Synthesis, antiviral and cytotoxic activity of 6-bromo-2,3-disubstituted-4(3H)-quinazolinones.

Article abstract of DOI:10.1248/bpb.26.1278

In the present study, a series of 6-bromo-2,3-disubstitued-4(3H)-quinazolinones was synthesized by condensation of 6-bromo-2-substituted-benzoxazin-4-one with trimethoprim, pyrimethamine and lamotrigine. The chemical structures of the synthesized compound

Full text of DOI:10.1248/bpb.26.1278

1
278
Biol. Pharm. Bull. 26(9) 1278—1282 (2003)
Vol. 26, No. 9
Synthesis, Antiviral and Cytotoxic Activity of 6-Bromo-2,3-disubstituted-
4
(3H)-quinazolinones
a
a
b
,c
Murugesan DINAKARAN, Periyaswamy SELVAM, Erik DECLERCQ, and Seshaiah Krishnan SRIDHAR*
a
Department of Pharamaceutical Chemistry, Periyar College of Pharmaceutical Science; Tiruchirappalli 620021,
b
Tamilnadu, India: Rega Institute of Medical Research, Katholieke University; Leuven, MinderBroedersstraat, B–3000,
Leuven, Belgium: and Department of Pharmaceutical Chemistry, C. L. Baid Metha College of Pharmacy; Jyothi Nagar,
c
Thorapakkam, Chennai 600096, Tamilnadu, India. Received March 14, 2003; accepted June 10, 2003
In the present study, a series of 6-bromo-2,3-disubstitued-4(3H)-quinazolinones was synthesized by conden-
sation of 6-bromo-2-substituted-benzoxazin-4-one with trimethoprim, pyrimethamine and lamotrigine. The
1
chemical structures of the synthesized compounds were confirmed by means of IR, H-NMR and mass spectral
and elemental analysis. The antiviral activity and cytotoxicity of the compounds were tested in E SM (Herpes
6
simplex-1 KOS, Herpes simplex-1 TK-KOS ACV, Herpes simplex-2 G, Vaccinia virus, Vesicular stomatitis virus,
Parainfluenza-3 virus, Reovirus-1, Sindbis virus, Coxsackie virus B4 and Punta Toro virus) and HeLa cell cul-
ture (Vesicular stomatitis virus, Coxsackie virus B4 and Respiratory syncyticla virus). Investigation of anti-HIV
activity was done against replication of HIV-1 (HTLV-III B LAI) in MT-4 cells. 6-Bromo-2-phenyl-3-[(4-amino-5-
(
4-chlorophenyl)-6-ethylpyrimidin-2-yl]-4(3H)-quinazolinone (4) exhibited the most potent antiviral activity with
a MIC of 1.92 mg/ml against vaccinia virus in E SM cell culture. The other compounds did not exhibit antiviral
6
activity nor afford significant cytoprotection to the E SM and HeLa cell culture when challenged with the
6
viruses. The study implies that 4 may possess activity against Pox viruses including variola. In the anti-HIV
study, 6-bromo-2-methyl-3-[(4-amino-5-(4-chlorophenyl)-6-ethylpyrimidin-2-yl]-4(3H)-quinazolinone (3) and 6-
bromo-2-phenyl-3-[(4-amino-5-(4-chlorophenyl)-6-ethylpyrimidin-2-yl]-4(3H)-quinazolinone (4) exhibited the
least cytotoxic concentration (0.424, 0.461 mg/ml) which is an index of the infective viability of mock infected MT-
4
cells with HIV-1. None of the compounds exhibited significant anti-HIV activity.
Key words quinazoline; antiviral; anti-HIV; cytotoxicity
4
(3H)-Quinazolinone derivatives were reported to possess Herpes simplex-1 TK-KOS ACV, Herpes simplex-2 G, Vac-
1
)
2)
3—7)
12)
analgesic, anti-inflammatory, antibacterial, antifungal,
anti-HIV,
anticancer, MAO inhibitory and central nervous system Toro virus) and HeLa cell culture (Vesicular stomatitis virus,
activities. 4(3H)-Quinazolinones with 3-substitution has Coxsackie virus B4 and Respiratory syncyticla virus). Inves-
cinia virus, Vesicular stomatitis virus, Parainfluenza-3 virus,
Reovirus-1, Sindbis virus, Coxsackie virus B4 and Punta
8
,9)
10)
11)
antihelminthic,
antiallergic,
antitumour,
1
3)
14)
1
5)
been reported to be associated with antimicrobial proper- tigation of anti-HIV activity was done against replication of
1
6—18)
ties.
The 3-substitution which were reported are various HIV-1 (HTLV-III B LAI) in MT-4 cells.
1
9)
20)
substituted phenyl ring moieties, bridged phenyl rings,
heterocyclic rings
tuted-4(3H)-quinazolinones were reported to possess an- are uncorrected. The IR spectra of the compounds were
timicrobial properties. The antibacterial, antifungal and anti- recorded on Bruker Vector-22 FT-IR with KBr pellets. H-
Chemistry The melting points were taken in open capil-
and aliphatic systems. 2,3-Substi- lary tube on a Thomas Hoover melting point apparatus and
2
1—23)
24)
2
5)
1
HIV screening of semi-derivatized conventional antibacterial NMR spectra were recorded on 500 MHz Bruker AMX 500
2
6,27)
28,29)
agents namely trimethoprim,
floxacin,
reported.
sulphadoxime,
nor- using DMSO-d as solvent. The chemical shifts are reported
6
3
0,31)
32)
32)
ciprofloxacin and lomefloxacin have been as parts per million downfield from tetramethylsilane
(Me Si). Mass spectra were recorded on Varian Atlas CH-7.
4
These observation lead to the conception that a new series Microanalyses for C, H, N were performed in Heraeus CHN
of 2-methyl/2-phenyl-4(3H)-quinazolinones with 2,4-di- Rapid Analyzer. All the compounds gave satisfactory chemi-
aminopyrimidine (trimethoprim and pyrimethamine) and 3,5- cal analyses (Ϯ0.4%). The purity of the compounds were
diaminotriazine (lamotrigine) substitutent in 3rd position checked by TLC on SiO gel (HF254, 200 mesh) coated glass
2
would exhibit potential cytoprotective and antiviral activity. plates using (4 : 1) CH OH : CHCl as mobile phase and visu-
3
3
The 2,4-diaminopyrimidines selected for the study possess alized by iodine vapours.
3
3)
dihydro folate reductase inhibitory property. Triazines have
Synthesis of 2-Methyl-6-bromo-3,1-benzoxazin-4-one
been reported to exhibit dihydro folate reductase inhibitory A mixture of 5-bromoanthranilic acid (0.1 mol) and acetic
3
4)
property. In continuation of our earlier work on 4(3H)- anhydride (0.2 mol) was refluxed under anhydrous conditions
quinazolinones, the present study deals with the synthesis of for 4 h. The excess acetic anhydride was distilled off under
a series of 6-bromo-2,3-disubstitued-4(3H)-quinazolinones reduced pressure. The product formed was filtered, vacuum
by condensation of 6-bromo-2-substituted-benzoxazin-4-one dried and recrystallised using absolute ethanol. Yieldϭ58%,
1
with trimethoprim, pyrimethamine and lamotrigine. The mp 180—181 °C. H-NMR (DMSO-d ) d: 8.04 (s, 1H; 5-H),
6
chemical structures of the synthesized compounds were con- 7.67 (d, Jϭ6.2 Hz, 1H; 7-H), 7.48 (d, Jϭ6.2 Hz, 1H; 8-H),
firmed by means of IR, H-NMR, mass spectral and elemen- 1.41 (s, 3H; 2-CH ). IR (KBr) cm : 1686 (CϭO), 1668
1
Ϫ1
3
tal analysis. The antiviral activity and cytotoxicity of the (CϭN), 1143 (C–O–C), 811, 774 (Ar–H), 616 (C–Br). EI-
compounds were tested in E SM (Herpes simplex-1 KOS, MS m/z: 240.19 (Calcd for C H BrNO : 240.06). Anal. Calcd
6
9
6
2
∗
To whom correspondence should be addressed. e-mail: sksridhar67@yahoo.com
© 2003 Pharmaceutical Society of Japan

September 2003
1279
for C H BrNO : C, 45.03; H, 2.52; N, 5.83. Found: C, 44.86; 7.49—7.65 (m, 9H; 2-C H & C H ), 3.43 (s, 2H; NH ), 2.51
9
6
2
6
5
6
4
2
H, 2.77; N, 6.11.
(q, Jϭ5.9 Hz, 2H; CH CH ), 2.23 (t, Jϭ5.9 Hz, 3H;
2 3
Ϫ1
Synthesis of 2-Phenyl-6-bromo-3,1-benzoxazin-4-one CH CH ). IR (KBr) cm : 3393 (NH ), 1681 (CϭO), 1657
2
3
2
To a solution of 5-bromoanthranilic acid (0.1 mol) in 50 ml (CϭN), 833, 813, 711 (Ar–H), 590 (C–Br). EI-MS m/z:
of dry pyridine, benzoyl chloride (0.2 mol) was added drop- 532.41 (Calcd for C H BrClN O: 532.87). Anal. Calcd for
2
6
19
5
wise with constant stirring at 15 °C. The reaction mixture C H BrClN O: C, 58.61; H, 3.59; N, 13.14. Found: C,
2
6
19
5
was cooled to 5 °C and aqueous sodium carbonate (15 ml, 58.34; H, 3.68; N, 12.92.
0% w/v) was added. The product formed was filtered, vac- 6-Bromo-2-methyl-3-[(5-amino-6-(2,3-dichlorophenyl)-
uum dried and recrystallised using absolute ethanol. 1,2,4-triazin-3-yl]-4(3H)-quinazolinone (5): Yieldϭ82%, mp
1
1
1
Yieldϭ76%, mp 119—120 °C. H-NMR (DMSO-d ) d: 8.32 240—241 °C. H-NMR (DMSO-d ) d: 8.52 (s, 1H; 5-H),
6
6
(
s, 1H; 5-H), 7.94 (d, Jϭ6.1 Hz, 1H; 7-H), 7.63 (d, Jϭ6.1 Hz, 8.14 (d, Jϭ6 Hz, 1H; 8-H), 7.71 (d, Jϭ6 Hz, 1H; 7-H),
H; 8-H), 6.84—7.11 (m, 5H; C H ). IR (KBr) cm : 1680 7.41—7.66 (m, 3H; phenyl), 3.56 (s, 2H; NH ), 2.15 (s, 3H;
CϭO), 1672 (CϭN), 1154 (C–O–C), 819, 744, 736 (Ar–H), 2-CH ). IR (KBr) cm : 3455 (NH ), 1681 (CϭO), 1645
Ϫ1
1
6
5
2
Ϫ1
(
3
2
6
3
4
11 (C–Br). EI-MS m/z: 302.32 (Calcd for C H BrNO : (CϭN), 1438 (NϭN), 830, 792, 737 (Ar–H), 604 (C–Br).
14 8 2
02.13). Anal. Calcd for C H BrNO : C, 56.66; H, 2.67; N, EI-MS m/z: 478.31 (Calcd for C H BrCl N O: 478.13).
1
4
8
2
18 11
2
6
.64. Found: C, 56.48; H, 2.92; N, 4.51.
Anal. Calcd for C H BrCl N O: C, 45.22; H, 2.32; N,
18 11 2 6
General Procedure for 6-Bromo-2,3-disubstituted- 17.58. Found: C, 45.41; H, 2.04; N, 17.74.
4
(3H)-quinazolinones Equimolar quantities (0.01 mol) of
6-Bromo-2-phenyl-3-[(5-amino-6-(2,3-dichlorophenyl)-
6
-bromo-2-substituted-3,1-benzoxazin-4-one and the pri- 1,2,4-triazin-3-yl]-4(3H)-quinazolinone (6): Yieldϭ87%, mp
1
mary amine (trimethoprim, pyrimethamine and lamotrigine) 210—211 °C. H-NMR (DMSO-d ) d: 8.82 (s, 1H; 5-H),
in glacial acetic acid (10 ml) was refluxed for 6 h. The reac- 8.36 (d, Jϭ7.1 Hz, 1H; 8-H), 8.08 (d, Jϭ7.1 Hz, 1H; 7-H),
6
tion content was cooled to room temperature and poured into 7.24—7.89 (m, 8H; 2-C H & phenyl), 3.42 (s, 2H; NH ). IR
6
5
2
Ϫ1
crushed ice. The product formed was filtered, vacuum dried (KBr) cm : 3393 (NH ), 1679 (CϭO), 1611 (CϭN), 1435
2
and recrystallised using absolute ethanol (Fig. 1).
(NϭN), 827, 799, 781 (Ar–H), 608 (C–Br). EI-MS m/z:
6
-Bromo-2-methyl-3-[(4-amino-5(3,4,5-trimethoxyben- 540.09 (Calcd for C H BrCl N O: 540.21). Anal. Calcd for
2
3
13
2
6
zyl)pyrimidin-2-yl)]-4(3H)-quinazolinone (1): Yieldϭ86%, C H BrCl N O: C, 51.14; H, 2.43; N, 15.56. Found: C,
2
3
13
2
6
1
mp 180—181 °C. H-NMR (DMSO-d ) d: 8.43 (s, 1H; 5-H), 50.98; H, 2.63; N, 15.39.
6
8
(
.09 (d, Jϭ7.1 Hz, 1H; 8-H), 7.82 (s, 1H; pyrimidinyl), 7.52
Antiviral Activity and Cytoxicity The antiviral activity
3
5—39)
d, Jϭ7.1 Hz, 1H; 7-H), 6.61 (s, 2H; C H (OCH ) ), 3.87(s, and cytotoxicity
of the synthesized compounds were
6
2
3 3
2
3
H; NH ), 3.68 (s, 9H; (OCH ) ), 2.51 (s, 2H; CH ), 1.23 (s,
2 3 3 2
Ϫ1
H; 2-CH ). IR (KBr) cm : 3385 (NH ), 1656 (CϭO), 1590
3
2
(
CϭN), 1120 (C–O–C), 818, 766, 714 (Ar–H), 636 (C–Br).
EI-MS m/z: 514.55 (Calcd for C H BrN O : 514.37). Anal.
2
3
22
5
4
Calcd for C H BrN O : C, 53.71; H, 4.31; N, 13.62. Found:
2
3
22
5
4
C, 53.94; H, 4.46; N, 13.51.
-Bromo-2-phenyl-3[(4-amino-5(3,4,5-trimethoxyben-
6
zyl)pyrimidin-2-yl)]-4(3H)-quinazolinone (2): Yieldϭ85%,
1
mp 164—165 °C. H-NMR (DMSO-d ) d: 8.66 (s, 1H; 5-H),
6
8
.02 (d, Jϭ7 Hz, 1H; 8-H), 7.72 (s, 1H; pyrimidinyl), 7.45—
.53 (m, 5H; C H ), 7.25 (d, Jϭ7 Hz, 1H; 7-H), 6.62 (s, 2H;
7
6
5
C H (OCH ) ), 3.75 (s, 2H; NH ), 3.58 (s, 9H; (OCH ) ),
6
2
3 3
2
3 3
Ϫ1
2
.51 (s, 2H; CH ). IR (KBr) cm : 3393 (NH ), 1681 (CϭO),
2
2
1
657 (CϭN), 1121 (C–O–C), 817, 765 (Ar–H), 595 (C–Br).
EI-MS m/z: 574.63 (Calcd for C H BrN O : 574.43). Anal.
2
8
24
5
4
Calcd for C H BrN O : C, 58.55; H, 4.21; N, 12.19. Found:
2
8
24
5
4
C, 58.37; H, 3.92; N, 12.26.
-Bromo-2-methyl-3-[(4-amino-5-(4-chlorophenyl)-6-eth-
ylpyrimidin-2-yl]-4(3H)-quinazolinone (3): Yieldϭ83%, mp
6
1
2
8
15—216 °C. H-NMR (DMSO-d ) d: 8.52 (s, 1H; 5-H),
6
.45 (d, Jϭ6.8 Hz, 1H; 8-H), 7.52—7.77 (m, 4H; C H ), 7.36
6
4
(
d, Jϭ6.8 Hz, 1H; 7-H), 3.52 (s, 2H; NH ), 2.54 (q, Jϭ6 Hz,
2
2
H; CH CH ), 2.24 (t, Jϭ6 Hz, 3H; CH CH ), 1.08 (s, 3H; 2-
2 3 2 3
Ϫ1
CH ). IR (KBr) cm : 3455 (NH ), 1669 (CϭO), 1645
3
2
(
(
CϭN), 827, 714 (Ar–H), 600 (C–Br). EI-MS m/z: 470.94
Calcd for C H BrClN O: 470.75). Anal. Calcd for
2
1
17
5
C H BrClN O: C, 53.58; H, 3.68; N, 14.88. Found: C,
2
1
17
5
5
3.85; H, 3.99; N, 14.69.
-Bromo-2-phenyl-3-[(4-amino-5-(4-chlorophenyl)-6-eth-
ylpyrimidin-2-yl]-4(3H)-quinazolinone (4): Yieldϭ84%, mp
6
1
1
8
94—195 °C. H-NMR (DMSO-d ) d: 8.67 (s, 1H; 5-H),
6
Fig. 1. Synthetic Scheme of 6-Bromo-2,3-disubstituted-4(3H)-quinazoli-
.18 (d, Jϭ7 Hz, 1H; 8-H), 7.97 (d, Jϭ7 Hz, 1H; 7-H), nones

1
280
Vol. 26, No. 9
tested in E SM (Herpes simplex-1 KOS, Herpes simplex-1
6
TK-KOS ACV, Herpes simplex-2 G, Vaccinia virus, Vesicu-
lar stomatitis virus, Parainfluenza-3 virus, Reovirus-1, Sind-
bis virus, Coxsackie virus B4 and Punta Toro virus) and
HeLa cell culture (Vesicular stomatitis virus, Coxsackie virus
B4 and Respiratory syncyticla virus). (E)-5-(2-Bromovinyl)-
2
Ј-deoxyuridine, (S)-9-(2,3-dihydroxypropyl)adenine and
(S)-9-(2,3-dihydroxypropyl)guanidine were used as stan-
dards. The minimum cytotoxic concentration (MCC) to
cause a microscopically detectable alteration of normal cell
morphology and minimum inhibitory concentration (MIC)
to reduce virus-induced cytopathogenicity by 50% were
recorded. The antiviral and cytotoxicity data are presented in
Table 1 and 2.
Anti-HIV Activity Cell Cultures: The MT-4 cells were
grown in RPM1-1640 DM (“Dutch Modification”) medium
(
Flow Laboratories, Irvine, Scotland), supplemented with
1
2
0% (v/v) heat-inactivated fetal calf serum (FCS) and
0 mg/ml gentamicin (E. Merck, Darmstadt, F.R.G.). The
cells were maintained at 37 °C in a humidified atmosphere of
5
% CO in air. Every 3—4 d, cells were spun down and
2
5
seeded at 3ϫ10 cells/ml in new cell culture flasks. At regu-
lar intervals, the MT-4 cells were analyzed for the presence
of mycoplasma and consistently found to be mycoplasma-
free.
4
0)
m
Virus: HIV-1 (strain HTLV-IIIB LAI) was obtained from
4
1)
the culture supernatant of HIV-1 infected MT-4 cell lines.
The Virus titer of the supernatant was determined in MT-4
cells. The virus stocks were stored at Ϫ70 °C until used.
Anti-HIV Assay: Flat bottom, 96-well plastic microtiter
plates (Falcon, Becton Dickinson, Mountain view, CA,
U.S.A.) were filled with 100 ml of complete medium using a
Titertek multidrop dispenser (Flow Laboratories). This eight
channel dispenser could fill a microtiter tray in less than 10 s.
Subsequently, stock solutions (10ϫ final test concentration)
of compounds were added in 25 ml volumes to two series of
triplicate wells so as to allow simultaneous evaluation of
their effects on HIV- and mock-infected cells. Serial five-fold
dilutions were made directly in the microtiter trays using a
Biomek 1000 robot (Beckman). Untreated control HIV-and
mock-infected cell samples was included for each com-
pound.
5
0 ml of HIV at 100 CCID medium was added to either
5
0
infected or mock-infected part of a microtiter tray. Exponen-
tially growing MT-4 cells were centrifuged for 5 min at
1
40ϫg and the supernatants were discarded. The MT-4 cells
5
resuspended at 6ϫ10 cells/ml in a flask that was connected
with an autoclavable dispensing cassette of Titertek mul-
tidrop dispenser. Under slight magnetic stirring 50 ml vol-
umes were then transferred to the microtiter tray wells. The
outer row wells were filled with 200 ml of medium. The cell
cultures were incubated at 37 °C in a humidified atmosphere
of 5% CO in air. The cells remained in contact with the test
2
compounds during the whole incubation period. Five days
after infection, the viability of mock and HIV-infected cells
were examined spectrophotometrically by the MTT method.
MTT Assay: The MTT assay was based on the reduc-
tion of the yellow colored 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) (Sigma Chemical Co.,
St. Louis, MO, U.S.A.) by mitochondrial dehydrogenase of
metabolically active cells to a blue formazan which can be
m

September 2003
1281
Table 2. Antiviral and Cytotoxicity Activity of Synthesized Compounds
HeLa cell cultures
MT-4 cell lines
HIV
b)
MIC (mg/ml)
Compound
a)
MCC (mg/ml)
c)
d)
Vesicular
stomatitis virus
Coxsackie
virus B4
Respiratory
syncyticla virus
IC50
(mg/ml)
CC50
(mg/ml)
1
2
3
4
5
400
80
80
80
80
Ͼ80
Ͼ16
Ͼ16
Ͼ16
Ͼ16
Ͼ16
Ͼ400
Ͼ400
48
Ͼ80
Ͼ16
Ͼ16
Ͼ16
Ͼ16
Ͼ16
Ͼ400
Ͼ400
240
Ͼ80
Ͼ16
Ͼ16
Ͼ16
Ͼ16
Ͼ16
Ͼ400
Ͼ400
16
Ͼ49.9
49.9
39.8
0.424
0.461
49.9
38.4
—
—
—
65.9
Ͼ39.8
Ͼ0.426
Ͼ0.461
Ͼ49.9
Ͼ38.4
—
6
80
BVDU
DHPA
Ribavirin
AZT
Ͼ400
Ͼ400
Ͼ400
—
—
—
0.0012
—
—
—
BVDUϭ(E)-5-(2-bromovinyl)-2Ј-deoxyuridine, DHPAϭ(S)-9-(2,3-dihydroxypropyl)-adenine and AZTϭazidothymidine. a) Minimum cytotoxic concentration, b) minimum
inhibitory concentration, c) effective concentration of compound achieving 50% protection of MT-4 cells against cytopathic effect of HIV and d) cytotoxic concentration of com-
pound required to reduce the viability of mock infected MT-4 cells by 50%.
measured spectrophotometrically. Therefore, to each well of RESULTS AND DISCUSSION
the microtiter plates 20 ml of a solution of MTT (7.5 mg/ml)
in phosphate-buffered saline was added using the Titertek
6-Bromo-2-phenyl-3-[(4-amino-5-(4-chlorophenyl)-6-eth-
Multidrop. The trays were further incubated at 37 °C in a ylpyrimidin-2-yl]-4(3H)-quinazolinone (4) exhibited the
CO incubator for one hour. A fixed volume of medium most potent antiviral activity with a MIC of 1.92 mg/ml
2
(
(
150 ml) was then removed from each cup using M96 washer against vaccinia virus in E SM cell culture. The other com-
6
ICN flow) without disturbing the MT-4 cells clusters con- pounds did not exhibit antiviral activity nor afford signifi-
taining the formazan crystals.
cant cytoprotection to the E SM and HeLa cell culture when
6
Solubilization of the formazan crystals was achieved by challenged with the viruses. The study implies that 4
adding 100 ml of 10% (v/v) Triton X-100 in acidified iso- may possess activity against Pox viruses including variola.
propanol (2 ml concentrated HCI per 500 ml solvent) using In the anti-HIV study, 6-bromo-2-methyl-3-[(4-amino-5-(4-
the M96 washer. Complete dissolution of the formazan crys- chlorophenyl)-6-ethylpyrimidin-2-yl]-4(3H)-quinazolinone
tals could be obtained after the trays had been placed on a (3) and 6-bromo-2-phenyl-3-[(4-amino-5-(4-chlorophenyl)-
plate shaker for 10 min. Finally, the absorbances were read in 6-ethylpyrimidin-2-yl]-4(3H)-quinazolinone (4) exhibited the
a eight-channel computer-controlled photometer (Multiskan least cytotoxic concentration (0.424, 0.461 mg/ml) which is
MCC, ICN flow) at two wavelengths (540, 690 nm). The ab- an index of the infective viability of mock infected MT-4
sorbance measured at 690 nm was automatically subtracted cells with HIV-1. None of the compounds exhibited signifi-
from the absorbance at 540 nm, so as to eliminate the effects cant anti-HIV activity.
of non-specific absorption.
A blank was carried out directly on the microtiter plates REFERENCES
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minimum of three wells. The 50% cytotoxic concentration
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50
that reduced the absorbance (OD540) of the Mock-infected
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