Molecular modeling studies and synthesis of novel methyl 2-(2-(4-Oxo-3-aryl-3,4-dihydroquinazolin-2-ylthio)acetamido)alkanoates with potential anti-cancer activity as inhibitors for methionine synthase

Article abstract of DOI:10.1248/cpb.c14-00158

Cobalamin-dependant cytosolic enzyme methionine synthase (MetS) catalyses the transfer of a methyl group from the methyltetrahydrofolate (MTHF) to homocysteine (Hcy) to produce methionine and tetrahydrofolate (THF). MetS is over-expressed in the cytosol of certain breast and prostate tumour cells. Methionine used as a source of one carbon atom for the building of the DNA of the tumour cells, structural protein and enzymes. In this study, we designed, synthesized and evaluated the cytotoxic activity of a series of substituted methyl 2-(2-(4-oxo-3-aryl-3,4-dihydroquinazolin-2-ylthio)acetamido)acetate and dipeptide that mimic the substructure of MTHF. These inhibitors were docked in to the MTHF binding domain in such the same way as MTHF in its binding domain. The free energies of the binding were calculated and compared to the IC ₅₀ values. This series has been developed by dicyclohexylcarbodiimide (DCC) and azide coupling methods of amino acid esters with carboxylic acid derivatives, respectively. Compound methyl 3-hydroxy-2-(2-(3-(4-methoxyphenyl)- 4-oxo-3,4-dihydroquinazolin-2-ylthio)acetamido)propanoate exhibited the highest IC₅₀ value 20 μg/mL against PC-3 cell line and scored the lowest free energy of the binding (-07.19 kJ/mol).

Full text of DOI:10.1248/cpb.c14-00158

July 2014
Regular Article
Chem. Pharm. Bull. 62(7) 675–694 (2014)
675
Molecular Modeling Studies and Synthesis of Novel Methyl 2-(2-(4-Oxo-
3-aryl-3,4-dihydroquinazolin-2-ylthio)acetamido)alkanoates with
Potential Anti-cancer Activity as Inhibitors for Methionine Synthase
,b
Ismail Mahmoud Elfekki,a Walid Fathalla Mohamed Hassan,*
Hosam Eldin Abd Elhamed Elshihawy,* Ibrahim Ahmed Ibrahim Ali,a and
,c
Elsayed Hussein Mostafa Eltamanya
c
a Department of Chemistry, Faculty of Science, Suez Canal University; Department of Organic Chemistry, Faculty
b
of Pharmacy, Suez Canal University; Ismailia 41522, Egypt: and Department of Physics and Math. Engineering,
Faculty of Engineering, Port-Said University; Port Said, Egypt.
Received February 16, 2014; accepted March 11, 2014
Cobalamin-dependant cytosolic enzyme methionine synthase (MetS) catalyses the transfer of a methyl
group from the methyltetrahydrofolate (MTHF) to homocysteine (Hcy) to produce methionine and tetrahy-
drofolate (THF). MetS is over-expressed in the cytosol of certain breast and prostate tumour cells. Methio-
nine used as a source of one carbon atom for the building of the DNA of the tumour cells, structural protein
and enzymes. In this study, we designed, synthesized and evaluated the cytotoxic activity of a series of substi-
tuted methyl 2-(2-(4-oxo-3-aryl-3,4-dihydroquinazolin-2-ylthio)acetamido)acetate and dipeptide that mimic
the substructure of MTHF. These inhibitors were docked in to the MTHF binding domain in such the same
way as MTHF in its binding domain. The free energies of the binding were calculated and compared to the
IC50 values. This series has been developed by dicyclohexylcarbodiimide (DCC) and azide coupling methods
of amino acid esters with carboxylic acid derivatives, respectively. Compound methyl 3-hydroxy-2-(2-(3-(4-
methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-ylthio)acetamido)propanoate exhibited the highest IC50 value
20µg/mL against PC-3 cell line and scored the lowest free energy of the binding (ꢀ207.19kJ/mol).
Key words cobalamin: methionine; methionine synthase; quinozaline; thioamide; chemoselective reaction
Methionine synthase (MetS) is one of the folic acid cycle There are a limited number of reports concerning the inhibi-
enzymes that catalyses the transfer of a methyl group from tion of MetS. A series of inhibitors of substituted 2,4-diami-
5-methyltetrahydrofolate to homocysteine to produce me- nopyrimidine derivatives that could bind the substrate-binding
thionine and tetrahydrofolate. The catalysis occurred via the pocket of dihydrofolate reductase was synthesized.7) 6-Substi-
coenzyme cobalamin cofactor (CH3-Cb).1) This enzyme has tuted pyrrolo- and thieno[2,3-d]pyrimidine derivatives were
been linked to the pathogenesis of anemias, neurodegen- identified as antifolates with selective membrane transport
erative disorders and cancer.2) Using mammalian methionine by proton-coupled folate transporter (PCFT) and folate re-
synthase, X-ray studies show that it is constructed of 1227 ceptors (FRs) over reduced folate carrier (RFC).8) A series
amino acid residues and has a molecular mass of 136kDa.3) of pyrido[2,3-d]pyrimidines was successfully synthesized
Cobalamin-dependent methionine synthase is made up of four and used by Gangjee et al. as selective and potent DHFR
modules 5-methyltetrahydrofolate and homocysteine binding inhibitors.9) In a strategy for discovering specific inhibitors
regions from the C-terminal of the protein and cobalamin and of MetS, some benzo-fused heterocycles have been designed
the S-adenosylmethionine binding regions from N-terminal.3) and synthesized that mimic the substructures of 5-methyltet-
Methionine is an essential amino acid necessary for normal rahydrofolate of MetS.10) Taking in consideration the structural
growth. Methionine is used as a methyl group donor, provid- requirements for the design of novel inhibitors that have high
ing one carbon units for DNA synthesis.4) Methionine depen- affinity to methyltetrahydrofolate (MTHF) receptor, a series
dence is defined as the inability of cancer cells to grow in a of dihydroquinazolinone derivatives have been synthesized.
methionine depleted medium even when replaced with the The structure requirement for the pharmacophoric portion
immediate precursor, homocysteine.5) Normal cells remain was maintained with the presence of quinazoline nucleus
unaffected by methionine restrictions if supplemented with which mimics the pteridine ring of MTHF with varying the
homocysteine, as they are able to synthesize methionine via side chain at positions 2 and 3 of quinazoline nucleus. Mean-
methylation of homocysteine in sufficient quantities. Most while the phenyl ring moiety was dislocated from its familiar
metastatic tumours, such as those originating in prostate, position at the side chain of the tested inhibitors in previous
lung and gastrointestinal tract, respond poorly to conventional work11,12) and in MTHF and relocated at N-3 of quinazoline
chemotherapy. Novel treatment strategies for advanced cancer nucleus to attempt the compensation of London interaction
are therefore needed. Dietary restriction of the essential amino contacts, including complimentary π–π stacking with the phe-
acid methionine offers promise as such a strategy, either alone nyl ring of phenylalanine 511.
or in combination with chemotherapy or other treatments.6)
The chemoselective reactions of heterocyclic thioamides
Although there are many approaches for developing inhibitors always attracted our research group. We earlier reported
for folic acid enzymes system collectively named antifolates. the chemoselective S- and N-alkylation of the model com-
pound 4-methyl-1-thioxo-1,2,4,5-tetrahydro[1,2,4]triazolo[4,3-
The authors declare no conflict of interest.
a]quinazolin-5-one with different electrophiles. These results
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: walid3369@yahoo.com;
hosam_sh_27@hotmail.com

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Vol. 62, No. 7
were supported by theoretical density functional theory
(DFT) computational calculations.13–15) We also invested these
findings in the syntheses of interesting heterocycles via
domino,15–17) rearrangement reactions18) and the preparation
of heterocyclic coupled amino acid esters.19,20) These results
motivated the development of a series of S-substituted amino
acid esters of biologically promising quinazoline ring system.
10000. Molecular dynamics (MD) at 300K were then per-
formed on the solvated system for a 50ps equilibration and
300ps of production employing a 1fs time step using OPLS
2005 force field, from which 100 structures were sampled at
1ps intervals and averaged. The final averaged structure was
then finally minimized.21)
Experimental Procedure. General Procedures Sol-
vents were purified and dried in the usual way. The boiling
range of the petroleum ether used was 40–60°C. Thin layer
Experimental
Molecular Modeling Procedure Molecular modeling was chromatography (TLC): silica gel 60 F254 plastic plates (E.
carried out on Schrodinger computational software worksta- Merck, layer thickness 0.2mm) detected by UV absorption.
tion using Maestro 9.3 graphic user interface (GUI) and Red Melting points were determined on a Buchi 510 melting-
Hat Linux nash Enterprise version V 4.1.18 on Batchmin V point apparatus and the values are uncorrected. NMR spectra
9.3 modeling engine. The atomic coordinates for the polypep- measured with Bruker AC 250 (250MHz). Tetramethylsilane
tide segments of MTHF and Hcy binding domains of MetS (TMS) (0.00ppm) as internal standard. Mass spectra were
enzyme extracted from human liver were obtained from the measured on a GC-MSQP 1000EX Shimadzu. Elemental
protein bank database PDB (Brookhaven protein database analyses were performed on a Flash EA-1112 instrument at
(PDB: 1Q8J)). Explicit calcium ion counter ions were included the Microanalytical laboratory, Faculty of Science, Suez Canal
instead of cadmium and positioned at a 4Å distance from the University, Ismailia, Egypt. Quinazoline derivatives 1, 5, 6
homocysteine binding site. The basic and acidic amino acids were prepared according to the method described in litera-
were neutralized at pH 7 2 by protonation of the terminal ture.17,22,23)
amino groups and basic amino acids such as histidine, lysine
General Preparation of 2-(3-Aryl-4-oxo-3,4-dihydro-
and arginine. Moreover, the terminal carboxylic acid groups quinazolin-2-ylthio)acetohydrazide (6) To a solution of
of amino acids and acidic amino acids such as glutamic acid, ester 5a (0.33g, 1.0mmol) in methyl alcohol (30mL), hydra-
aspartic acid, asparagine and glutamine were deprotonated. zine hydrate (2.4mL, 5mmol) was added. The reaction mix-
Prime 3.1 was used to check the energy content of the protein ture was refluxed for 4h, cooled and the resultant precipitate
segments and loops. The docking process involved the stan- was filtered off, washed with ethanol and ether then crystal-
dard precision docking (SP) in which ligand poses that were lized from aqueous ethanol to yield the hydrazide 6. White
expected to have unfavourable energies would be rejected crystals (0.29g, 89%); mp 195–196°C. 1H-NMR (300MHz,
from the conditioned set up of the hierarchal filter. The pre- dimethylsulfoxide (DMSO)) δ: 9.14 (1H, s, NH), 8.72 (2H, d,
sumption is that only active compounds will have available J=8.0Hz, ArH), 8.55 (1H, d, J=8.0Hz, ArH), 8.15–8.11 (1H,
poses that avoid these penalties. SP docking is appropriate m, ArH), 7.79–7.66 (2H, m, ArH), 7.68–7.61 (3H, m, ArH),
for screening ligands of unknown quality in large numbers. 4.21 (2H, s, SCH2CO), 1.92 (2H, m, NH2). Anal. Calcd for
SP is a soft docking programme that was adept at identifying C16H14N4O2S (326.4): C, 58.88; H, 4.32; N, 17.17 Found: C,
ligands that have a reasonable propensity to bind and 20% of 58.73; H, 4.28; N, 17.03.
the final poses produced from the SP docking were subjected
General Preparation of 3-Phenylaryl Quinazoline-
to the Extra Precision mode of Glide docking (XP) to perform 2,4(1H,3H)-dione (3) To a cold solution (−5°C) of hydra-
the more expensive docking simulation on worthwhile poses. zide 6 (0.33g, 1.0mmol) in AcOH (6mL), 1N HCl (3mL),
XP docking mode is harder than SP docking mode in that and water (25mL) was added a solution of NaNO2 (0.34g,
it penalizes the poses that violate established charges. Flex- 5.0mmol) in cold water (3mL). After stirring at −5°C for
ible docking was selected to generate conformations of all 1h. a thick precipitate was formed. The reaction mixture
possible ligand poses, which is more realistic as this occurs was extracted in cold ethyl acetate (30mL), washed with cold
in reality because the protein undergoes side chain and back 3% NaHCO3, H2O and finally dried (Na2SO4). A solution of
bone movement or both, upon ligand binding. Five and six- amino acid esters hydrochloride (1.0mmol) in ethyl acetate
membered rings were allowed to flip and amide bonds which (20mL) containing 0.2mL of triethyl amine was added to the
were not cis or trans configuration were penalised. Five thou- azide solution. The mixture was kept at −5°C for 24h, then
N HCl,
sand poses per ligand for the initial phase of docking with a at 25°C for another 24h, followed by washing with 0.5
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 dione 3a. White crystals (0.31g,
scoring window for keeping poses of 100kJ/mol were set up.
The best poses which fulfil those conditions were subjected to
energy minimization on the OPLS-AA nonbonded-interaction
grid with a distance dielectric constant of 2 and maximum
number of conjugate gradient steps of 10000 iterations. The
ligands of the poses selected by the initial screening were
subsequently minimized in the field of the receptor using a
standard molecular mechanics energy function (OPLS-AA
force field) in conjunction with a distance-dependant dielectric
model. Finally, the lowest energy poses obtained in this fash-
ion were subjected to a Monte Carlo procedure that examines
nearby torsion minima. The complex was minimized using
the conjugate gradients algorithm until an energy convergence
criterion of 0.1kJ/mol was reached with iteration cycle of
1
88%); mp 235–236°C. H-NMR (300MHz, DMSO) δ: 11.31
(1H, s, NH), 7.93 (1 H, d, J=7.8Hz, ArH), 7.65–7.51 (1H, d,
J=8.0Hz, ArH), 7.45–7.41 (3H, m, ArH), 7.27–7.15 (4H, m,
ArH). Anal. Calcd for C14H10N2O2 (238.2): C, 70.58; H, 4.23;
N, 11.76. Found: C, 70.45; H, 4.11; N, 11.53.
General Preparation of 2-(3-Aryl-4-oxo-3,4-dihydro-
quinazolin-2-ylthio)acetic Acid (2) To a solution ester
5a–c (1.0mmol) in methyl alcohol (30mL), was added sodium
hydroxide (0.112g, 2.0mmol) solution in 10mL H2O. The re-
action mixture was stirred for 3h till complete consumption

July 2014
677
of the ester (monitored by TLC). The reaction mixture was trum, m/z (Ir/%): 383.1.
evaporated under reduced pressure, diluted with water and
acidified by 1N HCl to pH 3. The separated whitish ppt was thio)acetamido)propanoate (8b): From
filtered off and washed several times with water, dried and White crystals (0.21g, 54%); mp 120–121°C. 1H-NMR
crystallized by DMF–H2O. (300MHz, CDCl3) δ: 8.68 (1H, d, J=7.2Hz, NH), 8.09 (1H, d,
Methyl
2-(2-(4-Oxo-3-phenyl-3,4-dihydroquinazolin-2-yl-
L
-AlaOCH3·HCl (0.14g).
2-(4-Oxo-3-phenyl-3,4-dihydroquinazolin-2-ylthio)acetic J=8.1Hz, ArH), 7.85 (1H, t, J=8.1Hz, ArH), 7.62–7.48 (4H, m,
Acid (2a): White crystals (0.31g, 88%); mp 245–246°C. ArH), 7.46–7.44 (3H, m, ArH), 4.31–4.27 (1H, m, CH), 3.92
1H-NMR (300MHz, DMSO) δ: 8.01 (1H, d, J=8.1Hz, (2H, s, SCH2), 3.59 (3H, s, OCH3), 1.33 (3H, d, J=7.2Hz, CH3).
ArH), 7.78 (1H, t, J=8.3Hz, ArH), 7.61–7.55 (4H, m, ArH), Anal. Calcd for C20H19N3O4S (397.4): C, 60.44%; H, 4.82%; N,
7.48–7.42 (3H, m, ArH), 4.18 (2H, s, SCH2CO). Anal. Calcd 10.57%; S, 8.07%. Found: C, 60.38%; H, 4.73%; N, 10.45%.
for C16H12N2O3S (312.3): C, 61.53; H, 3.87; N, 8.97; S, 10.27.
Found: C, 61.48; H, 3.65; N, 8.81.
Methyl 3-Hydroxy-2-(2-(4-oxo-3-phenyl-3,4-dihydroquina-
zolin-2-ylthio)acetamido)propanoate (8c): From L-SerOCH3·
2-(4-Oxo-3-p-tolyl-3,4-dihydroquinazolin-2-ylthio)acetic HCl (0.156g). White crystals (0.28g, 68%); mp 200–201°C.
Acid (2b): White crystals (0.31g, 88%); mp 235–236°C. 1H-NMR (300MHz, DMSO) δ: 8.59 (1H, d, J=7.8Hz, NH),
1H-NMR (300MHz, DMSO) δ: 8.03 (1H, d, J=8.1Hz, ArH), 8.05 (1H, d, J=8.1Hz, ArH), 7.82 (1H, t, J=8.3Hz, ArH),
7.91 (1H, t, J=8.1Hz, ArH), 7.49 (2H, d, J=8.0Hz, ArH), 7.61–7.55 (4H, m, ArH), 7.48–7.42 (3H, m, ArH), 5.07 (1H,
7.47–7.30 (4H, m, ArH), 4.16 (2H, s, SCH2CO), 2.41 (3H, s, t, J=5.7Hz, OH, D2O exchangeable), 4.37–4.31 (1H, m,
CH3). Anal. Calcd for C17H14N2O3S (326.4): C, 62.56; H, 4.32; CH), 3.93 (2H, s, SCH2), 3.69–3.55 (5H, m, OCH2, OCH3).
N, 8.58; S, 9.82. Found: C, 62.43; H, 4.15; N, 8.38.
13C-NMR (CDCl3) δ: 170.81 (C=O), 166.95 (C=O), 160.63
2-(3-(4-Methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2- (Cq), 156.85 (Cq), 147.14 (Cq), 135.77 (Cq), 134.85 (CHAr),
ylthio)acetic Acid (2c): White crystals (0.31g, 88%); mp 129.95 (CHAr), 129.38 (CHAr), 129.55 (CHAr), 126.50 (CHAr),
235–236°C. 1H-NMR (300MHz, DMSO) δ: 8.03 (1H, d, 125.99 (CHAr), 119.49 (Cq), 61.25 (OCH2), 53.83 (OCH3), 51.47
J=8.0Hz, ArH), 7.78 (1H, t, J=7.8Hz, ArH), 7.59 (1H, d, (CH), 36.48 (SCH2), Anal. Calcd for C20H19N3O5S (413.4): C,
J=7.8Hz, ArH), 7.45 (1H, t, J=7.9Hz, ArH), 7.33 (2H, d, 58.10%; H, 4.63%; N, 10.16%; S, 7.76%. Found: C, C, 57.94% ;
J=8.7Hz, ArH), 7.19 (2H, d, J=8.7Hz, ArH), 4.21 (2H, s, H, 4.56%; N, 9.96%;. Mass spectrum, m/z (Ir/%): 413.2.
SCH2CO), 3.85 (2H, s, OCH3). Anal. Calcd for C17H14N2O4S
(342.4): C, 59.64; H, 4.12; N, 8.18; S, 9.37. Found: C, 59.58; H, thio)acetamido)propanoate (8d): From β-AlaOCH3·HCl (0.14g).
4.01; N, 8.13.
White crystals (0.29g, 73%); mp 110–111°C. 1H-NMR
General Preparation of Methyl 2-(2-(4-Oxo-3-aryl-3,4- (300MHz, CDCl3) δ: 8.25 (1H, t, J=7.2Hz, NH), 8.05 (1H, d,
dihydroquinazolin-2-ylthio)acetamido)alkanoates (8–13) J=8.1Hz, ArH), 7.79 (1H, t, J=8.1Hz, ArH), 7.67–7.47 (4H, m,
Methyl
3-(2-(4-Oxo-3-phenyl-3,4-dihydroquinazolin-2-yl-
To a cold solution (−5°C) of the amino acid methyl ester ArH), 7.46–7.34 (3H, m, ArH), 3.93 (2H, s, SCH2), 3.52 (3H,
hydrochloride (1.0mmol) in acetonitrile (6mL) containing m, OCH2, OCH3), 3.30 (2H, q, J=6.4Hz, NCH2), 2.46 (2H,
triethyl amine (0.14mL, 1.0mmol), 2-(3-aryl-4-oxo-3,4- t, J=6.6Hz, CH2), Anal. Calcd for C20H19N3O4S (397.4): C,
dihydroquinazolin-2-ylthio)acetic acid 2a–c (1.0mmol), di- 60.44%; H %, 4.82%; N, 10.57%; S, 8.07%. Found: C, 60.36%;
cyclohexyl-carbodiimide (DCC) (0.207g, 1.0mmol) and H %, 4.70%; N, 10.49%.
hydroxybenzotriazole (HOBT) (0.135g, 1.0mmol) were added
Methyl
4-Methyl-2-(2-(4-oxo-3-phenyl-3,4-dihydroquina-
successively. The reaction mixture was stirred at 0°C for one zolin-2-ylthio)acetamido)pentanoate (8e): From L-LeuOCH3·
hour, at 5°C for one hour, then at room temperature for 8h. HCl 0.182g) and 8. White crystals (0.23g, 54%); mp
The reaction mixture was set aside overnight. The precipitated 215–216°C. 1H-NMR (300MHz, CDCl3) δ: 8.57 (1H, d,
dicyclohexylurea was filtered off and the filtrate was evaporat- J=7.8Hz, NH), 8.04 (1H, d, J=7.8Hz, ArH), 7.91 (1H, t,
ed under reduced pressure. The residue was dissolved in ethyl J=8.0Hz, ArH), 7.83–7.66 (4H, m, ArH), 7.53–7.30 (3H, m,
acetate, filtered and the filtrate was washed with 5% NaHCO3, ArH), 4.30–4.25 (1H, m, CH), 3.93 (1H, d, J=15.0Hz, SCH2),
1N HCl and saturated solution of sodium chloride then dried 3.87 (1H, d, J=15.0Hz, SCH2), 3.55 (3H, s, OCH3), 1.68–1.45
over anhydrous sodium sulphate. After evaporation of the sol- (2H, m, CH2), 1.22–1.13 (1H, m, CH), 0.97 (6H, d, J=6.0Hz,
vent, the remaining oily residue was triturated with petroleum 2CH3), 13C-NMR (CDCl3) δ: 173.17 (C=O), 167.29 (C=O),
ether (bp 40–60°C) at 0°C with scratching, afterwards the 161.08 (Cq), 157.29 (Cq), 147.61 (Cq), 136.23 (Cq), 135.26
formed solid was filtered off and crystallized from petroleum (CHAr), 130.41 (CHAr), 129.89 (CHAr), 129.52 (CHAr), 127.01
ether–ethyl acetate.
(CHAr), 126.46 (CHAr), 122.93 (CHAr), 119.96 (Cq), 52.31 (CH),
Methyl 2-(2-(4-Oxo-3-phenyl-3,4-dihydroquinazolin-2-yl- 51.02 (OCH3), 41.78 (CH2), 36.37 (SCH2), 24.63 (CH), 23.15
thio)acetamido)acetate (8a): From GlyOCH3·HCl (0.126g). (CH3), 21.69 (CH3). Anal. Calcd for C23H25N3O4S (439.5): C,
White crystals (0.29g, 77%); mp 234–235°C. 1H-NMR 62.85%; H, 5.73%; N, 9.56%; S, 6.92. Found: C, 62.81%; H,
(300MHz, DMSO) δ: 8.61 (1H, t, J=5.7Hz, NH), 8.04 (1H, 5.67%; N, 9.54%.
d, J=7.8Hz, ArH), 7.82 (1H, t, J=8.1Hz, ArH), 7.62–7.55
Methyl 3-Hydroxy-2-(2-(4-oxo-3-phenyl-3,4-dihydroquina-
(4H, m, ArH), 7.48–7.43 (3H, m, ArH), 3.92 (2H, s, SCH2), zolin-2-ylthio)acetamido)butanoate (8f): From L-ThrOCH3·
3.84 (2H, d, J=5.7Hz, NCH2), 3.57 (3H, s, OCH3). 13C-NMR HCl (0.156g). White crystals (0.31g, 74%); mp 155–156°C.
(CDCl3) δ: 170.52 (C=O), 167.79 (C=O), 161.16 (Cq), 157.17 1H-NMR (300MHz, CDCl3) δ: 8.37 (1H, d, J=8.4Hz, NH),
(Cq), 135.77 (Cq), 135.36 (CHAr), 130.46 (CHAr), 130.01 (CHAr), 8.09 (1H, d, J=7.8Hz, ArH), 7.83 (1H, t, J=8.0Hz, ArH),
129.91 (CHAr), 127.00 (CHAr), 126.62 (CHAr), 120.00 (Cq), 7.65–7.58 (4H, m, ArH), 7.51–7.44 (3H, m, ArH), 5.07 (1H,
52.21 (OCH3), 41.44 (CH2), 36.23 (SCH2). Anal. Calcd for d, J=5.1Hz, OH, D2O exchangeable), 4.31–4.27 (1H, m, CH),
C19H17N3O4S (383.4): C, 59.52%; H, 4.47%; N, 10.96%; S, 4.07–4.02 (3H, m, SCH2, CH), 3.59 (3H, s, OCH3), 1.04 (3H,
8.36%. Found: C, 59.44%; H, 4.39%; N, 10.86%. Mass spec- d, J=6.3Hz, CH3), Anal. Calcd for C21H21N3O5S, (427.5): C,

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Vol. 62, No. 7
59.00%; H, 4.95%; N, 9.83%; S, 7.50%. Found: C, 58.87%; H, (2H, t, J=7.1Hz, CH2), 2.42 (3H, s, CH3). Anal. Calcd for
4.91%; N, 9.69%. Mass spectrum, m/z (Ir/%): 428.1. C21H21N3O4S (411.5): C, 61.30%; H %, 5.14%; N, 10.21%; S,
Methyl 4-Hydroxy-1-(2-(4-oxo-3-phenyl-3,4-dihydroquina- 7.79%. Found: C, 61.28%; H %, 5.06%; N, 10.17%.
zolin-2-ylthio)acetyl)pyrrolidine-2-carboxylate (11): From L- Methyl 4-Methyl-2-(2-(4-oxo-3-p-tolyl-3,4-dihydroquina-
HOPrlOCH3·HCl (0.156g). White crystals (0.16g, 38%); zolin-2-ylthio)acetamido)pentanoate (9e): From L-LeuOCH3·
mp 103–104°C. 1H-NMR (300MHz, DMSO) δ: 8.05 (1H, HCl 0.182g). White crystals (0.25g, 56%); mp 159–160°C.
d, J=7.8Hz, NH), 7.81 (1H, t, J=7.8Hz, ArH), 7.65 (1H, 1H-NMR (300MHz, CDCl3) δ: 8.65 (1H, d, J=7.2Hz, NH),
d, J=8.0Hz, ArH), 7.62–7.56 (4H, m, ArH), 7.47–7.42 (3H, 8.03 (1H, d, J=8.1Hz, ArH), 7.86 (1H, t, J=7.8Hz, ArH),
m, ArH), 5.30 (1H, d, J=6.0Hz, CH), 4.42 (1H, brs, OH, 7.60 (1H, d, J=7.8Hz, ArH), 7.49 (1H, t, J=8.1Hz, ArH),
D2O exchangeable), 4.31 (1H, t, J=7.8Hz, CH), 4.11 (1H, d, 7.44 (2H, d, J=8.1Hz, ArH), 7.32 (2H, d, J=8.1Hz, ArH),
J=15.6Hz, SCH2), 3.98 (1H, d, J=15.6Hz, SCH2), 3.75–3.66 4.36–4.21 (1H, m, CH), 3.92 (1H, d, J=14.4Hz, SCH2), 3.59
(2H, m, CH2), 3.52 (3H, s, OCH3), 2.19–1.96 (2H, m, CH2). (3H, s, OCH3), 3.83 (1H, d, J=14.4Hz, SCH2), 2.41 (3H, s,
13C-NMR (CDCl3) δ: 172.58 (C=O), 166.25 (C=O), 161.13 CH3), 1.63–1.45 (2H, m, CH2), 0.97–0.95 (1H, m, CH), 0.84
(Cq), 157.21 (Cq), 147.67 (Cq), 136.27 (Cq), 135.32 (CHAr), (6H, d, J=7.2Hz, 2CH3). Anal. Calcd for C24H27N3O4S (453.6):
130.48 (CHAr), 130.04 (CHAr), 129.87 (CHAr), 126.97 (CHAr), C, 63.56%; H, 6.00%; N, 9.26%; S, 7.07. Found: C, 63.49%; H,
126.65 (CHAr), 126.49 (CHAr), 119.93 (Cq), 69.42 (CH), 58.33 5.93%; N, 9.14%.
(OCH3), 55.66 (SCH2), 52.25 (CH), 37.84 (CH2), 36.54 (CH2).
Anal. Calcd for C22H21N3O5S (439.5): C, 60.12%; H, 4.82%; N, zolin-2-ylthio)acetamido)butanoate (9f): From L-ThrOCH3·HCl
9.56%; S, 7.30%. Found: C, 60.03%; H, 4.78%; N, 9.45%. (0.156g). White crystals (0.17g, 41%); mp 174–175°C.
Methyl
2-(2-(4-oxo-3-p-tolyl-3,4-dihydroquinazolin-2-yl- 1H-NMR (300MHz, CDCl3) δ: 8.74 (2H, d, J=8.0Hz, ArH),
Methyl 3-Hydroxy-2-(2-(4-oxo-3-p-tolyl-3,4-dihydroquina-
thio)acetamido)acetate (9a): From GlyOCH3·HCl (0.126g). 8.58 (1H, d, J=8.0Hz, ArH), 8.18 (1H, d, J=8.0Hz, ArH),
White crystals (0.23g, 58%); mp 165–166°C. 1H-NMR 7.94 (1H, d, J=8.0Hz, NH), 7.69–7.59 (2H, m, ArH), 7.58–7.55
(300MHz, DMSO) δ: 8.60 (1H, t, J=7.2Hz, NH), 8.04 (1H, (3H, m, ArH), 5.12 (1H, brs, OH, D2O exchangeable), 4.65
d, J=8.1Hz, ArH), 7.84 (1H, t, J=8.1Hz, ArH), 7.63 (2H, (1H, d, J=6.0Hz, CH), 4.34–4.13 (3H, m, SCH2, CH), 3.71
d, J=8.0Hz, ArH), 7.50 (1H, t, J=8.1Hz, ArH), 7.47–7.30 (3H, s, OCH3), 1.42 (3H, d, J=7.2Hz, CH3), Anal. Calcd for
(3H, m, ArH), 3.93 (2H, s, SCH2), 3.86 (2H, d, J=5.7Hz, C22H23N3O5S, (441.5): C, 59.85%; H, 5.25%; N, 9.52%; S,
NCH2), 3.60 (3H, s, OCH3), 2.42 (3H, s, CH3). Anal. Calcd 7.26%. Found: C, 59.77%; H, 5.18%; N, 9.46%. Mass spec-
for C20H19N3O4S (397.4): C, 60.44%; H, 4.82%; N, 10.57%; S, trum, m/z (Ir/%): 442.1.
8.07%. Found: C, 60.15%; H, 4.63%; N, 10.43%. Mass spec-
trum, m/z (Ir/%): 397.2.
Methyl 4-Hydroxy-1-(2-(4-oxo-3-p-tolyl-3,4-dihydroquina-
zolin-2-ylthio)acetyl)pyrrolidine-2-carboxylate (12): From L-
Methyl
2-(2-(4-Oxo-3-p-tolyl-3,4-dihydroquinazolin-2-yl- HOPrlOCH3·HCl (0.156g). White crystals (0.30g, 67%);
1
thio)acetamido)propanoate (9b): From -AlaOCH3·HCl (0.14g). mp 201–202°C. H-NMR (300MHz, CDCl3) δ: 8.08 (1H, d,
L
White crystals (0.19g, 48%); mp 165–166°C. 1H-NMR J=7.8Hz, ArH), 7.83 (1H, t, J=7.8Hz, ArH), 7.67 (1H, d,
(300MHz, CDCl3) δ: 8.68 (1H, d, J=7.2Hz, NH), 8.06 (1H, J=7.8Hz, ArH), 7.49 (1H, t, J=7.8Hz, ArH), 7.47 (2H, d,
d, J=8.1Hz, ArH), 7.84 (1H, t, J=8.1Hz, ArH), 7.60 (1H, J=8.1Hz, ArH), 7.31 (2H, d, J=8.1Hz, ArH), 5.31 (1H, d,
d, J=8.1Hz, ArH), 7.47 (1H, t, J=8.1Hz, ArH), 7.37 (2H, d, J=5.3Hz, CH), 4.43 (1H, brs, OH, D2O exchangeable), 4.30
J=8.7Hz, ArH), 7.31 (2H, d, J=8.7Hz, ArH), 4.37–4.25 (1H, (1H, d, J=6.0Hz, CH), 3.81–3.72 (2H, m, SCH2, CH2), 3.58
m, CH), 3.90 (1H, s, SCH2), 3.59 (3H, s, OCH3), 2.41 (3H, s, (3H, s, OCH3), 2.41 (3H, s, CH3), 2.06–1.69 (2H, m, CH2).
CH3), 1.28 (3H, d, J=7.2Hz, CH3). Anal. Calcd for C21H21N3O4S Anal. Calcd for C23H23N3O5S (453.5): C, 60.91%; H, 5.11%; N,
(411.5): C, 61.30%; H, 5.14%; N, 10.26%; S, 7.79%. Found: C, 9.27%; S, 7.07%. Found: C, 60.76%; H, 5.04%; N, 9.16%.
61.21%; H, 5.08%; N, 10.18%.
Methyl 2-(2-(3-(4-Methoxyphenyl)-4-oxo-3,4-dihydroquina-
Methyl 3-Hydroxy-2-(2-(4-oxo-3-p-tolyl-3,4-dihydroquina- zolin-2-ylthio)acetamido)acetate (10a): From GlyOCH3·HCl
zolin-2-ylthio)acetamido)propanoate (9c): From L-SerOCH3· (0.126g). White crystals (0.23g, 57%); mp 188–189°C.
HCl (0.156g). White crystals (0.27g, 65%); mp 152–153°C. 1H-NMR (300MHz, DMSO) δ: 8.60 (1H, t, J=7.2Hz, NH),
1H-NMR (300MHz, CDCl3) δ: 8.59 (1H, d, J=7.2Hz, NH), 8.08 (1H, d, J=8.1Hz, ArH), 7.82 (1H, t, J=8.1Hz, ArH), 7.61
8.09 (1H, d, J=7.8Hz, ArH), 7.86 (1H, t, J=8.1Hz, ArH), 7.61 (1H, d, J=8.1Hz, ArH), 7.50 (1H, t, J=8.1Hz, ArH), 7.36 (2H,
(1H, d, J=7.8Hz, ArH), 7.47 (1H, t, J=8.1Hz, ArH), 7.38 (2H, d, J=9.0Hz, ArH), 7.11 (2H, d, J=9.0Hz, ArH), 3.93 (2H, s,
d, J=8.1Hz, ArH), 7.31 (2H, d, J=8.1Hz, ArH), 5.08 (1H, brs, SCH2), 3.87–3.84 (5H, m, NCH2, OCH3), 3.60 (3H, s, OCH3).
OH, D2O exchangeable), 4.37–4.33 (1H, m, CH), 3.94 (1H, Anal. Calcd for C20H19N3O5S (413.4): C, 58.10%; H, 4.63%; N,
s, SCH2), 3.70–3.57 (5H, m, CH2, OCH3), 2.41 (3H, s, CH3). 10.16%; S, 7.76%. Found: C, 57.85%; H, 4.51%; N, 10.01%.
Anal. Calcd for C21H21N3O5S (427.5): C, 59.00% ; H, 4.95%; Mass spectrum, m/z (Ir/%): 413.2.
N, 9.83%; S, 7.50%. Found: C, 58.73%; H, 4.85%; N, 9.69%.
Methyl 2-(2-(3-(4-Methoxyphenyl)-4-oxo-3,4-dihydroquina-
Mass spectrum, m/z (Ir/%): 428.1.
zolin-2-ylthio)acetamido)propanoate (10b): From L-AlaOCH3·
Methyl
3-(2-(4-Oxo-3-p-tolyl-3,4-dihydroquinazolin-2-yl- HCl (0.14g). White crystals (0.14g, 34%); mp 130–131°C.
thio)acetamido)propanoate (9d): From β-AlaOCH3·HCl (0.14g). 1H-NMR (300MHz, CDCl3) δ: 8.65 (1H, d, J=6.9Hz, NH),
White crystals (0.29g, 71%); mp 160–161°C. 1H-NMR 8.07 (1H, d, J=8.1Hz, ArH), 7.84 (1H, t, J=8.1Hz, ArH), 7.59
(300MHz, DMSO) δ: 8.23 (1H, t, J=7.2Hz, NH), 8.07 (1H, (1H, d, J=8.1Hz, ArH), 7.48 (1H, t, J=8.1Hz, ArH), 7.36 (2H,
d, J=7.8Hz, ArH), 7.83 (1H, t, J=7.8Hz, ArH), 7.55 (2H, d, d, J=8.7Hz, ArH), 7.11 (2H, d, J=8.7Hz, ArH), 4.31–4.26
J=8.0Hz, ArH), 7.49 (1H, t, J=7.8Hz, ArH), 7.46 (1H, d, (1H, m, CH), 3.91 (2H, s, SCH2), 3.85 (2H, s, OCH3), 3.59
J=8.0Hz, ArH), 7.30 (2H, d, J=8.0Hz, ArH), 3.81 (2H, s, (3H, s, OCH3), 1.28 (3H, d, J=7.2Hz, CH3). Anal. Calcd
SCH2), 3.55 (3H, s, OCH3), 3.42–3.32 (2H, m, NCH2), 2.52 for C21H21N3O5S (427.5): C, 59.00%; H, 4.95%; N, 9.83%; S,

July 2014
679
7.50%. Found: C, 58.91%; H, 4.73%; N, 9.65%.
J=9.0Hz, ArH), 7.11 (2H, d, J=9.0Hz, ArH), 5.17 (1H, d,
Methyl 3-Hydroxy-2-(2-(3-(4-methoxyphenyl)-4-oxo-3,4-di- J=6.0Hz, CH), 4.46 (1H, brs, OH, D2O exchangeable), 4.30
hydroquinazolin-2-ylthio)acetamido)propanoate (10c): From (1H, t, J=7.8Hz, CH), 4.11 (1H, d, J=15.6Hz, SCH2), 3.99
L-SerOCH3·HCl (0.156g). White crystals (0.29g, 66%); mp (1H, d, J=15.6Hz, SCH2), 3.85–3.73 (2H, s, CH2, OCH3), 3.54
192–193°C. 1H-NMR (300MHz, DMSO) δ: 8.59 (1H, d, (1H, t, J=7.8Hz, CH), 3.54 (3H, s, OCH3), 2.18–2.08 (2H, m,
J=6.9Hz, NH), 8.07 (1H, d, J=8.0Hz, ArH), 7.83 (1H, t, CH2), 2.01–1.91 (2H, m, CH2). Anal. Calcd for C23H23N3O5S
J=8.1Hz, ArH), 7.62 (1H, d, J=8.0Hz, ArH), 7.46 (1H, t, (453.5): C, 60.91%; H, 5.11%; N, 9.27%; S, 7.07%. Found: C,
J=8.0Hz, ArH), 7.37 (2H, d, J=8.9Hz, ArH), 7.12 (2H, d, 60.77%; H, 5.07%; N, 9.13%.
J=8.9Hz, ArH), 5.06 (1H, t, J=5.7Hz, OH, D2O exchange-
General Procedure for the Preparation of Thioacetyl
able), 4.34–4.26 (1H, m, CH), 3.94 (2H, s, SCH2), 3.85 (2H, Amino Acid Hydrazide (14a, 14d, 14e) To a solution of
s, OCH3), 3.68–3.60 (5H, m, OCH2, OCH3). Anal. Calcd for amino acide ester derivative 8a, 8d, 8e (1.0mmol) in methyl
C21H21N3O6S (443.5): C, 56.87% ; H, 4.77%; N, 9.48%; S, alcohol (30mL), hydrazine hydrate (2.4mL, 5mmol) was
7.23%. Found: C, 56.85%; H, 4.76%; N, 9.45%. Mass spec- added. The reaction mixture was refluxed for 4h, cooled and
trum, m/z (Ir/%): 443.2.
the resultant precipitate was filtered off, washed with ethanol
Methyl 3-(2-(3-(4-Methoxyphenyl)-4-oxo-3,4-dihydroquina- and ether then crystallized from aqueous ethanol to yield the
zolin-2-ylthio)acetamido)propanoate (10d): From β-AlaOCH3· hydrazide 14a, 14d and 14e.
HCl (0.14g). White crystals (0.32g, 76%); mp 212–213°C.
2-(2-(4-Oxo-3-phenyl-3,4-dihydroquinazolin-2-ylthio)-
1H-NMR (300MHz, CDCl3) δ: 8.78 (1H, dt, J=7.2Hz, NH), acetamido)acetohydrazide (14a): White crystals (0.32g, 83%);
1
8.02 (1H, d, J=8.0Hz, ArH), 7.84 (1H, t, J=8.1Hz, ArH), 7.58 mp 185–186°C. H-NMR (300MHz, DMSO) δ: 8.11 (1H, d,
(1H, d, J=8.0Hz, ArH), 7.51 (1H, t, J=8.0Hz, ArH), 7.38 (2H, J=7.9Hz, ArH), 8.03 (1H, d, J=8.0Hz, ArH), 7.82 (1H, t,
d, J=8.9Hz, ArH), 7.14 (2H, d, J=8.9Hz, ArH), 4.22 (2H, s, J=8.0Hz, ArH), 7.61–7.52 (4H, m, ArH), 7.50–7.43 (2H, m,
SCH2), 3.59 (3H, s, OCH3), 3.54 (2H, q, J=6.6Hz, NCH2), ArH), 5.13 (1H, t, J=6.1Hz, NH), 4.25–4.18 (3H, m, NH,
2.54 (2H, t, J=6.4Hz, CH2), 13C-NMR (CDCl3) δ: 172.19 (C= NH2), 3.93 (2H, s, SCH2), 3.87 (2H, s, NCH2). Anal. Calcd
O), 167.29 (C=O), 161.14 (Cq), 157.35 (Cq), 147.56 (Cq), 136.26 for C18H17N5O3S (383.4): C, 56.38%; H, 4.47%; N, 18.27%; S,
(Cq), 135.37 (CHAr), 130.44 (CHAr), 129.89 (CHAr), 129.55 8.36%. Found: C, 56.17%; H, 4.38%; N, 18.12%.
(CHAr), 127.04 (CHAr), 126.48 (CHAr), 119.99 (Cq), 51.82
3-(2-(4-Oxo-3-phenyl-3,4-dihydroquinazolin-2-ylthio)-
(OCH3), 36.54 (SCH2), 35.34 (CH2), 33.91 (CH2), Anal. Calcd acetamido)propanehydrazide (14d): White crystals (0.28g,
for C21H21N3O5S (427.5): C, 59.00%; H, 4.95%; N, 9.83%; S, 73%); mp 196–197°C. 1H-NMR (300MHz, DMSO) δ: 8.05
7.50%. Found: C, 58.81%; H %, 4.83%; N, 9.76%.
(1H, d, J=8.0Hz, ArH), 7.86 (1H, d, J=8.0Hz, ArH), 7.83
Methyl 2-(2-(3-(4-Methoxyphenyl)-4-oxo-3,4-dihydroquina- (1H, t, J=8.0Hz, ArH), 7.60–7.58 (4H, m, ArH), 7.47–7.45
zolin-2-ylthio)acetamido)-4-methylpentanoate (10e): From L- (2H, m, ArH), 4.09 (2H, s, SCH2), 3.91 (1H, t, J=7.2Hz, NH),
LeuOCH3·HCl 0.182g. White crystals (0.23g, 51%); mp 3.45–3.33 (5H, m, NHCH2, NH, NH2), 1.89 (2H, t, J=6.6Hz,
184–185°C. 1H-NMR (300MHz, CDCl3) δ: 8.65 (1H, d, CH2). Anal. Calcd for C19H19N5O3S (397.5): C, 57.42%; H,
J=6.9Hz, NH), 8.05 (1H, d, J=8.1Hz, ArH), 7.82 (1H, t, 4.82%; N, 17.62%; S, 8.07%. Found: C, 57.32%; H, 4.76%; N,
J=8.1Hz, ArH), 7.51 (1H, d, J=8.1Hz, ArH), 7.45 (1H, t, 17.55%.
J=8.1Hz, ArH), 7.34 (2H, d, J=9.0Hz, ArH), 7.12 (2H,
4-Methyl-2-(2-(4-oxo-3-phenyl-3,4-dihydroquinazolin-2-
d, J=9.0Hz, ArH), 4.51–4.32 (1H, m, CH), 3.85 (2H, d, ylthio)acetamido)pentanehydrazide (14e): White crystals
1
J=6.7Hz, SCH2), 3.81 (3H, s, OCH3), 3.59 (3H, s, OCH3), (0.40g, 92%); mp 165–166°C. H-NMR (300MHz, DMSO) δ:
1.63–1.45 (2H, m, CH2), 1.21–1.13 (1H, m, CH), 0.83 (6H, d, 9.15 (1H, brs, NH), 8.31 (1H, d, J=7.9Hz, ArH), 8.05 (1H, d,
J=6.0Hz, 2CH3). Anal. Calcd for C24H27N3O5S (469.6): C, J=8.1Hz, ArH), 7.81 (1H, t, J=8.1Hz, ArH), 7.58–7.55 (4H,
61.39%; H, 5.80%; N, 8.95%; S, 6.83. Found: C, 61.43%; H, m, ArH), 7.47–7.43 (2H, m, ArH), 4.25–4.18 (3H, m, CH,
5.64%; N, 8.81%.
Methyl 3-Hydroxy-2-(2-(3-(4-methoxyphenyl)-4-oxo-3,4-di- SCH2), 1.54–1.39 (3H, m, CH, CH2), 0.80 (6H, d, J=6.0Hz,
hydroquinazolin-2-ylthio)acetamido)butanoate (10f): From
2CH3). 13C-NMR (CDCl3) δ: 171.29 (C=O), 166.87 (C=
NH2), 3.96 (1H, d, J=15.0Hz, SCH2), 3.87 (1H, d, J=15.0Hz,
L
-
ThrOCH3·HCl (0.156g). White crystals (0.28g, 62%); mp O), 161.12 (Cq), 157.47 (Cq), 147.62 (Cq), 136.28 (Cq), 135.34
180–181°C. 1H-NMR (300MHz, CDCl3) δ: 8.36 (1H, d, (CHAr), 130.44 (CHAr), 130.02 (CHAr), 129.87 (CHAr), 127.02
J=8.7Hz, NH), 8.08 (1H, d, J=8.1Hz, ArH), 7.83 (1H, t, (CHAr), 126.50 (CHAr), 119.96 (Cq), 50.47 (CH), 41.78 (SCH2),
J=8.1Hz, ArH), 7.60 (1H, d, J=8.1Hz, ArH), 7.47 (1H, t, 36.63 (CH2), 24.59 (CH), 23.33 (CH3), 22.14 (CH3). Anal.
J=8.1Hz, ArH), 7.34 (2H, d, J=9.0Hz, ArH), 7.11 (2H, d, Calcd for C22H25N5O3S (439.5): C, 60.12%; H, 5.73%; N,
J=9.0Hz, ArH), 5.01 (1H, d, J=5.4Hz, OH, D2O exchangeable), 15.93%; S, 7.30%. Found: C, 60.01%; H, 5.68%; N, 15.81%.
4.36 (1H, d, J=6.5Hz, CH), 4.07–4.02 (3H, m, SCH2, CH), 3.85
General Procedure of Quinazoline Thioacetyl Dipeptide
(2H, s, OCH3), 3.60 (3H, s, OCH3), 1.03 (3H, d, J=6.3Hz, CH3). (16a, 16d, 16e) To a cold solution (−5°C) of hydrazide 14a–c
Anal. Calcd for C22H23N3O6S (457.5): C, 57.76%; H, 5.07%; N, (1.0mmol) in AcOH (6mL), 1N HCl (3mL), and water (25mL)
9.18%; S, 7.01%. Found: C, 57.64%; H, 4.83%; N, 9.03%. Mass was added a solution of NaNO2 (0.34g, 5.0mmol) in cold
spectrum, m/z (Ir/%): 457.2.
water (3mL). After stirring at −5°C for 1h. A thick precipi-
Methyl 1-(2-(3-(4-Methoxyphenyl)-4-oxo-3,4-dihydroquina- tate was formed. The reaction mixture was extracted in cold
zolin-2-ylthio)acetyl)pyrrolidine-2-carboxylate (13): From L- ethyl acetate (30mL), washed with cold 3% NaHCO3, H2O
PrlOCH3·HCl (0.156g). White crystals (0.27g, 61%); mp and finally dried (Na2SO4). A solution of amino acid esters
220–221°C. 1H-NMR (300MHz, DMSO) δ: 8.08 (1H, d, hydrochloride (1.0mmol) in ethyl acetate (20mL) containing
J=7.8Hz, NH), 7.81 (1H, t, J=7.8Hz, ArH), 7.68 (1H, d, 0.2mL of triethyl amine was added to the ethyl acetate azide
J=8.0Hz, ArH), 7.48 (1H, d, J=8.0Hz, ArH), 7.33 (2H, d, solution 15a–c. The mixture was kept at −5°C for 24h, then

680
Vol. 62, No. 7
at 25°C for another 24h, followed by washing with 0.5N HCl, and the IC50 values and hence there is inverse proportional
water, 3% solution of NaHCO3 and finally dried (Na2SO4). relationship between the IC50 values and the free binding en-
The solution was evaporated to dryness, and the residue was ergy.21)
recrystallized from petroleum ether–ethyl acetate to give the
corresponding quinazoline dipeptide 16a–c.
Biological Evaluation
Methyl 3-(2-(2-(4-Oxo-3-phenyl-3,4-dihydroquinazolin-2-yl-
Cytotoxicity Test. Determination of Potential Cytoxic-
thio)acetamido)acetamido)propanoate (16a): White crystals ity Using Sulforhodamine B (SRB) Assay This method
(0.20g, 46%); mp 130–131°C. 1H-NMR (300MHz, CDCl3) was carried out according to Skehan et al.24)
δ: 8.43 (1H, t, J=6.9Hz, NH), 8.09 (1H, d, J=8.0Hz, ArH),
The sensitivity of the human tumor cell lines to the synthe-
8.07–7.81 (2H, m, ArH), 7.64–7.57 (4H, m, ArH), 7.50–7.44 sized compounds was determined by the SRB assay. SRB is
(2H, m, ArH), 3.92 (2H, s, SCH2), 3.66 (2H, d, J=5.9Hz, a bright pink aminoxanthene dye with two sulphonic groups
NHCH2), 3.58 (3H, s, OCH3), 3.24 (2H, q, J=6.5Hz, NCH2), that bind to the amino groups of intracellular proteins under
2.43 (2H, t, J=6.5Hz, CH2). Anal. Calcd for C22H22N4O5S mild acidic conditions; and dissociate under basic conditions.
(454.5): C, 58.14%; H, 4.88%; N, 12.33%; S, 7.06%. Found: C, As the binding of SRB is stoichiometric the amount of dye
57.89%; H, 4.72%; N, 12.22%. extracted from stained cells is directly proportional to the cell
Methyl 2-(3-(2-(4-Oxo-3-phenyl-3,4-dihydroquinazolin-2-yl- mass.
thio)acetamido)propanamido)acetate (16d): White crystals
Procedure Cells were used when 90% confluence was
(0.19g, 42%); mp 145–146°C. 1H-NMR (300MHz, DMSO) reached in T25 flasks. Adherent cell lines were harvested
δ: 8.05 (1H, d, J=8.0Hz, ArH), 7.86 (1H, d, J=8.0Hz, with 0.025% trypsin. Viability was determined by trypan blue
ArH), 7.83 (1H, t, J=8.0Hz, ArH), 7.60–7.58 (4H, m, ArH), exclusion using the inverted microscope. Cells were seeded
7.47–7.45 (2H, m, ArH), 4.22 (2H, s, SCH2), 3.75–3.64 (5H, in 96-well microliter plates at a concentration of 5×104 cells/
m, CH2, OCH3), 3.54 (2H, q, J=6.6Hz, NHCH2), 2.54 (2H, well in a fresh medium and left to attach to the plates for
t, J=6.4Hz, CH2). Anal. Calcd for C22H22N4O5S (454.5): C, 24h. After 24h, cells were incubated with the appropriate
58.14%; H, 4.88%; N, 12.33%; S, 7.06%. Found: C, 57.83%; H, concentration of the synthesized compounds (100µg/well),
4.62%; N, 12.14%.
when completed to total of 200µL volume/well using fresh
Methyl 2-(4-Methyl-2-(2-(4-oxo-3-phenyl-3,4-dihydroquina- medium and incubation was continued for 24, 48 and 72h.
zolin-2-ylthio)acetamido)pentanamido)acetate (16e): White Control cells were treated with vehicle alone. For each drug
crystals (0.27g, 56%); mp 180–181°C. 1H-NMR (300MHz, concentration, 4 wells were used. The cells were fixed with
DMSO) δ: 8.36 (1H, t, J=8.1Hz, ArH), 8.24 (1H, d, J=7.8Hz, 50µL cold 50% trichloroacetic acid for 1h at 4°C. Wells were
NH), 8.08 (1H, d, J=8.1Hz, ArH), 7.84 (1H, t, J=8.1Hz, washed 5 times with distilled water and stained for 30min at
ArH), 7.61–7.57 (4H, m, ArH), 7.51–7.43 (2H, m, ArH), room temperature with 50µL 0.4% SRB dissolved in 1% ace-
4.28–4.18 (1H, m, CH), 4.01–3.67 (4H, m, SCH2, NHCH2), tic acid. The cells were then washed 4 times with 1% acetic
3.60 (3H, s, OCH3), 1.69–1.43 (3H, m, CH, CH2), 0.82 (6H, acid. The plates were air-dried and the dye was solubilized
d, J=6.0Hz, 2CH3). Anal. Calcd for C25H28N4O5S (496.6): C, with 100µL/well of 10mM Tris base (pH 10.5) for 5min on a
60.47%; H, 5.68%; N, 11.28%; S, 6.46%. Found: C, 60.35%; shaker (Orbital shaker OS 20, Boeco, Germany) at 1600rpm.
H, 5.54%; N, 11.16%.
The optical density (OD) of each well was measured spectro-
photometrically at 564nm with an ELISA microplate reader
(Meter tech. Σ 960, U.S.A.). The mean background absorbance
Theory–Calculation
The design of the molecules synthesized in this article was automatically subtracted and mean values of each drug
based on certain character of the structural similarity between concentration was calculated.
the synthesized compounds and the natural substrate.11,12,21)
Calculation The percentage of cell survival was calcu-
The synthesized compounds compete with the natural sub- lated as follows:
strate to its binding domain and this resulted in the inhibition
survival fraction =OD (treated cells) / OD (control cells)
of the enzyme reaction. The calculations based on this theory
relayed initially on calculating the free energy of binding of
The Response Parameters The IC50 values: (a parameter
designed and docked inhibitors in to the binding domain of reflects potency).
the methionine synthase and the free energy of the receptor
and the ligand individually (Eq. 1).
The concentration of tested compound required to produce
50% inhibition of cell growth was calculated using sigmoidal
dose response curve-fitting model.
IC50 Human tumor cell lines (PC-3) used in this study
were obtained from the American Type Culture Collection
ΔEbind = ΔEcomplex −(ΔEreceptor +ΔEligand
)
Eq. 1. The Free Energy of Binding
Comparison was established between the results obtained (ATCC, Minisota, U.S.A.). The tumor cell lines were main-
from the free energy of binding occurred in the docking stage tained at the National Cancer Institute, Cairo, Egypt, by serial
Table 1. The Inhibitory Constants and Percentage Inhibition Obtained from Free Cell Assay
Compound
x-Intercept
y-Intercept
Type of inhibition
Ki (µM)
% Inhibition/[I] (µM)
10a
10e
—
—
0.19
0.08
Non-competitive
Competitive
25.2
—
60 2/100
11 STMa)
6
a) STM: stimulant.

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681
The reaction pathway shows our results obtained by the chemoselective S-alkylation of quinazoline 1 with chloroacetic acid and methyl chloroacetate and the subsequent
hydrolysis and hydrazinolysis of the ester 5.
Chart 1. Synthetic Routes for Precursors 2–6
The reaction pathway shows the reaction of hydrazide 6 with amino acid esters under azide coupling condition to afford quinazoline 3 via an interesting rearrangement.
Chart 2. Attempted Synthesis of Azide Coupling of 6 with Amino Acid Esters
The reaction pathway shows quinazoline structure modification at sulfur atom by attachement of acetyl amino acid esters to produce 8–10 and 11–13. This was effi-
ciently formed by the reaction of carboxylic acid derivative 2 with amino acid esters in the presence of HOBT and DCC.
Chart 3. DCC Coupling Method of 2a–c with Amino Acid Esters

682
Vol. 62, No. 7
The reaction pathway shows quinazoline structure modification at sulfur atom by attachement of dipeptide linked with acetyl spacer. This was effeciently formed by the
reaction of carboxylic hydrazides 14a, 14a and 14e with amino acid esters under azide coupling condition.
Chart 4. Synthesis of Dipeptides 16a, 16d, 16e via Azide Coupling Method
Fig. 1. Image of MTHF and Its Interactions with the Conserved Residues of the Binding Site
Image of MTHF docked in to its binding domain of MetS where pterine ring docked deep inside the receptor and methylamino-benzoylamino]pentanedioic acid side
chain pointed towards the orifice of the receptor.
sub-culturing. Cells were maintained in vitro at 37°C in a 100 µM of l-homocysteine thiolactone (abbreviated M−H+).
5% carbon dioxide, 95% air humidified incubator. Cells were Media was supplemented with 10% 1kDa cut-off dialyzed
grown in RPMI media containing 100µM of l-methionine (ab- fetal calf serum (HL60, 20%), sodium pyruvate (1mM), L-
breviated M+H) or methionine-free RPMI media containing glutamine (2µM), hepes (24µM) and sodium bicarbonate (2µM)

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Table 2. The Calculated Energy Terms for the Interaction of a Series of Compounds with MTHF Binding Domain and Their Sensitivity, IC50 and
RMSD Values
Energy term (kJ/mol)
Sensitivity (PC-3)
(inhibition %)
IC50 (µg/mL)
Ligand
MTHF
RMSD (Å)
PC-3
ΔEbind
−122.36
—
ΔEcomplex
−255.00
—
ΔEreceptor
−190.64
—
ΔEligand
58.00
—
—
—
—
1.41
—
Lapatinib reference
3.4
8a
8d
−105.51
−60.93
−42.91
−8.91
−62.93
−122.62
−50.14
−150.19
+44.11
−0.19
−50.14
−190.09
−131.09
−120.19
−108.65
−170.49
−183.38
+19.81
−207.19
−177.19
−124.33
−104.59
−125.79
−123.83
+63.48
−202.72
−267.08
−275.87
−290.85
−341.15
−390.03
−321.00
−331.05
−301.55
−311.05
−321.00
−330.95
−321.95
−311.05
−280.03
−351.95
−359.61
−291.05
−381.85
−387.15
−307.15
−271.55
−311.75
−277.13
−281.05
−186.09
−290.24
−285.05
−318.42
−335.62
−306.11
−360.04
−280.94
−399.94
−390.94
−360.04
−200.94
−280.94
−280.94
−272.04
−283.04
−265.91
−380.94
−282.04
−280.94
−290.90
−270.04
−286.94
−252.04
−397.91
88.88
84.09
52.09
36.48
57.40
58.70
89.18
100.08
54.28
80.08
89.18
60.08
90.08
90.08
100.66
101.58
89.68
70.08
107.38
80.98
108.08
103.08
100.98
98.74
53.38
68.9
—
22.6
—
2.23
3.51
3.12
4.67
2.42
1.6
8f
—
—
8e
—
—
16a
11
—
—
71.4
—
—
8b
—
3.9
8c
68
34
1.9
16e
14e
14a
9a
—
—
5.81
5.91
3.9
—
—
—
—
73.1
—
20.6
—
1.93
2.33
2.01
3.10
2.88
1.55
4.88
1.41
2.09
3.01
4.88
3.01
4.44
5.81
9d
9f
—
—
12
67.2
—
—
9b
—
9c
67.8
—
30.7
—
9e
10a
10d
10f
13
69.3
—
20
—
30.6
—
—
—
10b
10c
10e
—
—
64.2
—
20.8
—
Cells in exponential growth were plated (500–1000×10−3 cells
per well) in complete M+H−, medium, containing l-methio-
The results of the IC50 values were listed down in Table 1.
Methionine Synthase Assay. Enzyme Purification
nine (100µM). After an initial incubation of 24h at 37°C the Adapted methodology from our recent publication was used
medium was changed to M−H− and cells were treated with for the isolation, purification and the assay of methionine syn-
compound concentrations between 100 and 0.1µM. The tested thase from rat liver.12) Frozen rat livers (200g) (−80°C) were
compounds were dissolved in DMSO. After 24h, the cells thawed on ice and rinsed in ice cold potassium phosphate
were washed twice with Hanks solution and transferred to buffer (50mM, pH 7.2). The livers were then chopped into
fresh M+H− medium (recovery). Cells were then incubated ice-cold homogenising buffer that was made using potassium
for a further 5d at 37°C before performing the MTT assay. phosphate buffer (500mL, 50mM, pH 7.2) supplemented with
Cell culture media was collected at appropriate times during protease inhibitors, (phenylmethylsulfonyl fluoride (PMSF)
recovery and stored at −80°C. Appropriate control experi- (26mg) dissolved in 1mL ethanol), Na-p-tosyl-L-lysine chlo-
ments were performed without the inhibitors.
romethyl ketone hydrochloride (35mg), trypsin (12.5mg)
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- and apotinin (2.6mg) each dissolved in potassium phosphate
mide (MTT) was used to assess the cytotoxicity of the tested buffer (1mL, 50mM, pH 7.2) prior to mixing into the ho-
compounds in cell culture. Following the above described mogenising buffer. The livers were then homogenised for six
treatments, media were recovered and the cells were treated short pulses using a blender taking care to ensure the homoge-
with fresh medium containing MTT (0.6mg/mL). The cells nate remained ice cold throughout. The resulting mixture was
were incubated for 4h at 37°C, the medium removed and centrifuged for 15min, at 5000rpm in a 4°C pre-cooled Beck-
replaced with DMSO (150µL) to dissolve the resulting crys- man centrifuge using a JA-20 rotor. The resulting supernatant
tals. The absorbance of this solution was recorded at 550nm liquid was centrifuged for a further 30min at 15000rpm at
in a plate reader. The final DMSO concentration was 0.1% 4°C. The supernatant liquid was further centrifuged for 1h, at
and each plate contained the blank group (complete medium 4°C at 28000rpm using a Beckman ultra centrifuge equipped
200µL/well and no cells), the control group (complete medium with a 28-swing rotor. The resulting cytosolic fraction was
200µL/well and untreated cells). After 96h of exposure time, collected and stored in ice for up to 24h awaiting purification.
20µL of MTT solution (5mg/mL) was added and incubated
in the dark at 37°C for 4h. The supernatant was removed and ration of the Resin Diethylaminoethyl (DEAE) cellulose
formazan crystals were dissolved in DMSO (150µL).
(205g) was stirred in sodium phosphate buffer (400mL, 0.2M,
Batch Chromatography on DEAE Cellulose. Prepa-

684
Vol. 62, No. 7
Fig. 2. Compound 10a Docked inside MTHF Binding Domain
Image of compound 10a docked in to the MTHF binding domain of MetS. The left hand side represents the outermost part of the enzyme and the orifice of the receptor
and the right hand site represents the innermost part of the enzyme.
pH 7.2) for 3min. The slurry was allowed to settle and the su- This solution was kept for up to 24h.
pernatant containing any fine partials was decanted. The resin
DEAE Regeneration and Storage Conditions The used
was re-dispersed in sodium phosphate buffer (400mL, 0.2M, DEAE cellulose was washed in sodium phosphate buffer
pH 7.2) the slurry again allowed to settle before decanting the (500mL, 20mM, pH 7.2) containing sodium chloride (0.5mM)
supernatant.
for one hour. The resin was then allowed to settle and the
The conditioned DEAE cellulose was then equilibrated with buffer decanted. The process was repeated twice using sodium
sodium phosphate buffer (400mL, 20mM, pH 7.2). In detail, phosphate buffer (250mL, 20mM, pH 7.2) containing 1M
the resin was suspended in sodium phosphate buffer, stirred sodium chloride. Finally the buffer was replaced with a 20%
for 20min, allowed to settle, and the supernatant decanted. ethanol/ water mixture and stirring continued for 15min. The
This process was repeated three times.
ethanol/ water wash was repeated three times in succession
Batch Chromatography of Cytosolic Fraction The rat before storing the DEAE cellulose at 4°C in 20% ethanol/
liver cytosol (approximately 200mL) was taken and stirred water (v/v).
with the previously equilibrated DEAE cellulose in sodium
Q-Sepharose FPLC Chromatography The Q-Sepharose
phosphate buffer (100mL, 20mM, pH 7.2) for one hour in a resin was conditioned prior to use with the separation buffers.
blacked out, cold cabinet. The supernatant containing any un- In detail, sodium phosphate buffer (20mM, pH 7.2), (low salt
bound proteins was removed by vacuum filtration. The DEAE buffer), was run through the column at a rate of 4mL/min for
cellulose was then washed twice with ice cold sodium phos- one hour followed by sodium phosphate buffer (20mM, pH
phate buffer (100mL, 20mM, pH 7.2) dispersing the red colour 7.2) containing sodium chloride (1M), (high salt buffer), for
from the resin. The bound proteins, including the desired 1h. Finally a stable baseline was recorded using the low salt
enzyme were then eluted from the resin by stirring the DEAE buffer.
cellulose in sodium phosphate buffer (200mL, 20mM, pH
The enzyme preparation obtained from the DEAE purifica-
7.2) containing sodium chloride (0.5M) for 15min in a dark, tion was diluted three fold with milli-Q water to reduce the
cold cabinet. The mixture was filtered under vacuum and the concentration of sodium chloride. The Q-sepharose packed
resin washed twice with the sodium chloride containing buffer XK16 column (15×2.5cm) was loaded with 300mL of dilute
(0.5 M, 2×50mL). The filtrates containing the enzyme were filtrate using a superloop (10mL) and flow rate of 4mL/min.
pooled and kept on ice in the dark awaiting further analysis. The column was then eluted at a flow rate of 2mL/min using

July 2014
685
Fig. 3. Compound 9c Docked inside MTHF Binding Domain
The left hand side represents the outermost part of the enzyme and the orifice of the receptor and the right hand site represents the innermost part of the enzyme.
the following gradient: Sodium phosphate buffer (20mM, pH water to make a working solution of BSA (0.0135µg/mL).
7.2) containing sodium chloride (1M) (high salt buffer) was The Bradford reagent (200µL) was added to each concentra-
increased to 0.5M NaCl over 50min. This was then increased tion making a final volume of 1mL. After incubation (15min
to 1M NaCl over 5min. This condition was held constant for at room temperature) aliquots of 200µL were transfer into
20min before washing back with sodium phosphate buffer a 96 well plate for reading. Protein samples were run along
(20mM, pH 7.2) (low salt) over 5min and holding constant for side the known concentrations of BSA. Serial dilutions of the
a further 20min to condition the resin for subsequent separa- unknown concentrations of protein were made to ensure their
tions. Fractions were collected at 12min intervals of approxi- absorbance readings fell within the range of the calibration
mately 50mL throughout the program.
Precautions were undertaken to ensure the enzyme solu-
line.
Preparation of Reagents Homocysteine solution (3.77mM)
tions remained cool and dark at all times. To this effect, frac- was prepared from D,L-homocysteine (5.1mg) in water (10mL).
tions were collected in pre-cooled tubes kept on ice. The col- Homocysteine solution was prepared daily.
umn and super loop were wrapped in foil throughout experi-
ments and the buffers were kept on ice during the separation.
The active fractions, identified using a radiometric methio-
Ascorbic acid solution (10mM) was prepared form ascorbic
acid (0.18g) in water (100mL).
5-Methyltetrahydrofolate (MTHF) solution (3.96mM) was
nine synthase assay, were pooled together and 50mL batches prepared from 5-methyltetrahydrofolic acid (25mg) in ascorbic
concentrated to one-fifth of the original volume using an Ami- acid solution (12.5mL, 10mM). 5-Methyltetrahydrofolate solu-
con ultra concentrator (30kDa cut-off size). The combined tion was stored in 1mL aliquots at −20°C.
concentrates were pooled and kept on ice. Glycerol was then
added to a final concentration of 20% v/v.
5-14C-Methyltetrahydrofolate (14C-MTHF) solution was
prepared from 5-14C-methyltetrahydrofolic acid barium salt
Protein Content Determination The protein content of (50 µCi) dissolved in ascorbic acid solution (1mL, 10mM).
all the collected samples was determined using a Bio-Rad 5-14C-Methyltetrahydrofolate solution was stored in 20µL ali-
protein assay kit. The Method used bovine serum albumin quots at −20°C.
(BSA) as the standard. The standard curve ranging from BSA
Dithiothreitol (DTT) solution (0.7µM) was prepared from
0 to 5.4µg. Assays were incubated for 15min to allow for the DTT (13mg) dissolved in water (120µL). Dithiothreitol solu-
formation of the chromophore. The absorptions of the samples tion was prepared daily.
were determined using an automatic plate reader at 600nm on
a 96 well plate.
Hydroxycobalamin (HO-Cbl) solution (1.8mM) was pre-
pared from hydroxycobalamin (2.5mg) dissolved in water
A stock solution of BSA (0.135µg/mL) was diluted with (1mL). Hydroxycobalamin solution was stored in 100µL ali-

686
Vol. 62, No. 7
Fig. 4. Compound 8c Docked inside MTHF Binding Domain
Image of compound 8c docked in to the MTHF binding domain of MetS in the primary structure of the enzyme. The left hand side represents the outermost part of the
enzyme and the orifice of the receptor and the right hand site represents the innermost part of the enzyme.
quots at −20°C.
When the assay was used to screen the activity of the in-
S-Adenosylmethionine (S-AdoMet) solution (1.2mM) was hibitors an aliquot of DMSO (5µL) vehicle was included and
prepared from S-adenosylmethionine iodine salt (5mg) dis- the buffer volume adjusted accordingly. Blank assays were
solved in water (7.9mL). S-Adenosylmethionine solution was prepared in the same way excluding the enzyme source and
stored in 500µL aliquots at −20°C.
General Assay Into clean, dry, labelled serum vials
substituting for an equivalent volume of assay buffer.
Enzyme Dose-Dependence of the Assay The enzyme
prepared in triplicate, MTHF (67.9nmol) made up of MTHF dose-dependence of the assays were performed using a fixed
(20µL, 3.96mM) and 14C MTHF (10µL, 2.47×10−3 MBq), were incubation time of 30min while varying the amount of en-
added. S-AdoMet (10µL, 1.2mM), DTT (10µL, 0.70µM), HO- zyme added. Aliquots of 10, 25, 75 and 100µL of purified
Cbl (10µL, 1.8mM), and potassium phosphate buffer (50mM, enzyme were used.
pH 7.2) (to give a total assay volume of 300µL) were added.
Time-Dependence of the Assays Determination of time-
The vials were excluded from light by wrapping in aluminium dependence was investigated using a fixed amount of enzyme
foil. Enzyme (50µL) was added to each vial in turn followed (50 µL) determined from the dose study at varying incubation
by sealing and flushing with nitrogen for 30s to create an- time. Assays were carried out at 0, 5, 10, 15, 30 and 45min.
aerobic conditions. The vials were pre-incubated for a period
Solvent Effect of the Aassay Solvent effects were de-
of 5min at 37°C. The reaction was initiated with the addition termined using the optimised conditions determined from
of homocysteine (40µL, 3.77mM). The total assay volume was the dose and time studies. The effect of 0, 2, 5 and 10µL of
300µL. Assay vials were then incubated for 45min at 37°C DMSO on enzyme activity was determined. The optimum
unless otherwise stated. The reactions were terminated with volume was found to be 5µL and was subsequently used for
ice cold milli-Q water (400µL) and the assay vials placed on all of the inhibition studies.
ice. The reaction mixture was then passed through AG1-X8
Preparation of Inhibitor Solutions Stock solutions (30mM)
resin columns (0.5×5cm) pre-conditioned with water (3mL) of all the compounds to be tested were prepared in DMSO.
followed by water (2mL) to elute [14C]-methionine. The 2mL Working solutions of the inhibitors were prepared by further
elute was collected and scintillation cocktail added (12mL) dilution of the stock with DMSO. The concentrations of these
before being quantified using a liquid scintillation counter.
working solutions were calculated to give the desired concen-

July 2014
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Fig. 5. Compound 10a Docked inside MTHF Binding van der Waals Radius
Image of compound 10a docked in to the MTHF binding domain of MetS with van der Waals radius. The left hand side represents the outermost part of the enzyme and
the orifice of the receptor and the right hand site represents the innermost part of the enzyme.
tration of inhibitor in the assay when 5µL of working solution −207.19kJ/mol and −124.33kJ/mol, respectively and the low-
was used per assay.
est IC50 values 22.6µg/mL, 20.6µg/mL, 20µg/mL and 20.8µg/
Time Inhibition Study Time-dependent assays of the mL against PC-3 cells, respectively. Compounds 8c and 9c
inhibitors were performed at 5, 10, 15, 30, 45, 60 and 90min exhibited moderate values of free energy of binding (150.19kJ/
keeping all other parameters fixed.
Enzyme Kinetics. Kinetic Parameters Kinetic param- and 30.7µg/mL (Table 1).
mol and −183.38kJ/mol) and moderate IC50 values 34µg/mL
eters (Vmax, Km and Ki) were calculated for both uninhibited
The structure assignment of both the S-substituted amino
1
and inhibited reactions using the following parameters: Inhibi- acid derivatives and dipeptide is based on H-, 13C-NMR and
tor concentrations of 500, 250 and 100µM. 5-Methyltetrahy- mass spectroscopy as well as physicochemical analysis. Both
1
drofolate concentrations: 200, 100, 80, 40, 20 and 10µM. The the H- and 13C-NMR clearly confirm the alkylation site for
1
concentrations of all other substrates remained constant. A all the isolated S-substituted derivatives. Thus the H-NMR
5min pre-incubation and 20min incubation time was used for spectrum of 8a exhibits a singlet signal at δ 3.92ppm typi-
all assays ensuring that substrate turnover was ≤20%.
cally associated with SCH2 group. The 1H-NMR spectrum
Kinetic Parameters of Methionine Synthase Prior to also shows an interesting triplet and doublet signals at δ 8.61
any inhibition studies the kinetic parameters of the enzyme and 3.84ppm associated with the NH and CH2 groups, respec-
reaction had to be established. This involved a series of exper- tively. This confirming the generation of amino acid coupling
iments to establish that all assays were performed within the that is common for all S-substituted α-amino acid esters 8–13.
linear region of the enzyme activity, and that the maximum The 13C-NMR spectrum of 8a shows signals at δ 52.21, 41.44,
turnover of the enzyme did not exceed 20% to avoid product 36.23ppm attributed to OCH3, NHCH2 and SCH2, respec-
inhibition and ensure that the concentration of the substrate tively.
was much greater than the enzyme. These experiments con-
sisted of time and dose-dependent assays.
Discussion
We now report the preparation of methyl 2-(2-(4-oxo-3-
aryl-3,4-dihydroquinazolin-2-ylthio)acetamido)alkanoates and
Results
Compounds 8a, 9a, 10a and 10f showed the lowest values dipeptide derivative. Quinazoline 1 was prepared either by the
of free energy of binding (−105.51kJ/mol, −190.09kJ/mol, reaction of aryl isothiocyanate with anthranilic acid in glacial

688
Vol. 62, No. 7
Fig. 6. Plots of Concentration Gradients versus Surviving Fractions for Compounds 8a, 8c, 9a, 9c, 10a and 10c
The plots of concentarion gradients used (0, 5, 12.5, 25, 50µg/mL) and the surviving fraction of the cancer cells.
acetic acid23) or could be obtained by the sequential reaction 2. The ester 5 was reacted with hydrazine hydrate in ethyl
of aniline derivatives with carbon disulfide followed by al- alcohol for 4h and gave hydrazide 6 (Chart 1). Compounds
kylation with dimethyl sulfate and finally refluxed with methyl 2 and 6 are excellent precursors for quinazoline structure
anthranilate in ethyl alcohol for 18h to afford the desired quin- modification at sulfur atom via amino acid coupling methods.
azoline 1.22) Structure modification of the model compound Azide coupling method was considered one of the important
1 could be achieved by the simple chemoselective alkylation methods to introduce amino acid derivatives from hydrazides.
reactions with electrophiles. However, the reaction of quinazo- It was also reported that this method decreases the degree of
line 1 with chloroacetic acid in the presence of NaOH gave a racemization in the amino acid coupling.25,26)
mixture of S-substituted carboxylic acid 2 contaminated with
Our first choice for structure modification of quinazoline
quinazoline dione 3 and benzanilde 4. On the other hand, the 1 by the introduction of acetyl amino acid esters at S-atom
reaction of 1 with methyl chloroacetate in the presence of a was the azide coupling method. The hydrazide 6 reacted with
NEt3 afforded the S-substituted ester 5 in an excellent yield. NaNO2 and HCl mixture at 0°C to presumably produce the
Saponification of ester 5 with KOH in methanol water mixture azide 7, which was extracted with ethyl acetate. The in situ
at room temperature for 4h gave S-substituted carboxylic acid generated azide derivative 7 was treated with amino acid

July 2014
689
Fig. 7. Compound 9c Docked inside MTHF Binding van der Waals Radius
Image of compound 9c docked in to the MTHF binding domain of MetS with van der Waals radius showing spheres of nonbonding interactions of the atoms of the
receptor and the ligand. The left hand side represents the outermost part of the enzyme and the orifice of the receptor and the right hand site represents the innermost part
of the enzyme.
ester hydrochloride and NEt3. Unfortunately, the amino acid reacted with NaNO2 and HCl mixture at 0°C to give the azide
ester derivative 8 was not formed and instead an interesting derivatives 15a, 15d and 15e, which was extracted with ethyl
rearrangement occurred to finally afford the dione 3 (Chart acetate. The in situ generated azide derivatives 15a, 15d and
2). The use of this rearrangement as an important synthetic 15e solution in ethyl acetate was reacted with amino acid
method together with rational mechanism explanation will methyl ester hydrochloride in the presence of triethyl amine
be discussed in another publication. Our alternative efforts to afford the dipeptides derivatives 16a, 16d and 16e in good
for structure modification of quinazoline by the introduction yield (Chart 4).
of acetyl amino acid esters at S-atom was efficiently formed
from the key substrate carboxylic acid derivative 2 via DCC least active compounds predicted by thermodynamic algo-
coupling method. rithms which showed the free energy of binding for compound
The enzyme free assay was performed on the most and
The DCC coupling is one of the major tools employed in lit- 10a −207.19kJ/mol and for compound 10e +63.48kJ/mol. The
erature to introduce peptide bonds by the reaction of carboxyl- results displayed that the percentage inhibition of the enzyme
ic acid with amino acid methyl ester.27) Hydroxybenzotriazole for compound 10a is 60 2/100 µM with inhibitory constant
(HOBT) is widely used as an additive to decrease racemiza- (Ki=25.2 µM). In contrary for compound 10e which exhib-
tion in the carbodiimide peptide coupling.28,29) Treatment of ited stimulating platform for the enzyme catalysed reaction as
carboxylic acid 2a–c with the amino acid esters hydrochloride shown in Table 1.
in the presence of coupling reagents DCC and HOBt afforded
amino acid ester S-alkanamide 8–13, respectively in good receptor and the designed inhibitors were docked in to the re-
yield (Chart 3). ceptor on the same docking pose of the MTHF substrate (Fig.
Molecular modeling investigations were done on the MTHF
Furthermore, our next target was the synthesis of dipeptide 1). The MTHF receptor is confined by 5 amino acids: aspartic
linked to quinazoline by thioacetyl spacer. The quinazoline acid 390, asparagine 411, aspartic acid 473, asparagine 508
amino acid esters 8a, 8d and 8e were boiled with hydrazine and arginine 516. The receptor is considered one of the biggest
hydrate in absolute ethanol to afford the hydrazides 14a, 14d receptors in size (20.7Å×12.2Å) (Fig. 1).
and 14e. The hydrazide derivatives 14a, 14d and 14e were
The ligand–protein complexes were subjected to intense

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Fig. 8. Compound 8c Docked inside MTHF Binding van der Waals Radius
Image of compound 8c docked in to the MTHF binding domain of MetS. with van der Waals radius showing spheres of nonbonding interactions of the atoms of the
receptor and the ligand. The left hand side represents the outermost part of the enzyme and the orifice of the receptor and the right hand site represents the innermost part
of the enzyme. Compound 4 makes hydrogen bond with asparagine 508 and arginine 518.
dynamic work to obtain the most suitable and stable confor- favourable binding.11) If the molecule of interest fits the bind-
mations and lowest in energy. The receptors do not recognize ing criteria and energy requests of the binding domain, then
their ligands in terms of molecular frame work, atom positions the receptor-molecule complex will have a lower energy than
or connectivities between them, but in terms of free energy the sum of the energies of the un-complexed protein and mol-
difference between unassociated and associated states of the ecule. The ligand showed a variation in the percentage inhibi-
ligand with the receptor. Hydrogen bonds, electrostatic inter- tion associated with a variation in the total binding enthalpy
actions, hydrophobic interactions and van der Waals interac- as shown in Table 2.
tions stabilize the associated ligand-receptor complex enthal-
Compounds 8c, 9c and 10a have been docked on the same
py.30) Subsequently the degree of freedom of the ligand and pose orientation of the natural substrate of the enzyme. The
receptor is reduced. It is not desirable to get active unstable root mean square deviations (RMSD) for them were 1.9Å,
conformation with high energy content compared to the global 1.55Å and 1.41Å, respectively (Figs. 2–4). This means that
minimum conformation. If the ligand structure is conforma- these compounds most likely will produce a competitive in-
tionally too unfavourable, the docking structure might become hibition and gradient concentration of these compounds will
too unstable in total energy.31) Therefore, it is necessary to displace MTHF from its domain and therefore inhibit the
examine the relative stability of the generated conformation enzyme reaction.12)
compared with all other possible conformations of the ligand.
Compound 10a docked vertically inside the pocket to avoid
The internal energy content, conformation, desolvation and the van der Waals repulsive interactions (Figs. 2, 5). The plau-
degree of freedom of the unassociated ligand and receptor will sible docking achieved RMSD value of 1.41Å which is closer
change after the docking occurred. Therefore, the free energy to the docking pose of the natural substrate MTHF. The com-
difference between the associated state and the unassociated pound achieved a low free energy of binding of −207.19kJ/
state was calculated from Eq. 1. Each algorithm of the binding mol and IC50 value 20µg/mL. This could be attributed to the
free energy for each ligand was compared to the IC50 value ligand fitted plausibly the cavity of the receptor and produced
of each ligand obtained. The comparison was accomplished favourable electrostatic interaction with serine 507, asparagine
by measuring ΔEbind with the more negative value, the more 323 and glutamic acid 320. This consequently contributed

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691
Table 3. Structure of MTHF and the Synthesized Compounds
The structure of the compounds
Fig. 9. Lineweaver–Burk Plot for Compounds 10a and 10e
Represents the inhibition plots of the enzyme reaction with different inhibitor
concentrations. The plots of compounds 10a and 10e showed non-competitive and
competitive inhibitions, respectively.
good in to the total free energy of binding and to the obtained
IC50 value (Fig. 6). The docking pose of compound 9c showed
that the ligand centre was slightly shifted outwards the bind-
ing domain. This probably could be ascribed to the free hy-
drophobic rotatable methyl group positioned at phenyl ring
and the branched serinyl group positioned at the side chain of
the quinazoline ring. This likely declined the Coulomb energy
of the total free energy of binding (−183.38kJ/mol) and IC50
value 30.7µg/mL due to the high energy barrier among the
methyl group and branched side chain of the ligand from one
side and the polar amino acids exist at the orifice of the bind-
ing domain (Figs. 3, 7). Compound 8c was docked horizon-
tally where the side chain and phenyl ring found at positions 2
and 3 of quinazoline ring protruded outside part of the recep-
tor to avoid the steric clashes (Figs. 4, 8). The docking score
started to diminish when the phenyl ring and the side chain of
the ligand moved away and the van der Waals force decreased.
There was an increase in steric clashes, which occurred with
the polar parts of the amino acids serine 507, asparagine 323
and glutamic acid 320. The docking score started to decrease
by 2kJ/mol. Such an energy penalty resulted in the reduction
in the binding affinity of the subsequent conformers of the
ligand 8 and hence reflected on the values of total energy of
binding (−150.19kJ/mol) and IC50 value 34µg/mL (Fig. 6).
The results of free cell assay suggested that the inhibi-
tors may bind at other binding sites as well as at the desired

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Table 3. SCtorunctitnuuredof MTHF and the Synthesized Compounds
The structure of the compounds
Table 3. Continued
Structur of MTHF and the Synthesized Compounds
The structure of the compounds

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693
Table 3. SCtorunctitnuuredof MTHF and the Synthesized Compounds
The structure of the compounds
1
V0
Km (1+[I] / Ki )
1
Ki
=
+
Vmax
[S] Vmax
Eq. 3. Lineweaver–Burk Equation for a Non-competitive Inhibitor
The non-competitive inhibition pattern exhibited by com-
pound 10a could be attributed to that ligand possibly bound
the enzyme whether or not it has already bound the substrate.
Meanwhile, the competitive inhibition pattern exhibited by
compound 13 probably certified to that the ligand competed
with the natural substrate (MTHF) to its binding domain.
Conclusion
MetS utilizes the most abundant form of the circulating fo-
lates, MTHF, as a substrate. MetS related pathways are impor-
tant in many biological processes including the synthesis of
proteins, lipids, polyamines and numerous AdoMet-dependent
transmethylation reactions. Inhibition or impairment of the
function of any one of these reactions may have fundamental
effects on the functioning and growth of the cells. In par-
ticular, impaired conversion of homocysteine to methionine
and MTHF to THF and could affect folate metabolism, DNA
sythesis, methionine and AdoMet syhthesis. Some cancer cells
showed methionine-dependence and depriving these cancer
cells of methionine could affect their proliferation. Methionine
synthase was found over expressed in 60% of the prostate
cancer therefore it has a kind of a link between inhibiting the
metabolic reaction of the enzyme and apoptosis.12) The syn-
thesized inhibitors were analogues of quinazoline derivatives
that inhibit the enzyme reaction and could be used as drug
candidates.
Acknowledgments Thanks for every person gave his
valuable contribution to this article.
References
1) Halpern B. C., Clark B. R., Hardy D. N., Halpern R. M., Smith R.
A., Proc. Natl. Acad. Sci. U.S.A., 71, 1133–1136 (1974).
2) Hoffman R. M., J. Tissue Cult. Association, 18, 421–428 (1982).
3) Goulding C. W., Postigo D., Matthews R., Biochemistry, 36, 8082–
8091 (1997).
4) Pavillard V., Drbal A., Swaine D. J., Phillips R. M., Double J. A.,
Nicolaou A., Biochem. Pharmacol., 67, 1587–1599 (2004).
5) Guo H., Lishko V. K., Herrera H., Groce A., Kubota T., Hoffman R.
M., Cancer Res., 53, 5676–5679 (1993).
6) Breillout F., Hadida F., Echinard-Garin P., Lascaux V., Poupon M.
F., Anticancer Res., 7 (4B), 861–867 (1987).
7) Nammalwar B., Bunce R., Berlin K., Med. Chem. Res., 54, 387–396
(2012).
8) Wang L., Cherian C., Kugel Desmoulin S., Mitchell-Ryan S., Hou
Z., Matherly L. H., Gangjee A., J. Med. Chem., 55, 1758–1770
(2012).
9) Gangjee A., Namjoshi O., Raghavan S., Queener S., Kisliuk R. L.,
Cody V., J. Med. Chem., 56, 4422–4441 (2013).
10) Banks E., Doughty S. W., Wheelhouse R., T., Nicolaou A., FEBS,
274, 287–299 (2007).
11) Elshihawy H., Hammad H., Med. Chem. Res., 22, 3405–3415 (2013).
12) Elshihawy H., Helal M., Said M., Hammad H., Bioorg. Med. Chem.,
22, 550–558 (2014).
5-methyltetrahydrofolate site were listed in Table 1. Methio-
nine synthase is a bisubstrate enzyme, it is possible that the
inhibitors also compete for the homocysteine binding domain
in addition to the 5-methyltetrahydrofolate binding domain,
or even bind at the binding site of the S-adenosylmethionine
reactivation cofactor. In this study we have only attempted the
evaluation of the inhibitory activity against the 5-methyltetra-
hydrofolate binding site keeping all other parameters constant.
The target compounds that were successfully synthesized
were tested for their inhibitory activity against highly purified
rat liver cobalamin-dependent methionine synthase. A pilot
study was undertaken on the synthesized series of substituted
quinazoline derivatives. The two compounds were tested using
an established in vitro radiometric enzyme assay.32–34) The
compounds were initially screened at 500µM then at gradient
concentrations of 250µM and 100µM. The tested compounds
exhibited typical non-competitive (10a) and competitive (10e)
inhibition patterns on Lineweaver–Burk plots (Eqs. 2, 3).
1
V0
K (1+[I] / K )
1
1


13) Fathalla W., Čajan M., Pazdera P., Molecules, 5, 1210–1223 (2000).
14) Fathalla W., Čajan M., Pazdera P., Molecules, 6, 557–573 (2001).
15) Fathalla W., Pazdera P., Marek J., J. Heterocycl. Chem., 39, 1139–
1144 (2002).
m
i
=
+




Vmax
[S] Vmax
Eq. 2. Lineweaver–Burk Equation for Competitive Inhibition

694
Vol. 62, No. 7
16) Fathalla W., Pazdera P., Marek J., J. Heterocycl. Chem., 39, 1145–
1152 (2002).
17) Fathalla W., Marek J., Pazdera P., J. Sulfur Chem., 29, 31–42 (2008).
18) Fathalla W., Ali I. A. I., Marek J., Pazdera P., J. Sulfur Chem., 33,
49–63 (2012).
25) Palaska E., Şahin G., Farmaco, 57, 101–107 (2002).
26) Ali I. A. I., Al-Masoudi I. A., Saeed B., Al-Masoudi N. A., La Colla
P., Heteroatom Chem., 16, 148–155 (2005).
27) Davis J. S., Mohammed A. K., J. Chem. Soc., Perkin Trans. 1, 1981,
2982–2990 (1981).
19) Fathalla W., Ali I. A. I., Heteroatom Chem., 18, 637–643 (2007).
28) König W., Geiger R., Chem. Ber., 103, 788–798 (1970).
20) Fathalla W., El Rayes S., Ali I. A. I., ARKIVOC, 13, 179–188 29) Pennington R. M., Fischer R. R., J. Biol. Chem., 256, 8963–8969
(2008).
(1981).
21) Cairns D., Michalitsi E., Jenkins T. C., Mackay S. P., Bioorg. Med.
Chem., 10, 803–807 (2001).
30) Sharp K., Fine R., Honig B., Science, 236, 1460–1463 (1987).
31) Taggi A., Hafez A., Dudding T., Lectka T., Tetrahedron, 58, 8351–
8356 (2002).
32) Waly M., Olteanu H., Banerjee R., Choi S., Mason J. B., Parker
B. S., Sukumar S., Shim S., Sharma A., Benzecry J. M., Power-
Charnitsky V. A., Deth R. C., Mol. Psychiatry, 9, 358–370 (2004).
33) Nicolaou A., Kenyon S. H., Gibbons J. M., Ast T., Gibbons W. A.,
Eur. J. Clin. Invest., 26, 167–170 (1996).
22) El-Azab A. S., Al-Omara M. A., Abdel-Aziz A. A.-M., Abdel-
Aziz N. I., El-Sayed M. A.-A., Aleisa A. M., Sayed-Ahmed M. M.,
Abdel-Hamide S. G., Eur. J. Med. Chem., 45, 4188–4198 (2010).
23) Kubicová L., Šustr M., Kráľová K., Chobot V., Vytlačilová J.,
Jahodář L., Vuorela P., Macháček M., Kaustová J., Molecules, 8,
756–769 (2003).
24) Skehan P., Storeng R., Scudiero D., Monks A., McMahon J., Vistica
D., Warren J., Bokesch H., Kenney S., Boyd M. R., J. Natl. Cancer
Inst., 82, 1107–1112 (1990).
34) Kenyon S. H., Waterfield C. J., Timbrell J. A., Nicolaou A.,
Biochem. Pharmacol., 63, 381–391 (2002).