Synthesis and anticancer activity of some 1,2,3-trisubstituted pyrazinobenzimidazole derivatives

Article abstract of DOI:10.3109/14756366.2013.858142

The synthesis of some new pyrazino[1,2-a]benzimidazole derivatives and investigation of their anticancer activities were aimed in this work. Thus, 2-acetylbenzimidazole was reacted with appropriate α-bromoacetophenones and potassium carbonate in acetone t

Full text of DOI:10.3109/14756366.2013.858142

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J Enzyme Inhib Med Chem, Early Online: 1–12
2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.858142
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ORIGINAL ARTICLE
Synthesis and anticancer activity of some 1,2,3-trisubstituted
pyrazinobenzimidazole derivatives
S¸eref Demirayak1 and Leyla Yurttas¸2
1
2
Department of Pharmaceutical Chemistry, Medipol University, Istanbul, Turkey and Department of Pharmaceutical Chemistry, Anadolu University,
_
Eski¸sehir, Turkey
Abstract
Keywords
The synthesis of some new pyrazino[1,2-a]benzimidazole derivatives and investigation of
their anticancer activities were aimed in this work. Thus, 2-acetylbenzimidazole was reacted
with appropriate a-bromoacetophenones and potassium carbonate in acetone to give
2-(2-acetyl-1H-benzimidazol-1-yl)-1-phenylethanone derivatives (3a–d). These diketone com-
pounds were reacted with varied benzylamines in acetic acid to obtain 2-benzyl-1-methylidene-
3-aryl-1,2-dihydropyrazino[1,2-a]benzimidazole derivatives (4a–t). The structures of the
obtained compounds were elucidated by using IR, 1H-NMR, 13C-NMR, MS spectral data and
elemental analyses results. Anticancer activities of the selected compounds were investigated
in National Cancer Institute, Bethesda, MD. 3c and 4n showed remarkable anticancer activity
comparing with standard drugs, melphalan and cisplatin.
1,2-disubstituted benzimidazole,
a,b-unsaturated carbonyl, anticancer
activity, pyrazino[1,2-a]benzimidazole
History
Received 8 July 2013
Revised 17 September 2013
Accepted 14 October 2013
Published online 24 January 2014
Introduction
Experimental section
Natural and synthetic a-methylene-g-lactone and quinone methide Chemistry
containing compounds are known with high anticancer activity by
All chemicals were purchased from Sigma-Aldrich Corp.
(St. Louis, MO) and Merck Chemicals (Merck KGaA,
Darmstadt, Germany). Melting points were determined using an
Electrothermal 9100 digital melting point apparatus
(Electrothermal, Essex, UK) and were uncorrected.
Spectroscopic data were recorded on the following instruments.
IR: Shimadzu 8400 FTIR spectrophotometer (Shimadzu, Tokyo,
mechanism acting as DNA alkylating agent1–5. For these com-
pounds, it was found that one of the structural requirement for
significant cytotoxicity was an O ¼ C–C ¼ CH2 system as part of
an ester, a ketone or lacton6. Natural compounds namely
elephantopin, eupatundin, vernolepin and helenalin including
a-methylene-g-lactone moiety in their structure (Figure 1) are
some of the compounds that have known with anticancer activities
and also high cytoxicities7–9. Toxic properties of these natural
compounds leaded medicinal chemists to synthesize new deriva-
tives including a,b-unsaturated carbonyl moiety as a pharmaco-
phoric group (Figure 2), which are thought to have high antitumor
activity with low cytotoxicity10.
1
Japan); H-NMR: Bruker DPX 500 NMR spectrometer (Bruker
Bioscience, Billerica, MA), in DMSO-d6, TMS as internal
standard; 13C-NMR: Bruker DPX 125 NMR spectrometer
(Bruker Bioscience), in DMSO-d6MS: AB SCIEX-3200
Q-TRAP LC/MS/MS MASS spectrometer (Fisons Instruments
Vertriebs GmbH, Mainz, Germany). Elemental analyses were
performed on a Leco TruSpec Micro CHN/CHNS elemental
analyzer (Leco, St. Joseph, MI).
Quinone methide moiety is also an important anticancer
active group, which has a,b-unsaturated carbonyl structure11–13
.
In studies, indole, benzimidazole and quinazoline rings have
been used as precursors of quinone methides because of their
excessive reactivity14–17. Reversibly binding to nucleophilic
2-(1-Hydroxyethyl)benzimidazole (1)
sites on telomeric DNA via Michael addition reaction is thought o-Phenylenediamine (100 mmol) and lactic acid (100 mmol) were
to be responsible for the anticancer activity of both of refluxed in 100 mL 4 N HCl solution for 8 h. The solution was
a-methylene-g-lactone and quinone methide chemical groups18–21
.
cooled, poured into ice water and neutralized with ammonia. The
In the light of these studies and as an extension of our previous precipitate was filtered and crystallised from ethanol–water.
works on anticancer active pyrazino[1,2-a]benzimidazole deriva- Yield: 71% m.p. 178–180 ꢀC (ref. 178.5–179.5 ꢀC)25.
tives22–24, we now report on the synthesis and the anticancer
activity testing of some 2-benzyl-1-methylidene-3-phenyl- 2-Acetylbenzimidazole (2)
1,2-dihydropyrazino[1,2-a]benzimidazole derivatives.
2-(1-Hydroxyethyl)benzimidazole (10 mmol) was dissolved in
100 mL acetic acid and heated to 90 ꢀC. The solution of
chromium trioxide (7.5 mmol) in 15 mL water was added
dropwise to this mixture and the temperature was fixed at
Address for correspondence: Leyla Yurttas¸, Department of
Pharmaceutical Chemistry, Anadolu University, Eskis¸ehir, Turkey. Tel:
90 ꢀC. After the addition, the mixture was cooled and poured into
water. The precipitate was filtered and extracted with chloroform.
þ90 222 335 05 80/3783. E-mail: lyurttas@anadolu.edu.tr

2
S¸. Demirayak & L. Yurttas¸
J Enzyme Inhib Med Chem, Early Online: 1–12
8.13 (2H, d, J: 8.09 Hz, Ar-H). 13C NMR (125 MHz, DMSO-d6):
O
O
ꢁ
27.56, 52.13, 112.39, 123.03, 124.57, 126.42, 129.43,
HO
O
O
130.36, 134.04, 135.21, 140.12, 143.36, 147.02, 193.82 and
194.10. For C17H14N2O2 calculated: 73.37% C, 5.07% H, 10.07%
N; found: 73.56% C, 5.02% H, 10.21% N. MS: m/z 279 (M þ 1).
O
O
O
O
O
OH
O
2-(2-Acetyl-1H-benzimidazol-1-yl)-1-(4-methoxyphenyl)ethanone
(3b)
O
O
Yield 65%. m.p. 141–142 ꢀC (ref. 141–142 ꢀC)27 IR (KBr)
ꢀmax (cm–1): 3052–3016 (Aromatic –C–H), 2983–2851
(Aliphatic –C–H), 1689 and 1674 (–C ¼ O), 1641–1463
Elephantopin
Eupatundin
1
(–C ¼ C, –C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 2.71 (3H, s,
OH
–COCH3), 3.72 (3H, s, –COCH3), 6.24 (2H, s, –CH2CO), 7.15–
7.24 (4H, m, Ar-H), 7.58 (2H, d, J: 8.15 Hz, Ar-H), 7.92 (2H, d, J:
8.14 Hz, Ar-H). 13C NMR (125 MHz, DMSO-d6): ꢁ 27.68, 52.94,
56.06, 113.08, 123.44, 124.52, 127.29, 130.07, 132.14, 139.52,
140.63, 142.45, 147.53, 150.12, 193.69 and 194.55. For
C18H16N2O3 calculated: 70.12% C, 5.23% H, 9.09% N; found:
70.16% C, 5. 25% H, 9.11% N. MS: m/z 309 (M þ 1).
H
H
O
O
O
O
H
O
H
OH
O
O
Vernolepin
Helenalin
Figure 1. Chemical structures of some natural a-methylene-g-lactones.
2-(2-Acetyl-1H-benzimidazol-1-yl)-1-(3-chlorophenyl)ethanone
(3c)
Yield 72%. m.p. 151–153 ꢀC. IR (KBr) ꢀmax (cm–1): 3068–3020
(Aromatic –C–H), 2993–2931 (Aliphatic –C–H), 1695 and 1675
(–C ¼ O), 1620–1452 (–C ¼ C, –C ¼ N). 1H-NMR (DMSO-d6):
ꢁ (ppm) 2.71 (3H, s, –COCH3), 6.24 (2H, s, –CH2CO), 7.39–7.42
(1H, m, Ar-H), 7.45–7.49 (1H, m, Ar-H), 7.69 (1H, t, J: 7.88 Hz,
Ar-H), 7.82 (1H, d, J: 8.24 Hz, Ar-H), 7.84–7.87 (1H, m, Ar-H),
7.91 (1H, d, J: 8.13 Hz, Ar-H), 8.06–8.09 (1H, m, Ar-H), 8.17
(1H, t, J: 1.82 Hz, J: 1.77 Hz, J: 1.81 Hz, Ar-H). 13C NMR
(125 MHz, DMSO-d6): ꢁ 27.98, 52.78, 112.66, 122.53, 124.90,
127.44, 128.02, 129.15, 132.28, 135.03, 135.17, 137.68, 138.19,
142.35, 147.21, 194.02 and 194.30. For C17H13ClN2O2 calcu-
lated: 65.29% C, 4.17% H, 8.96% N; found: 63.56% C, 4.02% H,
8.11% N. MS: m/z 312.9 (M þ 1).
O
O
O
O
O
O
O
O
O
O
Figure 2. Synthetic derivatives of a-methylene-g-lactones.
2-(2-Acetyl-1H-benzimidazol-1-yl)-1-(4-chlorophenyl)ethanone
(3d)
The solvent was evaporated at low pressure and the residue was
recrystallized from toluene.
Yield 71%. m.p. 161–162 ꢀC (ref. 161–162 ꢀC)22. IR (KBr) ꢀmax
(cm–1): 3045–3025 (Aromatic –C–H), 2933–2833 (Aliphatic –C–
Yield: 76% m.p. 188–190 ꢀC (ref. 188–189 ꢀC)26. IR(KBr)
ꢀmax (cm–1): 3288–2400 (–N–H), 1674 (–C ¼ O), 1600–1420
1
H), 1695 and 1674 (–C ¼ O), 1587–1452 (–C ¼ C, –C ¼ N). H-
1
NMR (DMSO-d6): ꢁ (ppm) 2.73 (3H, s, –COCH3), 6.24 (2H, s,
–CH2CO), 7.39–7.44 (2H, m, Ar-H), 7.70 (2H, d, J: 6.12 Hz, Ar-
H), 7.96–7.97 (1H, m, Ar-H), 8.10 (2H, d, J: 8.15 Hz, Ar-H), 8.22
(1H, d, J: 7.75 Hz, Ar-H). 13C NMR (125 MHz, DMSO-d6):
ꢁ 27.99, 52.62, 112.68, 122.52, 124.87, 127.10, 130.37, 131.35,
134.54, 138.22, 140.29, 142.36, 147.24, 193.97 and 194.31. For
C17H13ClN2O2 calculated: 65.29% C, 4.17% H, 8.96% N; found:
63.66% C, 4.05% H, 8.15% N. MS: m/z 313.1 (M þ 1).
(–C ¼ C, –C ¼ N). H-NMR (DMSO-d6) ꢁ: (ppm) 2.71 (3H, s,
–COCH3), 7.36–7.48 (2H, m, Ar-H), 7.86–8.14 (2H, m, Ar-H),
12.4 (1H, brs, N–H).
2-(2-Acetyl-1H-benzimidazol-1-yl)-1-arylethanone derivatives
(3a–d)
A mixture of the appropriate 2-acetylbenzimidazole (5 mmol),
2-bromoacetophenone (5 mmol) and potassium carbonate
(5 mmol) was stirred in acetone (50 mL) at room temperature.
Stirring was continued at room temperature until the disappear-
ance of the starting materials by TLC (4–6 h). The solvent was
evaporated at low pressure, and the residue was washed with
water and then ethanol. The raw product was recrystallized from
ethanol.
1-Methylidene-2-(4-substituted benzyl)-3-(4-substituted phenyl)-
pyrazino[1,2-a]benzimidazole (4a–t)
A mixture of the 3a–d (2 mmol) and an appropriate benzylamine
(2 mmol) was refluxed for 10 h in acetic acid (50 mL). At the end
of this time after cooling the mixture, ice water was poured into it
and the aqueous mixture was neutralized with sodium carbonate.
After removal of water, the obtained cohesive precipitate was
crystallized from ethanol.
2-(2-Acetyl-1H-benzimidazol-1-yl)-1-phenylethanone (3a)
Yield 75%. m.p. 167 ꢀC (ref. 166–168 ꢀC)27. IR (KBr) ꢀmax
(cmꢁ1): 3132–3061 (aromatic –C–H), 2937–2850 (aliphatic –C–
2-Benzyl-1-methylidene-3-phenyl-1,2-dihydropyrazino[1,2-a]
benzimidazole (4a)
1
H), 1686 and 1676 (–C ¼ O), 1593–1446 (–C ¼ C, –C ¼ N). H-
NMR (DMSO-d6): ꢁ (ppm) 2.71 (3H, s, –COCH3), 6.23 (2H, s,
–CH2CO), 7.36–7.48 (2H, m, Ar-H), 7.60 (1H, m, Ar-H),
7.72–7.82 (2H, m, Ar-H), 7.92 (2H, d, J: 7.95 Hz) Ar-H),
Yield 52%. m.p. 82–84 ꢀC. IR (KBr) ꢀmax (cm–1): 3057–3000
(Aromatic –C–H), 2926–2852 (Aliphatic –C–H), 1601–1471

DOI: 10.3109/14756366.2013.858142
1
1,2,3-Trisubstituted pyrazinobenzimidazole derivatives
3
(–C ¼ C, –C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 4.64 (2H, d, J: (–C ¼ C, –C ¼ N). 1H-NMR (DMSO-d6): ꢁ (ppm) 4.64 (2H,
6.22 Hz, Ar-CH2–), 6.62 (1H, s, Ar-H), 7.13 (1H, t, J: 6.28 Hz, d, J: 6.38 Hz, Ar-CH2–), 6.53 (1H, s, Ar-H), 7.22 (1H, t,
Ar-H), 7.23 (1H, t, J: 7.33 Hz, Ar-H), 7.32–7.38 (4H, m, Ar-H), J: 6.48 Hz, Ar-H), 7.35–7.40 (4H, m, Ar-H), 7.46–7.50 (5H, m,
7.45–7.50 (5H, m, Ar-H), 7.68 (2H, d, J: 8.02 Hz, Ar-H), 7.83 Ar-H), 7.69 (2H, d, J: 7.73 Hz, Ar-H), 7.83 (1H, d,
(1H, d, J: 8.16 Hz, ¼CH2), 8.35 (1H, d, J: 8.14 Hz, ¼CH2), 8.64 J: 8.18 Hz, ¼ CH2), 8.35 (1H, d, J: 8.10 Hz, ¼ CH2), 8.66 (1H, s,
(1H, s, pyrazinobenzimidazole C4–H). 13C NMR (125 MHz, pyrazinobenzimidazole C4–H). 13C NMR (125 MHz, DMSO-d6):
DMSO-d6): ꢁ 46.58, 101.17, 112.33, 113.49, 120.12, 121.89, ꢁ 45.74, 101.16, 112.47, 113.50, 121.92, 126.15, 127.78, 128.68,
126.13, 126.27, 127.75, 128.06, 128.4, 128.66, 129.63, 130.14, 129.56, 130.15, 130.27, 131.39, 132.53, 137.35, 138.98, 139.89,
131.40, 137.50, 139.04, 140.72, 143.24 and 144.64. For C24H19N3 143.20 and 144.64. For C24H18ClN3 calculated: 75.09% C, 4.73%
calculated: 82.49% C, 5.48% H, 12.03% N; found: 82.44% C, H, 10.95% N; found: 74.95% C, 5.02% H, 10.68% N. MS: m/z
5.17% H, 12.28% N. MS: m/z 350 (M þ 1).
383.7 (M þ 1).
2-(3-Methoxybenzyl)-1-methylidene-3-phenyl-1,2-dihydropyra-
zino[1,2-a]benzimidazole (4b)
2-Benzyl-1-methylidene-3-(4-methoxyphenyl)-1,2-dihydropyra-
zino[1,2-a]benzimidazole (4f)
Yield 50%. m.p. 105 ꢀC. IR (KBr) ꢀmax (cm–1): 3052–3024 Yield 51%. m.p. 136 ꢀC. IR (KBr) ꢀmax (cm–1): 3063–3012
(Aromatic –C–H), 2985–2886 (Aliphatic –C–H), 1573 – 1482 (Aromatic C–H), 2923–2876 (Aliphatic C–H), 1574–1486
1
1
(–C ¼ C, –C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 3.72 (3H, s, (–C ¼ C, –C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 3.81 (3H, s,
–CH3), 4.66 (2H, d, J: 6.02 Hz, Ar-CH2–), 6.56 (1H, s, Ar-H), –CH3), 4.64 (2H, d, J: 6.20 Hz, Ar-CH2–), 6.50 (1H, s, Ar-H),
7.10 (1H, t, J: 6.33 Hz, Ar-H), 7.18 (1H, t, J: 7.22 Hz, Ar-H), 7.03 (2H, d, J: 8.63 Hz, Ar-H), 7.09 (1H, t, J: 6.30 Hz, Ar-H),
7.28–7.41 (4H, m, Ar-H), 7.45–7.53 (4H, m, Ar-H), 7.62 (1H, d, 7.23 (1H, t, J: 7.17 Hz, Ar-H), 7.32–7.37 (3H, m, Ar-H),
J: 7.82 Hz, Ar-H), 7.78–7.80 (1H, m, Ar-H), 7.83 (1H, d, J: 7.46–7.49 (3H, m, Ar-H), 7.61 (2H, d, J: 8.59 Hz, Ar-H), 7.82
8.21 Hz, ¼CH2), 8.37 (1H, d, J: 8.0 Hz, ¼CH2), 8.67 (1H, s, (1H, d, J: 8.18 Hz, ¼CH2), 8.34 (1H, d, J: 7.11 Hz, ¼CH2),
pyrazinobenzimidazole C4–H). 13C NMR (125 MHz, DMSO-d6): 8.57 (1H, s, pyrazinobenzimidazole C4–H). 13C NMR (125 MHz,
ꢁ 46.82, 56.28, 102.52, 112.85, 113.68, 114.12, 115.23, 121.23, DMSO-d6): 46.07, 55.66, 102.41, 113.26, 115.23, 122.63,
121.87, 125.56, 126.47, 128.87, 129.79, 130.74, 131.45, 131.56, 128.42, 128.76, 129.02, 129.89, 130.22, 130.65, 131.49, 132.46,
132.74, 137.58, 139.10, 144.32 and 157.52. For C25H21N3O 136.54, 137.86, 138.72, 139.12, 143.57, 146.86 and 157.46.
calculated: 79.13% C, 5.58% H, 11.07% N; found: 79.17% C, For C25H21N3O calculated: 79.13% C, 5.58% H, 11.07% N;
5.61% H, 11.12% N. MS: m/z 380 (M þ 1).
found: 79.18% C, 5.59% H, 11.10% N. MS: m/z 380 (M þ 1).
2-(3-Chlorobenzyl)-1-methylidene-3-phenyl-1,2-dihydropyrazino
[1,2-a]benzimidazole (4c)
2-(3-Methoxybenzyl)-1-methylidene-3-(4-methoxyphenyl)-
1,2-dihydropyrazino[1,2-a]benzimidazole (4g)
Yield 54%. m.p. 74–75 ꢀC. IR (KBr) ꢀmax (cm–1): 3055–3030
(Aromatic –C–H), 2956–2902 (Aliphatic –C–H), 1585–1479
Yield 61%. m.p. 99 ꢀC. IR (KBr) ꢀmax (cm–1): 3046–3021
(aromatic –C–H), 2964–2859 (aliphatic –C–H), 1565–1446
(–C ¼ C, –C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 3.71 (3H, s,
1
1
(–C ¼ C, –C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 4.65 (2H, d,
J: 6.03 Hz, Ar-CH2–), 6.56 (1H, s, Ar-H), 7.25–7.55 (10H, m,
Ar-H), 7.69 (2H, d, J: 8.07 Hz, Ar-H), 7.84 (1H, d, J: 8.21 Hz,
¼CH2), 8.36 (1H, d, J: 8.0 Hz, ¼CH2), 8.66 (1H, s, pyrazino-
benzimidazole C4–H). 13C NMR (125 MHz, DMSO-d6): ꢁ 46.04,
102.52, 113.26, 114.41, 122.58, 127.23, 128.04, 129.19, 129.78,
130.03, 130.77, 131.48, 132.96, 138.46, 138.96, 139.56, 143.54
and 144.68. For C24H18ClN3 calculated: 75.09% C, 4.73% H,
10.95% N; found: 75.24% C, 4.77% H, 11.10% N. MS: m/z 383.6
(M þ 1).
–CH3), 3.81 (3H, s, –CH3), 4.60 (2H, d, J: 6.20 Hz, Ar-CH2–),
6.52 (1H, s, Ar-H), 6.80 (1H, d, J: 8.81 Hz, Ar-H), 7.03–7.10 (5H,
m, Ar-H), 7.25 (1H, t, J: 7.80 Hz, Ar-H), 7.35 (1H, t, J: 7.40 Hz,
Ar-H), 7.48 (1H, t, J: 7.64 Hz, Ar-H), 7.62 (2H, d, J: 8.46 Hz,
Ar-H), 7.82 (1H, d, J: 8.20 Hz, ¼CH2), 8.33 (1H, d, J:
8.00 Hz, ¼ CH2), 8.57 (1H, s, pyrazinobenzimidazole C4–H).
13C NMR (125 MHz, DMSO-d6): ꢁ 46.03, 55.69, 56.33, 102.40,
113.32, 113.46, 114.68, 121.69, 121.95, 123.44, 125.27, 126.75,
127.14, 127.76, 128.72, 130.61, 132.57, 135.24, 137.48, 141.54,
143.66, 145.58, 158.22 and 159.12. For C26H23N3O2 calculated:
76.26% C, 5.66% H, 10.26% N; found: 76.23% C, 5.69% H,
10.20% N. MS: m/z 410 (M þ 1).
2-(4-Methoxybenzyl)-1-methylidene-3-phenyl-1,2-dihydropyra-
zino[1,2-a]benzimidazole (4d)
Yield 63%. m.p. 102–103 ꢀC. IR (KBr) ꢀmax (cm–1): 3063–3015
(Aromatic –C–H), 2956–2898 (Aliphatic –C–H), 1554–1469
2-(3-Chlorobenzyl)-1-methylidene-3-(4-methoxyphenyl)-1,2-dihy-
dropyrazino[1,2-a]benzimidazole (4h)
1
(–C ¼ C, –C ¼ N). H-NMR (DMSO-d6): ꢁ 3.71 (ppm) (3H, s,
–CH3), 4.56 (2H, d, J: 6.25 Hz, Ar-CH2–), 6.57 (1H, s, Ar-H),
6.90 (2H, d, J: 8.72 Hz, Ar-H), 7.02 (1H, t, J: 6.28 Hz, Ar-H),
742–7.34 (4H, m, Ar-H), 7.47–7.50 (3H, m, Ar-H), 7.71 (2H, d, J:
7.38 Hz, Ar-H), 7.83 (1H, d, J: 8.17 Hz, ¼CH2), 8.35 (1H, d, J:
8.16 Hz, ¼CH2), 8.64 (1H, s, pyrazinobenzimidazole C4–H). 13C
NMR (125 MHz, DMSO-d6): 46.06, 56.25, 101.84, 113.48,
114.56, 122.87, 127.48, 128.64, 129.42, 129.89, 130.21, 130.79,
131.86, 132.74, 138.63, 138.97, 139.82, 143.43, 146.54 and
160.10. For C25H21N3O calculated: 79.13% C, 5.58% H, 11.07%
N; found: 79.19% C, 11.03% H, 11.08% N. MS: m/z 380 (M þ 1).
Yield 61%. m.p. 91–92 ꢀC. IR (KBr) ꢀmax (cm–1): 3061–3028
(Aromatic –C–H), 2961–2877 (Aliphatic –C–H), 1563–1486
1
(–C ¼ C, –C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 3.81 (3H, s,
–CH3), 4.65 (2H, d, J: 6.32 Hz, Ar-CH2–), 6.56 (1H, s, Ar-H),
7.04 (2H, d, J: 8.55 Hz, Ar-H), 7.22 (1H, t, J: 6.37 Hz, Ar-H),
7.28–7.38 (3H, m, Ar-H), 7.44–7.50 (2H, m, Ar-H), 7.57
(1H, m, Ar-H), 7.62 (2H, d, J: 8.55 Hz, Ar-H), 7.83 (1H, d,
J: 8.18 Hz, ¼CH2), 8.34 (1H, d, J: 8.15 Hz, ¼CH2), 8.58
(1H, s, pyrazinobenzimidazole C4–H). 13C NMR (125 MHz,
DMSO-d6): ꢁ 45.54, 57.45, 101.54, 110.62, 113.49, 115.89,
120.24, 121.46, 125.48, 126.36, 128.42, 129.12, 130.34, 131.45,
131.86, 132.85, 137.69, 139.73, 143.14, 144.37 and
159.46. For C25H20ClN3O calculated: 72.55% C, 4.87% H,
10.15% N; found: 72.52% C, 4.79% H, 10.10% N. MS: m/z
413.6 (M þ 1).
2-(4-Chlorobenzyl)-1-methylidene-3-phenyl-1,2-dihydropyrazino
[1,2-a]benzimidazole (4e)
Yield 65%. m.p. 104–106 ꢀC. IR (KBr) ꢀmax (cm–1): 3057–3032
(Aromatic –C–H), 2893–2845 (Aliphatic –C–H), 1562–1481

4
S¸. Demirayak & L. Yurttas¸
J Enzyme Inhib Med Chem, Early Online: 1–12
2-(4-Methoxybenzyl)-1-methylidene-3-(4-methoxyphenyl)-
1,2-dihydropyrazino[1,2-a]benzimidazole (4i)
For C25H20ClN3O calculated: 72.55% C, 4.87% H, 10.15% N;
found: 72.50% C, 4.83% H, 10.11% N. MS: m/z 413.6 (M þ 1).
Yield 55%. m.p. 148–149 ꢀC. IR (KBr) ꢀmax (cm–1): 3076–3012
(Aromatic –C–H), 2917–2837 (aliphatic –C–H), 1549–1453
2-(3-Chlorobenzyl)-1-methylidene-3-(3-chlorophenyl)-1,2-dihy-
dropyrazino[1,2-a]benzimidazole (4m)
1
(–C ¼ C, –C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 3.71 (3H, s,
–CH3), 3.81 (3H, s, –CH3), 4.55 (2H, d, J: 6.10 Hz, Ar-CH2–), Yield 72%. m.p. 105–106 ꢀC. IR (KBr) ꢀmax (cm–1): 3097–3020
6.53 (1H, s, Ar-H), 6.90 (2H, d, J: 8.40 Hz, Ar-H), 6.98 (1H, t, (aromatic C–H), 2840 (aliphatic C–H), 1591–1471 (C ¼ C,
1
J: 7.60 Hz, Ar-H), 7.05 (2H, d, J: 8.49 Hz, Ar-H), 7.35 (1H, t, J: C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 4.65 (2H, d, J: 6.03 Hz,
7.33 Hz, Ar-H), 7.40 (2H, d, J: 8.17 Hz, Ar-H), 7.47 (1H, t, Ar-CH2–), 6.56 (1H, s, Ar-H), 7.28–7.58 (9H, m, Ar-H), 7.67 (1H,
J: 7.58 Hz, Ar-H), 7.64 (2H, d, J: 8.51 Hz, Ar-H), 7.81 (1H, d, J: d, J: 7.76 Hz, Ar-H), 7.80 (1H, s, Ar-H), 7.84 (1H, d, J: 8.26 Hz,
8.19 Hz, ¼CH2), 8.33 (1H, d, J: 8.18 Hz, ¼CH2), 8.56 (1H, s, ¼CH2), 8.38 (1H, d, J: 8.12 Hz, ¼CH2), 8.76 (1H, s,
pyrazinobenzimidazole C4–H). 13C NMR (125 MHz, DMSO-d6): pyrazinobenzimidazole C4–H). 13C NMR (125 MHz, DMSO-
ꢁ 46.01, 55.79, 56.04, 101.26, 111.44, 113.43, 114.94, 115.51, d6): ꢁ 45.76, 101.66, 113.18, 113.60, 120.17, 122.07, 124.69,
120.06, 121.73, 126.02, 128.88, 129.71, 131.35, 132.45, 137.38, 126.27, 126.36, 127.18, 127.45, 128.01, 128.33, 128.47, 131.45,
143.14, 144.61, 159.62 and 160.29. For C26H23N3O2 calculated: 131.49, 131.94, 134.35, 135.02, 137.37, 141.15, 143.18, 143.62
76.26% C, 5.66% H, 10.26% N; found: 76.25% C, 5.71% H, and 144.65. For C24H17Cl2N3 calculated: 68.91% C, 4.10% H,
10.22% N. MS: m/z 410 (M þ 1).
10.05% N; found: 69.22% C, 4.07% H, 10.20% N. MS: m/z 419
(M þ 1).
2-(4-Chlorobenzyl)-1-methylidene-3-(4-methoxyphenyl)-1,2-dihy-
dropyrazino[1,2-a]benzimidazole (4j)
2-(4-Methoxybenzyl)-1-methylidene-3-(3-chlorophenyl)-1,2-dihy-
dropyrazino[1,2-a]benzimidazole (4n)
Yield 52%. m.p. 155 ꢀC. IR (KBr) ꢀmax (cm–1): 3063–3016
(aromatic –C–H), 2967–2837 (aliphatic –C–H), 1569–1416 Yield 64%. m.p. 88–89 ꢀC. IR (KBr) ꢀmax (cm–1): 3069–3029
1
(–C ¼ C, –C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 3.80 (3H, s, (Aromatic –C–H), 2837 (aliphatic –C–H), 1567–1459 (–C ¼ C,
–CH3), 4.63 (2H, d, J: 6.33 Hz, Ar-CH2–), 6.49 (1H, s, Ar-H), –C ¼ N). 1H-NMR (DMSO-d6): ꢁ (ppm) 3.72 (3H, s, –CH3), 4.62
7.04 (2H, d, J: 8.58 Hz, Ar-H), 7.17 (1H, t, J: 6.34 Hz, Ar-H), (2H, d, J: 6.20 Hz, Ar-CH2–), 6.54 (1H, s, Ar-H), 7.27–7.48 (9H,
7.34–7.40 (3H, m, Ar-H), 7.46–7.50 (3H, m, Ar-H), 7.61 (2H, d, m, Ar-H), 7.69 (2H, d, J: 8.12 Hz, Ar-H), 7.85 (1H, d, J: 8.11 Hz,
J: 8.62 Hz, Ar-H), 7.82 (1H, d, J: 8.22 Hz, ¼CH2), 8.33 (1H, d, J: ¼CH2), 8.35 (1H, d, J: 8.05 Hz, ¼CH2), 8.65 (1H, s,
8.14 Hz, ¼CH2), 8.57 (1H, s, pyrazinobenzimidazole C4–H). 13C pyrazinobenzimidazole C4–H). 13C NMR (125 MHz, DMSO-
NMR (125 MHz, DMSO-d6): ꢁ 45.77, 56.03, 101.31, 111.65, d6): ꢁ 45.96, 55.79, 101.62, 112.87, 113.55, 114.93, 120.14,
113.46, 115.51, 120.07, 121.79, 125.97, 126.04, 128.89, 129.55, 121.98, 124.76, 126.21, 126.34, 126.43, 127.44, 128.43, 129.79,
130.26, 131.23, 131.33, 132.52, 137.23, 139.91, 143.07, 144.61 131.45, 131.96, 132.09, 132.39, 135.02, 137.55, 141.26, 143.27,
and 160.31. For C25H20ClN3O calculated: 72.55% C, 4.87% H, 144.64 and 159.65. For C25H20ClN3O calculated: 72.55% C,
10.15% N; found: 72.53% C, 4.82% H, 10.13% N. MS: m/z 413.7 4.87% H, 10.15% N; found: 72.51% C, 4.82% H, 10.14% N. MS:
(M þ 1).
m/z 414.5 (M þ 1).
2-Benzyl-1-methylidene-3-(3-chlorophenyl)-1,2-dihydropyrazino
[1,2-a]benzimidazole (4k)
2-(4-Chlorobenzyl)-1-methylidene-3-(3-chlorophenyl)-1,2-dihy-
dropyrazino[1,2-a]benzimidazole (4o)
Yield 55%. m.p. 85–86 ꢀC. IR (KBr) ꢀmax (cm–1): 3059–3035 Yield 60%. m.p. 106–108 ꢀC. IR (KBr) ꢀmax (cm–1): 3052–3016
(Aromatic C–H), 2887 (aliphatic C–H), 1589–1465 (C ¼ C, (Aromatic –C–H), 2963–2841 (Aliphatic –C–H), 1590–1433
1
1
C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 4.66 (2H, d, J: 6.28 Hz, (–C ¼ C, –C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 4.65 (2H, d,
Ar-CH2–), 6.56 (1H, s, Ar-H), 7.13 (1H, t, J: 6.34 Hz, Ar-H), 7.23 J: 6.39 Hz, Ar-CH2–), 6.55 (1H, s, Ar-H), 7.21 (1H, t, J: 6.41 Hz,
(1H, t, J: 7.49 Hz, Ar-H), 7.32–7.44 (4H, m, Ar-H), 7.46–7.52 Ar-H), 7.38–7.44 (4H, m, Ar-H), 7.47–7.52 (4H, m, Ar-H), 7.67
(4H, m, Ar-H), 7.65 (1H, d, J: 7.74 Hz, Ar-H), 7.78–7.80 (1H, d, J: 7.74 Hz, Ar-H), 7.80 (1H, s, Ar-H), 7.84 (1H, d, J:
(1H, m, Ar-H), 7.84 (1H, d, J: 8.23 Hz, ¼CH2), 8.37 (1H, d, 8.18 Hz, ¼CH2), 8.36 (1H, d, J: 8.17 Hz, ¼CH2), 8.75 (1H, s,
J: 8.15 Hz, ¼CH2), 8.74 (1H, s, pyrazinobenzimidazole pyrazinobenzimidazole C4–H). 13C NMR (125 MHz, DMSO-d6):
C4–H). 13C NMR (125 MHz, DMSO-d6): 46.24, 102.48, 113.36, ꢁ 46.52, 101.25, 112.68, 113.96, 121.36, 123.16, 124.75, 126.37,
115.54, 122.28, 128.01, 128.13, 128.51, 129.04, 130.04, 126.89, 127.63, 128.29, 128.86, 128.94, 130.23, 131.53, 131.84,
130.47, 131.31, 133.52, 136.45, 137.88, 138.84, 139.17, 143.23 133.28, 135.69, 138.21, 140.25, 142.69, 143.58 and 144.85. For
and 146.45. For C24H18ClN3 calculated: 75.09% C, 4.73% C24H17Cl2N3 calculated: 68.91% C, 4.10% H, 10.05% N; found:
H, 10.95% N; found: 75.24% C, 4.97% H, 10.90% N. MS: m/z 69.10% C, 4.27% H, 10.10% N. MS: m/z 419 (M þ 1).
383.7 (M þ 1).
2-Benzyl-1-methylidene-3-(4-chlorophenyl)-1,2-dihydropyra-
2-(3-Methoxybenzyl)-1-methylidene-3-(3-chlorophenyl)-1,2-dihy-
dropyrazino[1,2-a]benzimidazole (4l)
zino[1,2-a]benzimidazole (4p)
Yield 54%. m.p. 174–176 ꢀC. IR (KBr) ꢀmax (cm–1): 3051–3012
Yield 62%. m.p. 115 ꢀC. IR (KBr) ꢀmax (cm–1): 3067–3012 (Aromatic –C–H), 2973–2850 (aliphatic –C–H), 1564–1469
1
(Aromatic –C–H), 2950–2837 (aliphatic –C–H), 1588–1451 (–C ¼ C, –C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 4.64 (2H, d,
1
(–C ¼ C, –C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 3.71 (3H, s, J: 6.30 Hz, Ar-CH2–), 6.54 (1H, s, Ar-H), 7.14 (1H, t, J: 6.40 Hz,
–CH3), 4.62 (2H, d, J: 6.23 Hz, Ar-CH2–), 6.52 (1H, s, Ar-H), Ar-H), 7.23 (1H, t, J: 7.33 Hz, Ar-H), 7.33–7.39 (3H, m, Ar-H),
7.35–7.52 (9H, m, Ar-H), 7.69 (1H, d, J: 7.68 Hz, Ar-H), 7.82 7.46–7.50 (3H, m, Ar-H), 7.53 (2H, d, J: 9.63 Hz, Ar-H), 7.73
(1H, s, Ar-H), 7.86 (1H, d, J: 8.16 Hz, ¼CH2), 8.37 (1H, d, J: (2H, d, J: 8.58 Hz, Ar-H), 7.83 (1H, d, J: 8.16 Hz, ¼CH2), 8.33
8.12 Hz, ¼CH2), 8.74 (1H, s, pyrazinobenzimidazole C4–H). 13C (1H, d, J: 8.18 Hz, ¼CH2), 8.70 (1H, s, pyrazinobenzimidazole
NMR (125 MHz, DMSO-d6): ꢁ 46.01, 56.04, 101.26, 111.44, C4–H). 13C NMR (125 MHz, DMSO-d6): ꢁ 45.69, 102.12, 113.54,
113.43, 114.95, 115.51, 120.06, 121.73, 126.02, 128.89, 129.72, 113.97, 122.89, 123.46, 124.15, 126.79, 126.87, 127.19, 128.47,
131.35, 132.46, 137.37, 143.14, 144.61 and 159.62. 128.84, 129.04, 130.47, 131.24, 131.87, 133.47, 135.59, 138.76,

DOI: 10.3109/14756366.2013.858142
1,2,3-Trisubstituted pyrazinobenzimidazole derivatives
5
140.33, 142.72, 143.14 and 144.46. For C24H18ClN3 calculated: pyrazinobenzimidazole C4–H). 13C NMR (125 MHz, DMSO-d6):
75.09% C, 4.73% H, 10.95% N; found: 75.20% C, 4.57% H, ꢁ 45.69, 101.46, 112.89, 113.45, 120.46, 122.68, 124.69, 127.21,
10.78% N. MS: m/z 383.7 (M þ 1).
127.78, 128.57, 128.89, 129.01, 131.63, 131.79, 132.21, 134.46,
135.34, 137.42, 141.63, 143.52, 143.88 and 144.17. For
C24H17Cl2N3 calculated: 68.91% C, 4.10% H, 10.05% N; found:
68.75% C, 3.92% H, 9.90% N. MS: m/z 419 (M þ 1).
2-(3-Methoxybenzyl)-1-methylidene-3-(4-chlorophenyl)-1,2-dihy-
dropyrazino[1,2-a]benzimidazole (4q)
Yield 59%. m.p. 118 ꢀC. IR (KBr) ꢀmax (cm–1): 3058–3029
(aromatic –C–H), 2964–2863 (aliphatic –C–H), 1586–1451
Anticancer activity tests
1
(–C ¼ C, –C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 3.72 (3H, s, The cytotoxic and/or growth inhibitory effects of the compounds
–CH3), 4.64 (2H, d, J: 6.72 Hz, Ar-CH2–), 6.52 (1H, s, Ar-H), were evaluated in vitro against approximately 60 human tumor
7.17 (2H, d, J: 8.45 Hz, Ar-H), 7.26 (1H, t, J: 6.75 Hz, Ar-H), cell lines derived from nine neoplastic diseases namely leukemia
7.25–7.34 (3H, m, Ar-H), 7.48–7.54 (2H, m, Ar-H), 7.59 (1H, m, (L, four or six cell lines), non-small cell lung cancer (NSCLC,
Ar-H), 7.64 (2H, d, J: 8.45 Hz, Ar-H), 7.88 (1H, d, J: 8.13 Hz, nine cell lines), colon cancer (CC, seven cell lines), central
¼CH2), 8.37 (1H, d, J: 8.19 Hz, ¼CH2), 8.71 (1H, s, nervous system cancer (CNSC, six cell lines), melanoma
pyrazinobenzimidazole C4–H). 13C NMR (125 MHz, DMSO- (M, eight or nine cell lines), ovarian cancer (OC, six or seven
d6): ꢁ 45.93, 56.08, 101.22, 113.12, 113.85, 114.76, 121.25, cell lines), renal cancer (RC, eight cell lines), prostate cancer (PC,
121.89, 123.65, 125.47, 126.89, 127.12, 127.82, 128.13, 129.65, two cell lines) and breast cancer (BC, six or eight cell lines). The
130.79, 132.02, 132.53, 135.22, 137.14, 141.13, 143.56, 144.48 evaluation of anticancer activity was performed at the National
and 158.74. For C25H20ClN3O calculated: 72.55% C, 4.87% H, Cancer Institute (NCI) of Bethesda, MD, USA, following the
10.15% N; found: 72.49% C, 4.83% H, 10.17% N. MS: m/z 413.6 in vitro screening program at 10-fold dilutions of five concentra-
(M þ 1).
tions ranging from 10–4 to 10–8 M. The percentage growth was
evaluated spectrophotometrically versus controls not treated with
test agents. A 48 h continuous drug exposure protocol was
followed and a sulforhodamine B protein assay was used to
estimate cell viability of growth. Three dose response parameters
(GI50, TGI and LC50) were calculated for each experimental
2-(3-Chlorobenzyl)-1-methylidene-3-(4-chlorophenyl)-1,2-dihy-
dropyrazino[1,2-a]benzimidazole (4r)
Yield 62%. m.p. 191–193 ꢀC. IR (KBr) ꢀmax (cm–1): 3056–3021
(aromatic –C–H), 2937–2864 (aliphatic C–H), 1557–1450
agent28–31
.
1
(–C ¼ C, –C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 4.66 (2H, d,
J: 6.42 Hz, Ar-CH2–), 6.53 (1H, s, Ar-H), 7.24 (1H, t, J: 6.51 Hz,
Ar-H), 7.36–7.40 (3H, m, Ar-H), 7.48–7.55 (3H, m, Ar-H), 7.54 Results and discussions
(2H, d, J: 8.51 Hz, Ar-H), 7.74 (2H, d, J: 8.63 Hz, Ar-H), 7.84
Chemistry
(1H, d, J: 8.19 Hz, ¼CH2), 8.35 (1H, d, J: 8.18 Hz, ¼CH2), 8.70
(1H, s, pyrazinobenzimidazole C4–H). 13C NMR (125 MHz, In this study, the syntheses of the new pyrazinobenzimidazoles
DMSO-d6): ꢁ 46.22, 101.54, 112.75, 113.41, 121.24, 122.52, (4a–t), have been carried out as shown in Scheme 1. First,
124.46, 126.78, 127.13, 128.28, 128.76, 129.38, 131.89, 132.67, o-phenylenediamine and lactic acid were reacted with 4 N HCl
132.83, 134.52, 135.29, 137.58, 141.29, 143.69, 143.92 and solution according to Phillips method to give 2-(1-hydroxyethyl)-
144.46. For C24H17Cl2N3 calculated: 68.91% C, 4.10% H, 10.05% benzimidazole. The obtained compound (1) was oxidized with
N; found: 68.66% C, 4.35% H, 10.20% N. MS: m/z 419 (M þ 1). chromium trioxide to give 2-acetyl benzimidazole (2) compound,
which was then reacted with appropriate a-bromoacetophenones
1
2-(4-Methoxybenzyl)-1-methylidene-3-(4-chlorophenyl)-1,2-dihy-
dropyrazino[1,2-a]benzimidazole (4s)
in the presence of potassium carbonate. IR, H-NMR and 13C-
NMR spectra were measured for the compounds 2 and 3a–d.
In the spectrum of the compound 2, bands were observed at about
3288–2400 cm–1 and 1674 cm–1 belonging to –N–H and –C ¼ O
bonds, respectively. Proton of the benzimidazole nitrogen was
observed at 12.4 ppm as broad singlet peak as expected. In the
spectrum of the compounds 3a–d, carbonyl bands were observed
1695–1674 cm–1 and methylene protons were observed at about
6.23–6.24 ppm. At the final step, 2-(2-acetyl-1H-benzimidazol-1-
yl)-1-(subtituted phenyl)ethanone derivatives (3a–d) and varied
benzylamines were refluxed in acetic acid to afford corresponding
2-(4-substituted benzyl)-1-methylidene-3-(4-substituted phenyl)-
pyrazino[1,2-a]benzimidazole (4a–t) derivatives. The resulting
products were yielded in the range of 50–72%. In the IR spectra of
the final compounds (4a–t), all bands were observed in expected
areas and in addition disappearance of stretching bands about
1695–1674 cm–1 belonging to ketone carbonyl was an evidence
for ring closure analyzed by IR spectroscopy. In the 1H-NMR
spectra of the final compounds, doublet peak was observed at
4.64–4.66 ppm belonging to –N–CH2 protons and the resonating
Yield 63%. m.p. 146 ꢀC. IR (KBr) ꢀmax (cm–1): 3059–3012
(Aromatic –C–H), 2954–2897 (aliphatic –C–H), 1565–1413
(–C ¼ C, –C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 3.71 (3H, s,
1
–CH3), 4.55 (2H, d, J: 8.21 Hz, Ar-CH2–), 6.56 (1H, s, Ar-H),
6.89 (2H, d, J: 8.58 Hz, Ar-H), 7.03 (1H, t, J: 8.24 Hz, Ar-H),
7.39–7.41 (3H, m, Ar-H), 7.48 (1H, t, J: 7.4 Hz, Ar-H), 7.54 (2H,
d, J: 8.46 Hz, Ar-H), 7.75 (2H, d, J: 8.48 Hz, Ar-H), 7.85 (1H, d,
J: 8.17 Hz, ¼CH2), 8.34 (1H, d, J: 8.14 Hz, ¼CH2), 8.68 (1H, s,
pyrazinobenzimidazole C4–H). 13C NMR (125 MHz, DMSO-d6):
ꢁ 45.87, 55.85, 101.54, 113.64, 113.89, 114.77, 120.58, 121.54,
125.63, 126.57, 127.56, 128.94, 129.96, 131.62, 132.05, 132.86,
133.29, 135.54, 137.73, 141.69, 143.12, 144.44 and 158.98. For
C25H20ClN3O calculated: 72.55% C, 4.87% H, 10.15% N; found:
72.48% C, 4.85% H, 10.12% N. MS: m/z 413.6 (M þ 1).
2-(4-Chlorobenzyl)-1-methylidene-3-(4-chlorophenyl)-1,2-dihy-
dropyrazino[1,2-a]benzimidazole (4t)
Yield 68%. m.p. 213–215 ꢀC. IR (KBr) ꢀmax (cm–1): 3041 of these protons as doublet was thought to be due to magnetic
(aromatic C–H), 2897 (aliphatic C–H), 1587–1460 (C ¼ C, anisotropy. The peaks seen at 7.83–8.38 ppm were assigned for
1
C ¼ N). H-NMR (DMSO-d6): ꢁ (ppm) 4.64 (2H, d, J: 6.42 Hz, methylene protons in the first position of pyrazinobenzimidazole
Ar-CH2–), 6.53 (1H, s, Ar-H), 7.24 (1H, t, J: 6.51 Hz, Ar-H), ring system. The signal belonging to the C4–H proton of the
7.36–7.40 (3H, m, Ar-H), 7.47–7.51 (3H, m, Ar-H), 7.54 (2H, d, pyrazinobenzimidazole condensed ring system was observed
J: 8.76 Hz, Ar-H), 7.74 (2H, d, J: 8.63 Hz, Ar-H), 7.84 (1H, d, much further downfield at about 8.64–8.75 ppm according to
J: 8.19 Hz, ¼CH2), 8.35 (1H, d, J: 8.18 Hz, ¼CH2), 8.70 (1H, s, the other aromatic protons. The other characteristic aromatic

6
S¸. Demirayak & L. Yurttas¸
J Enzyme Inhib Med Chem, Early Online: 1–12
OH
NH2
N
N
CH3
CH3
i
ii
+
COOH
H3C
N
H
N
H
O
OH
NH2
1
2
O
N
N
CH3
Br
iii
+
2
O
R
O
3a-d
R
N
N
4
R
R'
4
R
R'
NH2
iv
a
b
c
d
e
f
k
+
H
H
3a-d
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
H
l
H
H
H
H
3-OCH
3-Cl
4-OCH
4-Cl
3
3-OCH
3-Cl
4-OCH
4-Cl
3
R'
N
m
n
o
p
q
r
3
3
R'
4-OCH
4-OCH
4-OCH 3-Cl
4-OCH
4-OCH 4-Cl
H
3
H
g
h
i
3-OCH
3
3
3
3-OCH
3-Cl
4-OCH
4-Cl
3
3
3
4a-t
4-OCH
s
t
3
R
j
3
Scheme 1. The synthetic protocol of the compounds 3a–d and 4a–t.
The synthetic protocol of the compounds (4a–t). Reagents: (i) 4N HCl, reflux, 8 h, 71%; (ii) CrO3, CH3COOH, 90 ꢀC during the addition of chromium
trioxide solution addition then cooled, 76%; (iii) K2CO3, acetone, rt, 4–6 h, 65–75%; (iv) CH3COOH, reflux, 10 h, 50–72%.
protons were observed at expected areas, about 6.52–7.80 ppm. determined as growth percent values at 10–5 M concentration of
In the 13C NMR spectra of the compounds 3a–d, the characteristic the tested compounds. Results for each compound were reported
peaks were seen at about 193.82–194.55 ppm and 27.56– as a mean graph of the percent growth of the treated cells when
27.99 ppm belonging to –C ¼ O and –CH3 groups carbon. Peaks compared to the untreated control cells. The obtained growth
at about 45.54–46.82 ppm and 101.16–102.52 ppm belonging to percent values of the selected nine compounds were depicted in
–CH2– and ¼CH– carbon atoms were also seen in spectra of the Table 1.
final compounds (4a–t). The mass spectra of the compounds
As can be seen from the Table 1, according to the mean values
showed [M þ 1] peaks, in agreement with their molecular weight. 3c including 3-chloro phenyl moiety and 4n derived from 3c
All compounds gave satisfactory elemental analysis results in including 3-chloro phenyl and 4-methoxy benzyl moieties
correlation with the calculations. During the laboratory work, possessed significant low growth percentages among the other
another similar compound 2-(2-acetyl-1H-benzimidazol-1-yl)-1- tested compounds, which were found 24.53% and 22.62% (mean
(3-methoxyphenyl)ethanone was synthesized but 2-(substituted values), respectively. Among all of the cell lines, L cells were the
benzyl)-1-methylidene-3-(3-methoxyphenyl)-1,2-dihydropyra-
most susceptible cells against all tested compounds. Compounds
zino[1,2-a]benzimidazole derivatives which are planned to 3c and 4n had the lowest growth percentages, which were ꢁ7.27%
synthesize from this diketone compound could not be obtained and ꢁ0.37% (mean values) against L cells. Among the L cells,
purely. So, the indicated compounds did not include to this study. HL-60 (TB), SR and RPMI-8236 cell lines were defined as the
most susceptible cell lines against the compounds 3c and 4n. NCI-
H522 was found to be the most sensitive cell line through NSCLC
Anticancer activity evaluation
cells with the ꢁ15.98% and ꢁ10.93% growth percentages against
All final compounds (4a–t) and new diketone compound 3c were 3c and 4n compounds. The pointed two compounds induced
submitted to NCI for testing their anticancer activity according to growth percentage below 13% on HCT-116 and KM12 cell lines
in vitro drug screening protocol of the institute. The screening is a through CC cells. Against CNSC cell lines, 3c and 4n caused
two-stage process, beginning with the evaluation of all com- 8.27% and 10.72% growth percentages, and among these cell lines
pounds against the 60 cell lines at a single dose. 3c (NSC and among CNSC cancer cell lines and also all cell lines SF-539
748533), 4d (NSC 748537), 4e (NSC 748536), 4f (NSC 748535), was found to be one of most sensitive cell line with a growth value
4i (NSC 748540), 4j (NSC 748539), 4n (NSC 748541), 4p (NSC of ꢁ31.29%. The active two compounds were brought about
748534) and 4s (NSC 748538) were selected by NCI for the ꢁ32.33 to 90.58% growth percentage against M cell lines. The
anticancer tests for single dose testing. In vitro single dose highest activity was seen against MDA-MB-43 cell line, which
anticancer assay was performed in full NCI 60 cell panel was a M cancer cell line by compound 4n with a value of
representing L, NSCLC, CC, CNSC, M, OC, RC, PC and BC. ꢁ32.33%. Compounds 3c and 4n caused ꢁ31.30, ꢁ28.13%
In accordance with the protocol of the NCI, test results were growth percentage against OVCAR-3 and 4p caused ꢁ8.06%

DOI: 10.3109/14756366.2013.858142
1,2,3-Trisubstituted pyrazinobenzimidazole derivatives
7
Table 1. Sixty human tumor cell lines’ anticancer screening data at single dose assay as percent cell growth promotion of selected compounds.
Compounds
Cell lines
3c
4d
4e
4f
4i
4j
4n
4p
4s
L
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8236
SR
5.08
–28.87
–0.21
4.87
–11.83
–12.63
82.04
61.87
70.42
70.01
73.24
75.83
99.07
43.76
60.26
67.96
76.68
42.75
78.95
80.04
73.50
71.48
39.44
47.29
80.25
62.86
47.48
57.36
48.43
52.13
90.00
67.68
78.29
77.49
50.64
72.43
47.21
–25.03
0.53
8.13
–15.64
–17.39
78.93
60.15
59.12
58.46
35.25
23.70
86.32
53.04
59.25
64.80
30.93
–2.20
NSCLC
A549/ATCC
EKVX
HOP-62
15.25
17.06
55.75
75.62
104.70
36.61
48.22
4.22
84.92
75.16
134.64
86.79
97.32
63.37
97.04
90.34
72.68
87.16
81.17
125.50
75.61
92.71
80.87
96.97
91.76
73.54
96.00
62.59
136.46
95.87
102.38
81.31
102.47
90.84
64.47
79.22
73.42
130.11
58.30
96.17
78.37
113.70
88.60
72.77
87.43
80.76
142.98
77.68
98.52
85.05
117.07
88.76
68.19
14.32
8.80
70.56
70.79
105.64
73.65
76.86
73.41
96.01
88.22
66.35
84.54
71.27
118.31
77.44
80.83
54.18
100.79
81.32
57.28
41.19
77.90
94.08
29.39
44.64
4.58
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
–15.98
–10.93
CC
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
116.88
29.91
5.82
27.32
93.95
8.72
103.00
77.52
72.49
78.21
73.99
100.32
95.39
104.39
92.03
79.97
82.16
91.79
103.84
127.83
92.78
82.11
73.68
73.78
73.24
98.46
88.71
84.08
86.84
64.02
75.21
77.55
93.62
81.97
72.28
101.52
73.08
89.15
96.51
101.38
95.69
95.04
25.70
12.82
25.66
99.11
12.95
20.62
104.70
89.23
63.02
78.20
77.45
86.88
86.94
71.05
80.42
63.48
67.35
88.80
95.44
92.66
KM12
SW-620
23.03
CNSC
SF-268
SF-295
SF-539
SNB-19
SNB-75
U-251
13.16
8.35
–31.29
24.10
20.99
14.31
96.96
83.39
105.36
82.42
97.82
83.52
124.80
100.83
104.20
85.11
120.62
91.67
89.24
102.67
115.47
79.32
109.33
80.55
84.58
85.19
107.64
73.40
105.08
85.09
104.73
107.37
114.42
73.82
108.24
91.11
21.75
2.35
–14.95
20.37
16.46
18.35
90.33
89.78
96.39
85.27
–
96.09
94.91
104.61
79.14
103.24
92.84
90.92
M
LOX IMVI
MALME-3M
M14
MDA-MB-43
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
36.89
90.58
16.58
–27.42
61.92
67.72
–13.23
36.52
31.46
96.59
107.73
101.51
97.54
74.75
121.53
88.53
95.94
95.75
104.80
59.59
94.15
94.07
71.80
126.92
111.93
91.88
90.88
91.07
109.27
98.08
95.05
57.68
118.52
87.13
97.11
88.62
94.21
108.81
99.69
96.58
42.17
100.22
89.96
94.84
93.19
101.33
105.75
103.25
106.51
102.15
119.44
94.32
37.18
76.81
22.27
–32.33
28.21
67.67
–31.06
34.38
44.71
90.54
94.53
85.22
98.10
69.73
147.42
79.06
75.41
71.03
87.54
99.30
81.15
97.89
70.43
88.91
72.35
89.97
68.02
99.44
86.13
OC
IGROV1
–
69.76
94.76
79.86
77.82
95.19
69.61
105.52
75.59
111.35
103.76
101.70
92.27
96.65
85.39
80.07
100.51
88.25
77.38
107.39
74.40
93.80
91.53
86.75
79.22
68.58
107.63
80.39
97.63
101.10
103.90
91.41
83.74
89.61
–
106.30
92.16
–8.06
99.92
75.61
79.44
83.45
55.23
85.41
71.56
72.08
91.12
62.68
85.83
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
–31.30
22.93
47.52
10.07
13.86
17.49
–28.13
32.45
35.26
9.77
10.19
5.13
84.12
107.66
RC
786-0
A498
35.81
88.52
51.27
5.79
83.91
91.41
95.02
91.64
71.73
101.32
95.93
65.28
85.38
108.49
94.72
88.09
86.88
105.78
103.27
66.41
83.60
69.71
96.15
100.92
100.79
98.69
84.84
84.40
88.85
95.53
76.37
109.51
121.76
80.35
82.90
100.22
97.47
114.08
82.42
99.82
35.47
91.68
49.20
3.02
89.51
84.69
81.38
79.01
65.65
95.15
119.12
48.24
94.03
90.46
89.53
85.71
77.53
92.21
137.90
52.18
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
2.00
4.81
31.88
34.34
28.28
36.99
30.82
9.80
118.19
54.57
155.99
54.94
PC
DU-145
BC
5.27
93.14
132.14
102.71
94.60
92.76
15.29
103.20
103.14
MCF7
1.85
28.06
18.99
34.85
–11.32
32.66
82.25
109.80
108.22
119.02
58.31
85.59
106.86
196.08
117.67
67.12
86.26
99.97
116.05
–
59.87
–
79.30
94.86
91.32
143.00
46.13
89.00
77.25
101.40
105.86
97.11
57.03
97.02
4.64
36.28
15.47
–1.26
–22.52
32.02
62.95
85.37
87.01
142.39
66.04
59.06
53.99
90.09
103.87
97.82
48.05
80.79
MDA-MB-231a
HS 578T
BT-549
T-47D
MDA-MB468
91.60
107.90
(continued )

8
S¸. Demirayak & L. Yurttas¸
J Enzyme Inhib Med Chem, Early Online: 1–12
Table 1. Continued
Compounds
Cell lines
3c
4d
4e
4f
4i
4j
4n
4p
4s
Mean
Delta
Range
24.53
55.83
88.43
30.12
76.33
94.57
51.82
88.56
49.12
97.02
85.51
43.34
93.13
42.49
22.62
54.95
80.77
88.83
80.08
82.28
148.18
153.33
100.83
105.35
131.44
155.48
140.10
aMDA-MB-231/ATCC.
growth percentage against OVCAR-4, which were OC cell lines. concentration of the compound causing 50% decrease in net cell
Tested compounds showed the lowest activity to the RC cell lines growth, the TGI value (cytostatic activity) is the concentration of
with the growth percentages between 2 and 155.99% among all the compound resulting in total growth inhibition and LC50 value
tested cancer cell lines. As well as other types of cancer, (cytotoxic activity) is the concentration of the compound causing
compounds 3c and 4n was found to be the most active compounds net 50% loss of initial cells at the end of the incubation period of
with the respective values of 5.27 and 15.29% against PC. BT-549 48 h. A mean graph midpoint (MG-MID) was calculated for each
and T-47D cell lines were found to be the most sensitive cells subpanels of cancer types for the tested compounds and standard
through BC.
drugs cisplatin and melphalan, which are two of the commonly
Compounds 3a, 3b and 3d were synthesized, and 3a and 3d used chemotherapeutic agents by giving log10GI50, log10TGI and
were investigated for their anticancer activities in our previous log10LC50 (Table 3). The test method states that the compounds
study22. In addition similar diketone compounds and ring closure having log10GI50 values greater than ꢁ4 were considered as
products obtained from these diketone compounds were studied inactive. It can be seen that both of the compounds log10GI50
and anticancer activities of diketone compounds were found to be values are smaller than ꢁ4. The title compounds under investi-
higher than final compounds. This situation supports the finding gation (3c and 4n) exhibited remarkable anticancer activity
that intermediate diketone compound 3b had significant activity against all the tested cell lines representing nine different
compared with final products in this study. But increasingly, final subpanels with log10GI50 values between ꢁ7.61 and ꢁ5.18 for
compound 4n synthesized from 3c also showed as higher activity 3c and ꢁ6.70 and ꢁ4.81 for 4n. MDA-MB-43 (M cell line) and
as 3c, in contrast to other studies22–24. Other final products were HL-60 (TB) (L cell line) were found to be the most sensitive cell
found to be inactive with growth percentages greater than 80%. lines against compounds 3c and 4n with log10GI50 values of
Trying to explain the situation of the substituent effect is needed ꢁ7.61 and ꢁ6.70, respectively. According to subpanel average
but anticancer test assay was not performed for all compounds due log10GI50 values, L cancer was found to be the most sensitive
to NCI protocol. However, according to obtained test results, cancer type against both of the tested compounds 3c and 4n.
4-methoxybenzyl moiety on the second position pyrazinobenzi- Compound 3c possessed a value of log10GI50 ꢁ6.64, which was
midazole condensed ring system has increased activity a little bit smaller than standard drugs values (log10GI50-melphalan: ꢁ5.48,
with respect to chloro substitution. Another explanation is needed log10GI50-cisplatin: ꢁ6.39) against L. Furthermore, according to
for the lack of the activity of the final compounds except 4n (all cancer types) subpanel average log10GI50 values, both of the
when evaluated older studies. According to earlier studies, two compounds possessed smaller values than melphalan.
2-phenyl-attached pyrazinobenzimidazole compounds showed Therefore, we may conclude that both of our compounds under
good anticancer activity, but in this study, the new compounds investigation provide a notable activity level compared with
reported to have a benzyl group attached at the same position but standard drugs.
the activity shown is less than the former compounds. This
situation can be related with disconnecting of methylene group
Conclusions
(¼CH–) at the first position of the pyrazinobenzimidazole, which
is considered to be the active part of the molecule and binds to
biological receptor. Because of the molecular structure of benzyl
residue (conformations); alkylation reaction of the methylene
group with the nucleophilic sites of DNA can be avoided and so
activity is lacked.
Twenty
2-benzyl-1-methylidene-3-aryl-1,2-dihydropyrazi-
no[1,2-a]benzimidazole derivatives (4a–t) were synthesized
from four different diketone compounds (3a–d). The obtained
final compounds (4a–t) and diketone compounds (3a–d) were
offered to American NCI. Anticancer activities of the selected
compounds were evaluated in vitro against approximately
60 human cell lines derived from nine neoplastic diseases, and
the results were given as growth percentage values. Compound
3c namely 2-(2-acetyl-1H-benzimidazol-1-yl)-1-(3chloropheny-
l)ethanone and compound 4n derived from 3c namely 2-(4-
methoxybenzyl)-1-methylidene-3-(3-chlorophenyl)-1,2-dihydro-
pyrazino[1,2-a]benzimidazole were found to be as the most active
compounds with the lowest growth percentages 24.53 and
22.62%, respectively. In addition, log10GI50 value (log10 of
molar sample concentration resulting in 50% growth inhibition)
of the (MG-MID) compound 3c was found to be ꢁ6.32, which
was smaller than anticancer drug cisplatin (log10GI50: ꢁ6.20) and
log10GI50 value of the compound 4n was found to be ꢁ5.87,
which was smaller than anticancer drug melphalan (log10GI50:
ꢁ5.09). In connection with the result of this work and earlier
works, we plan to synthesize novel compounds including diketone
In the second stage of the anticancer screening tests, five-dose
assay was performed to the selected compounds against same 60
cell line at five different concentrations. The compounds that
reduced the growth of the cell lines to 32% or less (negative
number indicate kills) were considered in vitro active32.
Compounds 3c and 4n satisfied pre-determined threshold
growth inhibition criteria and further selected for NCI full panel
five-dose assay at 10-fold dilutions of five different concentra-
tions (0.01, 0.1, 1, 10 and 100 mM) Dose response curves obtained
from the NCI’s in vitro disease-oriented human tumor cells line of
compounds 3c and 4n on nine cancer disease at five concentra-
tions were shown in Figures 3 and 4. The results of tested
compounds were also given by three response parameters (GI50,
TGI and LC50) for each cell line from log concentration of %
growth inhibition curves on nine cancer diseases (Table 2). The
GI50 value (growth inhibitory activity) corresponds to the

DOI: 10.3109/14756366.2013.858142
1,2,3-Trisubstituted pyrazinobenzimidazole derivatives
9
Figure 3. Dose response curves (% growth verses sample concentration at NCI fixed protocol, mM) obtained from the NCI’s in vitro disease-oriented
human tumor cells line of compound (3c) on nine cancer diseases.

10 S¸. Demirayak & L. Yurttas¸
J Enzyme Inhib Med Chem, Early Online: 1–12
Figure 4. Dose response curves (% growth verses sample concentration at NCI fixed protocol, mM) obtained from the NCI’s in vitro disease-oriented
human tumor cells line of compound (4n) on nine cancer diseases.

DOI: 10.3109/14756366.2013.858142
1,2,3-Trisubstituted pyrazinobenzimidazole derivatives 11
Table 2. NCI DTP in vitro testing results of compounds 3c and 4n at five-dose assay.
3c values (mM)
4n values (mM)
Cell lines
Log10GI50
Log10TGI
Log10LC50
Log10GI50
Log10TGI
Log10LC50
L
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8236
–6.24
–6.95
–7.15
–6.18
–6.68
4–4.00
–6.14
–4.71
–4.05
–4.56
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
–5.75
–6.70
–6.54
–5.49
–6.38
4–4.00
–6.07
–4.80
–4.65
–4.85
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
NSCLC
A549/ATCC
EKVX
HOP-62
–5.71
–5.99
–6.38
–5.42
–5.35
–6.53
–5.79
–6.47
–7.11
4–4.00
4–4.00
–4.50
–4.13
4–4.00
–4.65
4–4.00
–5.74
–6.16
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
–5.38
–5.91
–5.71
–6.07
–5.18
–6.25
–5.06
–6.39
–6.52
4–4.00
–4.91
–4.36
–4.17
–4.45
–4.89
4–4.00
–4.99
–5.71
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
CC
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
–5.18
–6.38
–6.47
–6.74
–5.06
–6.05
ꢁ4.20
–5.78
4–4.00
4–4.00
4–4.00
–4.73
4–4.00
–4.87
4–4.00
4–4.00
4–4.00
4–4.00
–5.19
–5.91
–6.27
–6.24
–5.16
–5.60
–4.61
–5.49
–4.94
4–4.00
–4.12
–4.83
–4.14
–5.08
4–4.00
4–4.00
4–4.00
4–4.00
KM12
CNSC
SF-268
SF-295
SF-539
SNB-19
SNB-75
U-251
–6.35
–6.44
–6.62
–6.28
–6.50
–6.37
–4.29
–5.27
–6.11
4–4.00
–4.22
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
–6.08
–5.77
–6.15
–6.03
–6.23
–6.07
–4.49
–5.05
–5.30
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
M
LOX IMVI
MALME-3M
M14
MDA-MB-43
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
–6.35
–6.27
–6.84
–7.61
–6.45
–6.14
–7.02
–5.83
–6.27
4–4.00
4–4.00
4–4.00
–
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
–6.07
–6.08
–6.16
–6.65
–6.11
–4.98
–6.53
–4.98
–5.69
4.67
–4.09
4–4.00
4–4.00
–4.16
–4.26
–5.46
4–4.00
–4.32
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
–4.46
–4.02
4–4.00
– 4.93
4–4.00
–5.33
4–4.00
4–4.00
OC
IGROV1
–6.33
–6.76
–6.28
–6.56
–6.13
–6.79
–6.32
4–4.00
4–4.00
4–4.00
–4.29
4–4.00
–6.19
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
–6.22
–6.53
–5.69
–5.50
–5.48
–6.36
–5.61
–4.70
–
4–4.00
–5.02
4–4.00
–5.47
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
4–4.00
RC
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
–6.39
–5.31
–6.04
–6.00
–6.58
–6.29
–5.72
–6.27
–4.77
–4.08
4–4.00
–5.39
–6.06
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
–5.47
–4.81
–5.74
–5.82
–5.68
–5.87
–5.03
–5.80
–4.77
–4.04
4–4.00
–5.10
–5.09
–4.50
4–4.00
–4.52
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
PC
PC-3
DU-145
–6.39
–6.49
4–4.00
–5.00
4–4.00
4–4.00
–5.79
–5.74
4–4.00
–4.54
4–4.00
4–4.00
BC
MCF7
–6.50
–6.09
–6.53
–6.38
–6.46
–6.55
4–4.00
4–4.00
–
4–4.00
4–4.00
–5.56
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
–6.45
–6.06
–6.27
–5.55
–5.66
–5.93
4–4.00
4–4.00
4–4.00
4–4.00
–4.19
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
4–4.00
MDA-MB-231a
HS 578T
BT-549
T-47D
MDA-MB468
–4.70
aMDA-MB-231/ATCC.

12 S¸. Demirayak & L. Yurttas¸
J Enzyme Inhib Med Chem, Early Online: 1–12
Table 3. Antiproliferative activities of the compounds (log10GI50).
Comp
L
NSCLC
CC
CNSC
M
OC
RC
PC
BC
MG-MID
3c
4n
A
–6.64
–6.17
–5.48
–6.39
–6.08
–5.83
–5.17
–6.20
–6.08
–5.81
–5.11
–6.14
–6.43
–6.06
–5.12
–6.18
–6.53
–5.92
–5.08
–6.08
–6.45
–5.91
–5.18
–6.45
–6.08
–5.53
–4.99
–6.17
–6.44
–5.77
–4.49
–6.41
–6.42
–5.99
–4.79
–6.05
–6.32
–5.87
–5.09
–6.20
B
Growth inhibition dose (GI50; M) represents the concentration required for 50% inhibition of growth of the treated cells. The compounds, whose
log10GI50 was higher than ꢁ4 were considered as not active.
A: melphalan, B: cisplatin, L: leukemia, NSCLC: non-small cell lung cancer, CC: colon cancer, CNSC: central nervous system cancer, M: melanoma,
OC: ovarian cancer, RC: renal cancer, PC: prostate cancer, BC: breast cancer, MG-MID: mean-graph midpoint.
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