Synthesis and anticancer and anti-HIV testing of some pyrazino[1,2-a]benzimidazole derivatives

Article abstract of DOI:10.1016/S0223-5234(01)01313-7

In this study, some 1-methylene-2,3-diaryl-1,2-dihydropyrazino[1,2-a]benzimidazole and some 1-(2-arylvinyl)-3-arylpyrazino[1,2-a]benzimidazole derivatives were synthesised. The structure elucidation of the compounds was performed by IR, ¹H-NMR and MASS spectroscopic data and elemental analyses results. Anticancer and anti-HIV activities of the compounds were examined, however no anti-HIV activity was seen; highly notable anticancer activity was obtained. It was also observed that the compounds were more potent against leukaemia cell lines.

Full text of DOI:10.1016/S0223-5234(01)01313-7

European Journal of Medicinal Chemistry 37 (2002) 255–260
www.elsevier.com/locate/ejmech
Short communication
Synthesis and anticancer and anti-HIV testing of some
pyrazino[1,2-a]benzimidazole derivatives
S¸eref Demirayak *, Usama Abu Mohsen, Ahmet C¸ ag˘ri Karaburun
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Uni6ersity of Anadolu, 26470 Eskis¸ehir, Turkey
Received 12 April 2001; received in revised form 15 November 2001; accepted 15 November 2001
Abstract
In this study, some 1-methylene-2,3-diaryl-1,2-dihydropyrazino[1,2-a]benzimidazole and some 1-(2-arylvinyl)-3-
arylpyrazino[1,2-a]benzimidazole derivatives were synthesised. The structure elucidation of the compounds was performed by IR,
1H-NMR and MASS spectroscopic data and elemental analyses results. Anticancer and anti-HIV activities of the compounds were
examined, however no anti-HIV activity was seen; highly notable anticancer activity was obtained. It was also observed that the
´
compounds were more potent against leukaemia cell lines. © 2002 Published by Editions scientifiques et me´dicales Elsevier SAS.
Keywords: Pyrazino[1,2-a]benzimidazoles; Anticancer activity; Anti-HIV activity
1. Introduction
of some 1-methylene-2,3-diaryl-1,2-dihydropyrazino-
[1,2-a]benzimidazole derivatives (5a–s) and some 1-(2-
It has been known that a-methylene-g-lactone deriva-
tives, which are sesquiterpenic natural compounds [1–
7] (i.e. Helenalin I) and their structural analogues,
quinone methide derivatives [8–13] possess anticancer
activity. For these compounds, it can be said that the
most important structural requirement for cytotoxicity
is an OꢀCꢁCꢀCH2 residue, which is a part of an ester,
lactone or ketone [5]. The natural compounds, mito-
mycines II [14–20] are also thought to be acting as a
precursor of quinone methide; however, mitomycines
have limited use because of their toxicity. Efforts to
obtain less toxic compounds led to the new anti-tumour
agents of indole III [21–24], benzimidazole IV [25–32]
and quinazoline V [33–36] derivatives. It was reported
that these compounds interact with biological nucle-
arylvinyl)-3-arylpyrazino[1,2-a]benzimidazole
deriva-
tives (10a–h) was attempted. On the other hand, our
compounds should also be assumed as the structural
analogues of nonsteroidal antiestrogenic triarylethylene
ophiles such as
L-systein and sulfhydryl bearing en-
zymes in a Michael-type addition reaction and possess
cytotoxic and antitumour activity as an alkylating agent
[37,38]. Considering a-methylene-g-lactone residue as
the pharmacophoric group, we should lead to a much
simpler structure; a,b-unsaturated ketones [39–42] as a
bioisostere of this structure. In this study, the synthesis
* Correspondence and reprints.
E-mail address: sdemiray@anadolu.edu.tr (S¸. Demirayak).
Fig. 1.
´
0223-5234/02/$ - see front matter © 2002 Published by Editions scientifiques et me´dicales Elsevier SAS.
PII: S0223-5234(01)01313-7

256
S¸. Demirayak et al. / European Journal of Medicinal Chemistry 37 (2002) 255–260
The other group of compounds whose syntheses was
attempted was pyrazinobenzimidazole derivatives 10a–
h.
Reaction of 2-acetylbenzimidazole 1 with substituted
benzaldehydes 6a–d afforded 1-(benzimidazol-2-yl)-3-
aryl-2-propenone derivatives 7a–d, as described in the
literature [43]. 1-[1-(2-Aryl-2-oxoethyl)benzimidazol-2-
yl]-3-arylpropenones 9 were then synthesised by reac-
ting 7a–d with a-bromoacetophenones 8 in acetone in
the presence of potassium carbonate. Finally, heating 9
with ammonium acetate in acetic acid gave 10.
3. Results and discussion
3.1. Anticancer acti6ity
According to the test method, the compounds having
log10 GI50 (GI50: growth inhibition of 50%) values
greater than −4 are considered as inactive. It can be
seen that for all of our compounds, log10 GI50 values
are smaller than −4. Therefore, we may conclude that
all of our compounds provide a notable activity level.
Melphalan and cis-diaminodichloroplatinum, two of
Fig. 2.
derivatives VI including Tamoxifen and Clomiphene
which were known for their anticancer activity [37]
(Fig. 1).
2. Chemistry
For the synthesis of the aimed heterocycles, the reac-
tion sequences outlined in Figs. 2 and 3 were followed.
To obtain 1-(2-aryl-2-oxoethyl)-2-acetylbenzimidazoles
3, which are starting materials of the compounds 5,
2-acetylbenzimidazole 1 and a suitable a-bromoace-
tophenone 2 derivative was reacted in the presence of
potassium carbonate in acetone. 1-Methylene-2,3-di-
aryl-1,2-dihydropyrazino[1,2-a]benzimidazoles
5a–s
were obtained by reacting 3 and 4a–g in acetic acid.
Although the compounds 4a–e were commercially
available, 4-[(2-(piperidin-1-yl)ethoxy]aniline 4f and 4-
[2-(morpholin-4-yl)ethoxy]aniline 4g derivatives, the
starting materials of compounds 5m–s were obtained
by the hydrolyses of the ethers formed by reacting
4-hydroxyacetanilide
with
2-(piperidin-1-yl)ethyl-
chloride and 2-(morpholine-4-yl)ethylchloride, respec-
tively. These compounds were synthesised and used
without any further purification or structure
elucidation.
Fig. 3.

S¸. Demirayak et al. / European Journal of Medicinal Chemistry 37 (2002) 255–260
257
the commonly used chemotherapeutic agents, were used
as standard compounds. When the meangraph mid-
point (MG-MID) values of the compounds melphalan
and cis-diaminodichloroplatinum, i.e. −5.09 and
−6.20 respectively, are considered, it is observed that
our compounds provide high activity levels. It is espe-
cially noteworthy that MG-MID values of compounds
5e,f and i are between the values obtained from the
standard compounds. However, when the MG-MID
values, whose average values against various cancer
types were given, are taken into consideration four of
the compounds with MG-MID values lower than −5
attract attention, i.e. 5e,f,i,l. Each of these four com-
pounds is carrying a 1-methylene residue and may be
considered as a structural analogue of OꢀCꢁCꢀCH2
residue. Also, we should say that their structure resem-
bles Tamoxifen group drugs of oestrogen antagonist
activity. Nevertheless, the compounds 5n,o,r,s, carrying
a 2-substituted aminoethyl residue and especially pre-
pared to emphasise this similarity, were failed to sustain
the expected increase in activity. It is observed that the
activity levels of the a,b-unsaturated ketone analogue
compounds 9a,b,d,e,f,h and their cycled forms, com-
pounds 10a,b,c,e,g,h; could not reach the levels ob-
tained from the 1-methylene residue carrying
compounds 5. Nonetheless, a,b-unsaturated ketone
analogues 9, among which compounds 9b,e,h have ac-
tivity values almost approximate to −5, appear to be
more active than pyrazinone derivatives 10. With re-
gard to all these data, in the three groups of newly
synthesised compounds, no significant difference was
observed among substituents. When these data ob-
tained are examined according to their activity against
various cancer types, it is observed that both the stan-
dard and the tested compounds are effective against
leukaemia in lower concentrations; for 12 of the com-
pounds this value is lower than −5. The most note-
worthy compound is 5o, i.e. it is even more active than
melphalan against leukaemia.
VG Platform Mass spectrometer. Analyses for C, H, N
was within 90.4% of the theoretical values. 2-Acetyl-
benzimidazole [44] and a-bromoacetophenone [45]
derivatives were prepared according to the methods in
the literature. Some characteristics of the compounds
were given in Table 1.
4.1.1. General method
4.1.1.1.
1-Methylene-2,3-diaryl-1,2-dihydropyrazino-
[1,2-a]benzimidazoles (5a–s). A mixture of the suitable
1 (2 mmol) and an arylamine 4 (2 mmol) was refluxed
for 10 h in acetic acid (50 mmol). In the case of the
compounds 5b–e,g,j, the cooled solution was poured
into ice water and neutralised with sodium carbonate.
The precipitate was crystallised from ethanol. In the
case of the compounds 5a,f,h,i,k,l, the solution was
allowed to crystallise in a refrigerator. The crystallised
compound was recrystallised in acetic acid to give the
acetic acid salt of the compound. In the case of the
compounds 5m–s, the cooled solution was poured into
ice water and neutralised with sodium carbonate. The
precipitate formed was filtered and dissolved in
methanol. 1 mL concentrated hydrochloric acid was
added into the solution. The solvent was evaporated
under vacuum. The precipitate was crystallised from
methanol to give the hydrochloric acid salt of the
compound.
4.1.1.2. 1-[1-(2-Aryl-2-oxoethyl)benzimidazol-2-yl]-3-
arylpropenones (9a–h). A mixture of 7a–d (5 mmol), an
appropriate 8 (5 mmol) and potassium carbonate (6
mmol) in acetone (100 mL) was stirred at room temper-
ature for 6 h. The solvent was evaporated at low
temperature. The residue was washed with water and
then ethanol. The raw product was recrystallised from
ethanol.
4.1.1.3.
1-(2-Aryl6inyl)-3-arylpyrazino[1,2-a]benzimi-
dazole deri6ati6es (10a–h). A solution of suitable 9 (3
mmol) and ammonium acetate (30 mmol) in 50 mL of
acetic acid was refluxed for 1 h. The solution was
cooled, poured into ice water and neutralised with
sodium carbonate. The precipitate formed was filtered
and crystallised in ethanol.
3.2. Anti-HIV acti6ity
None of our compounds showed any anti-HIV
activity.
5a IR (KBr) wmax (cm−1): 3433–3252 (OꢁH), 2600–
4. Experimental protocols
2350 (N+ꢁH), 1708 (CꢀO), 1594–1479 (CꢀN, CꢀC),
1
1270 (CꢁO). H-NMR l (ppm): 1.89 (s, 3H), 7.00 (t,
4.1. Chemistry
1H), 7.33–7.51 (m, 10H), 7.73 (d, J=7.63 Hz, 2H),
7.85 (d, J=8.16 Hz, 1H), 8.39 (d, J=8.11 Hz, 1H),
8.58 (s, 1H), 8.83 (s, 1H), 12.00 (s, 1H). EIMS: m/z:
336.5 [M+1], 335.5 [M+], 334.5 [M−1, %100], 258,
167, 142. ESMS: m/z: 336.2 (%100).
Melting points were determined using an Electrother-
mal 9100 digital melting point apparatus and were
uncorrected. Spectroscopic data were recorded on the
following instruments. IR: Shimadzu 435 IR spec-
trophotometer; 1H-NMR: Bruker DPX 400 NMR spec-
trometer, in DMSO-d6, TMS as internal standard; MS:
5c IR (KBr)wmax (cm−1): 3443 (OꢁH), 1598–1490
1
(CꢀN, CꢀC), 1240 (CꢁO). H-NMR l (ppm): 3.76 (s,
3H), 7.00 (d, J=8.78 Hz, 2H), 7.37–7.68 (m, 8H), 7.72

258
S¸. Demirayak et al. / European Journal of Medicinal Chemistry 37 (2002) 255–260
Table 1
(ppm): 3.15–3.4 (m, 4H), 3.60 (t, 2H), 3.90–4.10 (m,
4H), 4.56 (t, 2H), 7.06 (d, J=8.86 Hz, 2H), 7.41 (d,
J=8.86 Hz, 2H), 7.43–7.52 (m, 2H), 7.59–7.70 (m,
4H), 7.75–7.81 (m, 1H), 7.94 (d, J=8.32 Hz, 1H), 8.67
(d, J=8.39 Hz, 1H), 9.12 (s, 1H), 9.51 (s, 1H), 12.03
(bs, 1H).
Some characteristics of the compounds
Compound m.p.
Yield Formulae
(%)
Molecular
weight
(°C)
5a
126–7 60
C
23H17N3·C2H4O2·
404.47
1/2H2O
5s IR (KBr) wmax (cm−1): 2660–2400 (N+ꢁH), 1598–
1500 (CꢀN, CꢀC), 1262 (CꢁO). 1H-NMR l (ppm):
3.30–3.45 (m, 4H), 3.71 (t, 2H), 3.75–3.85 (m, 4H),
4.46 (t, 2H), 6.95 (d, J=8.92 Hz, 2H), 7.29 (d, J=8.90
Hz, 2H), 7.37 (s, 1H), 7.45 (d, J=7.45 Hz, 2H),
7.49–7.51 (1H), 7.55–7.62 (1H), 7.66 (d, J=8.53 Hz,
2H), 7.99 (d, J=8.33 Hz, 1H), 8.51 (d, J=8.31 Hz,
1H), 9.05 (s, 1H), 9.13 (s, 1H), 11.19 (s, 1H).
9c IR (KBr) wmax (cm−1): 3490 (OꢁH), 1702–1660
(CꢀO), 1598–1450 (CꢀN, CꢀC). 1H-NMR l (ppm):
2.38 (s, 3H), 6.25 (s, 2H), 6.52 (bs, 1H), 7.02 (bs, 1H),
7.12 (d, J=8.50 Hz, 2H), 7.42–7.58 (m, 3H), 7.95–8.05
(m, 3H), 8.28 (d, J=17.05 Hz, 2H), 8.52 (m, 1H).
9e IR (KBr) wmax (cm−1): 3450 (OꢁH), 1696–1662
(CꢀO), 1610–1500 (CꢀN, CꢀC). 1H-NMR l (ppm):
6.22 (s, 2H), 6.56 (bs, 1H), 7.05 (bs, 1H), 7.40–7.65 (m,
3H), 7.71 (d, J=8.12 Hz, 2H), 7.80–7.90 (m, 2H), 8.11
(d, J=8.22 Hz, 2H), 8.32 (d, J=16.72 Hz, 2H), 8.45–
8.50 (m, 1H).
5b
5c
5d
5e
5f
5g
5h
5i
202–3 62
197–8 54
148–9 60
231–2 53
132–3 58
130–2 65
107–8 55
141–2 62
181–2 60
138–9 68
C24H19N3·2H2O
C24H19N3O·H2O
385.47
383.45
378.86
396.41
427.51
381.48
406.49
443.51
440.51
456.94
C
23H16ClN3·1/2H2O
C23H16N4O3
C24H19N3·2C2H4O2
C
25H21N3·H2O
C25H21N3O·C2H4O2
24H19N3O·C2H4O2
C
5j
5k
C25H21N3O2·5/2H2O
C23H16ClN3·2C2H4O2·
1/4H2O
C24H18ClN3O·C2H4O2
C30H30N4O·2HCl·H2O
C31H32N4O2·2HCl·H2O
5l
5m
5n
5o
5p
5r
5s
9b
9c
9d
9e
9f
9g
128–9 67
261–2 58
332–3 57
288–9 63
227–8 68
275–6 61
203–4 64
211–2 82
185–7 78
151–2 80
160–1 80
194–5 81
192–3 76
196–7 80
183–5 77
214–5 75
180–2 82
225–7 80
206–7 78
152–4 82
178–9 76
417.90
553.53
583.56
C30H29ClN4O·2HCl·H2O 587.98
C29H28N4O2·2HCl·H2O
C30H30N4O3·2HCl·H2O
C29H27ClN4O2·2HCl·H2O 589.95
555.50
585.53
C24H16Cl2N3O2·H2O
23H18N2O3·H2O
C23H18N2O4·H2O
22H15ClN2O3·H2O
C23H18N2O2S·H2O
467.32
388.41
404.41
408.83
404.47
420.47
424.89
581.32
351.39
367.39
371.80
367.45
383.45
399.87
C
C
9g IR(KBr)wmax (cm−1): 3470 (OꢁH), 1695–1655
(CꢀO), 1605–1450 (CꢀN, CꢀC). 1H-NMR l (ppm):
3.68 (s, 3H), 6.25 (s, 2H), 7.05 (t, 1H), 7.34–7.52 (m,
7H), 7.68 (d, J=8.61 Hz, 2H), 7.92 (d, J=16.21 Hz,
1H), 8.02 (d, J=7.59 Hz, 1H), 8.32 (d, J=16.18 Hz,
1H).
C
C
23H18N2O3S·H2O
22H15ClN2O2S·H2O
9h
10b
10c
10d
10e
10f
10g
10h
C24H15Cl2N3
C23H17N3O
C
C
23H17N3O2
22H14ClN3O
C23H17N3S
23H17N3OS
C23H14ClN3S
10b IR (KBr) wmax (cm−1): 1617–1450 (CꢀN, CꢀC).
1H-NMR l (ppm): 7.47–7.65 (m, 5H), 7.73 (d, J=8.32
Hz, 2H), 7.82 (d, J=8.12 Hz, 2H), 7.98 (d, J=16.20
Hz, 2H), 8.08 (d, J=8.32 Hz, 2H), 8.22 (d, J=8.16
Hz, 2H), 8.65 (s, 1H).
C
(d, J=7.63 Hz, 2H), 7.89 (d, J=8.16 Hz, 1H), 8.57 (d,
J=8.11 Hz, 1H), 8.91 (s, 1H), 9.02 (s, 1H). EIMS: m/z:
366 [M+1, %100], 365 [M+], 351, 334, 258, 244, 167.
5h IR (KBr)wmax (cm−1): 2600–2300 (N+ꢁH), 1705
10c IR (KBr) wmax (cm−1): 1630–1446 (CꢀN, CꢀC).
1H-NMR l (ppm): 3.81 (s, 3H), 6.61 (bs, 1H), 6.98 (bs,
1H), 7.12–7.60 (m, 6H), 7.88 (d, J=16.02 Hz, 1H),
8.02–8.10 (m, 3H), 8.28 (d, J=16.07 Hz, 1H), 8.71 (s,
1H).
1
(CꢀO), 1592–1480 (CꢀN, CꢀC), 1236 (CꢁO). H-NMR
l (ppm): 1.91 (s, 3H), 2.35 (s, 3H), 3.77 (s, 3H), 6.98 (d,
J=8.92 Hz, 2H), 7.29 (d, J=8.00 Hz, 2H), 7.43 (d,
J=8.89 Hz, 2H), 7.59 (d, J=8.10 Hz, 2H), 7.36–
7.55m, 2H, 7.08 (s, 1H), 7.86 (d, J=8.16 Hz, 1H), 8.39
(d, J=8.11 Hz, 1H), 8.36 (s, 1H), 8.73 (s, 1H), 12.00 (s,
1H).
10h IR (KBr) wmax (cm−1): 1627–1450 (CꢀN, CꢀC).
1H-NMR l (ppm): 7.04–7.56 (m, 8H), 7.90–8.12 (m,
4H), 8.26 (d, J=16.20 Hz, 1H), 8.81 (s, 1H).
4.2. Pharmacology
5k IR (KBr)wmax (cm−1): 2660–2240 (N+–H), 1704
4.2.1. Anticancer acti6ity
1
(CꢀO), 1597–1464 (CꢀN, CꢀC), 1255 (CꢁO). H-NMR
The cytotoxic and/or growth inhibitory effects of the
compounds were evaluated in vitro against approxi-
mately 60 human tumour cell lines derived from nine
neoplastic diseases, namely: leukaemia (L), non-small
cell lung cancer (NSCLC), colon cancer (CC), central
nervous system cancer (CNSC), melanoma (M),
ovarian cancer (OC), renal cancer (RC), prostate cancer
(PC), and breast cancer (BC). The evaluation of anti-
l (ppm): 1.92 (s, 3H), 7.03 (t, 1H), 7.35 (d, J=8.68 Hz,
2H), 7.33–7.56 (m, 7H), 7.78 (d, J=8.39 Hz, 2H), 7.87
(d, J=8.16 Hz, 1H), 8.40 (d, J=8.11 Hz, 1H), 8.62 (s,
1H), 8.88 (s, 1H), 12.00 (s, 1H). EIMS: m/z: 369 [M+],
334, 292, 258, 167, 166 (%100), 142.
5m IR (KBr) wmax (cm−1): 2650–2450 (N+–H),
1602–1500 (CꢀN, CꢀC), 1250 (CꢁO). 1H-NMR l

S¸. Demirayak et al. / European Journal of Medicinal Chemistry 37 (2002) 255–260
259
Table 2
Antiproliferative activities of the compounds (log GI50
)
Compound
L
NSCLC
CC
CNSC
M
OC
RC
PC
BC
MG-MID
5a
5b
5c
5d
5e
−4.50
−4.62
−4.64
−4.00
−5.39
−5.52
−4.54
−5.02
−5.76
−4.23
−4.42
−4.43
−5.51
−6.40
−5.44
−5.14
−4.70
−5.33
−4.74
−5.14
−4.80
−5.11
−4.49
−4.06
−4.61
−4.74
−5.02
−4.48
−5.48
−6.39
−4.36
−4.18
−4.86
−4.59
−5.23
−5.43
−4.82
−4.43
−4.96
−4.74
−5.22
−5.07
−4.76
−4.40
−4.54
−4.45
−4.65
−4.69
−4.38
−4.59
−4.32
−4.63
−4.25
−4.00
−4.09
−4.37
−4.32
−4.06
−5.17
−6.20
−4.65
−4.47
−4.75
−4.48
−5.19
−5.63
−4.73
−5.05
−5.50
−4.56
−4.78
−4.96
−5.04
−4.00
−4.82
−4.73
−4.70
−4.89
−4.51
−4.71
−4.49
−4.75
−4.38
−4.00
−4.12
−4.40
−4.50
−4.07
−5.11
−6.14
−4.29
−4.05
−4.91
−4.57
−5.00
−5.49
−4.38
−4.18
−4.89
−5.14
−5.03
−5.31
−4.80
−4.92
−4.50
−4.25
−4.53
−4.75
−4.42
−4.61
−4.22
−4.69
−4.21
−4.00
−4.10
−4.47
−4.17
−4.03
−5.12
−6.18
−4.27
−4.19
−4.68
−4.48
−4.87
−5.55
−4.45
−4.44
−5.24
−4.65
−4.75
−5.02
−4.85
−4.47
−4.87
−4.74
−4.60
−4.73
−4.54
−4.84
−4.46
−4.80
−4.13
−4.00
−4.22
−4.37
−4.47
−4.08
−5.08
−6.08
−4.27
−4.09
−4.62
−4.62
−5.14
−5.06
−4.39
−4.00
−4.91
−4.88
−4.87
−5.07
−4.66
−4.00
−4.41
−4.44
−4.67
−4.77
−4.36
−4.50
−4.32
−4.58
−4.26
−4.05
−4.15
−4.32
−4.27
−4.17
−5.18
−6.45
−4.27
−4.27
−4.69
−4.70
−5.38
−4.85
−4.22
−4.03
−4.81
−5.03
−4.74
−5.28
−4.70
−4.00
−4.44
−4.35
−4.64
−4.67
−4.42
−4.68
−4.29
−4.65
−4.28
−4.00
−4.05
−4.45
−4.02
−4.01
−4.99
−6.17
−4.23
−4.15
−4.65
−4.37
−4.81
−5.51
−4.41
−4.47
−4.63
−4.64
−4.53
−4.81
−4.89
−4.00
−4.68
−4.64
−4.50
−4.66
−4.42
−4.62
−4.31
−4.58
−4.10
−4.00
−4.06
−4.12
−4.00
−4.17
−4.49
−6.41
−4.45
−4.33
−4.66
−4.53
−5.08
−5.83
−4.79
−4.67
−5.54
−4.81
−4.78
−5.17
−4.97
−4.63
−4.73
−4.64
−4.62
−4.72
−4.53
−4.78
−4.46
−4.75
−4.28
−4.04
−4.26
−4.57
−4.54
−4.34
−4.79
−6.05
−4.37
−4.27
−4.72
−4.50
−5.14
−5.42
−4.54
−4.47
−5.17
−4.75
−4.82
−5.02
−4.90
−4.62
−4.72
−4.59
−4.64
−4.81
−4.50
−4.75
−4.42
−4.73
−4.28
−4.02
−4.18
−4.45
−4.41
−4.14
−5.09
−6.20
5f
5g
5h
5I
5j
5k
5l
5n
5o
5r
5s
9a
9b
9d
9e
9f
9h
10a
10b
10c
10e
10g
10h
A
B
Cells were exposed for 48 h to serial dilutions of compounds tested. Growth inhibition was evaluated by SRB assay. The optical density was read
with titertek microplate reader at 540 nm. Subsequent data analyses were performed with STATISTICA Microsoft software. Growth inhibition dose
(GI50; M) represents the concentrations which the percentage growth ii+50 as compared with control untreated cells (+100). The compounds,
whose log10 GI50 was higher than −4 were considered as not active. Leukaemia (L), non-small cell lung cancer (NSCLC), colon cancer (CC),
central nervous system cancer (CNSC), melanoma (M), ovarian cancer (OC), renal cancer (RC), prostate cancer (PC), breast cancer (BC).
Standard agents: A, melphalan; B, cis-diaminedichloroplatinum.
cancer activity was performed at the National Cancer
Institute of Bethesda, USA, following the in vitro
screening program, which is based upon the use of
multiple panels of 60 human tumour cell lines against
which our compounds were tested at 10-fold dilutions
of five concentrations ranging from 10−4 to 10−8 M.
The percentage growth was evaluated spectrophotome-
trically versus controls not treated with test agents. A
48 h continuous drug exposure protocol was followed
and a sulforhodamine B (SRB) protein assay was used
to estimate cell viability of growth [46,47]. The results
of the anticancer activity tests were given in Table 2.
served as basic control; the cultures were incubated at
37 °C in 5% carbon dioxide atmosphere for 6 days. The
tetrazolium salt XTT was added to all the wells and the
cultures were incubated to allow the development of
formazan colour by viable cells. Each well was analysed
spectrophotometrically to qualitate formazan produc-
tion and it was also viewed microscopically for detec-
tion of viable cells and confirmation of protective
activity. Drug treated virus-infected cells were com-
pared with drug treated non-infected cells.
4.2.2. Anti-HIV Acti6ity
Acknowledgements
The compound was dissolved in dimethylsulfoxide,
and then diluted 1:100 in culture medium before
preparing serial half log10 dilutions. T4 lymphocytes
(CEM cell line) were added and after a brief interval
HIV-1 was added resulting in a 1:200 final dilution of
the compound. Uninfected cells without the compound
Authors are grateful to Anadolu University Research
Fund for the support of this work and to V.L.
Narayanan and J.P. Bader of the National Institute of
Health, Maryland, Bethesda, USA, for anticancer and
anti-HIV screening.

260
S¸. Demirayak et al. / European Journal of Medicinal Chemistry 37 (2002) 255–260
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