Synthesis of methyl [3-alkyl-2-(2,4-dioxo-3,4-dihydro-2 H -quinazolin-1-yl)- acetamido] alkanoate

A series of methyl [3-alkyl-2-(2,4-dioxo-3,4-dihydro-2 H -quinazolin-1-yl)-acetamido] alkanoate 10-13a-f has been developed on the basis of the N-chemoselective reaction of 3-substituted quinazoline-2,4-diones 3 a-d with ethyl chloroacetate and azide coupling method with amino acid ester hydrochloride. The precursor quinazoline diones 3a-d chemoselective reactions were studied using DFT(B3LYP)/6-311G level of theory and were prepared by a new rearrangement method from the corresponding 2-(3-methyl-4-oxo-3,4-dihydroquinazolin-2-ylthio) acetohydrazide 6 .


Introduction
Receptor tyrosine kinases have a critical role in the development and progression of many types of cancer (e.g., breast, ovarian, colon, and prostate). 1The quinazoline nucleus is the scaffold of many antitumor drugs mainly acting as inhibitors of tyrosine kinase receptors (RTK).Anticancer agent Iressa (ZD1839) A is a clinically approved example of quinazoline-based inhibitors which is in phase III clinical trials for cancer used to inhibit the receptor tyrosine kinase of epidermal growth factor.

Results and Discussion
The hydrazide 6a-d reacted with a mixture of NaNO2 and HCl at 0 o C to presumably produce the azide.Then the in situ generated azide derivative was treated with triethyl amine in ethyl acetate to afford an interesting rearrangement and gave the dione 3a-d (Scheme 1).This procedure was discovered by our group on applying the amino acid azide coupling method to hydrazide 6a in the attempted preparation of methyl 2-(2-(4-oxo-3aryl-3,4-dihydroquinazolin-2-ylthio)acetamido) alkanoates. 20The precursor 3-phenylquinazoline-2,4-dione 3a was alternatively, prepared by refluxing anthranilic acid with phenyl isocyanate in acetonitrile for 4h.The produced urea derivative 2 was cyclized using NaOH in ethanol under reflux for 4h, Scheme 1.

Scheme 1
We further explored a suggested rationalized mechanism for formation of dione 3a-d.via azide I through tautomeric irreversible rearrangement which showed to start by Smiles rearrangement 38 as shown in Scheme 2.
A first step: acid catalyzed tautomerization of the azide derivative I giving rise to the enol II.Second step: oxygen nucleophilic attack on electrophilic carbon located at position 2 of the quinazoline system to give a five membered spiro-intermediate III leaving a negative charge on the nitrogen atom.0] The results obtained earlier show the formation of amides from resembling examples with the elimination of thiirane ring residue.A similar result was obtained by our group applying the azide coupling method to 2-[2-(4-phenyl [1,2,4]triazolo [4,3a]quinoxalin-1-ylsulfanyl) acetohydrazide giving an interesting rearrangement with the formation of 4phenyl [1,2,4]triazolo [4,3-a]quinoxaline-1(2H) thione.This latter reaction appears to confirm the formation of intermediates I and II, but differs in the cleavage of the spiro system probably due to the differences in the properties of quinazoline and triazoloquinoxaline ring systems.

Scheme 2
The structure assignment of quinazoline dione derivative 3c (R 1 = C 6 H 4 OCH 3 ) is based on 1 H NMR spectroscopy, as well as physicochemical analysis.The 1 H NMR spectrum of the dione 3c gave an interesting pattern and it gave the clear evidence for the presence of dione derivatives in the form of tautomeric mixture in a ratio of 1:1.Thus the 1  Structure modification of the model compound 3a-d could be simply achieved by chemoselective alkylation reactions with electrophiles.Thus, the reaction of quinazoline 3a-d with ethyl chloroacetate in the presence of NaH gave chemoselective N-substituted quinazoline derivatives 7a-d in good yield, Scheme 3.
The O/N reaction competitions in heterocyclic amides with electrophiles were considered as a major element for the synthesis of great variety of heterocyclic compounds of promising biological activities.Despite these types of reactions show great selectivity; the reason for the O-and N-atoms contributions were not yet described.Calculation of a number of parameters such as molecular orbital coefficients, charge distribution, and fukui functions of the model compound beside the experimental results might give us clear picture.The calculations were performed using MOLPRO program package.The optimized geometric structures were calculated using DFT(B3LYP)/6-311G level of theory.Taking into consideration the Fukui function reactivity indices; they give us information about which atoms in a molecule have a larger tendency to either loose or accept an electron.This function is used by a number of researchers to distinguish between soft and hard sites of different nucleophiles. 41For simplicity we will focus only on the values of Fukui function related only to the competing ambident nucleophilic sites.The following table shows with no doubts that N-atom is the most susceptible site for nucleophilic attacks, soft site of the ambident nucleophile with larger value of Fukui function, Table 1.
Table1.The Fukui indices of the atoms in molecule 3a calculated at DFT (B3LYP)/6311G level of theory using Mulliken population analysis The charge distribution on atoms in the model compound 3a as shown by Mulliken population analysis in the following table and the electron density represented by the following graphical presentation suggests that the oxygen atom is the hard site of the ambident nucleophile 3a, Table 2, figure 2. Total electron density of 3a HOMO orbital of 3a Figure 2 Figure 3 Finally, the graphical presentation of the HOMO orbitals shown, describes that the nitrogen atom has the highest-occupied molecular orbitals (HOMO) of high energy compared to that of oxygen atom, Figure 3.

Table 2. Mulliken population analysis 3a
To summarize the results obtained from computational studies we found out that the nitrogen atom of the ambident nucleophile 3a has larger Fukui function, lower electron density, polarizable, high chemical reactivity and is termed as the soft part of the ambident nucleophile.The obtained chemoselective N-alkylation reaction of 3a with ethyl chloroacetate was favoured due to interaction between HOMO at the nitrogen atom of the ambident nucleophile with high energy and the LUMO of the electrophile with low energy, resulting in a narrow energy gap and high reactivity to finally give N-alkylation.This result was deduced on the basis of Pearson`s hard soft-acid base (HSAB) principle.© ARKAT USA, Inc

Scheme 3
The esters 7a-d were reacted with hydrazine hydrate in ethyl alcohol for 4 h and gave hydrazide 8a-d, Scheme 3. Hydrazides 8a-d are excellent precursors for the quinazoline structure modification via azide coupling method.Azide coupling method is well recognized in the field of amino acid and protein chemistry via peptide bond formation leading to decrease in degree of racemization with easily removable by products.Thus, the reaction of hydrazides 8a-d with NaNO2 and HCl mixture at 0 o C principally gave the azide 9a-d.The in situ generated azide 9a-d solution in ethyl acetate subsequently reacted with amino acid methyl ester hydrochloride in the presence of triethyl amine to afford methyl 2-(2-(2,4-dioxo-3-substitutedquinazolin-1yl)acetamido) alkanoate 10-13 in moderate to good yields.The in situ generated azide 9a also reacted with hydroxy proline hydrochloride in the presence of NEt3 and gave the N-substituted hydroxy proline derivative 14 in 62 % yield, Scheme 3.
The amino acid ester derivative 10a was our key substrate to produce the quinazoline ring system linked to a dipeptide by a spacer as a representative example.Thus the reaction of hydrazine hydrate with 10a in ethanol under reflux condition for 4 h afforded the hydrazide 15.Reaction of hydrazide 15 with β-alanine methyl ester in the presence of acetic acid, hydrochloric acid and sodium nitrite to produce the corresponding dipeptide 16 via azide coupling method discussed earlier.O COOCH 3

Scheme 4
The structure of dipeptide 16 was confirmed by 1 H NMR and 13 C NMR.The 1 H NMR spectrum of 16 exhibits a multiplet signal at δ 3.52-3.46ppm associated with four protons of two NHCH2 groups.The 1 H NMR also gave signals at 7.00, 6.52, 3.98, 3.64 and 3.52-3.46ppm associated with NH, NH, NCH2, OCH3 and CH2 groups, respectively.The 13

General procedures for preparation of 2-(3-alkyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)acetohydrazide (8a-d).
To a solution of ester 7a-d (1.0 mmol) in ethyl alcohol (30 mL), hydrazine hydrate (2.4 mL, 5 mmol) was added.The reaction mixture was refluxed for 4 hours, cooled and the resultant precipitate was filtered off, washed with ethanol and ether then crystallized from aqueous ethanol to yield the hydrazide as a white crystals 8a-d.(10-13).To a cold solution (-5 o C) of hydrazide 8a-d (1.0 mmol) in AcOH (6 mL), 1 N HCl (3 mL), and water (25 mL) was added a solution of NaNO2 (0.07 g, 1.0 mmol) in cold water (3 mL).After stirring at -5 o C for 1 hour, a thick precipitate was formed.The reaction mixture was extracted in cold ethyl acetate (30 mL), washed with cold 3% NaHCO3, H2O and finally dried by (Na2SO4).A solution of amino acid esters hydrochloride (1.2 mmol) in ethyl acetate (20 mL) containing 0.2 mL of triethyl amine was added to the azide solution 9. The mixture was kept at -5 º C for 24 h., then at 25 º C for another 24 h., followed by washing with 0.5 N HCl, water, 3% solution of NaHCO3 and finally dried by (Na2SO4).The solution was evaporated to dryness, and the residue was recrystallized from petroleum ether/ ethyl acetate 3:1 to give the corresponding quinazoline amino acid ester derivatives 10-13.. AcOH (6 mL), 1 N HCl (3 mL), and water (25 mL) was added a solution of NaNO2 (0.34 g, 5.0 mmol) in cold water (3 mL).After stirring at -5 o C for 1 h.a thick precipitate was formed.The reaction mixture was extracted in cold ethyl acetate (30 mL), washed with cold 3% NaHCO3, H2O and finally dried (Na2SO4).A solution ofalanine hydrochloride (0.16 g, 1.1 mmol) in ethyl acetate (20 mL) containing 0.2 mL of triethyl amine was added to the ethyl acetate azide solution.The mixture was kept at -5 º C for 24 hrs., then at 25 º C for another 24 hrs., followed by washing with 0.5 N HCl, water, 3% solution of NaHCO3 and finally dried (Na2SO4).The solution was evaporated to dryness, and the residue was recrystallized from petroleum ether/ ethyl acetate to give the corresponding quinazoline dipeptide of dione derivative (16).White crystals (0.27 g, 62%), mp 173-174 0 C. 1 H NMR (300 MHz, CDCl3): 8.30 (1H, d, J 9.0 Hz, Ar-H), 7.54-6.98(8H, m, Ar-H), 7.0 (1H, bs, NH), 6.52 (1H, t, J 6.0 Hz, NH), 3.92 (2H, d, J 6.0 Hz , NCH2), 3.64 (3H, s, OCH3), 3.52-3.46(2H, m, NHCH2), 2.50-2.462 The initial structures of 3a was edited by an Avogadro package 53 The structure was optimized using the DFT calculations at the B3LYP level of theory employing 6-311G basis set.The optimized geometric structures of 3a calculated using DFT(B3LYP)/6-311G level of theory are presented in (Figure 1).Fukui functions.They were calculated for all atoms within structure 3a at the DFT(B3LYP)/6-311G level of theory using Mulliken population analysis.Applying finite difference approximation, it can be written as: 54 (nucleophilic attack) and (electrophilic attack).Herein, , and are defined as the atomic charges of the neutral, anionic, and cationic species, respectively.The nucleophilic and electrophilic Fukui functions are reported in (Table 1).The calculated Fukui functions for the charged species (N, N+1, and N-1) within the molecule 3a are reported in Table 1.It can be seen from Table 1 that the nitrogen atoms (N-4) is the most susceptible site for electrophilic attacks due to the largest values of which is -0.018.Frontier molecular orbitals (FMO).The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) are the most important orbitals in a molecule.These orbitals are named as the frontier molecular orbitals (FMOs).The HOMO orbital acts as an electron donor and the LUMO orbital acts as the electron acceptor.FMOs play key roles in interactions between the molecules.The frontier orbital gap (ELUMO-EHOMO) gives information about the chemical reactivity, kinetic stability and chemical hardness-softness of a molecule.A molecule with a small frontier orbital gap is generally associated with a high chemical reactivity, low kinetic stability and is also termed as soft molecule.On the contrary, the large LUMO-HOMO energy gap implies that the molecule has low chemical reactivity, high kinetic stability and is also termed as hard molecule.The soft molecules are more polarizable than the hard ones because they need small energy to excitation. 55
C NMR spectrrum showed signals at 172.8, 168.0, 167.4 and 135.7 ppm for (CO) groups, signals at 128.9, 128.3, 123.8, and 114.2 ppm for C-Ar group, signal at 51.8 ppm for NCH2 group, signal at 47.6 ppm for OCH3 group, signal at 35.0 ppm for NHCH2 group and signal at 33.5 ppm for CH2 group.