. After 5 hours, reaction was completed and reaction mixture was diluted with DCM and washed thoroughly with NaHCO3 solution and extracted to give light brown crude mass which was purified by column chromatography (Silica gel, mesh size 100-200) eluting (50% EtOAc/Pet ether) to obtain compound 108 (0.69 g, 89%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 11.79 (s, 1H), 9.21 (br.s, 1H), 8.64 (dd, J=9.2 Hz, 2.8 Hz, 1H), 8.61-8.60 (m, 1H), 8.31 (d, J=9.2 Hz, 1H), 3.45-3.42 (m, 4H), 3.23 (s, 3H), 2.59-2.52 (m, 1H), 1.11 (d, J=6.8 Hz, 6H). ESI-HRMS m/z 310.1390 (M+H+). Melting Point: 140° C.
Synthesis of 2-isopropyl-3-(2-methoxyethyl)-6-nitroquinazolin-4 (3H)-one (109): The compound was prepared by general procedure G provided in example 7 using compound 108 (0.5 g, 1.61 mmol), DMF (8 mL), ZnCl2 (0.88 g, 6.46 mmol), HMDS (2.71 mL, 12.93 mmol) to obtain compound 109 (0.39 g, 83%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.70 (d, J=6 Hz, 1H), 8.44-8.40 (m, 1H), 7.70 (d, J=8.8 Hz, 1H), 4.27 (t, J=5.2 Hz, 2H), 3.54 (t, J=5.2 Hz, 2H), 3.44-3.36 (m, 1H), 3.17 (s, 3H), 1.21 (d, J=6.4 Hz, 6H). ESI-HRMS m/z 292.1291 (M+H+). Melting Point: 136° C.
Synthesis of 6-amino-2-isopropyl-3-(2-methoxyethyl)quinazolin-4 (3H)-one (110): The compound was prepared by general procedure C provided in example 3 using compound 109 (0.22 g, 0.75 mmol), methanol (6 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 110 (0.17 g, 86%) as light brown solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 7.26 (d, J=7.6 Hz, 1H), 7.12 (d, J=2.8 Hz, 1H), 7.01 (dd, J=6 Hz, 2.8 Hz, 1H), 5.49 (s, 2H), 4.20 (t, J=5.6 Hz, 2H), 3.51 (t, J=5.6 Hz, 2H), 3.28-3.23 (m, 1H), 3.17 (s, 3H), 1.17 (d, J=6.8 Hz, 6H). ESI-HRMS m/z 262.1541 (M+H+). Melting Point: 170° C.
Synthesis of 1-(3-acetylphenyl)-3-(2-isopropyl-3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (111): The compound was prepared by general procedure D provided in example 4 using compound 110 (0.12 g, 0.45 mmol), dry THF (6 mL), 4-nitrophenylchloroformate (111 mg, 0.55 mmol), 3′-aminoacetophenone (0.075 g, 0.55 mmol), TEA (0.2 mL, 1.54 mmol) to obtain compound 111 (0.08 g, 41%) as light brown solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.09 (s, 1H), 8.99 (s, 1H), 8.31 (d, J=2.4 Hz, 1H), 8.10 (s, 1H), 7.79 (dd, J=8.7 Hz, J=2.4 Hz, 1H), 7.70 (d, J=7.8 Hz, 1H), 7.60 (d, J=7.8 Hz, 1H), 7.56 (d, J=8.7 Hz, 1H), 7.48-7.42 (m, 1H), 4.30 (t, J=5.4 Hz, 2H), 3.59 (t, J=5.4 Hz, 2H), 3.42-3.38 (m, 1H), 3.23 (s, 3H), 2.57 (s, 3H), 1.26 (d, J=6.6 Hz, 6H). ESI-MS m/z 423.3 (M+H+). Melting Point: 206° C.
Synthesis of 2-(4-fluorobenzamido)-N-(2-methoxyethyl)-5-nitrobenzamide (112): Compound 2 (0.8 g, 3.34 mmol) was taken in dry DCM (10 mL) and TEA (1.1 mL, 8.36 mmol) was added to it. Under cooling conditions, 4-fluorobenzoyl chloride (0.5 mL, 4.01 mmol) was added dropwise at 0° C. and reaction was monitored by checking TLC. After 3 hours, reaction was completed and reaction mixture was diluted with DCM and washed thoroughly with NaHCO3 solution and extracted to give light brown crude mass which was purified by column chromatography (Silica gel, mesh size 100-200) eluting (50% EtOAc/Pet ether) to obtain compound 112 (1.1 g, 91%) as white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 13.03 (s, 1H), 9.44 (s, 1H), 8.86 (d, J=9.3 Hz, 1H), 8.78 (s, 1H), 8.45 (d, J=9.3 Hz, 1H), 8.04-7.99 (m, 2H), 7.50-7.44 (m, 2H), 3.51 (br.s, 4H), 3.28 (s, 3H). Melting Point: 256° C.
Synthesis of 2-(4-fluorophenyl)-3-(2-methoxyethyl)-6-nitroquinazolin-4 (3H)-one (113): The compound was prepared by general procedure G provided in example 7 using compound 112 (0.7 g, 1.93 mmol), DMF (8 mL), ZnCl2 (1.3 g, 9.69 mmol), HMDS (4 mL, 19.38 mmol) to obtain compound 113 (0.59 g, 89%) as white solid. 1H NMR (600 MHz, CDCl3) δ in ppm 9.18 (d, J=3 Hz, 1H), 8.54 (dd, J=9 Hz, 2.4 Hz, 1H), 7.83 (d, J=9 Hz, 1H), 7.63-7.61 (m, 2H), 7.24-7.21 (m, 2H), 4.28 (t, J=4.8 Hz, 2H), 3.62 (t, J=5.4 Hz, 2H), 3.18 (s, 3H). ESI-HRMS m/z 344.1038 (M+H+). Melting Point: 156° C.
Synthesis of 6-amino-2-(4-fluorophenyl)-3-(2-methoxyethyl)quinazolin-4 (3H)-one (114): The compound was prepared by general procedure C provided in example 3 using compound 113 (0.25 g, 0.72 mmol), methanol (5 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 114 (0.19 g, 83%) as brown solid. 1H NMR (400 MHz, CDCl3) δ in ppm 7.55-7.51 (m, 3H), 7.46 (d, J=2.8 Hz, 1H), 7.17-7.13 (m, 2H), 7.09 (dd, J=8.8 Hz, 2.8 Hz, 1H), 4.17 (t, J=5.6 Hz, 2H), 3.56 (t, J=5.6 Hz, 2H), 3.15 (s, 3H). ESI-HRMS m/z 314.1304 (M+H+). Melting Point: 160° C.
Synthesis of 1-(3-acetylphenyl)-3-(2-(4-fluorophenyl)-3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (115): The compound was obtained by general procedure D provided in example 4 using compound 110 (0.12 g, 0.38 mmol), dry THF (6 mL), 4-nitrophenylchloroformate (91 mg, 0.45 mmol), TEA (0.159 mL, 1.14 mmol) to obtain compound 115 (0.08 g, 44%) as white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.18 (s, 1H), 9.04 (s, 1H), 8.43 (d, J=2.4 Hz, 1H), 8.12-8.11 (m, 1H), 7.83 (dd, J=8.7 Hz, 2.4 Hz, 1H), 7.73-7.67 (m, 3H), 7.64-7.60 (m, 2H), 7.49-7.44 (m, 1H), 7.40-7.34 (m, 2H), 4.10 (t, J=5.7 Hz, 2H), 3.45 (t, J=5.7 Hz, 2H), 3.04 (s, 3H), 2.58 (s, 3H). ESI-MS m/z 475.1 (M+H+). Melting Point: 220° C.
Synthesis of 2-(4-methoxybenzamido)-N-(2-methoxyethyl)-5-nitrobenzamide (116): Compound 2 (0.6 g, 2.50 mmol) was taken in dry DCM (10 mL) and TEA (0.77 mL, 5.52 mmol) was added to it. Under cooling conditions, 4-methoxybenzoyl chloride (0.4 mL, 3.01 mmol) was added dropwise at 0° C. and reaction was monitored by checking TLC. After 3 hours, reaction was completed and reaction mixture was diluted with DCM and washed thoroughly with NaHCO3 solution and extracted to give light brown crude mass which was purified by column chromatography (Silica gel, mesh size 100-200) eluting (60% EtOAc/Pet ether) to obtain compound 116 (0.8 g, 85%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 12.89 (s, 1H), 9.36 (br.s, 1H), 8.84 (d, J=8.8 Hz, 1H), 8.71 (d, J=2.4 Hz, 1H), 8.36 (q, J=6.8 Hz, 2.8 Hz, 1H), 7.87 (d, J=8.8 Hz, 2H), 7.09 (d, J=8.8 Hz, 2H), 3.80 (s, 3H), 3.49-3.45 (m, 4H), 3.24 (s, 3H). ESI-HRMS m/z 374.1329 (M+H+). Melting Point: 184° C.
Synthesis of 3-(2-methoxyethyl)-2-(4-methoxyphenyl)-6-nitroquinazolin-4 (3H)-one (117): The compound was prepared by general procedure G provided in example 7 using compound 116 (0.6 g, 1.60 mmol), DMF (8 mL), ZnCl2 (1.0 g, 8.04 mmol), HMDS (3.3 mL, 16.08 mmol) to obtain compound 117 (0.59 g, 87%) as white solid. 1H NMR (400 MHz, CDCl3) δ in ppm 9.15 (d, J=2.4 Hz, 1H), 8.50 (dd, J=9 Hz, 2.4 Hz, 1H), 7.80 (d, J=9.2 Hz, 1H), 7.54 (d, J=8.8 Hz, 2H), 7.02 (d, J=8.8 Hz, 2H), 4.32 (t, J=5.6 Hz, 2H), 3.87 (s, 3H), 3.59 (t, J=5.6 Hz, 2H), 3.16 (s, 3H). ESI-HRMS m/z 356.1225 (M+H+). Melting Point: 162° C.
Synthesis of 6-amino-3-(2-methoxyethyl)-2-(4-methoxyphenyl)quinazolin-4 (3H)-one (118): The compound was prepared by general procedure C provided in example 3 using compound 117 (0.25 g, 0.70 mmol), methanol (5 mL), pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 118 (0.18 g, 89%) as brown solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 7.46 (d, J=7.2 Hz, 2H), 7.31 (d, J=8.4 Hz, 1H), 7.18 (d, J=2.8 Hz, 1H), 7.05 (dd, J=6 Hz, 2.4 Hz, 1H), 6.99 (d, J=8.8 Hz, 2H), 5.62 (s, 2H), 4.05 (t, J=6 Hz, 2H), 3.78 (s, 3H), 3.36 (t, J=6 Hz, 2H), 2.99 (s, 3H). ESI-HRMS m/z 326.1478 (M+H+). Melting Point: 172° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-2-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (119): The compound was prepared by general procedure D provided in example 4 using compound 118 (0.12 g, 0.36 mmol), dry THF (6 mL), 4-nitrophenylchloroformate (89 mg, 0.44 mmol), 3′-aminoacetophenone (0.061 g, 0.44 mmol), TEA (0.15 mL, 1.10 mmol) to obtain compound 119 (0.075 g, 42%) as white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.16 (s, 1H), 9.03 (s, 1H), 8.40 (s, 1H), 8.11 (s, 1H), 7.81 (d, J=8.7 Hz, 1H), 7.71 (d, J=8.1 Hz, 1H), 7.62-7.55 (m, 4H), 7.49-7.43 (m, 1H), 7.07 (d, J=8.1 Hz, 2H), 4.15 (t, J=5.7 Hz, 2H), 3.83 (s, 3H), 3.46-3.42 (m, 2H) 3.04 (s, 3H), 2.58 (s, 3H). ESI-MS m/z 487.1 (M+H+). Melting Point: 220° C.
Synthesis of 2-(cyclohexanecarboxamido)-N-(2-methoxyethyl)-5-nitrobenzamide (120): Compound 2 (0.6 g, 2.50 mmol) was taken in dry DCM (10 mL) and TEA (0.9 mL, 6.27 mmol) was added to it. Cyclohexanecarbonyl chloride (0.6 mL, 5.01 mmol) was added dropwise at 0° C. and reaction was monitored by checking TLC. After 3 hours, reaction was completed and reaction mixture was diluted with DCM and washed thoroughly with NaHCO3 solution and extracted to give light brown crude mass which was purified by column chromatography (Silica gel, mesh size 100-200) eluting (50% EtOAc/Pet ether) to obtain compound 120 (0.75 g, 86%) as white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 11.83 (s, 1H), 9.30-9.26 (m, 1H), 8.72 (d, J=9 Hz, 1H), 8.66 (d, J=2.7 Hz, 1H), 8.37 (dd, J=6.9 Hz, 2.4 Hz, 1H), 3.50-3.40 (m, 4H), 3.29 (s, 3H), 2.39-2.32 (m, 1H), 1.92-1.88 (m, 2H), 1.77-1.66 (m, 3H), 1.47-1.30 (m, 4H), 1.29-1.16 (m, 1H). ESI-HRMS m/z 350.1698 (M+H+). Melting Point: 130° C.
Synthesis of 2-cyclohexyl-3-(2-methoxyethyl)-6-nitroquinazolin-4 (3H)-one (121): The compound was prepared by general procedure G provided in example 7 using compound 120 (0.55 g, 1.57 mmol), DMF (8 mL), ZnCl2 (1.07 g, 7.87 mmol), HMDS (3.3 mL, 15.75 mmol) to obtain compound 121 (0.45 g, 86%) as white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.80 (d, J=2.7 Hz, 1H), 8.51 (dd, J=6.3 Hz, 2.7 Hz, 1H), 7.79 (d, J=9 Hz, 1H), 4.34 (t, J=5.4 Hz, 2H), 3.62 (t, J=5.4 Hz, 2H), 3.24 (s, 3H), 3.15-3.08 (m, 1H), 1.92-1.88 (m, 3H), 1.82-1.78 (m, 3H), 1.74-1.66 (m, 2H), 1.46-1.33 (m, 2H). ESI-HRMS m/z 332.1614 (M+H+). Melting Point: 148° C.
Synthesis of 6-amino-2-cyclohexyl-3-(2-methoxyethyl)quinazolin-4 (3H)-one (122): The compound was prepared by general procedure C provided in example 3 using compound 121 (0.3 g, 0.90 mmol), methanol (5 mL) and pinch of 10% wet Pd—C to obtain compound 122 (0.25 g, 92%) as brown solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 7.30 (d, J=8.7 Hz, 1H), 7.15 (d, J=2.4 Hz, 1H), 7.04 (dd, J=8.4 Hz, 2.4 Hz, 1H), 5.53 (s, 2H), 4.23 (t, J=5.4 Hz, 2H), 3.56 (t, J=5.4 Hz, 2H), 3.23 (s, 3H), 2.99-2.89 (m, 1H), 1.85-1.72 (m, 5H), 1.62-1.51 (m, 2H), 1.42-1.25 (m, 3H). ESI-HRMS m/z 302.1852 (M+H+). Melting Point: 146° C.
Synthesis of 1-(3-acetylphenyl)-3-(2-cyclohexyl-3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (123): The compound was prepared by general procedure D provided in example 4 using compound 122 (0.12 g, 0.39 mmol), dry THF (7 mL), 4-nitrophenylchloroformate (0.12 g, 0.59 mmol), 3′-aminoacetophenone (0.06 g, 0.47 mmol), TEA (0.2 mL, 1.64 mmol) to obtain compound 123 (0.075 g, 41%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.01 (s, 1H), 8.92 (s, 1H), 8.24 (d, J=2.8 Hz, 1H), 8.04-8.03 (m, 1H), 7.73 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.66-7.63 (m, 1H), 7.56-7.53 (m, 1H), 7.49 (d, J=8.8 Hz, 1H), 7.41-7.37 (m, 1H), 4.23 (t, J=5.6 Hz, 2H), 3.54 (t, J=5.6 Hz, 2H), 3.19 (s, 3H), 3.00-2.93 (m, 1H), 2.52 (s, 3H), 1.83-1.72 (m, 4H), 1.67-1.50 (m, 3H), 1.38-1.16 (m, 3H). ESI-MS m/z 463.3 (M+H+). Melting Point: 202° C.
Synthesis of 2-(cyclopentanecarboxamido)-N-(2-methoxyethyl)-5-nitrobenzamide (124): Compound 2 (0.6 g, 2.50 mmol) was taken in dry DCM (10 mL) and TEA (1.0 mL, 7.52 mmol) was added to it. Cyclopentanecarbonyl chloride (1 mL, 10.03 mmol) was added dropwise at 0° C. and reaction was monitored by checking TLC. After 5 hours, reaction was completed and reaction mixture was diluted with DCM and washed thoroughly with NaHCO3 solution and extracted to give light brown crude mass which was purified by column chromatography (Silica gel, mesh size 100-200) eluting (50% EtOAc/Pet ether) to obtain compound 124 (0.75 g, 89%) as white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 11.82 (s, 1H), 9.28-9.25 (m, 1H), 8.70 (d, J=9.3 Hz, 1H), 8.66 (d, J=2.7 Hz, 1H), 8.37 (dd, J=8.4 Hz, 2.4 Hz, 1H), 3.53-3.46 (m, 4H), 3.32 (s, 3H), 2.87-2.79 (m, 1H), 1.94-1.87 (m, 2H), 1.79-1.56 (m, 6H). ESI-HRMS m/z 336.1560 (M+H+). Melting Point: 126° C.
Synthesis of 2-cyclopentyl-3-(2-methoxyethyl)-6-nitroquinazolin-4 (3H)-one (125): The compound was prepared by general procedure G provided in example 7 using compound 124 (0.6 g, 1.79 mmol), DMF (8 mL), ZnCl2 (0.97 g, 7.16 mmol), HMDS (3 mL, 14.32 mmol) to obtain compound 125 (0.48 g, 84%) as white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.79 (d, J=2.4 Hz, 1H), 6.51 (dd, J=9 Hz, 2.7 Hz, 1H), 7.77 (d, J=9 Hz, 1H), 4.35 (t, J=5.4 Hz, 2H), 3.62 (q, J=5.4 Hz, 2H), 3.57-3.52 (m, 1H), 3.24 (s, 3H), 2.06-1.94 (m, 4H), 1.82-1.64 (m, 4H). ESI-HRMS m/z 318.1449 (M+H+). Melting Point: 130° C.
Synthesis of 6-amino-2-cyclopentyl-3-(2-methoxyethyl)quinazolin-4 (3H)-one (126): The compound was prepared by general procedure C provided in example 3 using compound 125 (0.3 g, 0.94 mmol), methanol (5 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 126 (0.22 g, 81%) as brown solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 7.35 (d, J=8.4 Hz, 1H), 7.26 (d, J=7.2 Hz, 1H), 7.13 (dd, J=9 Hz, 2.4 Hz, 1H), 4.26 (t, J=5.7 Hz, 2H), 3.57 (t, J=5.7 Hz, 2H), 3.46-3.41 (m, 1H), 3.23 (s, 3H), 2.01-1.87 (m, 4H), 1.84-1.75 (m 2H), 1.70-1.58 (m, 2H). ESI-HRMS m/z 288.1704 (M+H+). Melting Point: 142° C.
Synthesis of 1-(3-acetylphenyl)-3-(2-cyclopentyl-3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (127): The compound was prepared by general procedure D provided in example 4 using compound 126 (0.12 g, 0.41 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.12 g, 0.62 mmol), 3′-aminoacetophenone (0.07 g, 0.52 mmol), TEA (0.2 mL, 1.72 mmol) to obtain compound 127 (0.079 g, 42%) as brown solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.08 (s, 1H), 9.00 (s, 1H), 8.30 (d, J=2.4 Hz, 1H), 8.10 (s, 1H), 7.78 (dd, J=8.8 Hz, 2.7 Hz, 1H), 7.70 (d, J=9 Hz, 1H), 7.60 (d, J=7.5 Hz, 1H), 7.54 (d, J=8.7 Hz, 1H), 7.48-7.42 (m, 1H), 4.31 (t, J=5.4 Hz, 2H), 3.60 (t, J=5.4 Hz, 2H), 3.54-3.44 (m, 1H), 3.24 (s, 3H), 2.57 (s, 3H), 2.04-1.86 (m, 4H), 1.82-1.73 (m, 2H), 1.69-1.61 (m, 2H). ESI-MS m/z 449.2 (M+H+). Melting Point: 204° C.
Synthesis of 2-amino-N-(2-methoxyphenyl)-5-nitrobenzamide (128): The compound was prepared by general procedure A provided in example 1 using compound 1 (1 g, 5.49 mmol), DMF (10 mL), HATU (2.2 g, 6.04 mmol), o-anisidine (0.7 mL, 6.04 mmol), TEA (1.9 mL, 13.73 mmol) to obtain compound 128 (1.2 g, 76%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.83 (s, 1H), 8.64 (d, J=2.4 Hz, 1H), 8.07 (dd, J=8.7 Hz, J=2.4 Hz, 1H), 7.68 (s, 2H), 7.54 (dd, J=7.8 Hz, J=1.8 Hz, 1H), 7.24-7.19 (m, 1H), 7.11-7.08 (m, 1H), 6.99-6.93 (m, 1H), 6.85 (d, J=9.3 Hz, 1H), 3.82 (s, 3H). ESI-HRMS m/z 288.0998 (M+H+). Melting Point: 178° C.
Synthesis of 3-(2-methoxyphenyl)-6-nitroquinazolin-4 (3H)-one (129): The compound was prepared by general procedure B provided in example 2 using compound 128 (1 g, 2.78 mmol), trimethylorthoformate (TMOF) (6 mL, 55.73 mmol) to obtain compound 129 (0.69 g, 83%) as pale yellow solid. 1H NMR (400 MHz, CDCl3) δ in ppm 9.18 (d, J=2.4 Hz, 1H), 8.55 (dd, J=9.2 Hz, J=2.8 Hz, 1H), 8.08 (s, 1H), 7.87 (d, J=9.2 Hz, 1H), 7.51-7.47 (m, 2H), 7.33-7.31 (m, 1H), 7.13-7.09 (m, 1H), 3.81 (s, 3H). ESI-HRMS m/z 298.0836 (M+H+). Melting Point: 164° C.
Synthesis of 6-amino-3-(2-methoxyphenyl)quinazolin-4 (3H)-one (130): The compound was prepared by general procedure C provided in example 3 using compound 129 (0.8 g, 2.69 mmol), methanol (10 mL), pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 130 (0.65 g, 90%) as light brown solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 7.83 (s, 1H), 7.52-7.39 (m, 3H), 7.25-7.22 (m, 2H), 7.14-7.07 (m, 2H), 5.72 (brs, 2H), 3.76 (s, 3H). ESI-HRMS m/z 268.1096 (M+H+). Melting Point: 169° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (131): The compound was prepared by general procedure D provided in example 4 using compound 130 (0.12 g, 0.44 mmol), dry THF (6 mL) and 4-nitrophenylchloroformate (0.13 g, 0.67 mmol), 3′-aminoacetophenone (0.075 g, 0.56 mmol), TEA (0.3 mL, 1.85 mmol) to obtain compound 131 (0.05 g, 27%) as light yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.18 (s, 1H), 9.01 (s, 1H), 8.37 (d, J=2.8 Hz, 1H), 8.10-8.09 (m, 1H), 8.07 (s, 1H), 7.89 (dd, J=8.8 Hz, 2.8 Hz, 1H), 7.77-7.72 (m, 2H), 7.61-7.59 (m, 1H), 7.54-7.49 (m, 1H), 7.47-7.43 (m, 2H), 7.26 (dd, J=8.4 Hz, 1.2 Hz, 1H), 7.14-7.10 (m, 1H), 3.77 (s, 3H), 2.57 (s, 3H).
Synthesis of 2-amino-N-(2-morpholinoethyl)-5-nitrobenzamide (132): The compound was prepared by general procedure A provided in example 1 using compound 1 (1 g, 5.49 mmol), DMF (10 mL), HATU (2.2 g, 6.04 mmol), 4-(2-aminoethyl)morpholine (0.8 mL, 6.04 mmol), TEA (1.9 mL, 13.73 mmol) to obtain compound 132 (0.9 g, 56%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.64 (t, J=5.7 Hz, 1H), 8.46 (d, J=2.7 Hz, 1H), 8.02 (dd, J=9.3 Hz, J=2.4 Hz, 1H), 7.73 (s, 2H), 6.78 (d, J=9.3 Hz, 1H), 3.57 (t, J=4.5 Hz, 4H), 3.39-3.36 (m, 2H), 2.51-2.48 (m, 2H), 2.43-2.39 (m, 4H). ESI-HRMS m/z 295.1411 (M+H+). Melting Point: 152° C.
Synthesis of 3-(2-morpholinoethyl)-6-nitroquinazolin-4 (3H)-one (133): The compound was prepared by general procedure B provided in example 2 using compound 132 (0.6 g, 2.03 mmol), trimethylorthoformate (TMOF) (4 mL, 40.79 mmol) to obtain compound 133 (0.42 g, 68%) as pale yellow solid. ESI-HRMS m/z 305.1247 (M+H+). Melting Point: 142° C.
Synthesis of 6-amino-3-(2-morpholinoethyl)quinazolin-4 (3H)-one (134): The compound was preapred by general procedure C provided in example 3 using compound 133 (0.25 g, 0.82 mmol), methanol (6 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 134 (0.15 g, 67%) as light brown solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.72 (s, 1H), 8.63 (d, J=1.5 Hz, 1H), 8.11 (s, 1H), 7.53-7.48 (m, 2H), 7.25 (dd, J=8.7 Hz, 2.4 Hz, 1H), 4.06 (t, J=6 Hz, 2H), 3.52-3.51 (m, 4H), 2.59 (t, J=3.9 Hz, 2H), 2.43 (m, 4H). ESI-HRMS m/z 275.1513 (M+H+). Melting Point: 186° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-morpholinoethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (135): The compound was prepared by general procedure E provided in example 5 using compound 134 (0.12 g, 0.43 mmol), dry THF (6 mL), 3-acetylphenylisocyanate (0.071 mL, 0.51 mmol), TEA (0.3 mL, 1.80 mmol) to obtain compound 135 (0.04 g, 21%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 11.00 (s, 1H), 9.74 (s, 1H), 8.33 (d, J=2.4 Hz, 2H), 8.27 (s, 1H), 8.16 (dd, J=9 Hz, 4 Hz, 1H), 7.96 (d, J=8.1 Hz, 1H), 7.69-7.63 (m, 2H), 7.48-7.43 (m, 1H), 4.10 (t, J=6 Hz, 2H), 3.54-3.51 (m, 4H), 2.61 (t, J=6 Hz, 2H), 2.57 (s, 3H), 2.45-2.42 (m, 4H). ESI-HRMS m/z 436.1987 (M+H+). Melting Point: 140° C.
Synthesis of 2-amino-N-(3-morpholinopropyl)-5-nitrobenzamide (136): The compound was prepared by general procedure A provided in example 1 using compound 1 (1 g, 5.49 mmol), DMF (10 mL), HATU (2.2 g, 6.04 mmol), 3-morpholinopropylamine (0.9 mL, 6.04 mmol), TEA (1.9 mL, 13.73 mmol) to obtain compound 136 (1 g, 59%) as yellow solid. ESI-HRMS m/z 309.1568 (M+H+). Melting Point: 126° C.
Synthesis of 3-(3-morpholinopropyl)-6-nitroquinazolin-4 (3H)-one (137): The compound was prepared by general procedure B provided in example 2 using compound 136 (0.6 g, 1.94 mmol), trimethylorthoformate (TMOF) (4 mL, 38.94 mmol) to obtain compound 137 (0.48 g, 88%) as pale yellow solid. ESI-HRMS m/z 319.1398 (M+H+). Melting Point: 118° C.
Synthesis of 6-amino-3-(3-morpholinopropyl)quinazolin-4 (3H)-one (138): The compound was prepared by general procedure C provided in example 3 using compound 137 (0.3 g, 0.94 mmol), methanol (8 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 138 (0.18 g, 66%) as light brown solid. ESI-HRMS m/z 289.1664 (M+H+). Melting Point: 148° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(3-morpholinopropyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (139): The compound was prepared by general procedure D provided in example 4 using compound 138 (0.12 g, 0.41 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.13 g, 0.62 mmol), 3′-aminoacetophenone (0.07 g, 0.52 mmol), TEA (0.3 mL, 1.71 mmol) to obtain compound 139 (0.04 g, 22%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.13 (s, 1H), 8.99 (s, 1H), 8.30 (s, 1H), 8.21 (s, 1H), 8.04 (s, 1H), 7.77-7.74 (m, 1H), 7.65-7.62 (m, 1H), 7.58-7.53 (m, 2H), 7.55 (dd, J=11.1 Hz, 6.9 Hz, 2H), 7.42-7.38 (m, 1H), 3.96 (t, J=3 Hz, 2H), 3.48-3.41 (m, 4H), 2.52 (s, 3H), 2.37-2.19 (m, 6H), 1.86-1.79 (m, 2H). ESI-HRMS m/z 450.2160 (M+H+). Melting Point: 178° C.
Synthesis of 2-amino-N-(2-(dimethylamino)ethyl)-5-nitrobenzamide (140): The compound was prepared by general procedure A provided in example 1 using compound 1 (1 g, 5.49 mmol), DMF (10 mL), HATU (2.2 g, 6.04 mmol), N,N-dimethylethylenediamine (0.7 mL, 6.04 mmol), TEA (1.9 mL, 13.73 mmol) to obtain compound 140 (0.8 g, 56%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.83 (d, J=2.7 Hz, 1H), 8.55 (dd, J=9 Hz, j=2.7 Hz, 1H), 8.52 (s, 1H), 7.86 (d, J=9 Hz, 1H), 4.11 (t, J=6 Hz, 2H), 2.57 (t, J=6 Hz, 2H), 2.19 (s, 6H). ESI-HRMS m/z 253.1304 (M+H+). Melting Point: 116° C.
Synthesis of 3-(2-(dimethylamino)ethyl)-6-nitroquinazolin-4 (3H)-one (141): The compound was prepared by general procedure B provided in example 2 using compound 140 (0.6 g, 2.37 mmol), trimethylorthoformate (TMOF) (4 mL, 38.94 mmol) to obtain compound 141 (0.49 g, 86%) as pale yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.83 (d, J=2.7 Hz, 1H), 8.55 (dd, J=9 Hz, J=2.7 Hz, 1H), 8.52 (s, 1H), 7.86 (d, J=9 Hz, 1H), 4.11 (t, J=6 Hz, 2H), 2.58 (t, J=6 Hz, 2H), 2.20 (s, 6H). ESI-HRMS m/z 263.1154 (M+H+). Melting Point: 115° C.
Synthesis of 6-amino-3-(2-(dimethylamino)ethyl)quinazolin-4 (3H)-one (142): The compound was prepared by general procedure C provided in example 3 using compound 141 (0.3 g, 1.14 mmol), methanol (8 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 142 (0.19 g, 72%) as light brown solid. Melting Point: 178° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-(dimethylamino)ethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (143): The compound was prepared by general procedure E provided in example 5 using compound 142 (0.12 g, 0.51 mmol), dry THF (8 mL), 3-acetylphenylisocyanate (0.065 mL, 0.61 mmol), TEA (0.3 mL, 1.71 mmol) to obtain compound 143 (0.109 g, 54%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.17 (s, 1H), 9.04 (s, 1H), 8.34 (d, J=2.4 Hz, 1H), 8.19 (s, 1H), 8.09 (brs, 1H), 7.79 (d, J=8.7 Hz, J=2.4 Hz, 1H), 7.69 (d, J=8.1 Hz, 1H), 7.62-7.57 (m, 2H), 7.46-7.41 (m, 1H), 4.06 (t, J=6 Hz, 2H), 2.60-2.56 (m, 5H), 2.20 (s, 6H). ESI-HRMS m/z 394.1879 (M+H+).
Synthesis of 2-amino-5-nitro-N-(2-(piperidin-1-yl)ethyl)benzamide (144): The compound was prepared by general procedure E provided in example 5 using compound 1 (1 g, 5.49 mmol), DMF (10 mL), HATU (2.2 g, 6.04 mmol), 1-(2-aminoethyl)piperidine (0.83 mL, 6.04 mmol), TEA (1.9 mL, 13.73 mmol) to obtain compound 144 (0.7 g, 62%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.58 (t, J=5.7 Hz, 1H), 8.41 (d, J=2.7 Hz, 1H), 7.99 (dd, J=9.0 Hz, J=2.4 Hz, 1H), 7.70 (s, 2H), 6.76 (d, J=9.3 Hz, 1H), 2.42-2.33 (m, 6H), 1.50-1.33 (m, 2H). ESI-HRMS m/z 293.1614 (M+H+). Melting Point: 148° C.
Synthesis of 6-nitro-3-(2-(piperidin-1-yl)ethyl)quinazolin-4 (3H)-one (145): The compound was prepared by general procedure B provided in example 2 using compound 144 (0.6 g, 2.05 mmol), trimethylorthoformate (TMOF) (4 mL, 39.56 mmol) to obtain compound 145 (0.52 g, 88%) as pale yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.79 (d, J=2.8 Hz, 1H), 8.50 (dd, J=9.2 Hz, J=2.8 Hz, 1H), 8.43 (s, 1H), 7.82 (d, J=9.2 Hz, 1H), 4.05 (t, J=6 Hz, 2H), 2.51 (t, J=6 Hz, 2H), 2.33 (m, 4H), 1.39-1.35 (m, 4H), 1.32-1.30 (m, 2H). ESI-HRMS m/z 303.1453 (M+H+). Melting Point: 120° C.
Synthesis of 6-amino-3-(2-(piperidin-1-yl)ethyl)quinazolin-4 (3H)-one (146): The compound was prepared by general procedure C provided in example 3 using compound 145 (0.3 g, 0.99 mmol), methanol (8 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 146 (0.189 g, 70%) as light brown solid.
Synthesis of 1-(3-acetylphenyl)-3-(4-oxo-3-(2-(piperidin-1-yl)ethyl)-3,4-dihydroquinazolin-6-yl)urea (147): The compound was prepared by general procedure E provided in example 5 using compound 146 (0.130 g, 0.47 mmol), dry THF (8 mL), 3-acetylphenylisocyanate (0.065 mL, 0.61 mmol), TEA (0.3 mL, 1.71 mmol) to obtain compound 147 (0.095 g, 46%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.53 (s, 1H), 8.35 (s, 1H), 8.20 (dd, J=8.8 Hz, 2 Hz, 1H), 8.00 (d, J=2.8 Hz, 1H), 7.96 (s, 1H), 7.90-7.87 (m, 2H), 7.61-7.56 (m, 2H), 7.38-7.34 (m, 1H), 4.07 (t, J=5.6 Hz, 2H), 2.69 (t, J=4.8 Hz, 2H), 2.59 (s, 3H), 2.45 (brs, 4H), 1.54-1.50 (m, 4H), 1.41-1.38 (m, 2H). ESI-HRMS m/z 434.2193 (M+H+).
Synthesis of 2-amino-5-nitro-N-(pyridin-4-yl)benzamide (148): The compound was prepared by general procedure A provided in example 1 using compound 1 (1 g, 5.49 mmol), DMF (10 mL), HATU (2.2 g, 6.04 mmol), 4-aminopyridine (0.62 mL, 6.04 mmol), TEA (1.9 mL, 13.73 mmol) to obtain compound 148 (1.02 g, 72%) as yellow solid. ESI-HRMS m/z 259.0833 (M+H+). Melting Point: 246° C.
Synthesis of 6-nitro-3-(pyridin-4-yl)quinazolin-4 (3H)-one (149): The compound was prepared by general procedure B provided in example 2 using compound 148 (0.6 g, 2.32 mmol), trimethylorthoformate (TMOF) (4 mL, 39.56 mmol) to obtain compound 149 (0.56 g, 90%) as pale yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.83 (d, J=2.4 Hz, 1H), 8.77 (dd, J=4.8 Hz, 1.2 Hz, 2H), 8.60-8.57 (m, 2H), 7.92 (d, J=8.8 Hz, 1H), 7.64 (dd, J=4.4 Hz, 1.6 Hz, 2H) Melting Point: 202° C.
Synthesis of 6-amino-3-(pyridin-4-yl)quinazolin-4 (3H)-one (150): The compound was prepared by general procedure C provided in example 3 using compound 149 (0.3 g, 1.11 mmol), methanol (8 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 150 (0.191 g, 72%) as light brown solid.
Synthesis of 1-(3-acetylphenyl)-3-(4-oxo-3-(pyridin-4-yl)-3,4-dihydroquinazolin-6-yl)urea (151): The compound was prepared by general procedure D provided in example 4 using compound 150 (0.130 g, 0.54 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.131 g, 0.65 mmol), 3′-aminoacetophenone (0.081 g, 0.60 mmol), TEA (0.3 mL, 1.71 mmol) to obtain compound 151 (0.115 g, 53%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.29 (s, 1H), 9.09 (s, 1H), 8.79 (d, J=6 Hz, 2H), 8.43 (d, J=2.4 Hz, 1H), 8.30 (s, 1H), 8.10 (brs, 1H), 7.91 (dd, J=9 Hz, J=2.7 Hz, 1H), 7.73 (s, 1H), 7.70-7.67 (m, 4H), 7.48-7.43 (m, 1H), 2.58 (s, 3H). ESI-HRMS m/z 400.1411 (M+H+).
Synthesis of 2-amino-5-nitro-N-(pyridin-3-yl)benzamide (152): The compound was prepared by general procedure A provided in example 1 using compound 1 (1 g, 5.49 mmol), DMF (10 mL), HATU (2.2 g, 6.04 mmol), 3-aminopyridine (0.62 mL, 6.04 mmol), TEA (1.9 mL, 13.73 mmol) to obtain compound 152 (1.02 g, 72%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.19 (d, J=2.8 Hz, 1H), 8.78 (dd, J=4.8 Hz, J=1.6 Hz, 1H), 8.70 (d, J=2.8 Hz, 1H), 8.59 (dd, J=9.2 Hz, J=2.8 Hz, 1H), 8.22 (s, 1H), 7.91 (d, J=8.8 Hz, 1H), 7.86-7.83 (m, 1H), 7.56-7.52 (m, 1H). ESI-HRMS m/z 259.0844 (M+H+). Melting Point: 266° C.
Synthesis of 6-nitro-3-(pyridin-3-yl)quinazolin-4 (3H)-one (153): The compound was prepared by general procedure B provided in example 2 using compound 152 (0.6 g, 2.32 mmol), trimethylorthoformate (TMOF) (4 mL, 39.56 mmol) to obtain compound 153 (0.56 g, 90%) as pale yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.88 (d, J=2.4 Hz, 1H), 8.80 (d, J=2.1 Hz, 1H), 8.74 (dd, J=4.8 Hz, J=1.2 Hz, 1H), 8.65 (s, 1H), 8.63 (d, J=2.7 Hz, 1H), 8.10-8.06 (m, 1H), 7.98 (d, J=9 Hz, 1H). Melting Point: 263° C.
Synthesis of 6-amino-3-(pyridin-3-yl)quinazolin-4 (3H)-one (154): The compound was prepared by general procedure C provided in example 3 using compound 153 (0.3 g, 1.11 mmol), methanol (8 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 154 (0.191 g, 72%) as light brown solid.
Synthesis of 1-(3-acetylphenyl)-3-(4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-6-yl)urea (155): The compound was prepared by general procedure D provided in example 4 using compound 154 (0.130 g, 0.54 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.131 g, 0.65 mmol), 3′-aminoacetophenone (0.081 g, 0.60 mmol), TEA (0.3 mL, 1.71 mmol) to obtain compound 155 (0.093 g, 48%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.81 (d, J=2.1 Hz, 1H), 8.71 (dd, J=4.8 Hz, J=1.5 Hz, 1H), 8.48 (s, 1H), 8.34 (d, J=2.4 Hz, 1H), 8.21-8.19 (m, 1H), 8.15-8.06 (m, 3H), 7.96 (d, J=8.7 Hz, 1H), 7.88 (d, J=8.1 Hz, 1H), 7.79-7.74 (m, 1H), 7.67-7.63 (m, 1H), 2.63 (s, 3H). ESI-HRMS m/z 400.1412 (M+H+).
Synthesis of 2-amino-5-nitro-N-(pyridin-2-yl)benzamide (156): The compound was prepared by general procedure A provided in example 1 using compound 1 (1 g, 5.49 mmol), DMF (10 mL), HATU (2.2 g, 6.04 mmol), 2-aminopyridine (0.62 mL, 6.04 mmol), TEA (1.9 mL, 13.73 mmol) to obtain compound 156 (0.98 g, 69%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 10.99 (s, 1H), 8.60 (d, J=2.4 Hz, 1H), 8.36-8.34 (m, 1H), 8.04-8.00 (m, 2H), 7.81-7.77 (m, 1H), 7.59 (brs, 2H), 7.14-7.11 (m, 1H), 6.80 (d, J=9.2 Hz, 1H). ESI-HRMS m/z 281.0650 (M+Na+). Melting Point: 180° C.
Synthesis of 6-nitro-3-(pyridin-2-yl)quinazolin-4 (3H)-one (157): The compound was prepared by general procedure B provided in example 2 using compound 156 (0.6 g, 2.32 mmol), trimethylorthoformate (TMOF) (4 mL, 39.56 mmol) to obtain compound 157 (0.52 g, 89%) as pale yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.20 (d, J=2.8 Hz, 1H), 8.77 (s, 1H), 8.63-8.61 (m, 1H), 8.56 (dd, J=9 Hz, 2.8 Hz, 1H), 7.94-7.88 (m, 3H), 7.44-7.41 (m, 1H). ESI-HRMS m/z 269.0677 (M+H+). Melting Point: 220° C.
Synthesis of 6-amino-3-(pyridin-2-yl)quinazolin-4 (3H)-one (158): The compound was prepared by general procedure C provided in example 3 using compound 157 (0.3 g, 1.11 mmol), methanol (8 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 158 (0.183 g, 71%) as light brown solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.64 (d, J=4.2 Hz, 1H), 8.23 (s, 1H), 8.07-8.01 (m, 1H), 7.80 (d, J=8.1 Hz, 1H), 7.56-7.51 (m, 1H), 7.46 (d, J=8.4 Hz, 1H), 7.30 (d, J=2.4 Hz, 1H), 7.13 (dd, J=8.7 Hz, 2.7 Hz, 1H), 5.79 (brs, 2H).
Synthesis of 1-(3-acetylphenyl)-3-(4-oxo-3-(pyridin-2-yl)-3,4-dihydroquinazolin-6-yl)urea (159): The compound was prepared by general procedure D provided in example 4 using compound 158 (0.130 g, 0.54 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.131 g, 0.65 mmol), 3′-aminoacetophenone (0.081 g, 0.60 mmol), TEA (0.3 mL, 1.71 mmol) to obtain compound 159 (0.095 g, 44%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.21 (s, 1H), 9.02 (s, 1H), 8.66 (dd, J=4.8 Hz, 1.2 Hz, 1H), 8.46 (s, 1H), 8.45 (s, 1H), 8.10-8.05 (m, 2H), 7.91-7.83 (m, 2H), 7.71 (d, J=8.8 Hz, 2H), 7.61-7.55 (m, 2H), 7.47-7.43 (m, 1H), 2.57 (s, 3H). ESI-HRMS m/z 400.1407 (M+H+).
Synthesis of 2-amino-N-(1-methylpiperidin-4-yl)-5-nitrobenzamide (160): The compound was prepared by general procedure A provided in example 1 using compound 1 (1 g, 5.49 mmol), DMF (10 mL), HATU (2.2 g, 6.04 mmol), 1-methylpiperidin-4-amine (0.75 mL, 6.58 mmol), TEA (1.9 mL, 13.73 mmol) to obtain compound 160 (1.007 g, 66%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.53 (d, J=7.5 Hz, 1H), 8.48 (d, J=2.7 Hz, 1H), 8.01 (dd, J=9.3 Hz, 2.7 Hz, 1H), 7.72 (s, 2H), 6.78 (d, J=9.3 Hz, 1H), 3.75-3.65 (m, 1H), 2.80-2.76 (m, 2H), 2.16 (s, 3H), 1.98-1.89 (m, 2H), 1.77-1.72 (m, 2H), 1.64-1.51 (m, 2H). ESI-HRMS m/z 279.1470 (M+H+).
Synthesis of 3-(1-methylpiperidin-4-yl)-6-nitroquinazolin-4 (3H)-one (161): The compound was preapred by general procedure B provided in example 2 using compound 160 (0.6 g, 2.32 mmol), trimethylorthoformate (TMOF) (4 mL, 38.23 mmol to obtain compound 161 (0.52 g, 89%) as pale yellow solid. 1H NMR (300 MHz, CDCl3) δ in ppm 9.13 (d, J=2.4 Hz, 1H), 8.50 (dd, J=8.8 Hz, 2.8 Hz, 1H), 8.26 (s, 1H), 7.80 (d, J=9.2 Hz 1H), 3.04-3.01 (m, 2H), 2.34 (s, 3H) 2.24-2.18 (m, 2H), 2.02-1.97 (m, 4H).
Synthesis of 6-amino-3-(1-methylpiperidin-4-yl)quinazolin-4 (3H)-one (162): The compound was prepared by general procedure C provided in example 3 using compound 161 (0.3 g, 1.11 mmol), methanol (8 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 162 (0.166 g, 62%) as light brown solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.10 (s, 1H), 7.37 (d, J=8.4 Hz, 1H), 7.22 (d, J=2.4 Hz, 1H), 7.07 (dd, J=8.7 Hz, 2.7 Hz, 1H), 5.66 (brs, 2H), 4.64-4.54 (m, 1H), 2.94-2.91 (m, 2H), 2.23 (s, 3H), 2.13-2.03 (m, 4H), 1.75-1.70 (m, 2H).
Synthesis of 1-(3-acetylphenyl)-3-(3-(1-methylpiperidin-4-yl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (163): The compound was prepared by general procedure D provided in example 4 using compound 162 (0.120 g, 0.46 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.110 g, 0.55 mmol), 3′-aminoacetophenone (0.075 g, 0.55 mmol), TEA (0.3 mL, 1.71 mmol) to obtain compound 163 (0.062 g, 32%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.42 (s, 1H), 9.26 (s, 1H), 8.37-8.34 (m, 2H), 8.11 (s, 1H), 7.83 (dd, J=8.5 Hz, 1.5 Hz, 1H), 7.71 (d, J=8.1 Hz, 1H), 7.63-7.58 (m, 2H), 7.47-7.42 (m, 1H), 4.62-4.55 (m, 1H), 2.92-2.89 (m, 2H), 2.57 (s, 3H), 2.21 (s, 3H), 2.07-2.04 (m, 4H), 1.79-1.76 (m, 2H). ESI-HRMS m/z 420.2036 (M+H+).
Synthesis of 2-amino-N-(2-(methylamino)ethyl)-5-nitrobenzamide (164): The compound was prepared by general procedure A provided in example 1 using compound 1 (1 g, 5.49 mmol), DMF (10 mL), HATU (2.2 g, 6.04 mmol), N-Methylethylenediamine (0.48 mL, 6.58 mmol), TEA (1.9 mL, 13.73 mmol) to obtain compound 164 (0.810 g, 62%) as yellow solid. ESI-HRMS m/z 239.1146 (M+H+).
Synthesis of 3-(2-(methylamino)ethyl)-6-nitroquinazolin-4 (3H)-one (165): The compound was prepared by general procedure B provided in example 2 using compound 164 (0.6 g, 2.51 mmol), trimethylorthoformate (TMOF) (4 mL, 38.23 mmol) to obtain compound 165 (0.48 g, 78%) as pale yellow solid.
Synthesis of 6-amino-3-(2-(methylamino)ethyl)quinazolin-4 (3H)-one (166): The compound was prepared by general procedure C provided in example 3 using compound 165 (0.3 g, 1.37 mmol), methanol (10 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 166 (0.147 g, 56%) as light brown solid.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-(methylamino)ethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (167): The compound was prepared by general procedure D provided in example 4 using compound 166 (0.120 g, 0.55 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.133 g, 0.66 mmol), 3′-aminoacetophenone (0.074 g, 0.54 mmol) was added followed by TEA (0.3 mL, 1.71 mmol) to obtain compound 167 (0.048 g, 23%) as yellow solid.
Synthesis of 2-amino-N-(1-methoxybutan-2-yl)-5-nitrobenzamide (168): The compound was prepared by general procedure A provided in example 1 using compound 1 (0.8 g, 4.39 mmol), DMF (8 mL), HATU (1.8 g, 4.83 mmol), 2-amino-1-methoxybutane (0.6 mL, 5.27 mmol), TEA (1.5 mL, 10.98 mmol) to obtain compound 168 (0.8 g, 68%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.51 (d, J=2.4 Hz, 1H), 8.43 (d, J=8.4 Hz, 1H), 8.02 (dd, J=9.1 Hz, 2.7 Hz, 1H), 7.73 (s, 2H), 6.79 (d, J=9.3 Hz, 1H), 4.07-4.00 (m, 1H), 3.44-3.40 (m, 1H), 3.38-3.30 (m, 1H), 3.26 (s, 3H), 1.65-1.41 (m, 2H), 0.88 (t, J=4.5 Hz, 3H). ESI-HRMS m/z 268.1289 (M+H+). Melting Point: 122° C.
Synthesis of 3-(1-methoxybutan-2-yl)-6-nitroquinazolin-4 (3H)-one (169): The compound was prepared by general procedure B provided in example 2 using compound 168 (0.8 g, 2.99 mmol), trimethylorthoformate (TMOF) (6.5 mL, 59.89 mmol) to obtain compound 169 (0.70 g, 84%) as pale yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.84 (d, J=2.7 Hz, 1H), 8.61 (s, 1H), 8.57 (dd, J=9 Hz, 2.7 Hz, 1H), 7.89 (d, J=8.7 Hz, 1H), 4.92-4.83 (m, 1H), 3.87-3.81 (m, 1H), 3.65-3.60 (m, 1H), 3.23 (s, 3H), 1.92-1.82 (m, 2H), 0.84 (t, J=7.5 Hz, 3H). ESI-HRMS m/z 278.1138 (M+H+). Melting Point: 104° C.
Synthesis of 6-amino-3-(1-methoxybutan-2-yl)quinazolin-4 (3H)-one (170): The compound was prepared by general procedure C provided in example 3 using compound 169 (0.3 g, 1.08 mmol), methanol (8 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 170 (0.23 g, 86%) as light brown solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.02 (s, 1H), 7.37 (d, J=8.7 Hz, 1H), 7.20 (d, J=2.7 Hz, 1H), 7.07 (dd, J=9.1 Hz, 2.4 Hz, 1H), 5.65 (s, 2H), 4.91-4.76 (m, 1H), 3.81-3.75 (m, 1H), 3.56 (q, J=6.3 Hz, 1H), 3.21 (s, 3H), 1.85-1.75 (m, 2H), 0.78 (t, J=7.2 Hz, 3H). ESI-HRMS m/z 248.1384 (M+H+). Melting Point: 140° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(1-methoxybutan-2-yl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (171): The compound was prepared by general procedure D provided in example 4 using compound 170 (0.10 g, 0.40 mmol), dry THF (6 mL), 4-nitrophenylchloroformate (0.12 g, 0.60 mmol), 3′-aminoacetophenone (0.065 g, 0.48 mmol), TEA (0.2 mL, 1.66 mmol) to obtain compound 171 (0.07 g, 42%) as off white solid. 1H NMR (300 MHz, CDCl3) δ in ppm 9.09 (s, 1H), 8.96 (s, 1H), 8.30 (d, J=2.4 Hz, 1H), 8.21 (s, 1H), 8.06-8.05 (m, 1H), 7.79 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.67-7.65 (m, 1H), 7.59-7.54 (m, 2H), 7.43-7.39 (m, 1H), 4.86-4.80 (m, 1H), 3.80-3.75 (m, 1H), 3.57-3.53 (m, 1H), 3.19 (s, 3H), 2.53 (s, 3H), 1.83-1.76 (m, 2H), 0.77 (t, J=7.2 Hz, 3H). ESI-MS m/z 409.2 (M+H+). Melting Point: 182° C.
Synthesis of 2-amino-N-butyl-5-nitrobenzamide (172): The compound was prepared by general procedure A provided in example 1 using compound 1 (0.8 g, 4.39 mmol), DMF (8 mL), HATU (1.8 g, 4.83 mmol), 1-butylamine (0.5 mL, 5.27 mmol), TEA (1.5 mL, 10.98 mmol) to obtain compound 172 (0.82 g, 79%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.62 (br.s, —NH), 8.43 (d, J=2.4 Hz, 1H), 7.96 (dd, J=9.2 Hz, 2.8 Hz, 1H), 7.70 (br.s, 2H), 6.74 (d, J=9.6 Hz, 1H), 3.18 (q, J=5.6 Hz, 2H), 1.50-1.42 (m, 2H), 1.33-1.25 (m, 2H), 0.86 (t, J=7.6 Hz, 3H). ESI-HRMS m/z 238.1173 (M+H+). Melting Point: 128° C.
Synthesis of 3-butyl-6-nitroquinazolin-4 (3H)-one (173): The compound was prepared by general procedure B provided in example 2 using compound 172 (0.6 g, 2.53 mmol), trimethylorthoformate (TMOF) (5.5 mL, 50.60 mmol) to obtain compound 173 (0.55 g, 88%) as pale yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.76 (d, J=2.4 Hz, 1H), 8.55 (s, 1H), 8.48 (dd, J=9 Hz, 2.4 Hz, 1H), 7.81 (d, J=9.6 Hz, 1H), 3.95 (t, J=7.2 Hz, 2H), 1.67-1.60 (m, 2H), 1.31-1.22 (m, 2H), 0.86 (t, J=7.2 Hz, 3H). ESI-HRMS m/z 248.1042 (M+H+). Melting Point: 104° C.
Synthesis of 6-amino-3-butylquinazolin-4 (3H)-one (174): The compound was prepared by general procedure C provided in example 3 using compound 173 (0.25 g, 1.01 mmol) was dissolved in methanol (5 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 174 (0.20 g, 91%) as light brown solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.03 (s, 1H), 7.37 (d, J=8.7 Hz, 1H), 7.20 (d, J=2.7 Hz, 1H), 7.06 (dd, J=8.7 Hz, 2.7 Hz, 1H), 5.64 (s, 2H), 3.91 (t, J=7.2 Hz, 2H), 1.68-1.58 (m, 2H), 1.33-1.23 (m, 2H), 0.90 (t, J=7.5 Hz, 3H). ESI-HRMS m/z 218.1293 (M+H+). Melting Point: 98° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-butyl-4-oxo-3,4-dihydroquinazolin-6-yl)urea (175): The compound was prepared by general procedure D provided in example 4 using compound 174 (0.12 g, 0.55 mmol), dry THF (6 mL), 4-nitrophneylchloroformate (0.133 g, 0.66 mmol), 3′-aminoacetophenone (0.090 g, 0.66 mmol), TEA (0.2 mL, 1.38 mmol) to obtain compound 175 (0.07 g, 33%) as off white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.09 (s, 1H), 8.96 (s, 1H), 8.29 (d, J=2.8 Hz, 1H), 8.22 (s, 1H), 8.05-8.04 (m, 1H), 7.77 (dd, J=6.8 Hz, 2.8 Hz, 1H), 7.66-7.64 (m, 1H), 7.58-7.53 (m, 2H), 7.41-7.37 (m, 1H), 3.91 (t, J=7.6 Hz, 2H), 2.52 (s, 3H), 1.65-1.58 (m, 2H), 1.30-1.21 (m, 2H), 0.86 (t, J=7.6 Hz, 3H). ESI-MS m/z 379.2 (M+H+). Melting Point: 196° C.
Synthesis of 2-amino-N-(1-methoxypropan-2-yl)-5-nitrobenzamide (176): The compound was prepared by general procedure A provided in example 1 using compound 1 (0.8 g, 4.39 mmol), DMF (8 mL), HATU (1.8 g, 4.83 mmol), 1-methoxy-2-propylamine (0.5 mL, 5.27 mmol), TEA (1.5 mL, 10.98 mmol) to obtain compound 176 (0.83 g, 75%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.46 (br.s, —NH), 8.44 (d, J=2.8 Hz, 1H), 7.96 (dd, J=9.2 Hz, 2.8 Hz, 1H), 7.68 (br.s, 2H), 6.74 (d, J=9.6 Hz, 1H), 4.17-4.10 (m, 1H), 3.38-3.34 (m, 1H), 3.26-3.23 (m, 1H), 3.22 (s, 3H), 1.08 (d, J=6.8 Hz, 3H). ESI-HRMS m/z 254.1130 (M+H+). Melting Point: 118° C.
Synthesis of 3-(1-methoxypropan-2-yl)-6-nitroquinazolin-4 (3H)-one (177): The compound was prepared by general procedure B provided in example 2 using compound 176 (0.6 g, 2.37 mmol), trimethylorthoformate (TMOF) (5.1 mL, 47.41 mmol) to obtain compound 177 (0.57 g, 91%) as pale yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.84 (s, 1H), 8.62 (s, 1H), 8.57 (d, J=9 Hz, 1H), 7.88 (d, J=9.3 Hz, 1H), 5.11-5.00 (m, 1H), 3.83-3.77 (m, 1H), 3.62-3.57 (m, 1H) 3.24 (s, 3H), 1.43 (d, J=6.9 Hz, 3H). ESI-HRMS m/z 264.0977 (M+H+). Melting Point: 100° C.
Synthesis of 6-amino-3-(1-methoxypropan-2-yl)quinazolin-4 (3H)-one (178): The compound was prepared by general procedure C provided in example 3 using compound 177 (0.6 g, 2.28 mmol), methanol (8 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 178 (0.48 g, 90%) as light brown solid. ESI-HRMS m/z 234.1229 (M+H+). Melting Point: 130° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(1-methoxypropan-2-yl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (179): The compound was prepared by general procedure D provided in example 4 using compound 178 (0.12 g, 0.51 mmol), dry THF (6 mL), 4-nitrophenylchloroformate (0.15 g, 0.60 mmol), 3′-aminoacetophenone (0.087 g, 0.64 mmol), TEA (0.3 mL, 2.12 mmol) to obtain compound 179 (0.07 g, 41%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.16 (s, 1H), 9.03 (s, 1H), 8.35 (s, 1H), 8.29 (s, 1H), 8.10 (s, 1H), 7.83 (d, J=8.7 Hz, 1H), 7.71 (d, J=7.5 Hz, 1H), 7.64-7.60 (m, 2H), 7.48-7.43 (m, 1H), 5.11-5.01 (m, 1H), 3.79 (t, J=8.4 Hz, 1H), 3.60-3.55 (m, 1H), 3.24 (s, 3H), 2.58 (s, 3H), 1.40 (d, J=6.9 Hz, 3H). ESI-HRMS m/z 395.1728 (M+H+). Melting Point: 146° C.
Synthesis of 2-amino-N-(2-isopropoxyethyl)-5-nitrobenzamide (180): The compound was prepared by general procedure A provided in example 1 using compound 1 (0.8 g, 4.39 mmol), DMF (8 mL), HATU (1.8 g, 4.83 mmol), 2-aminoethyl isopropyl ether (0.6 mL, 4.83 mmol), TEA (1.5 mL, 10.98 mmol) to obtain compound 180 (0.83 g, 68%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) 8.74 (br.s, 1H), 8.49 (d, J=2.7 Hz, 1H), 8.01 (dd, J=9.3 Hz, 1H), 7.74 (br.s, 2H), 6.79 (d, J=9.3 Hz, 1H), 3.62-3.53 (m, 1H), 3.48 (t, J=6 Hz, 2H), 3.38-3.33 (m, 2H), 1.05 (d, J=6.3 Hz, 6H). (ESI-HRMS m/z 268.1304 (M+H+). Melting Point: 88° C.
Synthesis of 3-(2-isopropoxyethyl)-6-nitroquinazolin-4 (3H)-one (181): The compound was prepared by general procedure B provided in example 2 using compound 180 (0.80 g, 2.99 mmol), trimethylorthoformate (TMOF) (6.5 mL, 59.89 mmol) to obtain compound 181 (0.70 g, 84%) as pale yellow solid. 1H NMR (300 MHz, d6-DMSO) 8.85 (d, J=2.7 Hz, 1H), 8.56 (dd, J=9 Hz, 2.7 Hz, 1H), 8.49 (s, 1H), 7.89 (d, J=9 Hz, 1H), 4.16 (t, J=5.4 Hz, 2H), 3.66 (t, J=5.4 Hz, 2H), 3.57-3.49 (m, 1H), 1.01 (d, J=6.3 Hz, 6H). ESI-HRMS m/z 278.1139 (M+H+). Melting Point: 82° C.
Synthesis of 6-amino-3-(2-isopropoxyethyl)quinazolin-4 (3H)-one (182): The compound was prepared by general procedure C provided in example 3 using compound 181 (0.6 g, 2.16 mmol), methanol (8 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 182 (0.40 g, 78%) as light brown solid. 1H NMR (300 MHz, d6-DMSO) 7.93 (s, 1H), 7.37 (d, J=8.4 Hz, 1), 7.21 (d, J=2.7 Hz, 1H), 7.06 (dd, J=8.7 Hz, 2.4 Hz, 1H), 5.64 (s, 2H), 4.04 (t, J=5.4 Hz, 2H), 3.61 (t, J=5.4 Hz, 2H), 3.55-3.47 (m, 1H), 1.00 (d, J=6.3 Hz, ESI-HRMS m/z 248.1406 (M+H+). Melting Point: 80° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-isopropoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (183): The compound was prepared by general procedure D provided in example 4 using compound 182 (0.12 g, 0.48 mmol), dry THF (6 mL), 4-nitrophenylchloroformate (0.15 g, 0.72 mmol), 3′-aminoacetophenone (0.082 g, 0.60 mmol), TEA (0.3 mL, 2 mmol) to obtain compound 183 (0.075 g, 37%) as off white solid. 1H NMR (300 MHz, d6-DMSO) 9.14 (s, 1H), 9.01 (s, 1H), 8.37 (d, J=2.4 Hz, 1H), 8.16 (s, 1H), 8.10 (brs, 1H), 7.81 (dd, J=8.8 Hz, 2.7 Hz, 1H), 7.70 (d, J=7.2 Hz, 1H), 7.64-7.59 (m, 2H), 7.48-7.43 (m, 1H), 4.11 (t, J=5.1 Hz, 2H), 3.64 (t, J=5.1 Hz, 2H), 3.56-3.48 (m, 1H), 2.57 (s, 3H), 1.01 (d, J=6 Hz, 6H). ESI-HRMS m/z 409.1890 (M+H+). Melting Point: 142° C.
Synthesis of 2-amino-N-cyclohexyl-5-nitrobenzamide (184): The compound was prepared by general procedure A provided in example 1 using compound 1 (1 g, 5.49 mmol), DMF (10 mL), HATU (2.2 g, 6.04 mmol), cyclohexylamine (0.65 mL, 6.58 mmol), TEA (1.9 mL, 13.73 mmol) to obtain compound 184 (0.982 g, 68%) as yellow solid. ESI-HRMS m/z 264.1346 (M+H+). Melting Point: 102° C.
Synthesis of 3-cyclohexyl-6-nitroquinazolin-4 (3H)-one (185): The compound was prepared by general procedure B provided in example 2 using compound 184 (0.6 g, 2.28 mmol), trimethylorthoformate (TMOF) (4 mL, 38.23 mmol) to obtain compound 185 (0.49 g, 80%) as pale yellow solid. ESI-HRMS m/z 274.1200 (M+H+).
Synthesis of 6-amino-3-cyclohexylquinazolin-4 (3H)-one (186): The compound was prepared by general procedure C provided in example 3 using compound 185 (0.3 g, 1.09 mmol), methanol (10 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 186 (0.160 g, 60%) as light brown solid. ESI-HRMS m/z 244.1454 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(3-cyclohexyl-4-oxo-3,4-dihydroquinazolin-6-yl)urea (187): The compound was prepared by general procedure D provided in example 4 using compound 186 (0.120 g, 0.49 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.119 g, 0.59 mmol), 3′-aminoacetophenone (0.086 g, 0.63 mmol), TEA (0.3 mL, 1.71 mmol) to obtain compound 187 (0.091 g, 46%) as yellow solid.
Synthesis of 2-amino-N-(2-methoxyethyl)-4-nitrobenzamide (189): The compound was prepared by general procedure A provided in example 1 using 2-amino-4-nitrobenzoic acid (compound 188) (1 g, 5.49 mmol), DMF (10 mL), HATU (2.2 g, 6.04 mmol), 2-methoxyethylamine (0.50 mL, 6.58 mmol), TEA (1.9 mL, 13.73 mmol) to obtain compound 189 (0.945 g, 72%) as yellow solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.59 (s, —NH), 7.66 (d, J=8.7 Hz, 1H), 7.57 (d, J=2.4 Hz, 1H), 7.28 (dd, J=8.4 Hz, 2.4 Hz, 1H), 6.84 (br.s, 2H), 3.45-3.38 (m, 4H), 3.26 (s, 3H). ESI-HRMS m/z 240.0996 (M+H+).
Synthesis of 3-(2-methoxyethyl)-7-nitroquinazolin-4 (3H)-one (190): The compound was prepared by general procedure B provided in example 2 using compound 189 (0.6 g, 2.28 mmol), trimethylorthoformate (TMOF) (4 mL, 38.23 mmol) to obtain compound 190 (0.49 g, 80%) as pale yellow solid. ESI-HRMS m/z 250.0834 (M+H+).
Synthesis of 7-amino-3-(2-methoxyethyl)quinazolin-4 (3H)-one (191): The compound was prepared by general procedure C provided in example 3 using compound 190 (0.3 g, 1.20 mmol), methanol (10 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 191 (0.158 g, 60%) as light brown solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.01 (s, 1H), 7.78 (d, J=8.7 Hz, 1H), 6.73 (dd, J=8.7 Hz, 2.1 Hz, 1H), 6.60 (d, J=2.1 Hz, 1H), 6.11 (br.s, 2H), 4.04 (t, J=5.1 Hz, 2H), 3.55 (t, J=5.1 Hz, 2H), 3.23 (s, 3H). ESI-HRMS m/z 220.1094 (M+H+). Melting Point: 132° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-7-yl)urea (192): The compound was prepared by general procedure D provided in example 4 using compound 191 (0.120 g, 0.49 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.119 g, 0.59 mmol), 3′-aminoacetophenone (0.086 g, 0.63 mmol), TEA (0.3 mL, 1.71 mmol) to obtain compound 192 (0.091 g, 46%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.23 (s, 1H), 9.05 (s, 1H), 8.18 (s, 1H), 8.06 (m, 1H), 8.02 (d, J=8.8 Hz, 1H), 7.83 (d, J=2 Hz, 1H), 7.68-7.65 (m, 1H), 7.59-7.57 (m, 1H), 7.49 (dd, J=8.8 Hz, 2 Hz, 1H), 7.44-7.40 (m, 1H), 4.08 (t, J=5.2 Hz, 2H), 3.56 (t, J=4.8 Hz, 2H), 3.21 (s, 3H), 2.54 (s, 3H). ESI-MS m/z 381.3 (M+H+).
Synthesis of 1-(4-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-7-yl)urea (193): The compound was prepared by general procedure E provided in example 5 using compound 191 (0.120 g, 0.49 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.119 g, 0.59 mmol), 4′-aminoacetophenone (0.086 g, 0.63 mmol), TEA (0.3 mL, 1.71 mmol) to obtain compound 193 (0.079 g, 38%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.30 (s, 1H), 9.24 (s, 1H), 8.17 (s, 1H), 8.01 (d, J=8.4 Hz, 1H), 7.88-7.82 (m, 3H), 7.56 (d, J=8 Hz, 2 Hz, 2H), 7.46 (d, J=8.4 Hz, 1H), 4.06 (t, J=4.8 Hz, 2H), 3.53 (t, J=4.4 Hz, 2H), 3.20 (s, 3H), 2.47 (s, 3H). ESI-MS m/z 381.2 (M+H+).
Synthesis of N-(2-((2-methoxyethyl)carbamoyl)-4-nitrophenyl)picolinamide (208): Picolinic acid (0.64 g, 5.20 mmol) was dissolved in dry DCM (10 mL) followed by addition of one drop of DMF. Oxalyl Chloride (0.66 mL, 7.80 mmol) was added to the reaction mixture under ice cold condition and nitrogen atmosphere. The reaction was stirred for 15 mins for the formation of acid chloride. Then the reaction mass was evaporated to dryness to remove excess oxalyl chloride and DCM. The crude is kept under inert atmosphere and dissolved in DCM (20 mL). In another flask, compound 2 (0.5 g, 2.09 mmol) was dissolved in DCM (10 mL) followed by addition of TEA (0.7 mL, 5.64 mmol). In this reaction medium, the previously prepared acid chloride was added dropwise and under ice cold condition. The reaction was monitored by checking TLC. Upon completion of the reaction, reaction mass was evaporated to dryness and washed with water and extracted with EtOAc to get the crude. The product was purified by flash chromatography (Silica gel, mesh size 100-200) eluting (50% EtOAc/Pet ether) to obtain compound 208 (0.26 g, 29%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 13.24 (s, 1H), 9.20 (d, J=5.2 Hz, 1H), 8.94 (d, J=9.2, 1H), 8.76-8.74 (m, 1H), 8.64 (d, J=2.8 Hz, 1H), 8.42 (dd, J=9.2 Hz, 2.8 Hz, 1H), 8.19-8.16 (m, 1H), 8.08-8.04 (m, 1H), 7.69-7.06 (m, 1H), 3.51-3.44 (m, 4H), 3.25 (s, 3H). ESI-HRMS m/z 345.1201 (M+H+). Melting point 224° C.
Synthesis of 3-(2-methoxyethyl)-6-nitro-2-(pyridin-2-yl)quinazolin-4 (3H)-one (209): The compound was prepared by general procedure G provided in example 7 using compound 208 (0.25 g, 0.72 mmol), DMF (5 mL), zinc chloride (0.40 g, 2.91 mmol), HMDS (1.21 mL, 5.80 mmol) to obtain compound 209 (0.165 g, 70%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.84 (d, J=2.8 Hz, 1H), 8.96-8.67 (m, 1H), 8.57-8.54 (m, 1H), 8.07-8.02 (m, 1H), 7.89-7.85 (m, 2H), 7.61-7.57 (m, 1H), 4.37 (t, J=6 Hz, 2H), 3.43 (t, J=6 Hz, 2H), 3.31 (s, 3H). ESI-HRMS m/z 327.1096 (M+H+). Melting point 144° C.
Synthesis of 6-amino-3-(2-methoxyethyl)-2-(pyridin-2-yl)quinazolin-4 (3H)-one (210): The compound was prepared by general procedure C provided in example 3 using compound 209 (0.155 g, 0.47 mmol), methanol (10 mL), pinch of Pd/C under hydrogen atmosphere to obtain compound 210 (0.075 g, 54%) as brown solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.63-8.61 (m, 1H), 7.99-7.94 (m, 1H), 7.77-7.74 (m, 1H), 7.51-7.48 (m, 1H), 7.40 (d, J=8.8 Hz, 1H), 7.26 (d, J=2 Hz, 1H), 7.11 (dd, J=8 Hz, 2.4 Hz, 1H), 4.32 (t, J=6.4 Hz, 2H), 3.38 (t, J=6 Hz, 2H), 2.94 (s, 3H). ESI-HRMS m/z 297.1352 (M+H+). Melting point 138° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-2-(pyridin-2-yl)-3,4-dihydroquinazolin-6-yl)urea (211): The compound was prepared by general procedure D provided in example 4 using compound 210 (0.065 g, 0.21 mmol), dry THF (5 mL), 4-nitrophenyl chloroformate (0.06 g, 0.32 mmol), 3-aminoacetophenone (0.039 g, 0.26 mmol), TEA (0.073 mL, 0.52 mmol) to obtain 211 as off white solid (0.021 g, 21%). 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.17 (s, 1H), 9.01 (s, 1H), 8.66-8.64 (m, 1H), 8.39 (d, J=2.4 Hz, 1H), 8.07 (t, J=1.6 Hz, 1H), 8.02-7.98 (m, 1H), 7.83-7.79 (m, 2H), 7.69-7.66 (m, 1H), 7.63 (d, J=8.8 Hz, 1H), 7.58-7.52 (m, 2H), 7.42 (t, J=8.0 Hz, 1H), 4.34 (t, J=6 Hz, 2H), 3.41 (t, J=6 Hz, 2H), 2.93 (s, 3H), 2.50 (s, 3H). ESI-HRMS m/z 458.1841 (M+H+). Melting point 230° C.
Synthesis of N-(2-((2-methoxyethyl)carbamoyl)-4-nitrophenyl)nicotinamide (212): Commercially available Nicotinic acid (0.64 g, 5.20 mmol) was dissolved in dry DCM (10 mL) followed by addition of one drop of DMF. Oxalyl chloride (0.66 mL, 7.80 mmol) was added to the reaction mixture under ice cold condition and nitrogen atmosphere. The reaction was stirred for 15 mins for the formation of acid chloride. Then the reaction mass was evaporated to dryness to remove excess oxalyl chloride and DCM. The crude is kept under inert atmosphere and dissolved in DCM (20 mL). In another flask, compound 2 (0.5 g, 2.09 mmol) was dissolved in DCM (10 mL) followed by addition of TEA (0.7 mL, 5.64 mmol). In this reaction medium, the previously prepared acid chloride was added dropwise and under ice cold condition. The reaction was monitored by checking TLC. Upon completion of the reaction, reaction mass was evaporated to dryness and washed with water and extracted with EtOAc to get the crude. The product was purified by flash chromatography (Silica gel, mesh size 100-200) eluting (50% EtOAc/Pet ether) to obtain compound 212 (0.213 g, 24%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 13.03 (s, 1H), 9.37 (s, 1H), 9.07 (s, 1H), 8.80-8.73 (m, 3H), 8.41 (d, J=8 Hz, 1H), 7.63-7.59 (m, 1H), 3.49-3.45 (m, 4H), 3.24 (s, 3H). ESI-HRMS m/z 345.1201 (M+H+). Melting point 180° C.
Synthesis of 3-(2-methoxyethyl)-6-nitro-2-(pyridin-3-yl)quinazolin-4 (3H)-one (213): The compound was prepared by general procedure G provided in example 7 using compound 212 (0.20 g, 0.58 mmol), DMF (5 mL), zinc chloride (0.40 g, 2.91 mmol), HMDS (1 mL, 4.65 mmol) to obtain compound 213 (0.163 g, 86%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.85 (d, J=2.4 Hz, 1H), 8.81 (dd, J=2.4 Hz, J=0.8 Hz, 1H), 8.72 (dd, J=5 Hz, J=1.6 Hz, 1H), 8.57-8.54 (m, 1H), 8.09-8.06 (m, 1H), 7.80 (d, J=8.8 Hz, 1H), 7.58-7.55 (m, 1H), 4.09 (t, J=5.6 Hz, 2H), 3.44 (t, J=5.6 Hz, 2H), 3.01 (s, 3H). ESI-HRMS m/z 327.1097 (M+H+). Melting point 170° C.
Synthesis of 6-amino-3-(2-methoxyethyl)-2-(pyridin-3-yl)quinazolin-4 (3H)-one (214): The compound was prepared by general procedure C provided in example 3 using compound 213 (0.150 g, 0.45 mmol), methanol (10 mL) to obtain compound 214 (0.06 g, 33%) as brown solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.72 (dd, J=2.2 Hz, J=0.8 Hz, 1H), 8.64 (dd, J=4.8 Hz, J=1.6 Hz, 1H), 8.00-7.97 (m, 1H), 7.51-7.47 (m, 1H), 7.35 (d, J=8.8 Hz, 1H), 7.20 (d, J=2.4 Hz, 1H), 7.06 (dd, J=8.4 Hz, J=2.4 Hz, 1H), 5.69 (brs, 2H), 4.00 (t, J=6 Hz, 2H), 3.391 (t, J=6 Hz, 2H), 2.98 (s, 3H). Melting point 152° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-2-(pyridin-3-yl)-3,4-dihydroquinazolin-6-yl)urea (215): The compound was prepared by general procedure D provided in example 4 using compound 214 (0.05 g, 0.16 mmol), dry THF (5 mL), 4-nitrophenyl chloroformate (0.06 g, 0.26 mmol), 3′-aminoacetophenone (0.030 g, 0.26 mmol), (0.059 mL, 0.42 mmol) to obtain compound 215 (0.037 g, 48%) as off white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.15 (s, 1H), 8.99 (s, 1H), 8.77 (d, J=1.6 Hz, 1H), 8.68 (dd, J=4.8 Hz, J=1.6 Hz, 1H), 8.40 (d, J=2.4 Hz, 1H), 8.07-8.02 (m, 2H), 7.79 (dd, J=8.8 Hz, J=2.8 Hz, 1H) 7.69-7.66 (m, 1H), 7.62-7.55 (m, 2H) 7.54-7.55 (m, 1H), 7.44-7.40 (m, 1H), 4.05 (t, J=5.6 Hz, 2H), 3.42 (t, J=5.6 Hz, 2H) 3.00 (s, 3H), 2.53 (s, 3H). ESI-HRMS m/z 458.184 (M+H+). Melting point 218° C.
Synthesis of N-(2-((2-methoxyethyl)carbamoyl)-4-nitrophenyl)isonicotinamide (216): Commercially available Isonicotinic acid (0.64 g, 5.20 mmol) was dissolved in dry DCM (10 mL) followed by addition of one drop of DMF. Oxalyl Chloride (0.66 mL, 7.80 mmol) was added to the reaction mixture under ice cold condition and nitrogen atmosphere. The reaction was stirred for 15 mins for the formation of acid chloride. Then the reaction mass was evaporated to dryness to remove excess oxalyl chloride and DCM. The crude is kept under inert atmosphere and dissolved in DCM (20 mL). In another flask, compound 2 (0.5 g, 2.09 mmol) was dissolved in DCM (10 mL) followed by addition of TEA (0.7 mL, 5.64 mmol). In this reaction medium, the previously prepared acid chloride was added dropwise under ice cold condition. The reaction was monitored by checking TLC. Upon completion of the reaction, reaction mass was evaporated to dryness and washed with water and extracted with EtOAc to get the crude. The product was purified by flash chromatography (Silica gel, mesh size 100-200) eluting (50% EtOAc/Pet ether) to obtain compound 216 (0.313 g, 33%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 13.10 (s, 1H), 9.40 (d, J=4.8 Hz, 1H), 8.84-8.79 (m, 3H), 8.75 (d, J=2.4 Hz, 1H) 8.43 (dd, J=5.4 Hz, J=2.4 Hz, 1H), 7.79 (dd, J=4.4 Hz, J=1.6 Hz, 2H), 3.49-3.46 (m, 4H), 3.24 (s, 3H). Melting point 206° C.
Synthesis of 3-(2-methoxyethyl)-6-nitro-2-(pyridin-4-yl)quinazolin-4 (3H)-one (217): The compound was prepared by general procedure G provided in example 7 using compound 216 (0.30 g, 0.87 mmol), DMF (5 mL), zinc chloride (0.48 g, 3.48 mmol), HMDS (1.5 mL, 6.96 mmol) to obtain compound 217 (0.210 g, 73%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.85-8.85 (m, 1H), 8.75 (dd, J=4.4 Hz, J=1.6 Hz, 2H), 8.56 (dd, J=10 Hz, J=2.8 Hz, 1H), 7.86 (dd, J=9.0 Hz, J=0.4 Hz, 1H), 7.63 (dd, J=4.4 Hz, J=1.6 Hz, 2H), 4.05 (t, J=5.6 Hz, 2H), 3.43 (t, J=5.6 Hz, 2H), 3.01 (s, 3H). Melting point 200° C.
Synthesis of 6-amino-3-(2-methoxyethyl)-2-(pyridin-4-yl)quinazolin-4 (3H)-one (218): The compound was prepared by general procedure C provided in example 3 using compound 217 (0.200 g, 0.61 mmol), methanol (10 mL), pinch of 10% Pd/C under hydrogen atmosphere to obtain compound 218 (0.065 g, 34%) as brown solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.69-8.67 (m, 2H), 7.56-7.55 (m, 2H), 7.35 (d, J=8.8 Hz, 1H), 7.20 (d, J=2.4 Hz, 1H), 7.06 (dd, J=8.8 Hz, J=2.4 Hz, 1H), 5.71 (brs, 2H), 4.00 (t, J=6 Hz, 2H), 3.83 (t, J=5.6 Hz, 2H), 2.98 (s, 3H). Melting point 42° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-2-(pyridin-4-yl)-3,4-dihydroquinazolin-6-yl)urea (219): The compound was prepared by general procedure D provided in example 4 using compound 218 (0.059 g, 0.19 mmol), dry THF (5 mL), 4-nitrophenyl chloroformate (0.06 g, 0.29 mmol), 3′-aminoacetophenone (0.032 g, 0.26 mmol), TEA (0.07 mL, 0.49 mmol) to obtain compound 219 (0.018 g, 20%) as off white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.16 (s, 1H), 8.99 (s, 1H), 8.71 (dd, J=4.4 Hz, J=1.6 Hz, 2H), 8.40 (d, J=2.4 Hz, 1H), 8.06 (t, J=2 Hz, 1H), 7.79 (dd, J=8.8 Hz, J=2.8 Hz, 1H), 7.68-7.66 (m, 1H), 7.61-7.59 (m, 3H), 7.58-7.56 (m, 1H), 7.44-7.40 (m, 1H), 4.04 (t, J=5.6 Hz, 2H), 3.41 (t, J=5.6 Hz, 2H), 3.00 (s, 3H), 2.53 (s, 3H). ESI-HRMS m/z 458.1828 (M+H+). Melting point 240° C.
Synthesis of N-(2-((2-methoxyethyl)carbamoyl)-4-nitrophenyl)pyrazine-2-carboxamide (220): Commercially available pyrazine-2-carboxylic acid (0.649 g, 5.22 mmol) was dissolved in dry DCM (10 mL) followed by addition of one drop of DMF. Oxalyl Chloride (0.66 mL, 7.80 mmol) was added to the reaction mixture under ice cold condition and nitrogen atmosphere. The reaction was stirred for 15 mins for the formation of acid chloride. Then the reaction mass was evaporated to dryness to remove excess oxalyl chloride and DCM. The crude is kept under inert atmosphere and dissolved in DCM (20 mL). In another flask, compound 2 (0.5 g, 2.09 mmol) was dissolved in DCM (10 mL) followed by addition of TEA (0.7 mL, 5.64 mmol) and allowed to stir at room temperature for 7 hours. In this reaction medium, the previously prepared acid chloride was added dropwise under ice cold condition. The reaction was monitored by checking TLC. Upon completion of the reaction, reaction mass was evaporated to dryness and washed with water and extracted with EtOAc to get the crude. The product was purified by flash chromatography (Silica gel, mesh size 100-200) eluting (50% EtOAc/Pet ether) to obtain compound 220 (0.175 g, 49%) as white solid.
Synthesis of 3-(2-methoxyethyl)-6-nitro-2-(pyrazin-2-yl)quinazolin-4 (3H)-one (221): The compound was prepared by general procedure G provided in example 7 using compound 220 (0.165 g, 0.47 mmol), DMF (5 mL), zinc chloride (0.26 g, 1.91 mmol), HMDS (0.83 mL, 3.89 mmol) to obtain compound 221 (0.125 g, 49%) as white solid.
Synthesis of 6-amino-3-(2-methoxyethyl)-2-(pyrazin-2-yl)quinazolin-4 (3H)-one (222): The compound was prepared by general procedure C provided in example 3 using compound 221 (0.118 g, 0.39 mmol), methanol (10 mL), a pinch of 10% Pd/C under hydrogen atmosphere to obtain compound 222 (0.08 mg, 78%) as dark green solid.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-2-(pyrazin-2-yl)-3,4-dihydroquinazolin-6-yl)urea (223): The compound was prepared by general procedure D provided in example 4 using compound 222 (0.70 g, 0.23 mmol), dry THF (5 mL), 4-nitrophenyl chloroformate (0.072 g, 0.35 mmol), 3′-aminoacetophenone (0.039 g, 0.28 mmvol), TEA (0.082 mL, 0.58 mmol) to obtain compound 223 as off white solid (0.04 g, 39%).
Synthesis of 2-(2-chloroacetamido)-N-(2-methoxyethyl)-5-nitrobenzamide (228): Compound 2 (1 gm, 4.18 mmol) was dissolved in dry DCM and TEA (1.16 mL, 8.36 mmol) was added followed by chloroacetyl chloride (0.39 ml, 5.01 mmole) dropwise at cooling and the reaction mixture was allowed to stir at room temperature for 1 hour. After completion of the reaction, it was worked up with ethyl acetate and water and purified by column chromatography (Silica gel, mesh size 100-200) eluting (50% EtOAc/Pet Ether) to obtain compound 228 (0.800 g, 60%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 12.26 (s, 1H), 9.26-9.24 (m, 1H), 8.64-8.62 (m, 2H), 8.36 (dd, J=9.2 Hz, 2.8 Hz, 1H), 4.44 (s, 2H), 3.48-3.40 (m, 4H), 3.24 (s, 3H). ESI-HRMS m/z 316.0703 (M+H+).
Synthesis of 2-(chloromethyl)-3-(2-methoxyethyl)-6-nitroquinazolin-4 (3H)-one (229): The compound was prepared by general procedure G provided in example 7 using compound 228 (0.8 g, 2.53 mmol), DMF (10 mL), ZnCl2 (1.4 g, 10.12 mmol), HMDS (4.24 mL, 20.24 mmol) to obtain compound 229 (0.60 g, 79%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.78-8.75 (m, 1H), 8.55-8.50 (m, 1H), 7.86-7.82 (m, 1H), 4.92 (s, 2H), 4.31 (t, J=5.2 Hz, 2H), 3.61 (t, J=5.2 Hz, 2H), 3.20 (s, 3H). ESI-HRMS m/z 298.0595 (M+H+).
Synthesis of 3-(2-methoxyethyl)-6-nitro-2-(pyrrolidin-1-ylmethyl)quinazolin-4 (3H)-one (230): Compound 229 (0.2 g, 0.74 mmol) and pyrrolidine (0.125 mL, 1.48 mmol) were dissolved in toluene and reaction mixture was refluxed for 2 hours. After completion of the reaction, it was washed with water and extracted with ethyl acetate to obtain light yellow crude mass which was then purified by column chromatography (Silica gel, mesh size 100-200) eluting (80% EtOAc/Pet Ether) to obtain compound 230 (0.159 g, 64%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.76 (d, J=2.8 Hz, 1H), 8.48 (dd, J=8.8 Hz, 2.8 Hz, 1H), 7.80 (d, J=9.2 Hz, 1H), 4.45 (t, J=6 Hz, 2H), 3.83 (s, 2H), 3.59 (t, J=5.6 Hz, 2H), 3.20 (s, 3H), 2.54-2.49 (m, 4H), 1.69-1.65 (m, 4H). ESI-HRMS m/z 333.1572 (M+H+).
Synthesis of 6-amino-3-(2-methoxyethyl)-2-(pyrrolidin-1-ylmethyl)quinazolin-4 (3H)-one (231): The compound was prepared by general procedure C provided in example 3 using compound 230 (0.150 g, 0.42 mmol), methanol (10 mL), pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 231 (0.158 g, 60%) as light brown solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 7.30 (d, J=8.8 Hz, 1H), 7.12 (d, J=2.8 Hz, 1H), 7.01 (dd, J=8.4 Hz, 2.4 Hz, 1H), 5.57 (s, 2H), 4.36 (t, J=6 Hz, 2H), 3.68 (s, 2H), 3.54 (t, J=5.6 Hz, 2H), 3.18 (s, 3H), 2.45-2.42 (m, 4H), 1.67-1.62 (m, 4H). ESI-HRMS m/z 303.1826 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-2-(pyrrolidin-1-ylmethyl)-3,4-dihydroquinazolin-6-yl)urea (232): The compound was prepared by general procedure D provided in example 4 using compound 231 (0.120 g, 0.39 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.094 g, 0.46 mmol), 3′-aminoacetophenone (0.062 g, 0.46 mmol), TEA (0.14 mL, 0.78 mmol) to obtain compound 232 (0.077 g, 42%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.09 (s, 1H), 8.97 (s, 1H), 8.29 (d, J=2.8 Hz, 1H), 8.06-8.05 (m, 1H), 7.75 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.67-7.65 (m, 1H), 7.56 (d, J=8.8 Hz, 2H), 7.43-7.39 (m, 1H), 4.42 (t, J=5.6 Hz, 2H), 3.76 (s, 2H), 3.58 (t, J=5.6 Hz, 2H), 3.20 (s, 3H), 2.53 (s, 3H), 2.51-2.49 (m, 4H), 1.69-1.64 (m, 4H). ESI-HRMS m/z 464.2317 (M+H+).
Synthesis of 2-((dimethylamino)methyl)-3-(2-methoxyethyl)-6-nitroquinazolin-4 (3H)-one (233): Compound 229 (0.2 g, 0.74 mmol) and dimethylamine (0.1 mL, 1.48 mmol) were dissolved in toluene and reaction mixture was refluxed for 2 hours. After completion of the reaction, it was washed with water and extracted with ethyl acetate to get yellow crude mass which was then purified by column chromatography (Silica gel, mesh size 100-200) eluting (5% CHCl3-MeOH) to obtain compound 233 (0.154 g, 75%) as yellow gummy product. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.77 (d, J=2.4 Hz, 1H), 8.49 (dd, J=9.2 Hz, 2.8 Hz, 1H), 7.82 (d, J=8.8 Hz, 1H), 4.47 (t, J=5.6 Hz, 2H), 3.63 (s, 2H), 3.59 (t, J=5.6 Hz, 2H), 3.20 (s, 3H), 2.20 (s, 6H). ESI-HRMS m/z 307.1405 (M+H+).
Synthesis of 6-amino-2-((dimethylamino)methyl)-3-(2-methoxyethyl)quinazolin-4 (3H)-one (234): The compound was prepared by general procedure C provided in example 3 using compound 233 (0.120 g, 0.39 mmol), methanol (5 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 234 (0.086 g, 80%) as light brown gummy product. 1H NMR (400 MHz, d6-DMSO) δ in ppm 7.31 (d, J=8.4 Hz, 1H), 7.13 (d, J=2.8 Hz, 1H), 7.01 (dd, J=8.4 Hz, 2.4 Hz, 1H), 5.59 (s, 2H), 4.37 (t, J=5.6 Hz, 2H), 3.54 (t, J=5.6 Hz, 2H), 3.49 (s, 2H), 3.18 (s, 3H), 2.15 (s, 6H). ESI-HRMS m/z 277.1667 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(2-((dimethylamino)methyl)-3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (235): The compound was prepared by general procedure D provided in example 4 using compound 234 (0.060 g, 0.21 mmol), dry THF (3 mL), 4-nitrophenylchloroformate (0.053 g, 0.26 mmol), 3′-aminoacetophenone (0.035 g, 0.26 mmol), TEA (0.14 mL, 0.52 mmol) to obtain compound 235 (0.062 g, 54%) as off white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.10 (s, 1H), 8.98 (s, 1H), 8.29 (d, J=2.4 Hz, 1H), 8.06-8.05 (m, 1H), 7.76 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.67-7.65 (m, 1H), 7.57-7.54 (m, 2H), 7.42-7.39 (m, 1H), 4.43 (t, J=5.6 Hz, 2H), 3.59-3.56 (m, 4H), 3.20 (s, 3H), 2.53 (s, 3H), 2.18 (s, 6H). ESI-HRMS m/z 438.2140 (M+H+).
Synthesis of 3-(2-methoxyethyl)-6-nitro-2-(piperidin-1-ylmethyl)quinazolin-4 (3H)-one (236): Compound 229 (0.2 g, 0.74 mmol) and piperidine (0.15 mL, 1.48 mmol) were dissolved in toluene and reaction mixture was refluxed for 2 hours. After completion of the reaction, it was washed with water and extracted with ethyl acetate to get light brown crude mass which was then purified by column chromatography (Silica gel, mesh size 100-200) eluting (50% EtOAc/Pet Ether) to obtain compound 233 (0.220 g, 85%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.76 (d, J=2.8 Hz, 1H), 8.48 (dd, J=9.2 Hz, 2.8 Hz, 1H), 7.80 (d, J=8.8 Hz, 1H), 4.45 (t, J=6.0 Hz, 2H), 3.66 (s, 2H), 3.61 (t, J=6.0 Hz, 2H), 3.21 (s, 3H), 2.42-2.37 (m, 4H), 1.48-1.42 (m, 4H), 1.37-1.309 m, 2H). ESI-HRMS m/z 347.1716 (M+H+).
Synthesis of 6-amino-3-(2-methoxyethyl)-2-(piperidin-1-ylmethyl)quinazolin-4 (3H)-one (237): The compound was prepared by general procedure C provided in example 3 using compound 236 (0.150 g, 0.47 mmol), methanol (10 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 237 (0.089 g, 65%) as light brown solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 7.30 (d, J=8.8 Hz, 1H), 7.12 (d, J=2.8 Hz, 1H), 7.01 (dd, J=8.4 Hz, 2.4 Hz, 1H), 5.57 (s, 2H), 4.36 (t, J=6 Hz, 2H), 3.68 (s, 2H), 3.54 (t, J=5.6 Hz, 2H), 3.18 (s, 3H), 2.45-2.42 (m, 4H), 1.67-1.62 (m, 4H). ESI-HRMS m/z 317.1976 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-2-(piperidin-1-ylmethyl)-3,4-dihydroquinazolin-6-yl)urea (238): The compound was prepared by general procedure D provided in example 4 using compound 237 (0.080 g, 0.25 mmol), dry THF (3 mL), 4-nitrophenylchloroformate (0.060 g, 0.30 mmol), 3′-aminoacetophenone (0.040 g, 0.30 mmol), TEA (0.07 mL, 0.50 mmol) to obtain compound 238 (0.077 g, 42%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.08 (s, 1H), 8.96 (s, 1H), 8.29 (d, J=2.4 Hz, 1H), 8.06-8.05 (m, 1H), 7.75 (dd, J=9.2 Hz, 2.8 Hz, 1H), 7.67-7.64 (m, 1H), 7.55 (d, J=8.4 Hz, 2H), 7.42-7.38 (m, 1H), 4.41 (t, J=6.0 Hz, 2H), 3.61-3.58 (m, 4H), 3.21 (s, 3H), 2.53 (s, 3H), 2.39-2.34 (m, 4H), 1.47-1.41 (m, 4H), 1.37-1.30 (m, 2H). ESI-HRMS m/z 478.2463 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-2-(piperidin-1-ylmethyl)-3,4-dihydroquinazolin-6-yl)urea hydrochloride (238a): Compound 238 (0.05 g, 0.104 mmol) was taken in dioxane (1 mL) and 4 M HCl in dioxane (0.8 mL) was added under ice cold condition and reaction was heated at 100° C. for 2 hour. During heating, at first compound dissolved to obtain a clear solution and then slowly solidified after 2 hours of constant heating. Reaction mass was cooled and evaporated in rotary evaporator to obtain compound 238 a (53 mg, 98%) as pale yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 10.04 (s, 1H), 9.77 (s, 1H), 9.56 (s, 1H), 8.35 (d, J=2.4 Hz, 1H), 8.07-8.07 (m, 1H), 7.86 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.66 (d, J=8.8 Hz, 2H), 7.56 (d, J=7.6 Hz, 1H), 7.43-7.39 (m, 1H), 4.64 (d, J=3.6 Hz, 2H), 4.16 (t, J=4.8 Hz, 2H), 3.62-3.56 (m, 4H), 3.21 (s, 3H), 3.15-3.05 (m, 2H), 2.53 (s, 3H), 1.85-1.79 (m, 4H), 1.70-1.63 (m, 1H), 1.51-1.40 (m, 1H). The crystal structure of compound 238a is provided in
Synthesis of 3-(2-methoxyethyl)-2-(morpholinomethyl)-6-nitroquinazolin-4 (3H)-one (239): Compound 229 (0.2 g, 0.74 mmol) and morpholine (0.13 mL, 1.48 mmol) were dissolved in toluene and reaction mixture was refluxed for 2 hours. After completion of the reaction, it was washed with water and extracted with ethyl acetate to obtain light yellow crude mass which was then purified by column chromatography (Silica gel, mesh size 100-200) eluting (50% EtOAc/Pet Ether) to obtain compound 239 (0.195 g, 75%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.76 (d, J=2.4 Hz, 1H), 8.49 (dd, J=9.2 Hz, 2.8 Hz, 1H), 7.81 (d, J=8.4 Hz, 1H), 4.46 (t, J=5.6 Hz, 2H) 3.72 (s, 2H), 3.62 (t, J=5.6 Hz, 2H), 3.54-3.52 (m, 4H), 3.28-3.27 (m, 4H), 3.21 (s, 3H). ESI-HRMS m/z 349.1513 (M+H+).
Synthesis of 6-amino-3-(2-methoxyethyl)-2-(morpholinomethyl)quinazolin-4 (3H)-one (240): The compound was prepared by general procedure C provided in example 3 using compound 239 (0.150 g, 0.43 mmol), methanol (10 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 240 (0.089 g, 65%) as light brown solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 7.30 (d, J=8.8 Hz, 1H), 7.13 (d, J=2.8 Hz, 1H), 7.01 (dd, J=8.8 Hz, 2.8 Hz, 1H), 5.59 (s, 2H), 4.36 (t, 5.6 Hz, 2H), 3.58-3.55 (m, 4H), 3.52-3.50 (m, 4H), 3.20 (s, 3H), 2.39-2.35 (m, 4H). ESI-HRMS m/z 319.1773 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-2-(morpholinomethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (241): The compound was prepared by general procedure D provided in example 4 using compound 240 (0.070 g, 0.22 mmol), dry THF (3 mL), 4-nitrophenylchloroformate (0.053 g, 0.26 mmol), 3′-aminoacetophenone (0.035 g, 0.26 mmol), TEA (0.14 mL, 0.52 mmol) to obtain compound 241 (0.055 g, 53%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.08 (s, 1H), 8.95 (s, 1H), 8.28 (d, J=2.4 Hz, 1H), 8.05-8.04 (m, 1H), 7.75 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.67-7.64 (m, 1H), 7.56 (d, J=8.8 Hz, 2H), 7.43-7.39 (m, 1H), 4.42 (t, J=5.6 Hz, 2H), 3.64 (s, 2H), 3.60 (t, J=5.6 Hz, 2H), 3.54-3.52 (m, 4H), 3.21 (s, 3H), 2.53 (s, 3H), 2.43-2.39 (m, 4H). ESI-HRMS m/z 480.2246 (M+H+).
Synthesis of 3-(2-methoxyethyl)-2-((4-methylpiperazin-1-yl)methyl)-6-nitroquinazolin-4 (3H)-one (242): Compound 229 (0.2 g, 0.74 mmol) and 1-methylpiperazine (0.142 mL, 1.48 mmol) were dissolved in toluene and reaction mixture was refluxed for 2 hours. After completion of the reaction, it was washed with water and extracted with ethyl acetate to obtain light yellow crude mass which was then purified by column chromatography (Silica gel, mesh size 100-200) eluting (5% CHCl3-MeOH) to obtain compound 242 (0.202 g, 75%) as white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.76 (s, 1H), 8.48 (d, J=8.8 Hz, 1H), 7.80 (d, J=8.8 Hz, 1H), 4.43 (d, J=5.6 Hz, 2H), 3.70 (s, 2H), 3.61 (t, J=5.6 Hz, 2H), 3.27-3.21 (m, 3H), 2.36-2.18 (m, 4H), 2.10 (s, 3H). ESI-HRMS m/z 362.1821 (M+H+).
Synthesis of 6-amino-3-(2-methoxyethyl)-2-((4-methylpiperazin-1-yl)methyl)quinazolin-4 (3H)-one (243): The compound was prepared by general procedure C provided in example 3 using compound 242 (0.150 g, 0.41 mmol), methanol (10 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 243 (0.110 g, 80%) as light brown solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 7.31 (d, J=8.8 Hz, 1H), 7.12 (d, J=2.8 Hz, 1H), 7.01 (dd, J=8.8 Hz, 2.8 Hz, 1H), 5.59 (s, 2H), 4.34 (t, J=6 Hz, 2H), 3.55 (t, J=6 Hz, 4H), 3.20 (s, 3H), 2.42-2.34 (m, 4H), 2.31-2.16 (m, 4H), 2.09 (s, 3H). ESI-HRMS m/z 332.2090 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-2-((4-methylpiperazin-1-yl)methyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (244): The compound was prepared by general procedure D provided in example 4 using compound 243 (0.070 g, 0.21 mmol), dry THF (3 mL), 4-nitrophenylchloroformate (0.053 g, 0.26 mmol), 3′-aminoacetophenone (0.035 g, 0.26 mmol), TEA (0.14 mL, 0.52 mmol) to obtain compound 244 (0.062 g, 54%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.17 (s, 1H), 9.04 (s, 1H), 8.28 (d, J=2.4 Hz, 1H), 8.06-8.04 (m, 1H), 7.75 (dd, J=8.8 Hz, 2.8 Hz, 1H), 7.56 (d, J=8.6 Hz, 1H), 7.43-7.39 (m, 1H), 4.39 (t, J=6 Hz, 2H), 3.63 (s, 2H), 3.59 (t, J=6 Hz, 2H), 3.21 (s, 3H), 2.53 (s, 3H), 2.44-2.40 (m, 4H), 2.34-2.26 (m, 4H), 2.11 (s, 3H). ESI-HRMS m/z 493.2566 (M+H+).
Synthesis of N-(2-fluorophenyl)-2-(6-nitro-4-oxoquinazolin-3 (4H)-yl)acetamide (251): The compound was prepared by general procedure H provided in example 8 using compound 81 (0.25 g, 0.90 mmol), toluene (5 mL), anhydrous AlCl3 (0.481 g, 3.6 mmol), 2-fluoroaniline (0.120 g, 1.08 mmol), triethylamine (0.3 mL, 2.43 mmol) to obtain compound 251 (0.184 g, 49%) as bright white crystal. ESI-HRMS m/z 343.0844 (M+H+).
Synthesis of 2-(6-amino-4-oxoquinazolin-3 (4H)-yl)-N-(2-fluorophenyl)acetamide (252): The compound was prepared by general procedure C provided in example 3 using compound 251 (0.34 g, 0.99 mmol), methanol (10 mL), pinch of Pd/C under hydrogen atmosphere to obtain compound 252 (0.182 g, 43%) as light brown solid. ESI-HRMS m/z 313.1101 (M+H+).
Synthesis of 2-(6-(3-(3-acetylphenyl)ureido)-4-oxoquinazolin-3 (4H)-yl)-N-(2-fluorophenyl)acetamide (253): The compound was prepared by general procedure D provided in example 4 using compound 252 (0.140 g, 0.44 mmol), dry THF (5 mL), 4-nitrophenyl chloroformate (0.14 g, 0.67 mmol), 3′-aminoacetophenone (0.073 g, 0.53 mmol), TEA (0.16 mL, 1.12 mmol) to obtain compound 253 as white amorphous solid (0.064 g, 30%). ESI-HRMS m/z 474.1580 (M+H+).
Synthesis of N-(2-methoxyphenyl)-2-(6-nitro-4-oxoquinazolin-3 (4H)-yl)acetamide (254): The compound was prepared by general procedure H provided in example 8 using compound 81 (0.25 g, 0.90 mmol), toluene (5 mL), anhydrous AlCl3 (0.481 g, 3.61 mmol), o-anisidine (0.134 g, 1.08 mmol), TEA (0.3 mL, 2.43 mmol) to obtain compound 254 (0.140 g, 44%) as white crystal. ESI-HRMS m/z 355.1045 (M+H+).
Synthesis of 2-(6-amino-4-oxoquinazolin-3 (4H)-yl)-N-(2-methoxyphenyl)acetamide (255): The compound was prepared by general procedure C provided in example 3 using compound 254 (0.12 g, 0.34 mmol), methanol (10 mL), pinch of Pd/C under hydrogen atmosphere to obtain compound 255 (0.1 g, 78%) as light brown solid. ESI-HRMS m/z 325.1304 (M+H+).
Synthesis of 2-(6-(3-(3-acetylphenyl)ureido)-4-oxoquinazolin-3 (4H)-yl)-N-(2-methoxyphenyl)acetamide (256): The compound was prepared by general procedure D provided in example 4 using compound 255 (0.085 g, 0.26 mmol), dry THF (4 mL), 4-nitrophenyl chloroformate (0.08 g, 0.39 mmol), 3′-aminoacetophenone (0.05 g, 0.31 mmol), TEA (0.1 mL, 0.65 mmol) to obtain compound 256 (0.054 g, 39%) as off white solid.
Synthesis of N-(2-bromophenyl)-2-(6-nitro-4-oxoquinazolin-3 (4H)-yl)acetamide (257): The compound was prepared by general procedure H provided in example 8 using compound 81 (0.25 g, 0.90 mmol), toluene (5 mL), anhydrous AlCl3 (0.481 g, 3.6 mmol), 2-bromoaniline (0.186 g, 1.08 mmol), TEA (0.3 mL, 2.43 mmol) to obtain compound 257 (0.180 g, 49%) as white solid. ESI-HRMS m/z 403.0037 (M+H+).
Synthesis of 2-(6-amino-4-oxoquinazolin-3 (4H)-yl)-N-(2-bromophenyl)acetamide (258): Compound 257 (0.160 g, 0.39 mmol) was dissolved in methanol and SnCl2·2H2O (0.716 g, 3.17 mmol) was added. Two drops of concentrated HCl was added and reaction mixture was heated at 80° C. for 2 hours. The reaction was monitored by checking TLC. Upon completion of the reaction, reaction mass was evaporated to dryness and washed with saturated NaHCO3 solution, and extracted with EtOAc to afford the yellow coloured crude mass. The compound was dissolved in DCM (5 mL) and pet ether was added dropwise to get precipitation which was filtered under vacuum to obtain compound 258 (0.075 g, 50%) as light yellow solid.
Synthesis of 2-(6-(3-(3-acetylphenyl)ureido)-4-oxoquinazolin-3 (4H)-yl)-N-(2-bromophenyl)acetamide (259): The compound was prepared by general procedure D provided in example 4 using compound 258 (0.040 g, 0.11 mmol), dry THF (4 mL), 4-nitrophenyl chloroformate (0.04 g, 0.17 mmol), 3′-aminoacetophenone (0.018 g, 0.13 mmol), TEA (0.037 mL, 0.27 mmol) to obtain compound 259 (0.015 g, 26%) as off white solid. ESI-HRMS m/z 534.0797 (M+H+).
Synthesis of 2-(6-nitro-4-oxoquinazolin-3 (4H)-yl)-N-(2-(trifluoromethoxy)phenyl)acetamide (260): The compound was prepared by general procedure H provided in example 8 using compound 81 (0.1 g, 0.36 mmol), toluene (5 mL), anhydrous AlCl3 (0.192 g, 1.44 mmol), 2-(trifluoromethoxy)aniline (0.077 g, 0.43 mmol), TEA (0.13 mL, 0.97 mmol) to obtain compound 260 (0.072 g, 49%) as white solid. ESI-HRMS m/z 409.0755 (M+H+).
Synthesis of 2-(6-amino-4-oxoquinazolin-3 (4H)-yl)-N-(2-(trifluoromethoxy)phenyl)acetamide (261): The compound was prepared by general procedure C provided in example 3 using compound 260 (0.12 g, 0.29 mmol), methanol (10 mL), pinch of Pd/C under hydrogen atmosphere to obtain compound 261 (0.059 g, 50%).
Synthesis of 2-(6-(3-(3-acetylphenyl)ureido)-4-oxoquinazolin-3 (4H)-yl)-N-(2-(trifluoromethoxy)phenyl)acetamide (262): The compound was prepared by general procedure D provided in example 4 using compound 261 (0.05 g, 0.15 mmol), dry THF (5 mL), 4-nitrophenyl chloroformate (0.04 g, 0.19 mmol), 3′-aminoacetophenone (0.022 g, 0.15 mmol), TEA (0.05 mL, 0.33 mmol) to obtain 262 (0.06 g, 84%) as white solid. ESI-HRMS m/z 540.1500 (M+H+).
Synthesis of 2-(6-nitro-4-oxoquinazolin-3 (4H)-yl)-N-(2-(trifluoromethyl)phenyl)acetamide (263): The compound was prepared by general procedure H provided in example 8 using compound 81 (0.25 g, 0.9 mmol), toluene (5 mL), anhydrous AlCl3 (0.481 g, 3.6 mmol), 2-(trifluoromethyl)aniline (0.174 g, 1.08 mmol) to obtain compound 263 (0.160 g, 45%) as white solid.
Synthesis of 2-(6-amino-4-oxoquinazolin-3 (4H)-yl)-N-(2-(trifluoromethyl)phenyl)acetamide (264): The compound was prepared by general procedure C provided in example 3 using compound 263 (0.08 g, 0.20 mmol), methanol (10 mL), one pinch of Pd/C under hydrogen atmosphere to obtain compound 264 (0.50 g, 68%) as light yellow solid.
Synthesis of 2-(6-(3-(3-acetylphenyl)ureido)-4-oxoquinazolin-3 (4H)-yl)-N-(2-(trifluoromethyl)phenyl)acetamide (265): The compound was prepared by general procedure D provided in example 4 using compound 264 (0.04 g, 0.11 mmol), dry THF (4 mL), 4-nitrophenyl chloroformate (0.04 g, 0.16 mmol), 3′-aminoacetophenone (0.02 g, 0.13 mmol), TEA (0.04 mL, 0.27 mmol) to obtain compound 265 as off white solid (0.015 g, 26%).
Synthesis of N-(4-methoxyphenyl)-2-(6-nitro-4-oxoquinazolin-3 (4H)-yl)acetamide (266): The compound was prepared by general procedure H provided in example 8 using compound 81 (0.25 g, 0.90 mmol), toluene (5 mL), anhydrous AlCl3 (0.481 g, 3.61 mmol), p-anisidine (0.134 g, 1.08 mmol), TEA (0.3 mL, 2.43 mmol) to obtain compound 266 (0.142 g, 45%) as white crystal. ESI-HRMS m/z 355.1045 (M+H+).
Synthesis of 2-(6-amino-4-oxoquinazolin-3 (4H)-yl)-N-(4-methoxyphenyl)acetamide (267): The compound was prepared by general procedure C provided in example 3 using compound 266 (0.21 g, 0.59 mmol), methanol (10 mL), pinch of Pd/C under hydrogen atmosphere to obtain compound 267 (0.15 g, 78%) as light brown solid. ESI-HRMS m/z 325.1304 (M+H+).
Synthesis of 2-(6-(3-(3-acetylphenyl)ureido)-4-oxoquinazolin-3 (4H)-yl)-N-(4-methoxyphenyl)acetamide (268): The compound was prepared by general procedure D provided in example 4 using compound 267 (0.085 g, 0.26 mmol), dry THF (4 mL), 4-nitrophenyl chloroformate (0.08 g, 0.39 mmol), 3′-aminoacetophenone (0.05 g, 0.31 mmol), TEA (0.1 mL, 0.65 mmol) to obtain compound 268 (0.054 g, 39%) as off white solid.
Synthesis of 2-acetamido-N-(1-methoxybutan-2-yl)-5-nitrobenzamide (278): Compound 168 (0.5 g, 1.87 mmol) was dissolved in DCM (10 mL). Then TEA (0.522 mL, 3.74 mmol) was added. Then acetyl chloride (0.267 mL, 3.74 mmol) was added dropwise under cooling conditions and the reaction mass was stirred for 8 hours. After completion of the reaction, DCM was evaporated out and washed with water and extracted with ethyl acetate and purified by column chromatography (Silica gel, mesh size 100-200) eluting (30% Ethyl acetate-Pet ether) to obtain compound 278 (0.462 g, 80%) as off white solid. ESI-HRMS m/z 310.1402 (M+H+).
Synthesis of 3-(1-methoxybutan-2-yl)-2-methyl-6-nitroquinazolin-4 (3H)-one (279): The compound was prepared by general procedure G provided in example 7 using compound 278 (0.4 g, 1.29 mmol), DMF (4 mL), ZnCl2 (0.703 g, 5.16 mmol), HMDS (2.16 mL, 10.32 mmol) to obtain compound 279 (0.278 g, 74%) as off white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.73 (d, J=2.4 Hz, 1H), 8.47 (dd, J=9.2 Hz, 2.8 Hz, 1H), 7.72 (d, J=8.8 Hz, 1H), 4.49-4.41 (m, 1H), 4.07-4.02 (m, 1H), 3.62-3.58 (m, 1H), 3.17 (s, 3H), 2.62 (s, 3H), 2.21-2.11 (m, 1H), 1.87-1.76 (m, 1H), 0.79 (t, J=7.6 Hz, 3H). ESI-HRMS m/z 292.1292 (M+H+).
Synthesis of 6-amino-3-(1-methoxybutan-2-yl)-2-methylquinazolin-4 (3H)-one (280): The compound was prepared by general procedure C provided in example 3 using compound 279 (0.150 g, 0.51 mmol), methanol (5 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 280 (0.102 g, 76%) as light brown solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 7.22 (d, J=8.4 Hz, 1H), 7.06 (d, J=2.8 Hz, 1H), 6.99 (dd, J=8.4 Hz, 2.4 Hz, 1H), 5.49 (brs, 2H), 4.31-4.23 (m, 1H), 3.57-3.54 (m, 1H), 3.16 (s, 3H), 2.45 (s, 3H), 2.23-2.11 (m, 1H), 1.80-1.68 (m, 1H), 0.74 (t, J=7.2 Hz, 3H). ESI-HRMS m/z 262.1559 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(3-(1-methoxybutan-2-yl)-2-methyl-4-oxo-3,4-dihydroquinazolin-6-yl)ureae (281): The compound was prepared by general procedure D provided in example 4 using compound 280 (0.080 g, 0.30 mmol), dry THF (3 mL), 4-nitrophenylchloroformate (0.073 g, 0.36 mmol), 3′-aminoacetophenone (0.048 g, 0.36 mmol), TEA (0.083 mL, 0.60 mmol) to obtain compound 281 (0.069 g, 54%) as off white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.03 (s, 1H), 8.97 (s, 1H), 8.21 (d, J=2.4 Hz, 1H), 8.06-8.05 (m, 1H), 7.73 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.66-7.63 (m, 1H), 7.56-7.54 (m, 1H), 7.47 (d, J=8.8 Hz, 1H), 7.42-7.38 (m, 1H), 4.39-4.31 (m, 1H), 4.08 (t, J=9.6 Hz, 1H), 3.60-3.56 (m, 1H), 3.17 (s, 3H, 2.53 (s, 6H), 2.24-2.15 (m, 1H), 1.82-1.73 (m, 1H), 0.77 (t, J=7.6 Hz, 3H). ESI-HRMS m/z 423.2030 (M+H+).
Synthesis of 2-acetamido-N-(1-methoxypropan-2-yl)-5-nitrobenzamide (282): Compound 176 (0.5 g, 1.97 mmol) was dissolved in DCM (10 mL). Then TEA (0.551 mL, 3.95 mmol) was added. Then Acetyl chloride (0.281 mL, 3.95 mmol) was added dropwise under cooling conditions and the reaction mass was stirred for 8 hours. After completion of the reaction, DCM was evaporated out and worked up with ethyl acetate and water and purified by column chromatography (Silica gel, mesh size 100-200) eluting (30% Ethyl acetate-Pet ether) to obtain compound 282 (0.437 g, 75%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 11.55 (s, 1H), 8.97 (d, 7.85 Hz, 1H), 8.64-8.59 (m, 2H), 8.36 (dd, J=9.3 Hz, 2.7 Hz, 1H), 3.47-3.42 (m, 2H), 3.29 (s, 3H), 2.17 (s, 3H), 1.17 (d, J=6.6 Hz, 3H). ESI-HRMS m/z 318.1074 (M+H+).
Synthesis of 3-(1-methoxypropan-2-yl)-2-methyl-6-nitroquinazolin-4 (3H)-one (283): The compound was prepared by general procedure G provided in example 7 using compound 282 (0.4 g, 1.35 mmol), DMF (4 mL), ZnCl2 (0.738 g, 5.42 mmol), HMDS (2.26 mL, 10.84 mmol) to obtain compound 283 (0.255 g, 68%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 8.72 (d, J=2.7 Hz, 1H), 8.45 (dd, J=9 Hz, 2.7 Hz, 1H), 7.72 (d, 9 Hz, 1H), 4.72-4.59 (m, 1H), 4.10-4.04 (m, 1H), 3.62-3.57 (m, 1H), 3.20 (s, 3H), 2.65 (s, 3H), 1.50 (d, J=3.9 Hz, 3H). ESI-HRMS m/z 278.1151 (M+H+).
Synthesis of 6-amino-3-(1-methoxypropan-2-yl)-2-methylquinazolin-4 (3H)-one (284): The compound was prepared by general procedure C provided in example 3 using compound 283 (0.150 g, 0.54 mmol), methanol (5 mL), pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 284 (0.086 g, 76%) as light brown solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 7.26 (d, J=8.7 Hz, 1H), 7.10 (d, J=2.4 Hz, 1H), 7.02 (dd, J=8.7 Hz, 2.7 Hz, 1H), 5.52 (s, 2H), 4.60-4.42 (m, 1H), 4.10-4.04 (m, 1H), 3.63-3.57 (m, 1H), 3.21 (s, 3H), 2.50 (s, 3H), 1.47 (d, J=6.6 Hz, 3H). ESI-HRMS m/z 248.1400 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(3-(1-methoxypropan-2-yl)-2-methyl-4-oxo-3,4-dihydroquinazolin-6-yl)urea (285): The compound was prepared by general procedure D provided in example 4 using compound 284 (0.070 g, 0.28 mmol), dry THF (3 mL), 4-nitrophenylchloroformate (0.068 g, 0.34 mmol), 3′-aminoacetophenone (0.046 g, 0.34 mmol), TEA (0.083 mL, 0.56 mmol) to obtain compound 285 (0.069 g, 54%) as off white solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 9.06 (s, 1H), 6.99 (s, 1H), 8.25 (d, J=2.4 Hz, 1H), 8.10 (brs, 1H), 7.76 (dd, J=8.7 Hz, 2.4 Hz, 1H), 7.70 (dd, J=8.1 Hz, 0.9 Hz, 1H), 7.60 (d, J=7.8 Hz, 1H), 7.50 (d, J=9 Hz, 1H), 7.48-7.42 (m, 1H), 4.57 (s, 1H), 4.12 (t, J=9.3 Hz, 1H), 3.63-3.59 (m, 1H), 3.22 (s, 3H), 2.57 (s, 6H), 1.50 (d, J=6.9 Hz, 3H). ESI-HRMS m/z 409.1877 (M+H+).
Synthesis of 2-(cyclohexanecarboxamido)-N-(1-methoxypropan-2-yl)-5-nitrobenzamide (286): Compound 176 (0.5 g, 1.97 mmol) was dissolved in DCM (10 mL). Then TEA (0.6 mL, 3.95 mmol) was added. Then cyclohexanecarbonyl chloride (0.57 g, 3.95 mmol) was added dropwise under cooling conditions and the reaction mass was stirred for 8 hours. After completion of the reaction, DCM was evaporated out and worked up with ethyl acetate and water and purified by column chromatography (Silica gel, mesh size 100-200) eluting (30% Ethyl acetate-Pet ether) to obtain compound 286 (0.446 g, 75%) as off white solid.
Synthesis of 2-cyclohexyl-3-(1-methoxypropan-2-yl)-6-nitroquinazolin-4 (3H)-one (287): The compound was prepared by general procedure G provided in example 7 using compound 286 (0.4 g, 1.35 mmol), DMF (4 mL), ZnCl2 (0.73 g, 5.42 mmol), HMDS (2.26 mL, 10.84 mmol) to obtain compound 287 (0.285 g, 75%) as off white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 8.72 (d, J=2.8 Hz, 1H), 8.44 (dd, J=8.8 Hz, 2.8 Hz, 1H), 7.70 (d, J=9.2 Hz, 1H), 4.68 (s, 1H), 4.10-4.01 (m, 1H), 3.64-3.60 (m, 1H), 3.17 (s, 3H), 3.00-2.93 (m, 1H), 1.93-1.80 (m, 2H), 1.78-1.72 (m, 2H), 1.70-1.55 (m, 3H), 1.49 (d, J=6.8 Hz, 3H), 1.44-1.33 (m, 2H), 1.33-1.28 (m, 1H). ESI-HRMS m/z 346.1772 (M+H+).
Synthesis of 6-amino-2-cyclohexyl-3-(1-methoxypropan-2-yl)quinazolin-4 (3H)-one (288): The compound was prepared by general procedure C provided in example 3 using compound 287 (0.200 g, 0.58 mmol), methanol (5 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 288 (0.127 g, 70%) as light brown solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 7.22d, J=8.4 Hz, 1H), 7.07 (d, J=2.8 Hz, 1H), 6.98 (dd, J=8.8 Hz, 2.8 Hz, 1H), 5.46 (s, 2H), 4.48 (brs, 1H), 4.06-3.99 (m, 1H), 3.65-3.61 (m, 1H), 3.16 (s, 3H), 2.83-2.75 (m, 1H), 1.87-1.84 (m, 1H), 1.79-1.71 (m, 3H), 1.66-1.63 (m, 1H), 1.58-1.52 (m, 2H), 1.45 (d, J=6.8 Hz, 3H), 1.38-1.30 (m, 2H), 1.24-1.16 (m 1H). ESI-HRMS m/z 316.2027 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(2-cyclohexyl-3-(1-methoxypropan-2-yl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (289): The compound was prepared by general procedure D provided in example 4 using compound 288 (0.080 g, 0.25 mmol), dry THF (3 mL), 4-nitrophenylchloroformate (0.061 g, 0.30 mmol), 3′-aminoacetophenone (0.040 g, 0.30 mmol), TEA (0.069 mL, 0.50 mmol) to obtain compound 289 (0.062 g, 52%) as off white solid. ESI-HRMS m/z 477.2504 (M+H+).
Synthesis of tert-butyl 4-((2-((2-methoxyethyl)carbamoyl)-4-nitrophenyl)carbamoyl)piperidine-1-carboxylate (296): Commercially available N-Boc piperidine-4-carboxylic acid (1.2 g, 5.22 mmol) was dissolved in dry DCM (10 mL) followed by addition of one drop of DMF. Oxalyl chloride (0.66 mL, 7.80 mmol) was added to the reaction mixture at ice cold condition and under nitrogen atmosphere. The reaction is stirred for 15 mins for the formation of corresponding acid chloride. Then, the reaction mass was evaporated to dryness to remove excess oxalyl chloride and DCM. The crude was kept at nitrogen atmosphere and dissolved in measured amount of DCM (20 mL). In another vessel, compound 2 (0.50 g, 2.09 mmol) was dissolved in DCM (10 mL) followed by addition of TEA (0.7 mL, 5.64 mmol) and dropwise addition of prepared acid chloride. The reaction was stirred for another 3 hours. The reaction was monitored by checking TLC. Upon completion of the reaction, reaction mass was evaporated to dryness and washed with saturated NaHCO3 and extracted with EtOAc to get the crude. The product was purified by flash chromatography (Silica gel, mesh size 100-200) eluting (60% EtOAc/Pet ether) to obtain compound 296 (0.31 g, 48.88%) as a white solid. ESI-HRMS m/z 451.219 (M+H+). Melting point 102° C.
Synthesis of tert-butyl 4-(3-(2-methoxyethyl)-6-nitro-4-oxo-3,4-dihydroquinazolin-2-yl)piperidine-1-carboxylate (297): The compound was prepared by general procedure G provided in example 7 using compound 296 (0.30 g, 0.66 mmol), DMF (5 mL), zinc chloride (0.63 g, 4.66 mmol), HMDS (0.83 mL, 3.99 mmol) to obtain compound 297 (0.17 g, 49%) as yellow solid. ESI-HRMS m/z 433.2090 (M+H+). Melting point 170° C.
Synthesis of tert-butyl 4-(6-amino-3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)piperidine-1-carboxylate (298): The compound was prepared by general procedure C provided in example 3 using compound 297 (0.16 g, 0.38 mmol), methanol (10 mL), a pinch of Pd—C under hydrogen atmosphere to obtain compound 298 (0.15 g, 78%) as light brown solid. ESI-HRMS m/z 403.2349 (M+H+). Melting point 174° C.
Synthesis of tert-butyl 4-(6-(3-(3-acetylphenyl)ureido)-3-(2-methoxyethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)piperidine-1-carboxylate (299): The compound was prepared by general procedure D provided in example 4 using compound 298 (0.14 g, 0.34 mmol), dry THF (5 mL), 4-nitrophenylchloroformate (0.10 g, 0.52 mmol), 3′-aminoacetophenone (0.043 g, 0.31 mmol), TEA (0.06 mL, 0.41 mmol) to obtain compound 299 (0.09 g, 39%) as off white solid. ESI-HRMS m/z 564.2821 (M+H+). Melting point 196° C.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-2-(piperidin-4-yl)-3,4-dihydroquinazolin-6-yl)urea (300): Compound 299 (0.068 g, 0.12 mmol) was dissolved in DCM (3 mL) and TFA (0.05 mL, 0.60 mmol) was added dropwise under ice cold condition at inert atmosphere. The reaction was stirred for 8 hrs. The reaction was neutralised by saturated NaHCO3 solution and extracted with EtOAc. The product was then purified by column chromatography (Silica gel, mesh size 100-200) eluting (70% EtOAc/Pet ether) to obtain compound 300 (0.009 g, 39.2%) as off white amorphous solid. ESI-HRMS m/z 464.2296 (M+H+).
Synthesis of 2-(2-chloro-2,2-difluoroacetamido)-N-(2-methoxyethyl)-5-nitrobenzamide (326a): Compound 2 (0.6 g, 2.50 mmol) was dissolved in dry pyridine (6 mL), Chlorodifluoroacetic acid (0.360 g, 2.75 mmol) was added and then POCl3 (0.281 mL, 2.75 mmol) was added dropwise under cooling conditions and the reaction mixture was allowed to stir at room temperature for 1 hour. After completion of the reaction, workup was done with EtOAc and 1(N) HCl soln. The product was then purified by column chromatography (Silica gel, mesh size 100-200) eluting (30% EtOAc/Pet ether) to obtain compound 326a as off white amorphous solid (0.634 g, 72%). 1H NMR (400 MHz, d6-DMSO) δ in ppm 13.56 (s, 1H), 9.48 (s, 1H), 8.81 (d, J=2.4 Hz, 1H), 8.54 (d, J=8.8 Hz, 1H), 8.46 (dd, J=9.2 Hz, 2.4 Hz, 1H), 3.48-3.43 (m, 4H), 3.24 (s, 3H).
Synthesis of 2-(chlorodifluoromethyl)-3-(2-methoxyethyl)-6-nitroquinazolin-4 (3H)-one (327a): The compound was prepared by general procedure G provided in example 7 using compound 326a (0.4 g, 1.14 mmol), DMF (5 mL), zinc chloride (0.621 g, 4.56 mmol), HMDS (1.9 mL, 9.12 mmol) to obtain compound 327a as off white amorphous solid (0.303 g, 80%). 1H NMR (400 MHz, DMSO-d6) δ in ppm 8.80 (d, J=2.4 Hz, 1H), 8.60 (dd, J=8.8 Hz, 3.2 Hz, 1H), 8.00 (d, J=8.8 Hz, 1H), 4.29 (t, J=6.8 Hz, 2H), 3.60 (t, J=6.8 Hz, 2H), 3.24 (s, 3H).
Synthesis of 3-(2-methoxyethyl)-6-nitro-2-(piperidine-1-carbonyl)quinazolin-4 (3H)-one (328a): Compound 327a (0.25 g, 0.75 mmol) and piperidine (0.15 mL, 1.50 mmol) were dissolved in dry toluene and refluxed for 2 hrs. After completion of the reaction, workup was done with EtOAc and water. The product was then purified by column chromatography (Silica gel, mesh size 100-200) eluting (40% EtOAc/Pet ether) to obtain compound 328a as off white amorphous solid (0.232 g, 86%). 1H NMR (400 MHz, CDCl3) δ in ppm 9.12 (d, J=2.8 Hz, 1H), 8.51 (dd, J=8.8 Hz, 2.8 Hz, 1H), 7.77 (d, J=8.8 Hz, 1H), 4.34 (t, J=5.2 Hz, 2H), 3.78-3.66 (m, 2H), 3.26 (s, 3H), 1.75-1.66 (m, 6H).
Synthesis of 6-amino-3-(2-methoxyethyl)-2-(piperidine-1-carbonyl)quinazolin-4 (3H)-one (329a): The compound was prepared by general procedure C provided in example 3 using compound 328a (0.200 g, 0.55 mmol), methanol (10 mL), a pinch of Pd—C under hydrogen atmosphere to obtain compound 329a (0.113 g, 62%) as light brown solid. 1H NMR (400 MHz, CDCl3) δ in ppm 7.48 (d, J=8.8 Hz, 1H), 7.43 (d, J=2.8 Hz, 1H), 7.06 (dd, J=8.8 Hz, 1H), 4.29 (t, J=5.6 Hz, 2H), 4.02 (brs, 2H), 3.72-3.68 (m, 2H), 3.26 (s, 3H), 1.71-1.63 (m, 6H).
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-4-oxo-2-(piperidine-1-carbonyl)-3,4-dihydroquinazolin-6-yl)urea (330a): The compound was prepared by general procedure D provided in example 4 using compound 329a (0.07 g, 0.14 mmol), dry THF (5 mL), 4-nitrophenylchloroformate (0.048 g, 0.16 mmol), 3′-aminoacetophenone (0.032 g, 0.16 mmol), TEA (0.06 mL, 0.28 mmol) to obtain compound 330a (0.04 g, 39%) as off white solid. 1H NMR (400 MHz, CDCl3) δ in ppm 8.31 (s, 1H), 8.22 (s, 1H), 8.04 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.94-7.93 (m, 1H), 7.83 (dd, J=8.4 Hz, 2.4 Hz, 1H), 7.70 (d, J=2.4 Hz, 1H), 7.59-7.55 (m, 1H), 7.43 (d, J=8.8 Hz, 1H), 7.37-7.33 (m, 1H), 4.27 (t, J=4.8 Hz, 2H), 3.81-3.73 (m, 2H), 3.67 (t, J=4.8 Hz, 2H), 3.42-3.35 (m, 2H), 3.25 (s, 3H), 2.59 (s, 3H), 1.88-1.65 (m, 6H).
Synthesis of 3-(2-methoxyethyl)-2-(4-methylpiperazine-1-carbonyl)-6-nitroquinazolin-4 (3H)-one (331a): Compound 327a (0.25 g, 0.75 mmol) and 1-methylpiperazine (0.16 mL, 1.50 mmol) were dissolved in dry toluene (5 mL) and refluxed for 2 hrs. After completion of the reaction, workup was done with EtOAc and water. The product was then purified by column chromatography (Silica gel, mesh size 100-200) eluting (40% EtOAc/Pet ether) to obtain compound 331a as off white amorphous solid (0.232 g, 82%).
Synthesis of 6-amino-3-(2-methoxyethyl)-2-(4-methylpiperazine-1-carbonyl)quinazolin-4 (3H)-one (332a): The compound was prepared by general procedure C provided in example 3 using compound 331a (0.200 g, 0.55 mmol), methanol (10 mL), a pinch of Pd—C under hydrogen atmosphere to obtain compound 332a (0.120 g, 58%) as light brown solid.
Synthesis of 1-(3-acetylphenyl)-3-(3-(2-methoxyethyl)-2-(4-methylpiperazine-1-carbonyl)-4-oxo-3,4-dihydroquinazolin-6-yl)urea (333a): The compound was prepared by general procedure D provided in example 4 using compound 332a (0.07 g, 0.14 mmol), dry THF (5 mL), 4-nitrophenylchloroformate (0.049 g, 0.16 mmol), 3′-aminoacetophenone (0.033 g, 0.16 mmol), TEA (0.06 mL, 0.28 mmol) to obtain compound 333a (0.043 g, 42%) as off white solid. 1H NMR (400 MHz, DMSO-d6) δ in ppm 9.19 (s, 1H), 9.01 (s, 1H), 8.35 (d, J=2.8 Hz, 1H), 8.07-8.05 (m, 1H), 7.81 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.68-7.65 (m, 1H), 7.59 (d, J=8.8 Hz, 1H), 4.10 (t, J=6.0 Hz, 2H), 3.64-3.58 (m, 2H), 3.53 (t, J=6.0 Hz, 2H), 3.40-3.35 (m, 2H), 3.18 (s, 3H), 2.53 (s, 3H), 2.40-2.37 (m, 2H), 2.35-2.30 (m, 2H), 2.20 (s, 3H).
Synthesis of 2-amino-N-morpholino-5-nitrobenzamide (337): The compound was prepared by general procedure A provided in example 1 using compound 1 (0.3 g, 1.64 mmol), DMF (6 mL), HATU (0.7 g, 1.81 mmol), 4-aminomorpholine (0.18 mL, 1.81 mmol), TEA (0.57 mL, 4.12 mmol) to obtain compound 337 (0.38 g, 86%) as yellow solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.69 (s, 1H), 8.38 (d, J=2 Hz, 1H), 8.02 (dd, J=9.4 Hz, 2.8 Hz, 1H), 7.58 (brs, 2H), 6.79 (d, J=9.6 Hz, 1H), 3.67-3.65 (m, 4H), 2.88-2.85 (m, 4H).
Synthesis of 3-morpholino-6-nitroquinazolin-4 (3H)-one (338): The compound was prepared by general procedure B provided in example 2 using compound 337 (0.35 g, 1.31 mmol), trimethylorthoformate (TMOF) (1.5 mL, 13.15 mmol) to obtain compound 338 (0.3 g, 83%) as pale yellow solid. 1H NMR (400 MHz, d6-CDCl3) δ in ppm 9.13 (d, J=2.4 Hz, 1H), 8.50 (dd, J=8.8 Hz, 2.8 Hz, 1H), 8.26 (s, 1H), 7.80 (d, J=9.2 Hz, 1H), 3.04-3.01 (m, 2H), 2.24-2.18 (m, 2H), 2.02-1.97 (m, 4H).
Synthesis of 6-amino-3-morpholinoquinazolin-4 (3H)-one (339): The compound was prepared by general procedure C provided in example 3 using compound 338 (0.250 g, 0.90 mmol), methanol (5 mL) and pinch of 10% wet Pd—C under hydrogen atmosphere to obtain compound 339 (0.19 g, 86%) as brown solid. 1H NMR (300 MHz, d6-DMSO) δ in ppm 7.91 (s, 1H), 7.38 (d, J=8.7 Hz, 1H), 7.21 (d, J=2.4 Hz, 1H), 7.06 (dd, J=8.4 Hz, 2.4 Hz, 1H), 5.72 (brs, 2H), 3.75-3.67 (m, 6H), 3.18-3.15 (m, 2H).
Synthesis of 1-(3-acetylphenyl)-3-(3-morpholino-4-oxo-3,4-dihydroquinazolin-6-yl)urea (340): The compound was prepared by general procedure D provided in example 4 using compound 339 (0.14 g, 0.56 mmol), dry THF (8 mL), 4-nitrophenylchloroformate (0.18 g, 0.85 mmol), 3′-aminoacetophenone (0.093 mg, 0.68 mmol), TEA (0.2 mL, 1.42 mmol) to obtain compound 340 (0.09 g, 39%) as off white solid. 1H NMR (400 MHz, d6-DMSO) δ in ppm 9.11 (s, 1H), 8.95 (s, 1H), 8.31 (d, J=2.8 Hz, 1H), 8.09 (s, 1H), 8.06-8.05 (m, 1H), 7.80 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.66 (dd, J=8.2 Hz, 2 Hz, 1H), 7.59-7.55 (m, 2H), 7.42-7.39 (m, 1H), 3.73-3.65 (m, 8H), 2.53 (s, 3H).
Synthesis of 6-amino-5-bromo-3-(2-methoxyethyl)-2-methylquinazolin-4 (3H)-one (344): Compound 106 (0.300 g, 1.28 mmol) was dissolved in AcOH (4 mL). Solution of bromine (0.079 mL, 1.53 mmol) in chloroform (2 mL) was added dropwise in the reaction mixture at 0° C. and reaction mass was allowed to stir at room temperature for 4 hrs. After completion of the reaction, reaction mass was washed with aq. NaSCN soln and extracted with ethyl acetate and purified by column chromatography (Silica gel, mesh size 100-200) eluting (70% EA-PE) to obtain compound 344 (0.280 g, 70%) as off white solid. ESI-HRMS m/z 312.0351 (M+H+).
Synthesis of 1-(3-acetylphenyl)-3-(5-bromo-3-(2-methoxyethyl)-2-methyl-4-oxo-3,4-dihydroquinazolin-6-yl)urea (345): The compound was prepared by general procedure D provided in example 4 using compound 344 (0.080 g, 0.25 mmol), dry THF (3 mL), 4-nitrophenylchloroformate (0.062 g, 0.30 mmol), 3′-aminoacetophenone (0.040 g, 0.30 mmol), TEA (0.094 mL, 0.68 mmol) to obtain compound 345 (0.048 g, 40%) as light brown solid. ESI-HRMS m/z 473.0829 (M+H+).
To select the compounds capable of inhibiting the ubiquitination of ATGL by COP1 by targeting the VP motif, confocal microscopy was performed with the provided molecules. If the compound was effective in inhibiting the interaction, there would be a reduction in the number of fat droplets in the cells after treatment. This is because the increased ATGL levels would hydrolyze the accumulated TAG in oleate induced HepG2 cells and bring about the aforementioned reduction. With this rationale in mind, HepG2 cells were induced to accumulate lipid droplets after treatment with 250 M of oleate and 10 μM of the specific compounds were added. The potential of the compounds to bring about a reduction in the number of fat droplets was then checked by comparison with oleate induced cells by counting number of droplets of approximately 20 cells from each treatment and calculating the average number of lipid droplets of each cell. The selected compounds were then subjected to dose dependent treatments and the ones which could maintain its potency to reduce fat droplets at lower doses were then selected for western blot analysis. The compound which could reduce the number of fat droplets in the cells are expected to raise the levels of ATGL since they are likely to deter COP1 from ubiquitinating ATGL. This increase will be visible only in the protein level and gene expression is likely to remain unchanged since ubiquitination is a post transcriptional modification. Thus, western blot was performed to check ATGL levels in the cells with the selected molecules.
HepG2 cells were treated with the compounds 9, 10, 11, 17, 18, 23, 24, 115, 123, 127, 139,107, 171, 179, 73, 187, 211, 215, 219, 223, 232, 238, 241, 244, 258, 299, 308, and 333a, (10 μM for initial screening and 50 nM, 100 nM, 200 nM, 500 nM, 1 μM and 5 μM for dose dependent assays) for 24 hours. After removing media from the cells, the wells were washed with 1×PBS twice to remove any remnant media. Cells were then lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 and protease inhibitor cocktail (Millipore, Billierica, MA, USA). Following centrifugation at 20,000 g for 20 minutes, the protein solution was extracted from the cells. Protein was estimated using Bradford assay. Bradford's reagent (BioRad) was diluted in 1:4 ratio in double distilled water. 2 μl of protein sample was added to 100 μl of the reagent and absorbance was measured at 595 nm. 30 g of protein was diluted in lysis buffer. 1× loading buffer diluted from 5× stock containing 250 mM Tris-HCl (pH 6.8), 10% SDS, 50% glycerol, 0.1% bromophenol Blue and 10% β-mercaptoethanol was added. The protein samples were then heated at 95° C. for 10 minutes, cooled and centrifuged at 12,000 g for 2 minutes prior to loading.
For western blotting, the proteins were resolved in 10% SDS PAGE (discontinuous buffer system). 1× running buffer containing SDS, Tris Base and Glycine was used to run the gel at 80V for approximately 2 hours. Transfer was done using PVDF membrane (Millipore) having pore size of 0.45 μm. 1× transfer buffer containing Tris-Base, Glycine and 20% methanol was used for wet transfer. Transfer was done at 90V for 3 hours. Following transfer, the PVDF membrane containing the proteins were washed in 1×PBST comprising of 1×PBS and 1% Tween 20 (Sigma Aldrich). The membrane was then incubated for 1 hour at room temperature in 5% skim milk powder to block the non-specific sites. Following multiple washes with 1×PBST to wash away any remaining blocking buffer, the required primary antibody (COP1 [Bethyl Laboraties], ATGL [Cell Signalling Technology] or Actin[Cell Signalling Technology]) prepared with 1×PBST, 1% Bovine Serum Albumin and 0.04% Sodium Azide was added to the membrane and incubated overnight at 4° C. The next day, the membrane was again washed multiple times with 1×PBST to remove any unbound primary antibody. The membrane was then incubated with goat anti-rabbit secondary antibody (Genei) for 1 hour at room temperature and washed again for multiple times with 1×PBST. The membrane was then developed using Clarity™ ECL Western Blotting Substrate (BioRad) and viewed in ChemiDoc (BioRad). The EC50 values of compounds 127, 139, 211, 215, 219, 223, 232, 238 and 299 are described in Table 6.
HepG2 cells were plated in confocal dishes (SPL, Genetix Biotech Asia Pvt. Ltd.). The cells were allowed to adhere and divide for 16 hours. 10 μM for initial screening and 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μM and 5 μM for dose dependent assays of the compounds 9 and 107 were dissolved in DMSO and added to the cells. 250 μM of oleate was used for induction. BSA (Sigma Aldrich) was used as a negative control. Post 24 hours of treatment, media was decanted from the cells and washed with 1×PBS solution to remove any remnant. 200 μl of staining solution containing 200 ng/ml BODIPY (Invitrogen) and 25 g/ml HOECKST342 (Invitrogen) were added to the cells and incubated at 37° C. for 30 minutes under dark conditions. Cells were washed 3 times with 1×PBS to remove excess stain. FLUOVIEW FV10i (Olympus) was used to visualise the cells.
To further strengthen the efficiency of the compounds 9 and 107, primary hepatocytes were isolated from mice and these compounds were treated in a dose dependent manner for 24 hours. Primary hepatocytes and adipose tissue explants were isolated from mice and subjected to compound treatment for 24 hours at the doses of 100 nM, 500 nM and 1 μM. Post cell harvesting, western blot was carried out with the lysate to check the ATGL level. ATGL and COP1 antibodies were used. Actin served the purpose of a loading control.
1. Hepatocytes—2-4 months old chow-fed black male mouse (C57bl/6) was sacrificed using chloroform (SRL) and was cleaned with 70% ethanol. Under aseptic conditions, the ventral side of the mouse was cut open, until the liver, portal vein (PV) and inferior vena cava (IVC) were sufficiently exposed. Blood was drawn from the heart in order to prevent backflow into liver while perfusion. The butterfly cannula was inserted into the PV and 20 ml of HBSS (Hank's Balanced Salt Solution; 5 mM KCl, 0.4 mM KH2PO4, 4 mM NaHCO3, 140 mM NaCl, 0.3 mM Na2HPO4, 6 mM Glucose, HEPES, 0.5 mM MgCl2·6H2O, 0.4 mM MgSO4·7H2O, 0.5 mM EDTA; not containing 1 mM CaCl2)) was allowed to pass through the liver (Perfusion) at a constant flow rate of 3 ml/min, maintained by Masterflex digital peristaltic pump (Cole-Parmer). The IVC was cut as soon as the passage of the buffer through the liver began, so that blood and perfusate from liver is drained through the IVC. The liver blanched and became pale in color upon this treatment.
After the passage of HBSS, 25 ml of Collagenase (Roche) solution (1 mg/ml) in HBSS (containing 1 mM CaCl2)) was allowed to pass through the liver at a constant flow rate of 2 ml/min. After this digestion, the flow was stopped, the cannula removed and the pale and soggy lobes of the liver were gently excised from the body. The gall bladder was removed from the isolated liver. The pieces of digested liver tissue were then minced on a 10 cm culture plate in HBSS (containing 1 mM CaCl2)). The resulting suspension was then passed through a 100 cell strainer (SPL) to allow hepatocytes to pass through to the filtrate and retain cellular clumps and undigested tissue. The filtrate was centrifuged at 50 g for 2 minutes at 4° C. The supernatant was discarded and the cellular pellet was carefully resuspended in DMEM. The resulting suspension was centrifuged at 50 g for 2 minutes at 4° C. The supernatant was discarded and the cellular pellet was carefully resuspended in required volume of DMEM for plating. The hepatocytes were plated according to experimental requirements and were maintained in an incubator at 37° C. with 5% CO2. Cells were washed once with HBSS and DMEM 6-7 hours after plating and the adhered hepatocytes were maintained and subjected to requisite treatments.
2. Adipocytes—2-4 months old chow-fed black male mouse (C57bl/6) was sacrificed using chloroform (SRL) and was cleaned with 70% ethanol. Under aseptic conditions, the ventral side of the mouse was cut open, and gonadal (epididymal) white adipose tissue were excised using scissors and suspended in 1×PBS supplemented with 0.1% BSA in a 10 cm culture plate. The attached blood vessels were removed and the adipose tissue was minced as much as possible. The suspension was centrifuged at 50 g for 2 minutes at 4° C. After centrifugation, the adipose tissue pieces suspended at the uppermost layer of the supernatant as emulsion were carefully extracted and plated in DMEM in 35 mm culture dishes. The explant tissue thus obtained was treated with different concentrations of compounds after 6-7 hours.
The major source of ATGL is the adipose tissue and, therefore, it is fitting to check if the compounds had any effect on the ATGL protein level in adipose tissue. Both 9 and 107 did not show any significant changes in ATGL protein in cultures mouse adipose explants after 24 hours of compound treatment. This partly hints at a lack of a striking regulation of ATGL turnover by COP1.
Purification of ATGL protein and obtaining COP1 overexpressing cell lysate: Myc-ATGL plasmid and Myc-DDK-COP1 plasmid (2 g each) were transfected separately in cultured HEK293A cells with the help of Lipofectamine2000 (Invitrogen). Cells were harvested in Lysis Buffer and protein solution was extracted as described previously. The Lysis Buffer used to harvest COP1 overexpressing cells had 0.1% SDS supplemented in it. The concentration of protein was estimated using Bradford assay. For source of E3 ligase, the total cell lysate from COP1 overexpressing HEK293A cells were used.
For purification of Myc-ATGL: 1 ml of Ni2+-NTA resin (Roche) was taken in a 15 ml centrifuge tube and centrifuged at 2000 RPM for 5 minutes. The upper ethanol layer was discarded and the pellet was washed in 1×PBS twice to remove any remaining traces of ethanol. The pellet was finally washed once with Lysis Buffer for equilibration. 15 mg of protein was used for affinity purification reaction in a reaction volume of 10 ml with Lysis Buffer (containing protease inhibitor cocktail). The tube was incubated overnight at 4° C. in a rotary shaker.
The next day, the solution was centrifuged at 2000 RPM for 10 minutes and the supernatant collected. Lysis Buffer (containing protease inhibitor cocktail twice the previous concentration) was used for washing the beads twice to remove non-specific and unattached reactants in a stringent condition. The beads were finally washed once with 1×PBS containing the same amount of protease inhibitor for equilibration. 300 μl of 500 mM Imidazole (Sigma Aldrich) solution in 1×PBS was added to the resin for elution of ATGL and centrifuged at 2000 RPM for 30 minutes and the eluted protein was collected. To the separated resin, 300 μl of 1M Imidazole solution was added and centrifuged at 2000 RPM for 30 minutes and the second elute was collected. 201 of eluted protein from each elution was boiled with 5 μl of Laemmli's buffer at 95° C. for 10 minutes and run in 10% SDS-PAGE gel. The supernatant collected before elution of ATGL and the proteins still attached to the Ni2+-NTA resin were also run in the SDS-PAGE. The resolved proteins were subsequently transferred on PVDF membranes and probed with anti-myc primary antibody to check the presence of purified ATGL in the elute. Also to check the yield of the purified protein, 40 μl of the elute was run in 10% SDS page and stained with Coomassie Brilliant Blue solution followed by destaining with methanol, glacial acetic acid and water in a 50:40:10 ratio.
For identifying the E2 ubiquitin-conjugating enzyme responsible for ATGL ubiquitination by COP1, an in vitro ubiquitinylation kit (Enzo Life Sciences) was used. The assay was reconstituted as per manufacturer's protocol with the panel of E2 conjugating enzymes provided and other required components at the mentioned concentrations. The reaction was carried out for 6 hours following which it was quenched as per directions with the provided 2× non-reducing gel buffer and then analysed by western blotting.
Having identified the E2 ubiquitin conjugating enzyme as UbcH6, ubiquitination assay was set up in vitro as described previously with 5 μM of the compound 9, 107, 171, 179 and 73 followed by quenching and analysis by western blotting.
Ubiquitination of a target protein is carried out in a three step process involving three different enzymes: E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme and E3 ubiquitin ligase. In humans, there is only one E1 ubiquitin activating enzyme and multiple E2 and E3 enzymes. COP1 is an E3 ubiquitin ligase and the E2 enzyme responsible for ATGL ubiquitination is not known. In order to assert the specificity of the compounds of the present disclosure to inhibit COP1 and decrease ubiquitination of ATGL, the in vitro ubiquitinylation reaction was reconstituted with purified ATGL protein, total cell lysate of COP1 overexpressing HEK293A cells as source of E3 ubiquitin ligase, UbcH6 as E2 enzyme, 5 μM of the compounds 9, 107, 171, 179 and 73 and other components required to carry out the ubiquitinylation reaction as per the manufacturer's instructions.
COP1 is an E3 ubiquitin ligase and ATGL is one of its targets which gets ubiquitinated and ultimately degraded via proteasomal mediated pathway. Thus, the molecules inhibiting COP1 by targeting the VP motif of ATGL are actually expected to bring about a reduction in the ubiquitination levels of ATGL. The compounds of the present disclosure have shown a reduction in the lipid droplet count with a corresponding increase in ATGL protein levels while gene expression remained unaltered. However, it is of utmost importance to check the changes taking place at the ubiquitination level of ATGL upon treatment with the compounds.
To this end, an immunoprecipitation assay was performed wherein HepG2 cells overexpressing myc-ATGL were transfected with HA-Ubiquitin and treated with 5 μM of the compounds 9, 10, 107, 215, 219 and 238. MG-132, a proteasomal inhibitor, was added 4 hours before harvesting the cells. Immune complexes were pulled down with anti-myc antibody and immunoblot was done using anti-HA antibody. The resultant smears on the blot reflect the ubiquitination status of ATGL.
COP1 is capable of auto ubiquitinating its own self and getting degraded via the proteasomal pathway. To probe into this, HepG2 cells were co-transfected with HA tagged Ubiquitin and myc-flag tagged COP1. The transfected cells were treated with 5 μM of compounds 9, 10, 107 and 238 for 24 hours. MG-132, a proteasomal inhibitor, was added 4 hours before harvesting the cells. Immune complexes were pulled down with anti-myc antibody and immunoblot was done using anti-HA antibody. Interestingly, the compounds being COP1 inhibitors could markedly reduce COP1's auto ubiquitination property as shown in
COP1 is an E3 ubiquitin ligase with ATGL as one of its targets for ubiquitination and subsequent proteasomal degradation by 26S proteasome. Therefore, it is only legit for ATGL levels to decrease upon COP1 overexpression. To this end, HepG2 cells were transfected with 500 ng of myc-DDK-COP1 plasmid using Lipofectamine 2000 and treated with 5 μM of the compounds 9 and 10 for 24 hours. 48 hours post transfection, cells were harvested and western blot was carried out. Indeed, myc DDK-COP1 construct were transfected in HepG2 cells, ATGL level was dampened significantly. On treatment of the compounds of the present disclosure in COP1 overexpressing cells for 24 hours, ATGL level was sufficiently restored.
Ubiquitination of ATGL by COP1 is a post translational event, therefore, it is expected that the mRNA level of ATGL will not change compared to control upon treatment with the compound of the present disclosure which inhibit COP1. To establish this, HepG2 cells were treated with 5 μM and 10 μM of the compounds 9, 107, 171, 179 and 73 for 24 hours. RNA was isolated using TRIzol reagent followed by chloroform treatment and precipitation with isopropanol. cDNA was obtained from the isolated RNA and qPCR was carried out using SyBr green reagent. COP1 and ATGL gene expression levels were checked. 18s was used as control.
6-8 weeks old healthy female C57BL/6 mice (average weight: 25 grams) were taken for the study. These were then divided into three groups comprising of three mice per group (Control, compound 107 fed for 8 hours and compound 107 fed for 16 hours). Mice were fed with 30 mg/kg of compound 107 orally. The compound was dissolved in 25% DMSO and 75% PBS. The control group was fed only with the solvent in which the compound was dissolved. Post 8 hours and 16 hours of feeding, mice were sacrificed and a portion of the excised liver tissue was homogenized in lysis buffer containing protease inhibitor cocktail. The homogenate was centrifuged at 20,000 g for 30 minutes following which the supernatant containing the protein lysate was collected. The lysate was then diluted accordingly and protein estimation was carried out by Bradford Assay. This was followed by Western Blotting wherein the levels of ATGL and COP1 were checked. Actin was used as the loading control.
For checking the change in the ubiquitination status of ATGL on compound treatment, Myc-ATGL expressing HepG2 cells (ATGL overexpressing cell line) was transfected with 2 μg of a plasmid harboring the HA tagged ubiquitin gene using Lipofectamine 2000 reagent (Invitrogen) in OptiMEM media (HiMedia). Fresh DMEM complete media was added after 8-10 hours of transfection. After roughly 24 hours of transfection, 5 μM of the compounds 9, 10 and 107 was added and cells were harvested post 24 hours of treatment. 4 hours prior to cell harvesting, 10 μM of proteasomal inhibitor MG132 (Calbiochem, Merck Millipore) was added to the cells. Proteins were isolated from the cells and protein concentration was estimated as described previously. 30 μl of PureProteome Protein A/G Mix Magnetic Beads (Merck Millipore) was taken in a 1.5 ml microcentrifuge tube, placed in a Magna Rack (Merck Millipore) and allowed to adhere to the side of the tube. 200 μg of protein was used for immunoprecipitation reaction in a reaction volume of 1 ml. 2 μl of anti-Myc primary antibody (Cell Signaling Technology) was added and the tube was incubated overnight at 4° C. in a rotary shaker. The next day, the tube was placed back in the Magna Rack and the beads were allowed to attach to the tube. To remove non-specific and unattached reactants, 1×PBS and 0.1% Triton-X was used for washing. 2× loading dye and Lysis Buffer was added and heated at 95° C. for 10 minutes to allow the immune complex to dislodge from the magnetic beads. Western Blot was performed subsequently as described before with anti-HA primary antibody for immunoblotting (Cell Signaling Technology). The activity trends of the synthesized compounds are disclosed in Table-5.
In
For checking the autoubiquitination status of COP1, HepG2 cells were cotransfected with 2 μg each of Myc-DDK-COP1 and HA-Ub plasmids using Lipofectamine 2000 reagent (Invitrogen) in OptiMEM media (HiMedia). The rest of the procedure is similar to that done for Myc-ATGL.
Intensity of ATGL and thus its level compared to control will increase up to 1.6 fold with COP1 knockout using siCOP1. Accordingly, EC50 was calculated for COP1 modulator. The fold increase of ATGL was measured with densitometry analysis in ImageJ software for different doses (10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1000 nM, 5000 nM) of following compounds: 127, 139, 211, 215, 219, 223, 232, 238 and 299. These value obtained were divided with the ATGL level that will go up to maximum 1.6 fold with siCOP1. Thus, the values obtained was expressed in percentage and was plotted against log[conc] of the respective compounds to obtain the EC50 value in graphpad software.
Initial validation of the compounds has been done through (i) reduction of lipid droplets in or without presence of compounds, (ii) increase in ATGL level in or without presence of compounds through W-B (western blot) analysis. The compounds with desired properties from the above mentioned screening procedure were further assayed for their ability to ubiquitination of ATGL via immunoprecipitation assay (IP) in or without presence of compounds. Compound 6 was the initial hit as fulfilled the first two criteria (reduction of lipid droplet and increase in ATGL level). As compound 6 comprises terminal —OMe group in meta position in urea linked phenyl moiety, the inventors started investigating and derivatizing compounds keeping oxygen atom in that meta position assuming that hetroatom plays a role in ligand-protein interaction for showing potency. The investigation showed ketone function in compounds 9 and 10 is significant. Compound without the ketone function failed to increase ATGL level. Compound with amide group such as compounds 17, 18, 19, and 20 showed ability to increase ATGL level. Quinazolinone ring N-3 position was also substituted with substituted alkyl and aromatic moiety. Some compounds such as 89, 93, 135, 139, 143, 147, 163, 103, 155, and 159, showed some sort of increase in ATGL level and reduction in lipid droplets. Compound 175 was made, where no heteroatom is present in N-3 linker. Compound 175 did not increase ATGL level and reduced lipid droplets. Some aliphatic substituents at C-2 position of the quinazolinone ring such as isopropyl, cyclohexyl, cyclopentyl, piperidinyl, pyridinyl, pyrazinyl were also incorporated, which led to increase in the ATGL level.
1.56 g NaH2PO4·2H2O was dissolved in 0.5 L water in a 1 L beaker. After adjusting pH to 7.4 using NaOH solution, the volume was made up to 1 L. Equal volumes of sodium phosphate buffer (10 mM, pH 7.4) and n-octanol were added to a separation funnel and mixed thoroughly by shaking and inverting the funnel several times. The two layers were allowed to separate for overnight and then dispensed in two separate glass bottles. 10 mM stock solution was prepared in 100% DMSO and stored at 4° C. 495 μL of organic phase (1-octanol) was added to each well of a 2 mL deep well plate, followed by 495 μL of buffer and 10 μL of test compound was added. The plate was incubated for 3 hr at room temperature on a plate shaker at 500 rpm. After incubation, the samples were allowed to equilibrate for 20 min and then centrifuged at 4000 rpm for 30 min for complete phase separation and analysed by LC-UV. The results obtained for the tested compounds is provided in table 7.
Log D=Log (area of octanol/area of buffer)
Lipophilicity assay revealed that compounds 123, 139, 215 and 219 are hydrophilic in nature whereas, rest of the compounds 73, 179, 238, 330a 160, 241, 253 and 211 have ideal lipophilicity with respect to the control compounds Quercetin, Metroprolol and Propranolol. The Log D value of the compound privides insight into possible oral bioavilibility of the compounds with compounds showing ideal lipophilicity may be suitable candidate for oral dosage.
1 mM Stock of test compound was prepared from 10 mM initial stock solution of compounds by diluting 10 μL of 10 mM stock with 90 μL of DMSO. Then 10 μL of 1 mM stock was diluted with 90 L of DMSO to give 100 μM concentration. The frozen plasma was thawed at room temperature and centrifuged at 1400 rpm at 4° C., for 15 minutes. Approximately 9000 of the clear supernatant fraction was transferred to a separate tube and was used for the assay. Final working stock of 1 μM was prepared by diluting 3 μL of 100 μM with 297 μL of plasma. Plasma containing the test compound was incubated for 120 min at 37° C. in shaker with 500 rpm. 50 μL of aliquot of sample at 0, 15, 30, 60 and 120 minutes were precipitated with 150 μL of acetonitrile containing internal standard and centrifuged at 4000 rpm at 4° C. for 20 minutes. 120 μL of supernatant was diluted with 120 μL of water and analyzed by LC-MS/MS. The results obtained for various compounds is provided in Table 8.
Plasma stability assay of synthesized compounds after 2 hours revealed that compounds 9, 73 and 179 have lower plasma stability whereas, compounds 123, 238, 241, 330a, 253, 211, 139, 215 and 219 have good plasma stability with respect to the control compound Propantheline. Plasma stability assay provides insight into the compound stability in plasma and the percentage concentration of the compounds remaining after certain time interval. The assay provides information regarding compound degradation in plasma.
1 mM stock solution of test compound was prepared in DMSO and diluted with Acetonitrile:Water (1:1) to get a 100 μM working concentration. 100 mL of Milli Q water was added to K2HPO4 (1.398 g) and KH2PO4 (0.27 g) to get final pH 7.4 solution of potassium phosphate buffer. 3.333 mg/mL microsomal suspension was prepared by diluting 499.95 μL of 20 mg/mL microsomal stock to 2500.05 μL with buffer. 532.5 μL of 16 mM NADPH stock was added to 2467.5 μL of potassium phosphate buffer to get 2.84 mM working stock. 75 μL of 3.333 mg/mL working stock of liver microsomes and 85 μL of buffer was added to 2.5 μL of test compounds (100 μM). The above mixture was pre incubated for 15 minutes at 37° C. After pre incubation, 32.5 μL of the mixture was added to 17.5 μL of buffer, this was incubated for 60 minutes at 37° C. [60 min Without Cofactor (NADPH)]. 16.25 μL of the pre incubated mixture and 8.75 μL of cofactor was added to 150 μL of acetonitrile containing internal standard [0 min Sample]. 62 μL of cofactor was added to remaining pre incubation mixture [Incubation mixture]. 25 μL of incubation mixture at 0, 5, 15, 30, 60 min and 60 min without cofactor were precipitated with 150 μL of acetonitrile containing internal standard, vortexed and centrifuged at 4000 rpm at 4° C. for 20 minutes. 120 μL of supernatant was diluted with 120 μL of water and analyzed by LC-MS/MS [sample preparation]. The results obtained for the compounds is provided in Table 9.
In-Vitro evaluation of metabolic stability using Human Liver Microsomes (HLM) study revealed that compounds 9, 330a, 253, 215, and 219 have lower clearance. Compounds 211, and 139 have medium clearance and compounds 238, and 241 have higher clearance with respect to the control compound verapamil HCl. The liver microsomal stability assay primarily provides insight into the Phase I metabolism or biotransformation of the compounds.
HepG2 cells were plated in Seahorse cell culture microplates (24 well). Cells were treated with 5 μM of compounds 9, 107, 238 and 238a for 24 hours. The calibrator plate was dipped in the calibrator solution and incubated overnight at 37° C. in a non-CO2 incubator. The next day, cells were washed with Agilent Seahorse XF Base Medium, Minimal DMEM twice, making sure to leave at least 50 μl of media in the wells and incubated for 1 hour at 37° C. in a non-CO2 incubator. Following this, oxygen consumption rate was measured in the Seahorse instrument.
The compounds of the present disclosure having Structure I have several advantages including:
| Number | Date | Country | Kind |
|---|---|---|---|
| 202011027502 | Jun 2020 | IN | national |
This application is a national-stage application under 35 U.S.C. § 371 of International Application PCT/IN2021/050621, filed Jun. 25, 2021, which claims benefit of priority to Indian Patent Application No. 202011027502, filed Jun. 29, 2020.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IN2021/050621 | 6/25/2021 | WO |
