Synthesis and inhibitory activity against Epstein-Barr virus of some new 1,2,3,4-tetrahydropyrimidine-2-thiones

Article abstract of DOI:10.1080/10426500802043152

1,2,3,4-Tetrahydropyrimidine-2-thiones 4a-n were synthesized through the reaction of aromatic aldehydes 1a-n, ethyl acetoacetate (2) and thiourea (3). The structures of all newly synthesized heterocyclic compounds elucidated by the use of IR, 1H NMR, mass

Full text of DOI:10.1080/10426500802043152

Phosphorus, Sulfur, and Silicon, 183:2956–2967, 2008
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500802043152
Synthesis and Inhibitory Activity Against Epstein-Barr
Virus of Some New 1,2,3,4-Tetrahydropyrimidine-2-
thiones
Fawzy A. Attaby,1 Mostafa M. Ramla,2 and T. Harukuni3
1Chemistry Department, Faculty of Science, Cairo University, Giza,
Egypt
2Chemistry of Natural and Microbial Products Department, National
Research Center, Dokki, Giza, Egypt
3Molecular Biochemistry Department, Kyoto Prefectural University of
Medicine, Kawarmachi-dori, Kamigyo-Ku, Kyoto, Japan
1,2,3,4-Tetrahydropyrimidine-2-thiones 4a–n were synthesized through the reac-
tion of aromatic aldehydes 1a–n, ethyl acetoacetate (2) and thiourea (3). The struc-
tures of all newly synthesized heterocyclic compounds elucidated by the use of IR,
1H NMR, mass spectra, and elemental analyses. The inhibitory activity against the
Epstein-Barr Virus early antigen (EBA-VA) of all newly synthesized heterocyclic
compounds were evaluated.
Keywords 1,2,3,4-tetrahydropyrimidinethione; antigen; ethylaceto-acetate; Epstein-
Barr Virus; inhibitory activity; and thiourea
INTRODUCTION
The reported biological activities of polysubstituted pyrimidine for the
treatment of hypoxemia,1 neuronosis,2 neuropathy,3 as well as their
antitumor effect,4 and their physiological activity5 stimulated our in-
terest to synthesize several derivatives of these ring systems, and to
develop derivatives to investigate in our medicinal chemistry program.
In the last two decades, uracil, and oxopyrimidine derivatives have
been investigated extensively in relation to their antiviral and anti-
tumoral properties6; but the pyrimidinethiones have less attention in
Received 26 December 2007; accepted 10 March 2008.
The authors are grateful to Molecular Biochemistry Department, Kyoto Prefectural
University of Medicine, Kawarmachi-dori, Kamigyo-Ku, Kyoto, Japan for biological
evaluation.
Address correspondence to Fawzy A. Attaby, Faculty of Science, Chemistry Depart-
ment, Cairo University, Giza 12613, Egypt. E-mail: fattaby@hotmail.com
2956

1,2,3,4-Tetrahydrpyrimidinethiones and Inhibitory Activity
2957
spite of their synthetic potentiality,7−11 so we report here a method to
prepare a new derivatives of 1,2,3,4-tetrahydrpyrimidinethiones and
evaluate their inhibitory activity against the Epstein-Barr Virus early
antigen (EBA-VA).
RESULTS AND DISCUSSION
Chemistry
Although several publications12−15 have been appeared concerning the
synthesis of tetrahydropyrimidinethiones by direct Biginelli conden-
sation using ethyl acetoacetate as β-ketoester component of classical
Biginelli reaction12 with aromatic aldehydes and thiourea, no approach
using heterocyclic aldehydes has been reported. Thus, it has been found
that a ternary mixture of heterocyclic aldehydes 1a–e, ethyl acetoac-
etate 2 and thiourea 3 in absolute ethanol containing a catalytic amount
of concentrated hydrochloric acid gave reaction products 4a–e whose
structures were elucidated by considering the data of elemental analy-
ses, IR, 1H NMR, as well as mass spectra (cf. Experimental section and
Scheme 1).
The formation of 4a–e seemed to proceed via either reaction of
N,Nꢀ-(2-arylmethylene)bis(thiourea) 5, formed through the reaction of
thiourea (3) and heterocyclic aldehydes 1a–e, with ethyl acetoacetate
(2) or ethyl 3-(thiocarbamoylamino)but-2-enoate 6, formed through the
CH3
EtOOC
Ethanol
HCl
NH
CHO
CH3COCH2COOEt
Ar
NH2CSNH2
+
+
Ar
1a-n
2
N
H
S
3
R
4a-n
1, 4a, Ar=indol-3-yl; R=H
b, Ar=Pyridin-3-yl; R=H
1, 4h, Ar=2,5-Dimethoxyphenyl; R=H
1, 4i, Ar=4-Florophenyl; R=H
1, 4j, Ar=4-Methylphenyl; R=H
1, 4k, Ar=3-Nitrophenyl; R=H
1, 4l, Ar=2-Florophenyl; R=H
c, Ar=1,3-Benzodioxol-5-yl; R=H
d, Ar=5-Methyl-2-furyl; R=H
e, Ar=2-Thienyl; R=H
f, Ar=5-Methyl-2-thienyl; R=CH3
g, Ar=2,4-Dimethoxyphenyl; R=H
SCHEME 1
1, 4m, Ar=2-Hydroxyphenyl; R=H
1, 4n, Ar=4-Hydroxyphenyl; R=H

2958
F. A. Attaby et al.
Ar CHO
NH2CSNH2
+
3
1a-n
S
Ar
S
2
H2N
NH
NH
NH2
CH3
5
EtOOC
NH
Ar
N
H
S
COOEt
CH3
4a-n
S
H2N
NH
1a-n
6
CH3COCH2COOEt
NH2CSNH2
+
2
3
SCHEME 2
reaction of thiourea (3) and ethyl acetoacetate (2), with aldehydes 1a–e
(cf. Scheme 2).11–15
Surprisingly, 1-(thiophen-2-yl)ethanone (1f) reacted with thiourea
(3) and ethyl acetoacetate (2) under the same experimental condi-
1
tions to afford a reaction product 4f. The H NMR spectrum of this
reaction product revealed the signals of thiophene, CH3 at 5-position
of thiophene in addition to that of COOCH2CH3 protons (cf. Exper-
imental section). Moreover, its mass spectrum gave parent peak at
m/z = 296 that corresponded to the molecular weight of the molec-
ular formula C13H16N2O2S2of the assigned structure 4f and peak
at m/z = 281, which corresponded to the fragment at M-15 (loss of
CH3 at 6-position) and this confirm that the reaction proceeded by
the same above-mentioned mechanism (cf. Scheme 2). Considering

1,2,3,4-Tetrahydrpyrimidinethiones and Inhibitory Activity
2959
CH3
NH
EtOOC
S
S
O
NH2CSNH2
CH COCH COOEt
+
3
2
+
N
H
S
CH3
H3C
1f
2
3
4f
SCHEME 3 ethyl 4,6-dimethyl-4-(2-thienyl)-22thioxo-1,2,3,4-tetrahydropy-
rimidine-5-carboxylate
the above-mentioned data in addition to that of elemental analyses
4f could be formulated as ethyl 4,6-dimethyl-4-(2-thienyl)-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (cf. Scheme 3).
Similarly, aromatic aldehydes 1g–n reacted with ethyl acetoacetate
(2) and thiourea (3) as a ternary mixture in ethanolic hydrochloric
acid to give the corresponding tetrahydropyridinethione derivatives
4g–n, respectively. The structures of these reaction products were elu-
1
cidated by considering the data of elemental analyses, IR, H NMR,
and mass spectra (cf. Experimental section). It is important to refer
that the structures 4g–n formed through the same mechanistic path-
way (cf. Scheme 2).
Pharmacological Activity
The present study aimed to synthesize a new series of tetrahydropy-
rimidinethione having heterocyclic or substituted benzene ring directly
linked to the 4-position of the pyrimidine moiety. We have studied the
inhibitory activity of the newly synthesized heterocyclic compounds
against the Epstein-Barr Virus early antigen (EBA-VA) activation in-
troduced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and a struc-
ture activity relationship was deduced.
Compounds 4c, 4d, 4g, and 4h exhibit promising activity at concen-
tration (mol. Ratio/TPA) 1000 (cf. Figures 1−4).
CONCLUSIONS
Structure activity correlation of the obtained results revealed that the
addition of both benzene ring (containing methoxy group, dimethoxy
groups, dioxal group) and 5-methyl-2-furyl to the 4-position of pyrimi-
dine moiety increases the activity (% of TPA Reduction) more than the
addition of fluoro, nitro, hydroxy groups over the pyrimidine moiety. The

2960
F. A. Attaby et al.
Figure 1 Inhibitory activity of the newly synthesized heterocyclic compounds
at concentration 1000 of TPA.
overall results coincided with the previous conclusion that O-purenyl
structure or group was revealed to possess remarkable Inhibitory ac-
tivity against TPA induced EBV-EA potency.
EXPERIMENTAL
Melting points of the newly synthesized heterocyclic compounds were
determined by capillary method and are uncorrected. The IR spectra
1
were recorded on a Perkin-Elmer model 529 in nujol mull. H NMR
spectra of a few selected compounds were recorded on a 300 MHZ
Bruker WH-200 pulsed FT.NMR spectrometer using DMSO-d6 as sol-
vent and tetramethylsilane as an internal standard. All chemical shift
values are expressed in δ scale, mass spectra of some of the selected
compounds were recorded on a JEOL JMS-D-300mass spectrometer
operating at 70 V.
Synthesis of 2-Thioxo-1,2,3,4-tetrahydropyrimidinethiones
4a–n—General Procedure
To a solution of thiourea (3.8 g, 0.05 mol), appropriate aromatic or hete-
rocyclic aldehydes (0.05 mol), ethyl acetoacetate (0.05 mol), in absolute
ethanol (20 mL) a few drops (8 drops) of 37% HCl was added. The reac-
tion mixture was refluxed for 7−10 h, and then allowed to stand for 24 h
for crystallization. The product was filtrated and washed with ethanol,
dried, and recrystallized from the proper solvent.

1,2,3,4-Tetrahydrpyrimidinethiones and Inhibitory Activity
2961
60
50
40
30
20
10
0
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
Compound Number
Figure 2 Inhibitory activity of the newly synthesized heterocyclic compounds
at concentration 500 of TPA.
82
80
78
76
74
72
70
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
Compound Number
Figure 3 Inhibitory activity of the newly synthesized heterocyclic compounds
at concentration 100 of TPA.

2962
F. A. Attaby et al.
100
90
80
70
60
50
40
30
20
10
0
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
Compound Number
Figure 4 Inhibitory activity of the newly synthesized heterocyclic compounds
at concentration 10 of TPA.
Ethyl 1,2,3,4-Tetrahydro-4-(1H-indol-3-yl)-6-methyl-2-
thioxopyrimi-dine-5-carboxylate (4a)
Recrystallized from methanol m.p. 245−250◦C as dark brown solid;
1HNMR (300MHz, DMSO-d6): 1.22(t, J = 5.5 Hz, 3H, CH3), 1.57(s, 3H,
CH3), 4.19(q, J = 5.7 Hz,2H, CH2), 5.20(d, 1H, d, J = 3.75 Hz,H-4),
7.18(m, 4H aromatic indol), 10.22(s, 1H, NH); IR (cm−1): 3325(NH),
1705(C O), 1583 (C S). MS m/z(%): 315(90), 299(33), 286(12), 273(76);
anal calcd. for C16H17N3O2S: C,60.93; H,5.43; N,13.32; S,10.17; found
C, 60.98; H, 5.10; N, 14.01; S, 10.22.
Ethyl 1,2,3,4-Tetrahydro-6-methyl-4-(pyridine-3-yl)-2-
thioxopyrimi-dine-5-carboxylate (4b)
Recrystallized from methanol m.p. 75−79◦C as yellow solid; 1HNMR
(300MHz, DMSO-d6): 1.30 (t, J = 5.9 Hz, 3H, CH3), 1.71(s, 3H, CH3),
4.22(q, J = 6.1 Hz, 2H, CH2), 5.13(d, 1H, d, J = 3.45 Hz, H-4), 7.62,
8.65(m, 5H, pyridine); IR (cm−1): 3385(NH), 1720(C O), 1580 (C S).
MS m/z(%): 262(100), 247(13), 231(72); anal. calcd. for C13H15N3O2S:
C, 56.31, H, 5.41; N, 15.15; S, 11.55; found: C, 56.27; H, 5.38; N, 15.19;
S, 11.22.
Ethyl 4-(1,3-Benzodioxol-5-yl)-6-methyl-2- thioxo-1,2,3,4-
tetrahydro- pyrimidine-5-carboxylate (4c)
Recrystallized from methanol m.p. 172−175◦C as white solid;
1HNMR (300MHz, DMSO-d6): 1.22(t, J = 5.9 Hz, 3H, CH3), 1.81(s, 3H,

1,2,3,4-Tetrahydrpyrimidinethiones and Inhibitory Activity
2963
CH3), 4.15(q, J = 6.5 Hz,2H, CH2), 5.34(d, 1H, d, J = 3.80 Hz, H-
4), 5.91(s, 2H, CH2), 6.46–6.54 (m, 3H, ArH’s); IR (cm−1): 3405(NH),
1729(C O), 1570(C S). MS m/z (%): 320(100), 305(77) 288(53), 275(22);
anal. calcd. for C15H16N2O4S: C, 56.24; H, 5.03; N, 8.74; S, 10.01; found
C, 56.98; H,5.52; N, 8.44; S, 10.11.
Ethyl 1,2,3,4-Tetrahydro-6-methyl-4-(5-methyl-2-furyl)-2-
thioxopyrimidine-5-carboxylate (4d)
Recrystallized from methanol m.p. >350◦C as colorless solid;
1HNMR (300MHz, DMSO-d6): 1.19(t, J = 4.9 Hz, 3H, CH3), 1.41(s,
3H, CH3), 2.22(s, 3H, CH3), 4.55(q, J = 5.8 Hz, 2H, CH2), 5.13(d,
1H, d, J = 3.28 Hz, H-4), 5.82(dd, J = 6.1 Hz, 1H, CH), 5.92(dd,
J = 6.3 Hz, 1H, CH); IR (cm−1): 3407(NH), 1708(C O), 1587(C S); MS
m/z(%): 280(100), 251(60) 207(87); anal. calcd. for C13H16N2O3S: C,
55.69; H, 5.75; N, 9.99; S, 11.44; found C, 55.52; H, 6.01; N, 10.01;
S, 11.43.
Ethyl 1,2,3,4-Tetrahydro-6-methyl-4-(2-thienyl)-2-
thioxopyrimidine-5-carboxylate (4e)
Recrystallized from methanol m.p. 166 −171◦C as colorless solid;
1HNMR (300MHz, DMSO-d6): 1.09(t, J = 6.9 Hz, 3H, CH3), 1.91(s, 3H,
CH3), 2.42(s, 3H, CH3), 4.35(q, J = 7.3 Hz, 2H, CH2), 5.32(d, 1H, d,
J = 3.67 Hz, H-4), 6.42–6.95 (m, 3H, thiophene); IR (cm−1): 3397(NH),
1700(C O), 1574(C S); MS m/z(%): 282(100), 267(20); anal. calcd. for
C12H14N2O2S2: C, 51.04; H, 5.00; N, 9.92; S, 22.71; found C, 51.01; H,
5.91; N, 10.31; S, 23.03.
Ethyl 1,2,3,4-Tetrahydro-4,6-dimethyl-4-(thiophene-2-yl)-2-
thioxopyrim-idine-5-carboxylate (4f)
Recrystallized from methanol m.p. 195−198◦C as a yellow solid;
1HNMR (300MHz, DMSO-d6): 1.09(t, J = 6.1 Hz, 3H, CH3), 1.61(s,
3H, CH3), 1.92(s, 3H, CH3), 4.35(q, J = 6.3 Hz, 2H, CH2), 5.22(d, 1H, d,
J = 3.55 Hz, H-4), 6.52-6.85 (m, 3H, thiophene); IR (cm−1): 3347(NH),
1702(C O), 1584(C S); MS m/z(%): 296(100), 281(10) 265(74); anal.
calcd. for. C13H16N2O2S2: C, 52.58; H, 5.44; N, 9.45; S, 21.64; found C,
51.92; H, 5.21; N, 10.01; S, 21.03.

2964
F. A. Attaby et al.
Ethyl 1,2,3,4-Tetrahydro-4-(2,4-dimethoxyphenyl)-6-methyl-2-
thioxo-pyrimidine-5-carboxylate (4g)
Recrystallized from ethanol m.p. 120−125◦C as a brown solid;
1HNMR (300MHz, DMSO-d6): 1.11(t, J = 5.01 Hz, 3H, CH3), 1.27(s,
3H, CH3), 3.81(s, 6H, 2-OCH3), 4.22(q, J = 5.7 Hz, 2H, CH2); 5.17(d,
1H, d, J = 3.54 Hz, H-4), 6.16–6.84(m, 3H, ArH’s). IR (cm−1): 3330(NH),
1717(C O), 1572(C S). MS m/z (%): 336(100), 321(37) 305(13), 261(41);
anal. calcd. for C16H2ON2O4S: C, 57.09; H, 6.01; N, 8.31; S, 9.51; found:
C, 57.11; H, 6.12; N, 8.33; S, 9.49.
Ethyl 1,2,3,4-Tetrahydro-4-(2,5-dimethoxyphenyl)-6-methyl-2-
thioxo-pyrimidine-5-carboxylate (4h)
Recrystallized from ethanol m.p. 214−217◦C as a orange solid;
1HNMR (300MHz, DMSO-d6): 1.27(t, J = 4.6 Hz, 3H, CH3), 1.72(s,
3H, CH3), 3.60(s, 6H, 2-OCH3), 4.40(q, J = 5.3 Hz, 2H, CH2), 5.12(d,
1H, d, J = 3.65 Hz, H-4), 6.49-6.67(m, 3H, ArH’s); IR (cm−1): 3327(NH),
1723(C O), 1582(C S); MS m/z(%): 336(89), 321(15) 305(60), 261(11);
anal. calcd. for C16H2ON2O4S: C, 57.12; H, 5.99; N, 8.33; S, 9.53; found
C, 57.35; H, 6.01; N, 8.28; S, 9.56.
Ethyl-4-(4-Fluorophenyl)-1,2,3,4-tetrahydro-6-methyl-2-
thioxopyrimi-dine-5-carboxylate (4i)
Recrystallized from ethanol m.p. 193−195◦C as a white solid;
1HNMR (300 MHz, DMSO-d6): 1.18(t, J = 5.1 Hz, 3H, CH3), 1.66(s,
3H, CH3), 4.31(q, J = 6.3 Hz,2H, CH2), 5.46(d, 1H, d, J = 3.75
Hz, H-4), 6.88(m, 4H, ArH’s); IR (cm−1): 3390(NH), 1711(C O),
1585(C S); MS m/z(%) 294(100), 279(55) 265(20), 221(79); anal. calcd.
for C14H15FN2O2S: C,57.10; H, 5.16; F, 6.43; N, 9.55; S, 10.91; found C,
57.22; H, 5.01; F, 6.39; N, 9.63; S, 10.90.
Ethyl-1,2,3,4-tetrahydro-6-methyl-2-thioxo-4-(4-
methylphenyl)pyrimi-dine-5-carboxylate (4j)
1
Recrystallized from ethanol m.p. 185−188◦C as a yellow solid; H
NMR (300 MHz, DMSO-d6): 1.28(t, J = 6.2 Hz, 3H, CH3), 1.53(s, 3H,
CH3), 2.33(s, 3H, CH3), 4.01(q, J = 6.4 Hz, 2H, CH2), 5.49(d, 1H, d, J =
3.87 Hz, H-4), 6.94(m, 4H, ArH’s); IR (cm−1): 3310(NH), 1721(C O),
1579(C S). MS m/z(%): 290(100), 275(15) 261(12), 217(90); anal. calcd.
for: C15H18N2O2S : C,62.04; H, 6.25; N, 9.65; S, 11.04; found C, 61.98;
H, 5.89; N, 9.83; S, 11.13.

1,2,3,4-Tetrahydrpyrimidinethiones and Inhibitory Activity
2965
Ethyl 1,2,3,4-Tetrahydro-6-methyl-4-(3-nitrophyenyl)-2-
thioxopyrim-idine-5-carboxylate (4k)
Recrystallized from ethanol m.p. 212−215◦C as white powder solid;
1HNMR (300MHz, DMSO-d6): 1.27(t, J = 5.6 Hz, 3H,CH3), 1.92(s,
3H, CH3), 4.22(q, J = 5.4 Hz, 2H, CH2), 5.01(d, 1H, d, J = 3.36
Hz, H-4), 7.76–8.01(m, 5H, ArH’s); IR (cm−1): 3319(NH), 1701(C O),
1572(C S). MS m/z(%): 321(14), 285(35) 275(100), 241(66); anal. calcd.
For C14H15N3O4S: C, 53.01; H, 4.71; N, 13.09; S,10.01; found C, 53.01;
H, 4.60; N, 12.92; S, 9.99.
Ethyl-4-(2-Fluorophenyl)-1,2,3,4-tetrahydro-6-methyl-2-
thioxopyrim-idine-5-carboxylate (4l)
Recrystallized from mixture ethanol ethylacetate m.p. 87−90◦C as a
yellow solid; 1H NMR (300MHz, DMSO-d6): 1.12(t, J = 6.1Hz, 3H, CH3),
1.33(s, 3H, CH3), 4.01(q, J = 5.8 Hz, 2H, CH2), 5.19(d, 1H, d, J = 3.41
Hz, H-4), 6.82–7.10(m, 5H, ArH’s); IR (cm−1): 3298(NH), 1701(C O),
1580(C S); MS m/z(%): 294(44), 279(5) 265(20), 221(29); anal. calcd.
for C14H15FN2O2S: C, 57.13; H, 5.14; F, 6.45; N, 9.52; S, 10.89; found C,
56.94; H, 5.34; F, 5.87; N, 10.01; S, 9.99.
Ethyl 1,2,3,4-Tetrahydro-4-(2-hydroxyphenyl)-6-methyl-2-
thioxopyrim-idine-5-carboxylate (4m)
Recrystallized from methanol-water m.p. 199−203◦C as a yellow
solid; 1H NMR (300MHz, DMSO-d6): 1.37(t, J = 6.8 Hz, 3H, CH3),
1.90(s, 3H, CH3), 4.67(q, J = 6.9Hz, 2H, CH2) 5.32(d, 1H, d, J = 3.22
Hz, H-4), 6.65–6.95(m, 5H, ArH’s); IR (cm−1): 3366(NH), 1716(C O),
1570(C S); MS m/z(%): 292(100), 277(55) 261(30), 245(16); anal. calcd.
for C14H16N2O3S: C, 57.52; H, 5.52; N, 9.58; S, 10.97; found C, 57.01;
H, 5.33; N, 9.60; S, 10.96.
Ethyl 1,2,3,4-Tetrahydro-4-(4-hydroxyphenyl)-6-methyl-2-
thioxopyrim-idine-5-carboxylate (4n)
1
Recrystallized from ethanol m.p. 87−90◦C as a red solid; HNMR
(300MHz, DMSO-d6): 1.12(t, J = 5.3 Hz, 3H, CH3), 2.01(s, 3H, CH3),
4.50(q, J = 4.4 Hz,2H, CH2), 5.16(d, 1H, d, J = 3.70 Hz, H-4), 6.62(dd,
J = 7.7 Hz, 2H), 6.91(dd, J = 7.9 Hz,2H); IR (cm−1): 3156(NH),
1729(C O), 1577(C S); MS m/z(%): 292(100), 277(40); anal. calcd. for
C14H16N2O3S: C, 57.50; H, 5.59; N, 9.61; S, 10.99; found: C, 57.52; H,
5.53; N, 9.58; S, 10.96.

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F. A. Attaby et al.
Epstein-Barr Virus Activation Test
Materials and Methods
The newly synthesized heterocyclic compounds were tested for
Epstein-Barr virus (EBV) genome carrying lymphoblastoid cells (Raji
cells derived from Burkitt’s lymphoma) were cultured in 10% fetal
bovine serum (FBS) in RPMI-1640 medium (Sigma, MS, USA). Spon-
taneous activation of EBV-EA (early antigen) in our sub-line Raji cells
less than 0.1%. The inhibition of EBV-EA activation was assayed using
Raji cells (virus non producer type) as described previously.16 The indi-
cator cells (Raji cell, 1 × 106/ml) were incubated at 37◦C for 48 hrs in 1
ml of medium containing n-butyric acid (4 mmol), TPA (32 pmol 20 ng
in DMSO, 2 µl) as inducers and various amounts of test compounds
in 5 µl DMSO. Smears were made from the cell suspension, and the
activated cells that were stained by EBV-EA positive serum from na-
sopharyngeal carcinoma (NPC) patients were detected by an indirect
immunofluoresence technique.17 In each assay, at least 500 cells were
counted, and the number of stained cells (positive cells) present was
recorded. Triplicate assays were performed for each compound the av-
erage of EBV-EA induction of the test compounds was expressed as a
relative ratio to the control experiment (100%), which was carried out
only with n-butyric acid plus TPA. The viability of treated Raji cells
was assayed by the trypan Blue staining method.
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