Synthesis of quinazolindionyl amino acid and dipeptide derivatives as possible antitumour agents

Some physiologically active amino acid and dipeptide esters were coupled to 3-(2-hydroxyethyl)-2,4-dioxo-(1H,3H)-quinazoline at N-1 via acetyl/propionyl link. The C 2 /C 3 anchor segments were introduced by either alkylation or Michael addition reactions with ethyl chloroacetate or methyl acrylate respectively. The occurrence of these two reactions on nitrogen N-1 rather than oxygen atom was confirmed by spectral values ( 1 H and 13 C NMR). Amide bond formation was performed by the azide activation procedure at 0 o C to avoid Curtius rearrangement. 21 of the newly synthesized compounds exhibited IC 50 ’s in the range of 5.63 -26.9 µg/mL relative to doxorubicin (3.23 µg/mL) when tested against HepG2cell line.


Introduction
Cancer remains one of the most feared diseases all over the world, even with the huge efforts exerted on all levels to reduce and overcome it.2][3][4][5] Chemotherapy is a main method for the treatment of almost all types of cancer solely or with combination with another treatment approach.7][8][9] Quinazoline containing compounds are derived from some commercially available heterocyclic pharmaceuticals used in treatment of several types of cancer.][12][13][14][15][16][17][18][19] In continuation of our encouraging results on possible antitumour -active quinazoline compounds 20 , the present manuscript deals with the synthesis of a series of 2,4-quinazolindione derivatives incorporating physiologically active amino acids and dipeptides at position-1, (schemes 1and 2) to evaluate their anticancer effect against human liver carcinoma cell line (HepG2), (table 1).The start compound 3-(2-hydroxyethyl)-2,4dioxo-(1H,3H)-quinazoline (1) was found to be a suitable scaffold for derivatization in this study.

Results and Discussion
The key compound 1 was obtained from condensation of methyl benzoate carbamate derivative with ethanol amine according to the method described in literature. 21In order to facilitate the introduction of the amino acid at N-1 of the quinazoline nucleus ethyl [3-(2-hydroxyethyl)-2,4-dioxo-(1H,3H)quinazolin-1-yl]acetate (2) was prepared by alkylation of 1 with ethyl chloroacetate in the presence of anhydrous potassium carbonate.Moreover, to investigate the effect of the length of the link between quinazoline nucleus and the amino acids on the anticancer activity some propionyl amino acid derivatives were also synthesized (Scheme 1).
The methyl propionate derivative 5 was prepared via Michael addition of compound 1 on methyl acrylate.The reaction was conducted in different conditions to determine the most appropriate result (Scheme 2).It is obvious that compound 5 was obtained in low yield when the reaction was carried out in methanol or DMSO in the presence of triethyl amine, sodium methoxide or piperidine.However, better yield (55.6%) was achieved when the reaction was conducted in DMF with slight excess (20%) of triethyl amine and methyl acrylate.
The chemical structures of the ester derivatives 2 and 5 were assigned by elemental analysis, 1 H NMR and 13 C NMR spectra (see the experimental part). 1 H NMR spectrum of compound 2 showed a singlet signal at 4.90 ppm corresponding to the two protons of the acetate moiety NCH2CO and the 13 C NMR spectrum of this compound displayed a signal at 44.9 ppm attributable to the carbon of the same group.Moreover, the 1 H NMR spectrum of the propionyl derivative 5 showed the characteristic signals of the methyl propionate moiety as a triplet signal at 4.37 ppm for the two protons of the β-carbon of the propionate (NCH2), the two protons of the α-carbon (CH2CO) appeared as triplet signal at 2.79 ppm, and the 13 C NMR spectrum exhibited signal at 31.8 ppm attributable to the α-carbon of the propionate moiety.Spectral values published in literature for quinazolin-2,4-dione derivatives displayed the protons of NCH2CO (in the range 4.3-5.0ppm) and 13 C (in the range 44.9-46.33][24][25][26] These data revealed the occurrence of alkylation and Michael addition reactions on nitrogen atom (N-1) rather than oxygen atom.These results are based on density functional theory DFT reactivity studies which showed that the N-site has higher nucleophilicity compared to O-site. 24RKAT USA, Inc  Hydrazinolysis of the ester derivatives 2 and 5 in alcohol with approximately five to six equivalents of hydrazine hydrate afforded the hydrazide derivatives 3 and 6 respectively in good yields (Scheme 1).The two hydrazides 3 and 6 were characterized by their higher melting points and lower Rf values relative to the parent ester derivatives. 1 H NMR spectra of the synthesized hydrazides 3 and 6 showed the characteristic signals of the hydrazide group as two singlet signals at 9.31 ppm and 9.12 ppm for the proton of NH and at 4.27 ppm and 4.17 ppm for the two protons of NH2 respectively.Also, 13 C NMR spectra displayed the disappearance of the two signals of the ethyl ester group at 62.0 ppm and 14.1 ppm and the signal of OCH3 carbon of the ester at 52.0 ppm which confirms the formation of the hydrazides.
The chemical structures of the amino acid ester derivatives 4a-d and 7a-e were assigned by elemental analyses, 1 H and 13 C NMR.The 1 H NMR spectra of these compounds showed the common signals at 8.53-6.83ppm, multiplet signals between 8.19-6.98ppm, multiplet signals in the range 4.67-3.84ppm and singlet signals in the range between 3.69-3.54ppm attributed to the protons of CONH group of the peptide bond, aromatic protons of the quinazoline nucleus, the α-protons of the amino acids and the three protons of OCH3 of the ester groups respectively. 13 Based upon the high anticancer activity of the two esters 4a and 4b, they were converted to the corresponding hydrazides with the aim to prepare some dipeptide and hydrazone derivatives with promising antitumour action.The amino acid hydrazide derivatives 9a and 9b were prepared by hydrazinolysis of the corresponding esters 4a and 4b in methanol with six equivalents of hydrazine hydrate (Scheme2).These hydrazides are characterized by their physical data with respect to the parent esters.Moreover, the chemical structures of these hydrazides were elucidated from elemental analyses, 1 H NMR and 13 C NMR spectra (see experimental part).
The dipeptide derivatives 11a-c have been prepared from the methionine hydrazide derivative 9b via the azide coupling method (Scheme 3).The hydrazide 9b was treated with nitrous acid at low temperature (-5 o C), to form the corresponding azide.This azide also, was found to be unstable at room temperature, which in turn was allowed to react with the amino acid methyl esters and worked up as described for the synthesis of the amino acid methyl ester derivatives 4a-d to afford the dipeptide methyl ester derivatives 11a-c in 31.2-41.8%yields. 1 H NMR spectra of the synthesized dipeptides revealed two doublets in the range 8.45-6.84ppm for the two NH protons of the two peptide bonds.Also, exhibited other two multiplets in the range 4.75-4.42ppm for the two α-CH protons of the two amino acids of the dipeptide.The 13 C NMR spectra of compounds 11a-c showed five signals between 172.8-150.5 ppm attributable to the five carbonyl carbons.The signals of the two α-CH carbons of the two amino acids appeared between 51.7-46.9ppm in addition to the other signals corresponding to protons and carbons of quinazoline nucleus and the individual two side chains of the amino acids of the dipeptides (see the experimental part).
Hydrazide-hydrazones have wide interest because of their diverse biological applications [28][29][30] including their significant role as antitumor agents. 31Based on these findings, and in trying to find new potent anticancer agents; some quinazoline and quinazoline amino acid hydrazide-hydrazone derivatives with different aromatic aldehydes were prepared.
The hydrazone derivatives 8a-d were obtained from condensation of hydrazide derivatives 3 with the aromatic aldehydes: benzaldehyde, m-nitrobenzaldehyde, anisaldehyde and vanillin.On the other hand, the amino acid hydrazone derivatives 10a-d were also prepared by condensation of hydrazide derivatives 9a, or 9b with benzaldehyde and m-nitrobenzaldehyde.The chemical structures of the synthesized hydrazones 8a-d and 10a-d were assigned by 1 H-NMR, 13 C-NMR, and elemental analysis (see experimental part). 1 H-NMR spectra of these compounds showed their existence as an equilibrium mixture from cis-E and trans-E conformers in DMSO solution which is in agreement with previous studies on similar hydrazones. 20he synthesized quinazolindione derivatives were tested for their antiproliferative activity against HepG-2 (Table 1).Out of twenty-five screened novel synthesized compounds, twenty-one exhibited IC50's in the range of 5.63-26.9µg/mL, compared to the reference drug doxorubicin (IC50 3.23 µg/mL).It is evident that the benzylidene hydrazone 8c (IC50 of 5.63 µg/mL) has comparable activity to doxorubicin.Moreover, the presence of free acidic proton at N-1 dramatically decreased the activity (compound 1 IC50 > 100 µg/mL), and the length of the link between the amino acid/dipeptide and quinazoline nucleus has no appreciable effect (compounds 4a-d and 7a-e).
Generally, the anticancer activity of the current tested compounds of N-1 series is more potent than the corresponding our previously published N-3 derivatives. 20

Conclusions
The key compound 3-(2-hydroxyethyl)-2,4-dioxo-(1H,3H)-quinazoline (1) is a suitable scaffold for derivatization at the N-1 site to afford drugs with remarkable anticancer activity.It is hoped that the potential antiproliferative effect of the synthesized series could offer an effective treatment that is less likely to cause resistance, recurrence of cancer and less toxic to normal tissues than available drugs.Based upon the comparable activity of 3-nitrobenzylidene hydrazone derivative 8c with doxorubicin, it is necessary to extend this study to synthesize other hydrazones, bearing different substituents on the benzylidene nucleus.The azide method seemed to be suitable activation procedure for the formation of pure amino acid and dipeptide derivatives, not contaminated with the by-product urea derivatives, when carried out at 0 o C. 1 H and 13 C NMR data supported the occurrence of alkylation and Michael addition at nitrogen (N-1) and not oxygen atom.

General method for the synthesis of [3-(2-hydroxyethyl)-2,4-dioxo-(1H,3H)-quinazolin-1-yl]acetyl amino acid methyl esters (4a-d).
To a cold solution (-5 o C) of hydrazide 3 (0.45 g, 1.6 mmol) in acetic acid (12 mL), hydrochloric acid (5N, 6 mL), and water (50 mL), was added portion wise under stirring a cold solution (0 o C) of sodium nitrite (0.14 g, 2.0 mmol) in water (6 mL).After stirring at the same temperature for 30 minutes, the azide was extracted with cold ethyl acetate, and washed successively with cold water, 5 % NaHCO3 and water.After drying over anhydrous sodium sulfate, the azide was used directly without further purification in the next step.Amino acid methyl ester hydrochlorides (1.8 mmol), was stirred in ethyl acetate (50 mL) with triethyl amine (0.2 mL) at 0 o C for 20 minutes.The formed triethyl amine hydrochloride was filtered off and the filtrate was added to the previously prepared cold dried solution of the azide.Afterwards the mixture was kept 12 hrs in the refrigerator and then at room temperature for another 12 hrs.The reaction mixture was washed with 0.1N HCl, water, 5% NaHCO3 and water then dried over anhydrous sodium sulfate.The solvent was evaporated in vacuum and the residue was crystallized from ethyl acetate-petroleum ether to give [3-(2-hydroxyethyl)quinazolin-2,4-dione-1-yl]acetyl amino acid methyl esters (4a-d).

General method for the synthesis of [3-(2-hydroxyethyl)-2,4-dioxo-(1H,3H)-quinazolin-1-yl]acetyl dipeptide methyl esters (11a-c).
To a cold solution (-5 o C) of hydrazide 9b (0.65 g, 1.6 mmol) in acetic acid (12 mL), hydrochloric acid (5N, 6 mL), and water (50 mL), was added portion wise under stirring a cold solution (0 o C) of sodium nitrite (0.14 g, 2.0 mmol) in water (6 mL).After stirring at the same temperature for 30 minutes, the azide was extracted with cold ethyl acetate, and washed successively with cold water, 5 % NaHCO3 and water.After drying over anhydrous sodium sulphate, the azide was used directly without further purification in the next step.The amino acid methyl ester hydrochlorides (1.8 mmol), was stirred in ethyl acetate (50 mL) with triethyl amine (0.2 mL) at 0 o C for 20 minutes.The formed triethyl amine hydrochloride was filtered off and the filtrate was added to the previously prepared cold dried solution of the azide.The reaction mixture was treated as described above under the synthesis of the amino acid methyl ester derivatives 4a-d to afford the dipeptide ester derivatives 11a-c.

In vitro antiproliferative activity
Cytotoxicity of the newly synthesized compounds was tested against human liver carcinoma cell line (HepG2) using the method of Skehan et al. 32 A preliminary investigation was performed at 100 μg/mL.Based upon this study the inactive compounds were excluded.The data of the in vitro antiproliferative activity are presented in Table 1.

Supplementary Material
Copies of 1 H and 13 C NMR spectra of the new compounds can be found in the supplementary material file.
C NMR spectrum displayed four signals in the range 172.8-150.0ppm corresponding to the four-carbonyl carbon (CO), aromatic carbon signals between 139.4-109.3ppm, in addition to the signals of the α-carbons of the amino acids at 51.7-51.5 ppm and the signals of OCH3 carbons in the range 54.6-50.2ppm.Other signals corresponding to protons and carbons of the remaining groups are reported in the experimental part.
Different conditions of Michael addition of compound 1 on methyl acrylate.