Cytotoxicity and anti-HIV evaluations of some new synthesized quinazoline and thioxopyrimidine derivatives using 4-(thiophen-2-yl)-3,4,5,6- tetrahydrobenzo[h]quinazoline-2(1H)-thione as synthon

Article abstract of DOI:10.1007/s12039-012-0242-4

A series of dihydrobenzo[h]quinazoline derivatives 5-19 were synthesized using arylmethylene 2, thiopyrimidine 3 and 2-(4-(thiophen-2-yl)-5,6- dihydrobenzo[h]quinazolin-2-ylthio) acetic acid (4) as a starting materials. The biological screening showed that many of these compounds have good anticancer and antiviral activities. The structure assignments of the new compounds based on chemical and spectroscopic evidence. The detailed synthesis, spectroscopic data, and pharmacological properties are reported. Indian Academy of Sciences.

Full text of DOI:10.1007/s12039-012-0242-4

c
J. Chem. Sci. Vol. 124, No. 3, May 2012, pp. 693–702. ꢀ Indian Academy of Sciences.
Cytotoxicity and anti-HIV evaluations of some new synthesized
quinazoline and thioxopyrimidine derivatives using
4-(thiophen-2-yl)-3,4,5,6-tetrahydrobenzo[h]quinazoline-2(1H)-thione
as synthon
YAHIA A MOHAMEDa, ABD EL-GALIL E AMRb,c,∗, SALWA F MOHAMEDc,
MOHAMED M ABDALLAd, MOHAMED A AL-OMARe and SAMIRA H SHFIKc
aChemistry Department, Faculty of Science, El-Azhar University, Cairo 11787, Egypt
bDrug Exploration and Development Chair (DEDC), College of Pharmacy, King Saud University,
Riyadh 11451, Saudi Arabia
cApplied Organic Chemistry Department, National Research Centre, Dokki, Cairo 12622, Egypt
dResearch Unit, Saco Pharm. Co., 6th October City, Cairo 15324, Egypt
ePharmaceutical Chemistry Department, College of Pharmacy, King Saud University, Riyadh 11451,
Saudi Arabia
e-mail: aamr1963@yahoo.com
MS received 24 April 2011; revised 14 September 2011; accepted 22 November 2011
Abstract. A series of dihydrobenzo[h]quinazoline derivatives 5–19 were synthesized using arylmethylene 2,
thiopyrimidine 3 and 2-(4-(thiophen-2-yl)-5,6-dihydrobenzo[h]quinazolin-2-ylthio) acetic acid (4) as a starting
materials. The biological screening showed that many of these compounds have good anticancer and antiviral
activities. The structure assignments of the new compounds based on chemical and spectroscopic evidence.
The detailed synthesis, spectroscopic data, and pharmacological properties are reported.
Keywords. Arylmethylene; thiopyrimidine; dihydrobenzo[h]quinazolin-2-ylthioacetic acid; anticancer;
antiviral and cytotoxic evaluations.
1. Introduction
derivatives containing quinazoline moiety. Some of
the synthesized compounds were screened for their
anticancer and antiviral activities due to structure simi-
larity between our newly synthesized compounds and a
clinically used agents in these fields.
The pyrimidinoyl and thioxopyrimidinoyl groups are
of great importance in treating biological system.
Analgesic, anti-arrhythmic, anticancer, antipyretic
and anti-inflammatory activities are observed in some
pyrimidinoyl and thioxopyrimidinoyl derivatives.1–5
Also, in our previous work we prepared certain new
substituted heterocyclic derivatives which exhibit
2. Experimental
analgesic, anti-inflammatory, antiparkinsonian, and 2.1 Chemistry
androgenic-anabolic activities.6–11 In addition, the
Melting points were determined on open glass capil-
heterocyclic nitrogen derivatives exhibited a gene-
ral ionophoric potency for divalent cations12 and used
a novel thiocyanate-selective membrane sensor.13
Recently, anticancer and biological screening for some
of synthesized heterocyclic compounds was
reported.14–19 In view of these observations and in
continuation of our previous work in heterocyclic
chemistry, we have synthesized here of some new
laries using an Electrothermal IA 9000 digital melting
point apparatus. Elemental analyses were performed on
Elementar, Vario EL, Microanalytical Unit, National
Research Center, Cairo, Egypt and were found within
0.4% of the theoretical values. Infrared spectra were
recorded on Carlzeise Spectrophotometer model “UR
10” spectrophotometer using the KBr disc technique.
1H-NMR spectra were recorded on Varian Gemini
270 MHz spectrometer (DMSO-d6) and the chemical
shifts are given in δ (ppm) downfield from tetramethyl-
silane (TMS) as an internal standard. The mass spectra
∗For correspondence
693

694
Yahia A Mohamed et al.
were measured using a Finnigan SSQ 7000 mass spec- NMR (270 MHz, DMSO-d6): δ 1.91 (s, 3H, CH3), 2.94,
trometer. Follow up of the reactions and checking the 3.18 (2t, 4H, 2CH2 of quinazoline ring), 7.27–8.10
purity of the compounds was made by TLC on silica (m, 7H, Ar–H + thiophene-H), 10.38 (s, 1H, NH, D2O
gel-aluminum sheets (Type 60 F254, Merck, Darmstadt, exchangeable). MS m/z (%): 321 (M+, 100); Analysis
Germany).
for C18H15N3OS: Calcd. C, 67.27; H, 4.70; N, 13.07; S,
9.98. Found C, 67.23; H, 4.65; N, 13.03; S, 9.92.
2.1a 4-(Thiophen-2-yl)-5,6-dihydrobenzo[h]quinazo-
lin-2-amine (5): To a boiling solution of compound 2 2.1d N-(4-(Thiophen-2-yl)-5,6-dihydrobenzo[h]qui-
(2.4 g, 0.01 mol) in ethanolic potassium hydroxide nazolin-2-yl)methanesulphonamide (8): A mixture of
(100 ml, 1%), guanidine hydrochloride (0.95 g, compound 5 (2.79 g, 0.01 mol) and methylsulpho-
0.01 mol) was added. The reaction mixture was refluxed nylchloride (1.14 g, 0.01 mol) in dry pyridine (20 ml)
for 5 h, then allowed to cool and the solid formed was was heated at 70–75◦C on a waterbath for 4 h. After
filtered off, dried and crystallized to give compound cooling, the reaction mixture was acidified with diluted
5 in yield: 83%; m.p: 220◦C (EtOH); IR (KBr) νmax
:
hydrochloric acid and the obtained solid was filtered
3314, 3209 cm−1 (NH2). The IR spectrum is also devoid off, washed with hot water, dried and crystallized to
of the band corresponding to ν (C=O) present in the give compound 8 in yield: 67%; mp: 145◦C (EtOH);
1
IR spectrum of 2. H NMR (270 MHz, DMSO-d6): δ IR (KBr) νmax: 3344 cm−1 corresponding to ν(NH).
2.84, 2.97 (2t, 4H, 2CH2 of quinazoline ring), 6.56 (s, 1H NMR (270 MHz, DMSO-d6): δ 2.94–3.13 (2t, 4H,
2H, NH2, D2O exchangeable) 7.17–7.75 (m, 6H, Ar–H 2CH2 of quinazoline ring), 3.57 (s, 3H, CH3), 7.27–
+ thiophene-H), 8.12 (d, 1H, C5-thiophene). MS m/z 7.84 (m, 6H, Ar–H + thiophene), 8.15 (d, 1H, C5
(%): 281 (M++2, 100) corresponding to the molecular thiophene), 11.18 (s, 1H, NH, D2O exchangeable). MS
formula C16H13N3S. Analysis: Calcd. C, 68.79;H, 4.69; m/z (%): 357 (M+, 100) corresponding the molecular
N, 15.04; S, 11.48. Found C, 68.74;H, 4.65; N, 14.98; formula C17H15N3O2S2; Analysis: Calcd. C, 57.12; H,
S, 11.43.
4.23; N, 11.76; S,17.94. Found C, 57.06; H, 4.15; N,
11.70; S,17.88.
2.1b 4,5,6,7-Tetrachloro-2-(4-thiophen-2-yl-3,4,5,6-
tetrahydro-benzo[h]quinazolin-2-yl)-isoindole-1,3-dione 2.1e 4-(Thiophen-2-yl)-5,6-dihydrobenzo[h]quinazo-
(6): A mixture of 5 (2.79 g, 0.01 mol) and 3,4,5,6- line-2(1H)-thione (9): A mixture of compound 3
tetrachlorophthalic anhydride (2.85 g, 0.01 mol) was (2.98 g, 0.01 mol) and sodium hydride (0.8 g, 0.02 mol)
heated under fusion condition for 0.5 h., left to cool. in dimethylformamide (40 ml) was stirred at 70–80◦C
The residue was triturated with ethanol and the obtained for 1 h. then cooled to room temperature, chlorodiethyl
solid was filtered off, dried and crystallized to give ether (1 g, 0.01 mol) was added and heated at 90◦C
compound 6 in yield: 61%; m.p: 252◦C (AcOH); IR for 5 h. The reaction mixture was evaporated under
(KBr) νmax: 1737 cm−1 corresponding to ν (C=O).1H reduced pressure, the obtained residue was triturated
NMR (270 MHz, DMSO-d6): δ 3.00–3.10 (m, 4H, with water and the obtained solid was filtered off,
2CH2 of quinazoline), 7.30–8.20 (m, 11H, Ar–H + dried and crystallized to give compound 9 in yield:
thiophene-H). MS m/z (%): 547 (M+, 27) correspond- 72%; m.p: 220◦C (EtOH); IR (KBr) νmax: 3416 (NH),
1
ing to the molecular formula C24H11Cl4N3O2S and 1189 cm−1 (C=S); H NMR (270 MHz, DMSO-d6):
base peak at m/z 546 (100); Analysis: Calcd. C, 52.67; δ 2.70, 3.20 (2t, 4H, 2CH2 of quinazoline), 7.00–8.10
H, 2.03; Cl, 25.91; N, 7.68; S, 5.86. Found C, 52.61; (m, 7H, Ar–H + thiophene), 8.54 (s, 1H, NH, D2O
H,1.95; Cl, 25.86; N, 7.61; S, 5.78.
exchangeable). MS m/z (%): 296 (M+, 95) correspond-
ing the molecular formula C16H12N2S2 and base peak at
238 (100); Analysis: Calcd. C, 64.83; H, 4.08; N, 9.45;
S, 21.64.Found C, 64.76; H, 4.03; N, 9.39; S, 21.58.
2.1c N-(4-(Thiophen-2-yl)-5,6-dihydrobenzo[h]quina-
zolin-2-yl)acetamide (7): A solution of 5 (2.79 g,
0.01 mol) in acetic anhydride (10 ml) was heated
refluxed for 2 h. After cooling, the reaction mixture 2.1f 1-(4-(Thiophen-2-yl)-2-thioxo-1,2,5,6-tetrahydro-
was poured onto ice water. The separated solid was benzo[h]quinazolin-3(4H)-yl)ethanone (11): A mix-
collected by filtration, washed with water, dried and ture of compound 3 (2.98 g, 0.01 mol), acetylchloride
crystallized to give compound 7, in yield: 72%; mp: (0.78 g, 0.01 mol) in pyridine (30 ml) was refluxed for
115◦C (EtOH); IR (KBr) νmax: 3207 and 1661 cm−1 2 h. The reaction mixture was poured onto acidified
corresponding to ν(NH) and ν (C=O), respectively. 1H water (pH ∼ 3), the solid formed was filtered off,

Cytotoxicity and anti-HIV evaluations of some new synthesized quinazoline and thioxopyrimidine derivatives
695
washed with water, dried and crystallized to give com- H,4.55; N, 9.02; S, 20.66. Found C, 65.73; H,4.50; N,
pound 11 in yield: 60%; m.p: 195◦C (AcOH); IR (KBr) 8.96; S, 20.61.
νmax: 3257 and 1671 cm−1 corresponding to ν(NH)
and ν(C=O, acetyl), respectively. 1H NMR (270 MHz,
2.1i Ethyl 2-(4-(thiophen-2-yl)-5,6-dihydrobenzo[h]-
DMSO-d6): δ 2.30-2.90 (m, 7H, 2CH2+ CH3), 6.10 (s,
quinazolin-2-ylthio)acetate (14): A solution of com-
1H, CH of pyrimidine proton), 7.00–7.70 (m, 7H, Ar–
pound 4 (3.54 g; 0.01 mol) in ethanol (50 ml) contain-
H + thiophene-H), 11.80 (s, 1H, NH, D2O exchange-
ing few drops of sulphuric acid was refluxed for 4 h,
able). MS m/z (%): 340 (M+, 10) corresponding the
left to cool. The separated solid was filtered off, washed
molecular formula C18H16N2OS2 and base peak at 297
with water, dried and crystallized to give compound
(100); Analysis: Calcd. C, 63.50; H, 4.74; N, 8.23; S,
14 in yield: 73%; m.p: 92◦C (EtOH); IR (KBr) νmax
:
18.84. Found C, 63.46; H, 4.69; N, 8.17; S,18.68.
1744 cm−1 corresponding to ν(C=O, ester). H NMR
(270 MHz, CDCl3): δ 1.27 (t, 3H, CH3), 2.93, 3.18 (2t,
4H, 2CH2 of quinazoline ring), 4.00 (s, 2H, CH2), 4.16
(q, 2H, CH2), 6.00 (s, 1H, CH-pyrimidine), 7.15 (t, 1H,
Ar–H ), 7.25 (t, 1H, Ar–H ), 7.37 (m, 2H, Ar–H), 7.56
(m, 2H, Ar–H ), 8.30 (d, 1H, C5-thiophene). MS m/z
(%) 382 (M+, 25) corresponding the molecular formula
C20H18N2O2S2 and base peak at 309 (100). Analysis:
Calcd. C, 62.80; H, 4.74; N, 7.32; S, 16.77. Found C,
62.74; H, 4.69; N, 7.27; S,16.72.
1
2.1g S-[4-(Thiophen-2-yl)-3,4,5,6-tetrahydrobenzo[h]-
quinazolin-2-yl]-2-chloroethanethioate (12): A mix-
ture of 3 (2.98 g, 0.01 mol) and chloroacetylchloride
(1.12 g, 0.01 mol) in dry acetone (50 ml) in the pre-
sence of anhydrous K2CO3 (2.76 g, 0.02 mol) was
refluxed on a waterbath for 24 h. The reaction mixture
was evaporated under reduced pressure and the residue
was triturated with water. The separated solid was fil-
tered off, washed with water, dried and crystallized to
give compound 12 in yield: 71%; m.p: 175◦C (MeOH);
IR (KBr) νmax: 1721 cm−1 corresponding to ν(C=O). IR
spectrum taken as representative was devoid of bands
corresponding to ν(NH) and ν(C=S) present in spec-
trum of the parent compound 3.1H NMR (270 MHz,
DMSO-d6): δ 1.90, 2.70 (2t, 4H, 2CH2 of quinazoline
ring), 4.10 (s, 2H, CH2), 6.00 (s, 1H, CH of pyrimidine
proton), 6.90–7.80 (m, 7 H, Ar–H + thiophene-H) and
12.00 (s, 1H, NH, D2O exchangeable). MS m/z (%):
374 (M+, 15) corresponding the molecular formula
C18H15ClN2OS2 and base peak at 372 (100); Analysis:
Calcd. C, 57.67; H, 4.05; Cl, 9.46; N, 7.47; S, 17.11.
Found C, 57.63; H,3.98; Cl, 9.41; N, 7.43; S, 17.06.
2.1j 2-(4-(Thiophen-2-yl)-5,6-dihydrobenzo[h]quina-
zolin-2-ylthio)acetohydrazide (15): A mixture of 14
(3.82 g, 0.01 mol) and hydrazine hydrate (3.1 ml 80%,
0.05 mol) in absolute ethanol (20 ml) was stirred for 6 h.
The obtained solid was filtered off and crystallized to
give compound 15 in yield: 69%; m.p: 180◦C (EtOH);
IR (KBr) νmax: 3269, 3154 cm−1 (NH, NH2), 1653 cm−1
1
(C=O). H NMR (270 MHz, DMSO-d6): δ 2.93, 3.16
(2t, 4H, 2CH2 of quinazoline ring), 3.91 (s, 2H, CH2),
4.26 (s, 2H, NH2, D2O exchangeable), 7.26–7.87 (m,
6H, Ar–H + thiophene-H), 8.20 (d, 1H, C5-thiophene)
9.31 (s, 1H, NH, D2O exchangeable). MS m/z (%):
368 (M+, 30) corresponding to the molecular formula
C18H16N4OS2 with base peak at 309. Analysis: Calcd.
C, 58.67; H, 4.38; N, 15.21; S, 17.40. Found C, 58.61;
H, 4.32; N, 15.15; S,17.32.
2.1h 2-(Methylthio)-4-(thiophen-2-yl)-5,6-dihydroben-
zo[h]quinazoline (13): A mixture of compound 3
(2.98 g, 0.01 mol) and iodomethan (1.41 g, 0.01 mol) in
ethanolic solution of sodium ethoxide (prepared from
sodium metal 0.23 g, 10 mg-atom) in 20 ml ethanol was
heated under reflux for 2 h. After acidification with
acetic acid, the formed solid was collected by filtration
and crystallized to give compound 13 in yield: 66%;
m.p: 125◦C (MeOH); IR (KBr) νmax: 1592 cm−1 cor-
responding to ν(C=N). The spectrum is also devoid
of the bands corresponding to ν(NH) and ν(C=S)
present in the IR spectrum of 3.1H NMR (270 MHz,
CDCl3): δ 2.60 (s, 3H, SCH3), 2.94, 3.19 (2t, 4H, 2CH2
2.1k Synthesis of compounds (16) and (17): A mix-
ture of compound 15 (3.7 g, 0.01 mol) and phthalic
anhydride or 3,4,5,6-tetrachlorophthalic anhydride
(0.01 mol) in glacial acetic acid (50 ml) was heated
under reflux for 6 h. The reaction mixture was evapo-
rated under reduced pressure; the obtained residue was
solidified with ether, filtered off, and crystallized from
the proper solvent to give 16 and 17, respectively.
of quinazoline ring) and 7.10–8.30 (m, 6H, Ar–H + 2.1l
N-(1,3-Dioxoisoindolin-2-yl)-2-(4-(thiophen-2-
thiophene-H), 8.38 (d, 1H, C5 thiophene). MS m/z (%): yl)-5,6-dihydrobenzo[h]quinazolin-2-ylthio)-acetamide
310 (M+, 100, base peak) corresponding to the molec- 16: Yield 74%; mp. 190◦C (AcOH/H2O); IR (KBr)
ular formula C17H14N2S2. Analysis: Calcd. C, 65.77; νmax: IR spectrum of compound 16 demonstrated the

696
Yahia A Mohamed et al.
presence of the bands at: 3268 cm−1 corresponding to 2H, CH2), 7.24–8.25 (m, 12H, Ar–H + thiophene-
ν(NH), 1752, 1672 cm−1 corresponding to ν(C=O). H + CH=N), 11.62 (1s, 1H, NH). Analysis for
1H NMR (270 MHz, DMSO-d6): δ 2.90–3.20 (m, 4H, C25H19FN4OS2 (474.57): Calcd.: C. 63.27; H, 4.04; N,
2CH2 of quinazoline ring), 4.01 (s, 2H, CH2), 7.23– 11.81; S, 13.51. Found: C, 63.21; H, 3.97; N, 11.74; S,
8.40 (m, 11H, Ar–H + thiophene-H) 9.85 (s, 1H, NH). 13.47.
MS m/z (%): 495 (M+-3; 1) corresponding to molecu-
lar formula C26H18N4O3S2, a base peaks at m/z 309
(100). Analysis: Calcd. C. 62.63; H, 3.64; N, 11.24; S,
2.2 Biological screening in vitro cytotoxicity
12.86. Found: C, 62.57; H, 3.57; N, 11.18; S, 12.80.
The cytotoxicity of the newly synthesized compounds
against cancer cell lines in vitro was performed
with the MTT assay according to the Mosmann’s
2.1m
N-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2-
method.20 The MTT assay is based on the reduction
of the soluble 3-(4,5-methyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide (MTT) into a blue-purple for-
mazan product, mainly by mitochondrial reductase
activity inside living cells. The cells used in cytotoxi-
city assay were cultured in RPMI 1640 medium supple-
mented with 10% fetal calf serum. Cells suspended in
the medium (2 × 104/mL) were plated in 96-well cul-
ture plates and incubated at 37◦C in a 5% CO2 incuba-
tor. After 12 h, the test sample (2 μL) was added to the
cells (2Ý 104) in 96-well plates and cultured at 37◦C
for 3 days.
The cultured cells were mixed with 20 μL of MTT
solution and incubated for 4 h at 37◦C. The supernatant
was carefully removed from each well and 100 μL
of DMSO were added to each well to dissolve the
formazan crystals which were formed by the cellular
reduction of MTT. After mixing with a mechanical plate
mixer, the absorbance of each well was measured by
a microplate reader using a test wavelength of 570 nm.
The results were expressed as the IC50, which inducing
a 50% inhibition of cell growth of treated cells when
compared to the growth of control cells. Each experi-
ment was performed at least 3 times. There was a good
reproducibility between replicate wells with standard
errors below 10%.
yl)-2-(4-(thiophen-2-yl)-5,6-dihydrobenzo[h]-quinazolin-
2-ylthio)acetamide 17: Yield 78%; mp. 150◦C
(AcOH/H2O); IR (KBr) νmax: spectrum of com-
pound 17 demonstrated the presence of the bands at:
3168 cm−1 corresponding to ν(NH), 1747, 1713 cm−1
corresponding to ν (C=O). MS m/z (%): 637
(M++1, 2), corresponding to molecular formula
C26H14Cl4N4O3S2 with base peak at: m/z 214 (100).
Analysis: Calcd.: C. 49.07; H, 2.22; N, 8.80; S, 10.08.
Found: C, 49,01; H, 2.16; N, 8.74; S, 10.01.
2.1n Synthesis of compounds (18) and (19): A
mixture of the acid hydrazide 15 (3.82 g, 0.01 mol)
and phenylisothiocyanate or p-fluorobenzaldehyde
(0.01 mol) in absolute ethanol (50 ml) was refluxed for
3–7 h. After cooling, the separated solid was filtered off
and crystallized from the proper solvent to afford the
corresponding compounds 18 and 19, respectively.
2.1o N-phenyl-2-(2-(4-(thiophen-2-yl)-5,6-dihydroben-
zo[h]quinazolin-2-ylthio)acetyl)hydrazin0-carbothio-
amide 18: Yield 60%; mp. 195◦C (EtOH); IR (KBr)
νmax: spectrum of compound 18 showed absorption
bands at 3347, 3266 cm−1 corresponding to ν(NH),
1680 cm−1 corresponding to ν(C=O). 1H NMR
(270 MHz, DMSO-d6): δ 2.90, 3.12 (2t, 4H, 2CH2
quinazoline), 4.09 (s, 2H, CH2), 7.23–7.85 (m, 10
H, Ar–H + thiophene-H) 8.28(d, 1H, C5 thiophene)
9.29, 9.66 and 10.32 (3s, 3H, 3NH). Analysis for
C25H21N5OS3 (503.66): Calcd.: C. 59.62; H, 4.20; N,
13.09; S, 19.10. Found: C, 59.55; H, 4.15; N, 13.04; S,
19.05.
2.2a HIV inhibitor activities (reverse transcrip-
tase inhibitors) with therapeutic windows (cells and
viruses): The established human cells, laboratory-
derived virus isolates (including drug-resistant virus
isolates), and low-passage clinical virus isolates used
in these evaluations have previously been described in
detail.21 These cells were maintained in RPMI 1640
medium supplemented with 10% fetal bovine serum,
2 mM glutamine, penicillin (100 U/ml), and strepto-
mycin (100 μg/ml). Fresh human cells were obtained
from the American Red Cross (Baltimore, Md.).
2.1p N’-(4-fluorobenzylidene)-2-(4-(thiophen-2-yl)-
5,6-dihydrobenzo[h]quinazolin-2-ylthio)aceto-hydra-
zide 19: Yield 62%; mp. 225◦C (EtOH); IR (KBr)
νmax: spectrum of compound 19 showed absorption
bands at: 3247, 1662 cm−1 corresponding to ν(NH) and
1
(C=O), respectively. H NMR (270 MHz, DMSO-d6): 2.2b Antiviral and cross-resistance assays: The
δ 2.82, 3.00 (2t, 4H, 2CH2 of quinazoline), 4.01 (s, inhibitory activities of the compounds against HIV were

Cytotoxicity and anti-HIV evaluations of some new synthesized quinazoline and thioxopyrimidine derivatives
697
evaluated by microtiter anti-HIV assays with CEM- containing 10% heat-inactivated fetal bovine serum
SS cells or fresh human peripheral blood mononuclear (FBS), 2 mM L-glutamine, and non-essential amino
cells (PBMCs); these assays quantify the ability of a acids. Stable Huh-7 cells containing the self-replicating,
compound to inhibit HIV-induced cell killing or HIV sub-genomic HCV replicon, which was identical in
replication. Quantification was performed by the tetra- sequence to the I377neo/NS3-3ꢁ/wt replicon described
zolium dye XTT assay (CEM-SS, 174×CEM, MT2, by Lohmann et al.23 were selected and maintained in
and AA5 cell-based assays), which is metabolized to the presence of 0.25 mg/ml G418 (Invitrogen, Carlsbad,
a coloured formazan product by viable cells, RT assay CA) and were used for anti-HCV assays. Peripheral
(U937- and PBMC-based assays), and/or p24 enzyme- blood mononuclear cells (PBMC) were isolated from
linked immunosorbent assay (monocyte-macrophage fresh donor blood and cultured in RPMI-1640 medium
assays). Antiviral and toxicity data are reported as the (JRH Biosciences).
quantity of drug required for inhibiting virus-induced
cell killing or virus production by 50% (EC50).
2.2e Determination of anti-HCV activity and cyto-
toxicity: Determination of 50% inhibitory concentra-
tion (IC50), 90% inhibitory concentration (IC90), and
2.2c In vitro assays of anti-HIV activity: Purified RT
assays of the newly synthesized compounds were tested
50% cytotoxic concentration (CC50) of all the tested
for RT inhibitory activity against purified recombinant
compounds in HCV replicon cells was performed as
HIV-1 RT using the cell-free Quan-T-RT assay system
described . Pre-clinical profile of VX-950, a potent,
(Amersham Corp., Arlington Heights, IL), which uti-
selective, and orally bioavailable inhibitor of hepatitis C
lizes the scintillation proximity assay (SPA) principle,
virus (NS3-4A serine protease). Briefly, 1 × 104 repli-
as described in detail.22
con cells per well were plated in 96-well plates. On the
In the assay, a DNA/RNA template is bound to SPA
following day, replicon cells were incubated at 37◦C for
beads via a iotin/streptavidin linkage. The primer DNA
the indicated period of time with antiviral agents seri-
is a 16-mer oligo(T), which has been annealed to a
ally diluted in DMEM plus 2% FBS and 0.5% dimethyl-
poly(rA) template. The primer-template is bound to
sulphoxide (DMSO). Total cellular RNA was extracted
a streptavidin-coated SPA bead. [3H]TTP (thymidine
using an RNeasy-96 kit (QIAGEN, Valencia, CA), and
-5’- triphosphate) is incorporated into the primer by
the copy number of HCV RNA was determined using
reverse transcription. In brief, [3H]TTP, at a final con-
a quantitative RT-PCR (QRT-PCR) assay. Combination
centration of 0.5 μCi/sample, was diluted in RT assay
of a hepatitis C virus NS3-NS4A protease inhibitor
buffer (49.5 mM Tris-HCl, pH 8.0, 80 mM KCl, 10 mM
and alpha interferon synergistically inhibits viral RNA
MgCl2, 10 mM dithiothreitol, 2.5 mM EDTA, 0.05%
replication and facilitates viral RNA clearance in repli-
Nonidet P-40) and added to annealed DNA/RNA bound
con cells. Each datum point represents the average of
to SPA beads. The compound being tested was added to
five replicates in cell culture. The cytotoxicity of the
the reaction mixture at 0.001–100 μM concentrations.
tested compounds was measured under the same exper-
Addition of 10 mU of recombinant HIV RT and incu-
imental settings using a tetrazolium (MTS)-based cell
bation at 37◦C for 1 h resulted in the extension of the
viability assay. For the cytotoxicity assay with human
primer by incorporation of [3H]TTP. The reaction was
hepatocyte cell lines, 1 × 104 parental Huh-7 cells per
stopped by addition of 0.2 ml of 120 mM EDTA. The
samples were counted in an open window using a Beck-
man LS 7600 instrument and IC50[RT] values (concentra-
tion at which the compound inhibits recombinant RT by
well or 4 × 104 HepG2 cells per well were used. To
determine cytotoxicity of the tested compound against
resting PBMC, 1 × 105 cells per well were incubated
with the tested compound in RPMI-1640 medium (no
50%) were calculated by comparing the measurements
serum) for 48 h, and the cell viability was determined by
to an untreated.
the MTS-based assay. To determine cytotoxicity of the
tested compound against proliferating PBMC, 1 × 105
2.2d Hepatitis C virus (HCV) NS3-4A protease cells per well in RPMI-1640 medium were added to a
inhibitor activities: The success of human immun- 96-well plate, which was pre-coated with anti-human
odeficiency virus (HIV) protease inhibitors in treating CD3 antibody (Accurate Chemical and Scientific Cor-
HIV-infected patients has raised the hope that inhibitors poration, Westbury, NY). The cells were incubated
of HCV NS3-4A serine protease could also become with the tested compound and anti-human CD28 anti-
effective therapy options for hepatitis C patients.
body (Pharmingen/BD Biosciences, San Jose, CA) for
Parental Huh-7 and HepG2 cells were cultured 72 h at 37◦C, and the cell growth was determined by
in Dulbecco’s modified Eagle’s medium (DMEM) [3H]thymidine update between the 48th and 72nd h.

698
Yahia A Mohamed et al.
with 3,4,5,6-tetrachlorophthalic anhydride under fusion
O
O
S
C
S
CHO
S
H2N
NH2
to yield 4,5,6,7-tetrachloro-2-(4-thiophen-2-yl-3,4,5,6-
tetrahydrobenzo[h]quinazolin-2-yl)isoindole-1,3-dione
(6). Also, amino-pyrimidine 5 was treated with acetic
anhydride or methyl sulphonylchloride to afford the
corresponding N-(4-thiophen-2-yl-5,6-dihydrobenzo-
[h]quinazolin-2-yl)-acetamide (7) and N-(4-thiophen-
2-yl-5,6-dihydro-benzo[h]quinazolin-2-yl)methanesul-
phonamide (8), respectively (scheme 2).
EtOH/KOH
EtOH/KOH
1
2
H
N
S
N
N
S
ClCH2COOH
EtOH/KOH
NH
COOH
S
In addition, thioxopyrimidine 3 was reacted with
chlorodiethyl ether in the presence of sodium hydride
afforded 4-thiophen-2-yl-5,6-dihydro-1H-benzo[h]qui-
nazoline-2-thione (9) rather than the S-alkylated form
10. 2-Thiopyrimidine 3 underwent alkylation giving S-
alkylation or N-alkylation depending on the react-
ion conditions. So, when compound 3 was refluxed
in acetylchloride or chloro-acetylchloride afforded
the corresponding 1-(4-thiophen-2-yl-2-thioxo-1,4,5,6-
tetrahydro-2H-benzo[h]-quinazolin-3-yl)ethanone (11)
and S-[4-(thiophen-2-yl)-3,4,5,6-tetrahydrobenzo[h]-
quinazolin-2-yl] 2-chloroethanethioate (12), respec-
tively. On the other hand, compound 3 was reacted with
methyl iodide to afford 2-methylsulfanyl-4-thiophen-
2-yl-5,6-dihydrobenzo[h]quinazoline (13) (scheme 3).
On the other hand, When esterification of the car-
boxylic acid derivative 4 with refluxed ethanol in the
presence of concentrated sulphuric acid afforded ethyl
2-(4-(thiophen-2-yl)-5,6-dihydrobenzo[h]-quinazolin-2-
ylthio)acetate (14), which was treated with hydrazine
hydrate in ethanol to produce (4-thiophen-2-yl-3,4,5,
S
3
4
Scheme 1.
3. Results and discussion
3.1 Chemistry
In our previous work,6–19 we reported the synthe-
sis and a preliminary biological activity screening
of several thiopyrimidine derivatives based on aryl-
methylene 2, 4-thiophen-2-yl-3,4,5,6-tetrahydro-1H-
benzo[h]quinazoline-2-thione (3) and 2-(4-(thiophen-
2-yl)-5,6-dihydrobenzo[h]quinazolin-2-ylthio) acetic
acid (4), which were synthesized according to the
literature procedures24 (scheme 1).
Arylmethylene 2 was allowed to react with guani-
dine hydrochloride in ethanolic potassium hydroxide to
yield the corresponding 4-thiophen-2-yl-5,6-dihydro-
benzo[h]quinazolin-2-ylamine (5), which was reacted
Cl
Cl
Cl
O
O
Cl
Cl
Cl
Cl
O
N
N
O
Cl
N
S
O
fusion
6
H
N
N
CH3
NH
N
NH2
Ac2O
reflux
H2N
C NH2.HCl
N
S
O
2
N
S
EtOH/KOH
7
5
O
H
N
N
S
CH3
O
CH3SO2Cl
N
S
dry pyridine
8
Scheme 2.

Cytotoxicity and anti-HIV evaluations of some new synthesized quinazoline and thioxopyrimidine derivatives
699
6-tetrahydrobenzo[h] quinazolin-2-ylsulfanyl)-acetohy-
drazide 15. The reaction of the hydrazide 15 with
phthalic anhydride or 3,4,5,6-tetrachlorophthalic anhy-
dride in glacial acetic acid furnished pyrrolobenzene
derivatives 16 and 17, respectively. One the other hand,
treatment of hydrazide 15 with phenyl isothiocyanate or
p-florobenzaldehyde in ethanol under reflux afforded
thiosemicarbazide 18 and hydrazone 19, respectively
(scheme 4).
H
N
S
N
S
O
NH
N
S
S
10
9
NaH
ClCH2CH2OC2H5
3.2 Biological activity
H
H
N
Some of the newly synthesized compounds were tested
for their cytotoxicity on different cancer cell lines and
virus. The obtained results were summarized in the
following tables 1–4.
N
S
S
CH3COCl
NH
N
S
CH3
O
S
3
11
3.2a Anticancer activity: Regarding the anticancer
activity careful examining of the data obtained from
table 1, compounds 2, 3, 4, 6, 9, 11, 12, 13, 14, 15,
16 and 19 showed potent cytotoxic activity against
Leukemia (HL60, U937 and K562), melanoma (G361
and SK-MEL-28), neuroblast (GOTO and NB-1) and
normal cell carcinoma (W138) cell lines. The most
active compounds are 2, 6, 9, 13, 15 and 19. It is worth
to mention that most of the tested compounds showed
potent activities where they have minimal IC50. From
table 2, the same compounds showed cytotoxic activity
against cervical carcinoma (KB), CNS (SF-268), colon,
CH3I
NaOEt
ClCH2COCl
K2CO3
N
S
N
SCH3
Cl
NH
S
O
N
S
12
13
Scheme 3.
O
N
S
N
S
NHNH2
N2H4.H2O
EtOH
EtOH
N
S
N
COOEt
4
H2SO4
S
15
14
or
PhN=C=S
EtOH
F
CHO
X
X
O
O
O
X
X
AcOH
O
X
O
N N
O
X
X
N
N
S
N
S
NH
R
H
N
S
O
X
S
16, X= H
17, X= Cl
18, R= -NH-CSNHPh
19, R= -N=CH-C6H4F-p
Scheme 4.

700
Yahia A Mohamed et al.
Table 1. The activities of the tested compounds on some cancer cell lines.
IC50
Normal cell
carcinoma
Leukaemia
U937
Melanoma
SK-MEL-28
Neuroblast
Product
HL60
K562
G361
GOTO
NB-1
W138
2
5.4
E-03
3.5
4.4
E-03
6.4
1.4
E-03
6.4
1.5
E-03
6.8
5.2
E-03
7.9
7.5
E-03
9.7
4.7
E-03
9.7
6.4
E-03
9.7
3
E-03
8.3
E-03
8.9
E-03
8.7
E-03
9.0
E-03
7.2
E-03
8.7
E-03
8.8
E-03
7.9
E-03
7.7
E-03
9.8
E-03
6.6
E-03
8.7
E-03
8.5
E-03
4.3
E-03
9.8
E-03
-
4
6
E-03
2.4
E-03
6.4
E-03
7.8
E-03
8.8
E-03
6.7
E-03
6.4
E-03
7.8
E-03
5.3
E-03
3.5
E-03
6.4
E-03
9.8
E-03
8.9
E-03
9.8
E-03
7.7
E-03
9.8
E-03
5.3
E-03
7.4
E-03
5.7
E-03
8.0
E-03
7.0
E-03
9.8
E-03
7.9
E-03
7.6
E-03
3.5
E-03
7.3
E-03
9.7
E-03
9.7
E-03
7.8
E-03
7.6
E-03
4.0
E-03
7.5
E-03
3.5
E-03
4.2
E-03
6.5
E-03
9.7
E-03
8.0
E-03
9.0
E-03
2.8
E-03
8.3
E-03
5.1
E-03
4.7
E-03
4.5
E-03
9.7
E-03
7.6
E-03
9.8
E-03
9.2
E-03
6.4
E-03
3.2
E-03
3.1
E-03
8.5
E-03
6.8
E-03
3.2
E-03
7.6
E-03
8.7
E-03
9.6
E-03
6.8
9
8.4
E-03
6.8
E-03
9.7
E-03
8.7
E-03
7.6
E-03
1.4
E-03
9.0
E-03
7.9
11
12
13
14
15
16
19
E-03
E-03
E-03
E-03
E-03
E-03
E-03
E-03
adenocarcinoma (RKOP27), ovarial carcinoma (SKOV- 3.2b Anti-HIV activity: Regarding the anti-HIV
3) and non-small-cell lung cancer (NCI H-460) can- activities, compounds 2, 3, 4, 6, 9, 11, 12, 13, 14, 15,
cer cell lines. The most active compounds are 2, 4 and 16 and 19 showed potent anti-HIV activities and the
11. Also the tested compounds showed potent activities descending order of the activity is 6, 13, 3, 12, 19,
where they have minimal IC50.
14, 19, 15, 2, 9, 16, 4 and 11 (table 3). Concerning
the mechanism of action as anti-HIV, these compounds
were reverse transcriptase inhibitors.
Table 2. Cytotoxicity of some new compounds on some
new cancer cell lines.
Table 3. HIV inhibitor activities (reverse transcriptase
inhibitors) with therapeutic windows.
IC50
Product
EC50/Mm
IC50/μM
Therapeutic index
Product
KB
SK OV-3 SF-268 NCl H460 RKOP27
2
3
4
6
0.00525
0.00722
6.1
2.6
17.4
2.11
8.1
22.6
2.9
2.33
4.4
33000
32122
2
3
4
6
4.9 E-03 5.9 E-03 3.0 E-03 2.4 E-03 3.3 E-03
5.6 E-03 5.4 E-03 6.7 E-03 7.6 E-03 9.8 E-03
3.9 E-03 4.7 E-03 5.8 E-03 5.9 E-03 5.0 E-03
9.0 E-03 9.0 E-03 6.0 E-03 9.6 E-03 9.5 E-03
6.5 E-03 9.8 E-03 9.4 E-03 4.9 E-03 6.5 E-03
3.0 E-03 4.0 E-03 4.0 E-03 4.8 E-03 4.9 E-03
7.8 E-03 9.8 E-03 7.6 E-03 6.6 E-03 8.7 E-03
8.8 E-03 6.9 E-03 5.0 E-03 6.0 E-03 6.0 E-03
7.8 E-03 5.4 E-03 5.4 E-03 6.7 E-03 9.0 E-03
4.3 E-03 9.8 E-03 5.6 E-03 7.6 E-03 9.8 E-03
7.8 E-03 9.4 E-03 7.8 E-03 6.7 E-03 9.0 E-03
4.9 E-03 9.8 E-03 7.6 E-03 6.5 E-03 8.7 E-03
0,00084
784,532
1.89 × 105
44432
2.44 × 10−4
9
0.00778
9
11
12
13
14
15
16
19
5.44 × 10−4
3.71 × 10−4
6.24 × 10−5
10−4
8.34 × 105
3.66 × 105
2.15 × 106
2.33 × 105
92160
11
12
13
14
15
16
19
0.00871
2.3
0.00691
5.6
16.2
3.4
40
103654

Cytotoxicity and anti-HIV evaluations of some new synthesized quinazoline and thioxopyrimidine derivatives
701
Table 4. Hepatitis C virus (HCV) NS3-4A protease inhibitor activities.
IC50 ( SD)μM
IC90 ( SD)μM
Window
index
Product
after 48 h
after 48 h
CC50 ( SD)μM
2
4
11
14
16
0.318 0.042
0.188 0.022
0.288 0.031
0.244 0.035
0.145 0.020
2.78 0.077
0.501 0.033
1.89 0.066
0.897 0.053
0.301 0.021
2738 8.5
516 4.5
1788 6.5
716 6.8
215 3.2
2201.450
2723.450
2399.600
2578.500
2829.540
3.2c Anti-HCV activity: Regarding the anti-HCV a starting materials. The biological screening showed
activities, compounds 2, 4, 11, 14, and 16 showed that many of these compounds have good anticancer
potent anti-HCV activities and the descending order of and antiviral activities.
the activity is 16, 4, 14, 11 and 2 (table 4). Concerning
the mechanism of action as anti-HCV, these compounds
Acknowledgements
were HCV NS3-4A protease inhibitor activities.
The Authors thank the Deanship of Scientific Research
at King Saud University for funding the work through
3.3 Structure activity relationship (SAR)
the research group project No. RGP-VPP-099.
•
From tables 1 and 2, all the tested compounds
showed activity against leukemia (HL60, U937 and
K562), melanoma (G361 and SK-MEL-28), neu-
roblast (GOTO and NB-1), normal cell carcinoma
(W138), cervical carcinoma (KB), CNS (SF-268),
colon adenocarcinoma (RKOP27), ovarial carcinoma
(SKOV-3) and non-small-cell lung cancer (NCI H-
460) cancer cell lines. In addition, the presence
of pyrimidine ring fused with tetrahydronaphthaline
and carboxylic acid as substituted due to the increase
the anticancer activity.
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•
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•
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•
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