Synthesis and evaluation of antitumour activities of novel fused tri-And tetracyclic uracil derivatives

Article abstract of DOI:10.3184/174751916X14798125870610

Simple one-pot syntheses of indenopyrrolopyrimidines and indolopyrrolopyrimidines were achieved via the cyclocondensation of 6-Aminouracils and, respectively, ninhydrin and isatin in the presence of catalytic amounts of glacial acetic acid. Similarly, 5,6-diaminouracil derivatives were used as starting materials for the synthesis of indenopteridines and indolopteridines via their reaction with ninhydrin and isatin, respectively. The synthesised compounds were evaluated for antitumour activity against a human hepatocellular carcinoma cell line (HepG2), some showing antitumour activity comparable with 5-fluorouracil and imatinib.

Full text of DOI:10.3184/174751916X14798125870610

JOURNAL OF CHEMICAL RESEARCH 2016
VOL. 40 DECEMBER, 771–777
RESEARCH PAPER 771
Synthesis and evaluation of antitumour activities of novel fused tri- and
tetracyclic uracil derivatives
Samar ElKalyoubi* and Eman Fayed
Department of Organic Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Nasr City, Cairo, Egypt
Simple one-pot syntheses of indenopyrrolopyrimidines and indolopyrrolopyrimidines were achieved via the cyclocondensation of
6-aminouracils and, respectively, ninhydrin and isatin in the presence of catalytic amounts of glacial acetic acid. Similarly, 5,6-diaminouracil
derivatives were used as starting materials for the synthesis of indenopteridines and indolopteridines via their reaction with ninhydrin and
isatin, respectively. The synthesised compounds were evaluated for antitumour activity against a human hepatocellular carcinoma cell
line (HepG2), some showing antitumour activity comparable with 5-fluorouracil and imatinib.
Keywords: 6-aminouracils, 5,6-diaminouracil, indenopyrrolopyrimidines, indolopyrrolopyrimidines, indenopteridines, indolopteridines
In recent years, pyrimidine derivatives have gained a lot of
attention due to their various biological activities including
antifungal, antibacterial, antitumour, antitubercular and herbicidal
properties.1–3 Kidder and Dewey have noted that the biological
activity of pyrimidine differs with different substitutions.4
Pyrrolopyrimidines such as imatinib are significant anticancer
drugs which act as protein kinase inhibitors.5 On the other hand,
many alkaloids6 and drugs7,8 contain isatin as a main active
ingredient. Researches indicate that different derivatives of
isatin have a number of bioactivities including antibacterial,9
the presence of acetic acid for 10–15 min. On the other hand,
dihydroxyindolopyrrolopyrimidines 7–9 were obtained by
refluxing isatin and 6-aminouracils 1 in the presence of acetic
acid for 2–2.5 h as shown in Scheme 1.
The formation of 4–6 and 7–9 (Scheme 2) occurred via the
initial attack of the nucleophilic C-5 of the uracil moiety, via
its enol form, at the C-2 position of ninhydrin or at the C-3
position of isatin to yield non-isolable tricyclic intermediates.
The final tetracyclic products 4–6 and 7–9 were formed via
intramolecular cyclisation of the tricyclic intermediates by the
attack of the 6-amino group of uracil, respectively, at C-1 of the
carbonyl group of ninhydrin and at C-2 of the carbonyl group
of isatin.
13
antifungal,10 antiviral,11 anti-HIV,12 anti-mycobacterial, anticancer
14
anti-inflammatory and anticonvulsant properties.15 Indane
derivatives also possess a wide array of biological activities
including antimicrobial16,17 and anti-inflammatory action,18–20 and
antagonistic inhibition.21–23
The structures of dihydroxyindenopyrrolopyrimidines 4–6
and dihydroxyindolo-pyrrolpyrimidines 7–9 were assigned
partly on the basis of the position of the hydroxyl groups, NH
absorptions and the disappearance of NH2 stretching band in
Our plan was to investigate the (1:1) reaction of 6-amino- and
5,6-diaminouracils with ninhydrin and isatin. Previous studies
of similar reactions in which 2 equiv. of 6-aminouracils were
reacted with 1 equiv. of isatin24,25 or with 1 equiv. of ninhydrin24
in refluxing EtOH (plus added p-toluenesulfonic acid)25 yielded
pentacyclic spiro-pyrimidoquinolines25 or pentacyclic spiro-
pyridopyrimidines.24 In the event, our procedure produced
different tetracyclic heterocycles.
Here we report the synthesis of several examples of
indenopyrrolopyrimidines, indolopyrrolo-pyrimidines, indenop-
teridines and indolopteridines by the reaction of 6-amino- and
5,6-diaminouracils with ninhydrin and isatin. The new compounds
were evaluated as a possible antitumour agent.
1
the IR spectra. Further support was given by their H NMR
spectra, which showed signals characteristic of two OH groups
in 4–6 at δ 6.89–6.56 ppm and δ 6.08–5.82 ppm and in 7–9 at
δ 11.11–10.89 and 10.73–10.66 ppm. The phenyl group in 4–6
showed signals at δ 7.97–6.53 ppm and in 7–9 at δ 7.56–6.87.
The disappearance of both NH2 signals at δ 5.00–6.00 ppm
at positions C-5 and C-6 further confirmed the formation
of the new compounds. Compounds 7–9 showed signals at
δ 11.11–10.89 and 10.73–10.66 ppm for the NH groups.
Refluxing ninhydrin with 5,6-diaminouracil 3 in the
presence of acetic acid for 10–15 min led to the formation
of indenopteridines 10–14 through the formation of the
intermediate Schiff’s base. This reaction was faster than that
with isatin (reaction time 2–3 h). This can be explained by
an increase in the positive charge on carbon atom at position
2 in ninhydrin due to elimination of H2O in acidic medium
which facilitates the nucleophilic attack of more basic amino
groups at this position. On the other hand, refluxing isatin with
5,6-diaminouracils 3 in ethanol in the presence of TEA
involves the formation of Schiff’s bases 15–17. On the basis of
the resonating structures of diaminouracils 3, the –NH2 group at
position C-5 is expected to be more basic, attacking at the carbon
atom at position 3 of isatin moiety. Refluxing diaminouracils 3
with isatin in acetic acid afforded the indolopteridines 18 and
19 via the formation of Schiff’s base intermediates followed by
cyclodehydration (Scheme 4). Refluxing compound 15 in acetic
acid for 2h afforded compound 20 in good yield as shown in
Scheme 3.
Results and discussion
Thenitrosationof6-aminouracils126–34 yielded5-nitrosoderivatives
2 which upon reduction with (NH4)2S afforded 5,6-diaminouracils
3–e.30–34 6-Aminouracils and 5,6-diaminouracils used as direct
starting materials for the synthesis of our target compounds.
Reaction of ninhydrin with sulfur-, oxygen-, nitrogen- and
carbon-based nucleophiles takes place very quickly at C-2
due to the strong activity of this carbon centre which is located
between two 1,3-carbonyl groups, 30 whereas isatin, which has
two adjacent 1,2-carbonyl groups situated at the C-2 and C-3
positions, reacts with nucleophiles at the more reactive C-3
position.35,36 Reaction at these reactive centres with 6-aminouracils
1 and 5,6-diaminouracils 3 yielded indenopyrrolopyrimidines
4–6, indolopyrrolopyrimidines 7–9, indenopteridines 10–14 and
indolopteridines 18–20, respectively.
Dihydroxyindenopyrrolopyrimidine derivatives 4–6 were
synthesised by refluxing ninhydrin and 6-aminouracils 1 in
* Correspondent. E-mail: s.elkalyoubi@hotmail.com

772 JOURNAL OF CHEMICAL RESEARCH 2016
H
O
O
N
NC
H
S
H
NH
EtONa
N
N
X
OR
+
Reflux
NH2
NH2
N
NH
R
EtOOC
O
CH3
R
1a -d
1e
R = H, Me, Benz, 2 -Cl .Benz
ninhydrin/ AcOH
isatin/ AcOH
O
O
O
HO
HO
H
H
O
N
N
NH
OH
N
H
OH
N
H
X
N
R
N
R
7 - 9
4 - 6
X = O, R = H, Me; X = S, R = Me
R = Me, benzyl, 2 - Cl.benzyl
Scheme 1
O
H
N
NH2
N
X
H+
R
H+
3
H
H
O
O
O
O
H
X
H
HO
N
N
+
NH
O
+
HN
HO
N
R
N
N
R
X
O
H
O
H
HO
O
HO
H
X
H
N
H
N
HO
O
NH
N
R
N
H
HO
NH
X
N
R
O
- H2O
- H2O
O
O
HO
HO
H
X
H
X
N
N
N
H
O
N
H
OH
N
H
OH
N
R
N
R
7 - 9
4 - 6
Scheme 2

JOURNAL OF CHEMICAL RESEARCH 2016 773
O
N
O
N
H
X
N
H
X
N
NaNO2 / HCl
N
O
NH2
NH2
R
R
1
2
(NH4)2S
O
O
NH2
H
NH2
H
N
N
OR
NH2
N
R
NH2
3a - d
N
R
S
O
3e
R = H, Me, Benz., 2 -Cl.Benz.
ninhydrin/AcOH
isatin/AcOH
isatin
EtOH/ TEA
O
O
O
O
H
O
N
N
H
X
N
H
X
N
N
N
N
AcOH
N
H
N
H
N
R
N
N
R
N
R
NH2
O
10 - 14
15 - 17
18 - 20
X = O,S; R = H, Me, benzyl, 2 -Cl.benzyl
R = Me, benzyl, 2 - Cl.benzyl
X = O,S; R = H, Me
Scheme 3
O
O
H
N
NH2
NH2
HO
+
HO
O
N
X
H+
H+
R
H+
3
H
H
O
O
O
O
H
H
X
NH2
NH
H2N
HO
N
N
O
+
H
O+
H
+
N
HN
N
R
N
R
X
H
O
- H2O
- H2O
H
O
H
O
O
N
H
X
N
H
N
N
O
O
NH
N
H
X
NH
N
R
N
R
O
O
O
H
N
N
H
X
N
N
N
N
X
N
R
N
H
N
R
10 - 14
18 - 20
Scheme 4

774 JOURNAL OF CHEMICAL RESEARCH 2016
Table 1 Growth inhibition (%) of human hepatocellular carcinoma cell line (HepG2) by decreasing concentration (0.8 to 0.0162 µM) of compounds 4–6,
9, 10, 13, 14, 17–19 (Schemes 1 and 3)
Growth Inhibition /%
Compound concentration / µM
Compounds
P value
0.062
0
0
3.62
6.26
1.15
3.96
0
0.125
0
0.25
1.68
2.13
20.58
20.48
7.57
25.39
0
0.5
1
2
4
8
4
5
6
9
10
13
14
17
18
19
9.53
8.52
32.79
29.57
16.33
35.11
0
13.54
3.92
26.82
18.44
14.07
45.16
40.35
31.09
50.48
0.98
21.88
10.25
49.53
32.58
22.85
61.84
56.18
44.68
65.62
2.82
34.97
28.46
60.22
53.41
39.18
75.03
68.05
54.86
78.27
10.59
62.59
43.83
79.36
68.35
57.3
< 0.001a
< 0.001
< 0.001
< 0. 001
< 0.001
< 0.001
< 0.001a
< 0.001
< 0.001a
< 0.001
0.86
12.85
11.84
3.41
10.68
0
1.28
0
7.63
84.57
79.64
63.03
89.51
26.64
71.81
59.18
87.15
0
0
4.17
5.82
1.21
18.41
Significant value: P ≤ 0.01, aChi-square test conducted among inhibition rates of active concentrations only.
Table 2 The antiproliferative activity against a human hepatocellular
carcinoma cell line (HepG2) of ten of the synthesised compounds
4–6,9,10,13,14,17–19 (Schemes 1 and 3)
The structures of indenopteridines 10–14 were assigned on
the basis of the position of NH and carbonyl absorptions and
the disappearance of NH2 stretching in the IR spectra. Further
Compounds
IC50 / µM
Compounds
IC50 / µM
1
support was given by their H NMR spectra, which showed
4
5
6
9
3.67 0.15
7.04 0.22
1.29 0.07
1.61 0.13
3.05 0.19
1.8 0.13
13
14
17
18
19
0.98 0.11
>8
3.09 0.18
5.61 0.35
1.04 0.12
0.76 0.14
signals at δ 8.23–7.14 ppm which are characteristic of the
protons of a phenyl group and signals at δ 12.22–10.21 ppm
which are characteristic of a NH group. The disappearance
of both NH2 signals at δ 5.00–6.00 ppm for positions C-5 and
C-6 further confirmed the formation of the new compounds.
The structures of 6-amino-5-indoloaminopyrimidines 15–17
10
Imatinib
5-FU
1
were characterised by H NMR spectra which showed the
appearance of NH2 signals at δ 6.19–5.54 ppm, NH signals
at δ 12.37–10.20 ppm and signals for the phenyl group at
δ 8.30–7.35 ppm. Indolopteridines 18–20 showed signals at
δ 12.63–11.89 for the NH groups and signals atδ 8.23–7.12 ppm
for the protons of the phenyl group. The mechanism of formation
of 10–14 and 18–20 is shown in Scheme 4.
Data analysis
The percentage cell viability was calculated using the equation:
percentage of cell viability = [1 − (ODt/ODc)] × 100, where
ODt is the mean optical density of wells treated with the tested
compound and ODc is the mean optical density of untreated
cells. Comparison of the test compounds was made using the
IC50 value, i.e., the concentration of an individual compound
leading to 50% cell death that was estimated from graphical
plots of surviving cells versus compound concentrations.
Biological investigation
Cytotoxic activity
Usingascontrolsthewell-knownanticancerdrugs5-flourouracil
(5-FU) and Imatinib (2-substituted aminopyrimidine derivative;
Gleevec®), the in vitro growth inhibition (%) and inhibitory
growth activity (as measured by IC50) of the synthesised
compounds were investigated, using crystal violet colourimetric
viability assay. The results are shown in Tables 1 and 2 which
reveal that all the tested compounds showed high variation in
inhibitory growth and activities towards the tumour cell line in
a concentration dependent manner.
Conclusions
On the basis of our work, we can conclude that most of
the indenopyrrolopyrimidines, indolopyrrolopyrimidines,
indenopteridines and indolopteridines that we have synthesised
showed good anticancer activities against a HepG2 cell line.
Compounds 13 and 19, with an IC50 of 0.98 and 1.04 μM,
respectively, were found to be the most active.
Compound 6 had good inhibitory activities with an IC50 value
of 1.29 μM and it is clear that compounds 4, 9 and 13 were
very active at 8 μM against human hepatocellular carcinoma
(HepG2) cell line with inhibition ratios between 70% and 90%.
The highest activity against human hepatocellular carcinoma
(HepG2) cell line was shown by compound 13 with an IC50
value 0.98 μM, comparable with the reference drug imatinib.
However, compound 14 was almost inactive as shown in Table 2.
Compounds 4, 10 and 17 were also found to be active at 8
μM against human hepatocellular carcinoma (HepG2) cell line
with inhibition ratios - 68.35, 63.03 and 71.81%, respectively.
The highest detected inhibitory activities against human
hepatocellular carcinoma (HepG2) cell line was shown by
compound 13, comparable to the reference drug imatinib,
followed by 19, 16 then 9 with IC50 values of 0.98, 1.04,
1.29 and 1.61, activities of all the compounds with different
concentrations was statistically significant, P < 0.001.
Experimental
All melting points were determined with an Electrothermal
Melting-Temperature II apparatus and were uncorrected. Element
analyses were performed at the Regional Center for Mycology and
Biotechnology at Al-Azhar University. The infrared (IR) spectra
were recorded using potassium bromide disc technique on Nikolet IR
200 FTIR. Mass spectra were recorded on a DI-50 unit of Shimadzu
GC/MS-QP 5050A at the Regional Center for Mycology and
Biotechnology at Al-Azhar University. NMR spectra were obtained
on a Bruker 400 MHz spectrometer (1H NMR at 400 MHz, 13C NMR
at 100 MHz) in DMSO-d6 using TMS as an internal standard at the
Applied Nucleic Acid Research Center, Zagazig University, Egypt. All
reactions were monitored by TLC using precoated plastic sheets silica
gel (Merck 60 F254). Spots were visualised by irradiation with UV light
(254 nm). The solvent systems were either chloroform:methanol (9:1)
or ethyl acetate:toluene (1:1).

JOURNAL OF CHEMICAL RESEARCH 2016 775
6- Aminouracil, 6-amino-1-methyluracil and 6-amino-1-methyl-2-
4b,9b-Dihydroxy-1-methyl-9b,10-dihydroindeno[2′,1′:4,5]
thiouracil (1a, 1b and 1e)26–34
pyrrolo[2,3-d]pyrimidine-2(1H)-thio-4,5(1H,3H,4bH)-dione
(6):
Yield 71%; m.p. > 300 °C; IR (νmax, cm–1) KBr: 3400 (br., OH), 3198
Compounds 1a, 1b and 1e were prepared according to the reported
methods26–34 by the addition of urea, methylurea and/or methyl
thiourea (0.1 mol) to ethyl cyanoacetate (0.1 mol) in absolute ethanol
(290 mL) containing sodium (0.2 mol). The mixture was refluxed for
10–12 h, allowed to cool to r.t. and acidified with acetic acid (pH 6).
The resulting precipitate was washed with distilled water and dried in a
desiccator overnight.
1a: Yield 69%; m.p. > 360 °C (lit. ≥ 360 °C).
1b: Yield 63%; m.p. 309–310 °C (lit. 300 °C).
1e: Yield 59%; m.p. 266 °C (lit. 260 °C).
(NH), 3052 (CH Ar), 2959 (CH aliph.), 1695, 1647 (C=O), 1537
1
(C=C); H NMR (400 MHz, DMSO-d6) δ 11.84 (s, 1H, NH), 9.43 (s,
1H, NH), 7.93–7.91 (d, 1H, ArH), 7.85–7.81 (m, 1H, ArH), 7.71–7.69 (d,
1H, ArH), 7.60–7.56 (m, 1H, ArH), 6.89 (s, 1H, OH), 6.08 (s, 1H, OH),
3.53 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ 35.6, 83.0, 88.5,
92.3, 122.8, 130.2, 134.3, 135.8, 149.4, 153.9, 155.8, 172.0, 176.6, 197.1;
MS m/z (%): 317 ([M]+, 31), 315 (6), 299 ([M – H2O], 25), 256 (24),
240 (20), 224 (32), 192 (46), 184 (40), 160 (51), 128 (35), 104 (100), 76
(96); Anal. calcd for C14H11N3O4S: C, 52.99; H, 3.49; N, 13.24; found:
C, 53.08; H, 3.53; N, 13.37%.
6- Amino-1-benzyl- and 6-amino-1-[2-chlorobenzyl]uracil (1c, 1d)30–33
Dihydroxy-1-substituted-indolopyrrolopyrimidines (7–9); general
procedure
The compounds 1c and 1d were prepared according to the reported
methods.30,32,33
A mixture of 6-amino-1-substituted-uracils (1) (1.2 mmol) and isatin
(1.2 mmol) in acetic acid (5 mL) was heated under reflux for 2–2.30 h.
The formed hot precipitate was filtered, washed with ethanol and
crystallised from DMF/ethanol (1:3).
1c: Yield 72%; m.p. 285–286 °C (lit. 283 °C).
1d: Yield 66%; m.p. 295–296 °C (lit. 295 °C).
5,6- Diaminouracil, 5,6-diamino-1-substituted-uracils or -2-thiouracil
(3a–e)30,34
1- ( 2 - C h l o r o b e n z y l ) - 4 b , 9 a - d i h y d r o x y - 4 b , 9 , 9 a , 1 0 -
Compounds 3a–e were prepared by the same reported method.33,34 The
nitroso analogues 2a–e of compounds 1a–e [obtained by the addition
of a mixture of acetic acid (3 mL) and sodium nitrite (4.60 mmol) in
water (2.0 mL) to compound 1a–e suspended in water (20 mL) with
stirring at room temperature. The formed coloured nitroso analogue
2a–e was filtered, washed with water and dried in a desiccator]
(8.12 mmol) was added over 15 min to (NH4)2S solution (40.0 mL)
at 70–80 °C with stirring. The resulting precipitate was collected by
filtration, washed with ether and dried in a vacuum desiccator to afford
the diaminouracil analogues 3a–e.
tetrahydropyrimido[5′,4′:4,5]pyrrolo-[2,3-b]
indolo-2,4(1H,3H)-
dione (7): Yield 66%; m.p. > 300 °C; IR (νmax, cm–1) KBr: 3406 (OH),
3177 (NH), 3055 (CH Ar), 2808 (CH aliph.), 1710, 1652 (C=O), 1526
1
(C=C), 754 (o-substituted phenyl); H NMR (400 MHz, DMSO-d6)
δ 11.69 (s, 1H, NH), 11.20 (s, 1H, NH), 11.01 (s, 1H, OH), 10.66 (s,
1H, OH), 9.42 (s,1H, NH), 7.56–6.97 (m, 8H, ArH), 5.39–5.24 (dd, 2H,
NCH2); MS m/z (%): 400 ([M]2+, 1), 398 ([M]+, 2), 380 ([M – H2O], 2),
342 (72), 271 (14), 181 (31), 125 (100), 102 (24), 76 (14); Anal. calcd for
C19H15ClN4O4: C, 57.22: H, 3.79; N, 14.05; found: C, 57.43; H, 3.81; N,
14.13%.
5,6-Diaminouracil 3a: Yield 58%; m.p. 262 °C dec. (lit. > 260 °C dec.).
5,6-Diamino-1-methyluracil 3b: Yield 73%; m.p. 258 °C dec. (lit.
257 °C dec.).
5,6-Diamino-1-benzyluracil 3c: Yield 91%; m.p. 253 °C (lit.
250–252 °C).
5,6-Diamino-1-[2-chlorobenzyl]uracil 3d: Yield 85%; m.p. 250 °C
(lit. 245–248 °C).
5,6-Diamino-1-methyl-2-thiouracil 3e: Yield 68%; m.p. 242 °C
(lit. 240 °C).
1-Benzyl-4b,9a-dihydroxy-4b,9,9a,10-tetrahydropyrimido[5′,4′:4,5]
pyrrolo[2,3-b]indolo-2,4 (1H,3H)-dione (8): Yield 68%; m.p. > 300°C;
IR (νmax, cm–1) KBr: 3579, 3339 (OH), 3154 (NH), 3065 (CH Ar), 2860
1
(CH aliph.), 1720, 1654 (C=O), 1527 (C=C); H NMR (400 MHz,
DMSO-d6) δ 11.95 (s, 1H, NH), 11.69 (s, 1H, NH), 11.11 (s,1H, OH), 10.89
(s,1H, OH), 9.39 (s, 1H, NH), 7.40–6.87 (m, 9H, ArH), 5.42–5.27 (dd, 2H,
NCH2); 13C NMR (100 MHz, DMSO-d6) δ 50.4, 83.5,98.3, 116.7, 121.0,
123.9, 126.5, 128.5, 135.5, 136.5, 146.7, 150.0, 153.0, 157.8, 160.8, 172.0,
181.5; MS m/z (%): 364 ([M]+, 5), 348 (4), 302 (7), 276 (8), 263 (7), 208
(18), 119 (100), 106 (19), 92 (60); Anal. calcd for C19H16N4O4: C, 62.63; H,
4.43; N, 15.38; found: C, 62.80; H, 4.49; N, 15.47%.
4b,9a-Dihydroxy-1-methyl-4b,9,9a,10-tetrahydropyrimido[5′,4′:4,5]
pyrrolo-[2,3-b]indolo-2,4 (1H,3H)-dione (9): Yield 59%; m.p. >
300°C; IR (νmax, cm–1) KBr: 3524, 3385 (OH), 3159 (NH), 3025 (CH Ar),
2939, 2846 (CH aliph.), 1746, 1688 (C=O), 1555 (C=C); 1H NMR (400
MHz, DMSO-d6) δ 11.95 (s, 1H, NH), 11.48 (s, 1H, NH), 10.89 (s,1H,
OH), 10.73 (s,1H, OH), 9.25 (s, 1H, NH),7.26–6.93 (m, 4H, ArH), 3.42 (s,
3H, CH3); MS m/z (%): 288 ([M]+, 8), 270 ([M – H2O], 8), 249 (10), 188
(9), 119 (52), 92 (36), 61 (100); Anal. calcd for C13H12N4O4: C, 54.17; H,
4.20; N, 19.44; found: C, 54.25; H, 4.26; N, 19.58%.
4b,9b-Dihydroxy-1-substituted-9b,10-tetrahydroindeno[2′,1′:4,5]
pyrrolo[2,3-d]pyrimidine-2,4,5 (1H,3H,4bH)-triones (4 and 5) and
4b,9b-dihydroxy-1-methyl-9b,10-dihydroindeno[2′,1′:4,5]pyrrolo[2,3-d]
pyrimidine-2(1H)-thio-4,5(1H,3H,4bH)-dione (6); general procedure
A mixture of 6-amino-1-substituted-uracils (1) (1.2 mmol) and
ninhydrin (1.2 mmol) in acetic acid (5 mL) was heated under reflux
for 10–15 min. The resulting precipitate was filtered hot, washed with
ethanol and crystallised from DMF/ethanol (1:3).
4b,9b-Dihydroxy-9b,10-dihydroindeno[2′,1′:4,5]pyrrolo[2,3-d]
pyrimidine-2,4,5(1H,3H,4bH)-trione (4): Yield 73%; m.p. > 300 °C;
IR (νmax, cm–1) KBr: 3445 3326 (br.OH), 3210 (NH), 3093 (CH arom.),
1
1-Substituted-2H-indeno[2,1-g]pteridine-2,4,6-(1H,3H)-triones
2900 (CH aliph.), 1702, 1599 (C=O), 1567 (C=C); H NMR (400
(10–14); general procedure
MHz, DMSO-d6) δ 11.14 (s, 1H, NH), 10.05 (s, 1H, NH), 8.70 (s, 1H,
NH), 7.97–7.91 (d, 1H, ArH), 7.81–7.77 (m, 1H, ArH), 7.68–7.66 (d, 1H,
ArH), 7.57–7.53 (m, 1H, ArH), 6.56 (s, 1H, OH-9b), 5.82 (s, 1H, OH-
4b); MS m/z (%): 287 ([M]+, 20), 283 (27), 271 (28), 245 (24), 181 (59),
168 (38), 125 (100), 105 (47), 76 (60); Anal. calcd for C13H9N3O5: C,
54.36; H, 3.16; N, 14.63; found: C, 54.52; H, 3.21; N, 14.79%.
4b,9b-Dihydroxy-1-methyl-9b,10-dihydroindeno[2′,1′:4,5]
pyrrolo[2,3-d]pyrimidine-2,4,5(1H, 3H,4bH)-trione (5): Yield 79%;
m.p. > 300 °C; IR (νmax, cm–1) KBr: 3442, 3393 (br., OH), 3250, 3219
(NH), 3051 (CH Ar), 2990 (CH aliph.), 1700, 1654 (C=O), 1549
(C=C); 1H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H, NH), 9.24 (s,
1H, NH), 7.91–7.89 (d, 1H, ArH), 7.83–7.79 (m, 1H, ArH), 7.69–7.67 (d,
1H, ArH), 7.58–7.57 (m, 1H, ArH), 6.74 (s, 1H, OH), 5.89 (s, 1H, OH),
3.12 (s, 3H, CH3); MS m/z (%): 301 ([M]+, 6), 283 ([M – H2O], 9), 244
(17), 168 (41), 104 (100), 76 (88); Anal. calcd for C14H11N3O5: C, 55.82;
H, 3.68; N, 13.95; found: C, 55.97; H, 3.74; N, 14.04%.
A mixture of 5,6-diamino-1-substituted uracils (3) (1.00 mmol) and
ninhydrin (1.00 mmol) in acetic acid (5 mL) was heated under reflux
for 10–15 min. The resulting precipitate was filtered hot, washed with
ethanol and crystallised from DMF/ethanol (1:3).
2H-Indeno[2,1-g]pteridine-2,4,6-(1H,3H)-trione (10): Yield 81%;
m.p. > 300°C; IR (νmax, cm–1) KBr: 3193, 3260 (NH), 3089 (CH Ar),
2813 (CH aliph.), 1714, 1642 (C=O), 1562 (C=C); 1H NMR (400 MHz,
DMSO-d6) δ 11.35 (s, 1H, NH), 10.29 (s, 1H, NH), 7.87–7.81 (m, 3H,
ArH), 7.74–7.70 (m, 1H, ArH); MS m/z (%): 266 ([M]+, 1.10), 239 (27),
182 (8), 154 (13), 135 (100), 98 (59), 75 (25), 74 (52); Anal. calcd for
C13H6N4O3: C, 58.65; H, 2.27; N, 21.05; found: C, 58.83; H, 2.24; N,
21.19%.
1-Methyl-2H-indeno[2,1-g]pteridine-2,4,6-(1H,3H)-trione
(11):
Yield 79%; m.p. > 300 °C; IR (νmax, cm–1) KBr: 3176 (NH), 3071 (CH
1
Ar), 2827 (CH aliph.), 1684, 1652 (C=O), 1500 (C=C); H NMR

776 JOURNAL OF CHEMICAL RESEARCH 2016
(400 MHz, DMSO-d6) δ 10.54 (s, 1H, NH), 7.99–7.97 (d, 1H, ArH),
7.86–7.82 (m, 2H, ArH), 7.74–7.71 (m, 1H, ArH), 3.59 (s, 3H, CH3); MS
m/z (%): 280 ([M]+, 97), 209 (66), 182 (100), 154 (51), 130 (32), 102
(28), 76 (23); Anal. calcd for C14H8N4O3: C, 60.00; H, 2.88; N, 19.99;
found: C, 60.14; H, 2.91; N, 20.04%.
1-Methyl-2H-indeno[2,1-g]pteridine-2-thio-4,6-(1H,3H)-dione
(12): Yield 82%; m.p. > 300 °C; IR (νmax, cm–1) KBr: 3210 (NH), 3093
(CH Ar), 2900 (CH aliph.), 1702, 1599 (C=O), 1567 (C=C); 1H NMR
(400 MHz, DMSO-d6) δ 12.22 (s, 1H, NH), 8.03–7.73 (m, 4H, ArH),
3.68 (s, 3H, CH3); MS m/z (%): 296 ([M]+, 65), 280 (15), 236 (35), 209
(34), 182 (100), 154 (77), 126 (54), 102 (55), 75 (30); Anal. calcd for
C14H8N4O2S: C, 56.75; H, 2.72; N, 18.91; found: C, 56.89; H, 2.70; N,
19.07%.
(18), 104 (33), 98 (36), 97 (26), 92 (82), 84 (26), 76 (44), 57 (16), 55
(28); Anal. calcd for C13H11N5O2S: C, 51.82; H, 3.68; N, 23.24; found:
C, 51.98; H, 3.71; N, 23.43%.
1-Substituted-indolopteridines (18–20); general procedure
Method A: A mixture of 5,6-diamino-1-substituted-uracils (3)
(1.00 mmol) and isatin (1.00 mmol) in acetic acid (5.0 mL) was heated
under reflux for 2–3 h. The resulting precipitate was filtered when hot,
washed with ethanol and crystallised from DMF/ethanol (1:3) to give
compounds 18 and 19.
Method B: Compound 15 (1.00 mmol) in acetic acid (5 mL) was
heated under reflux for 2 h. The resulting precipitate was filtered when
hot, washed with ethanol and crystallised from DMF/ethanol (1:3) to
give compound 20.
1-(2-Chlorobenzyl-1,10-dihydro-2H-indolo[3,2-g]pteridine-2,3(3H)-
dione (18): Yield 68% (Method A); m.p. > 300 °C; IR (νmax, cm–1) KBr:
3250, 3183 (NH), 3050 (CH Ar), 2814 (CH aliph.), 1705, 1638 (C=O),
1562 (C=C); 1H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H, NH), 10.79
(s,1H, NH), 8.23–8.21 (d, 1H, ArH), 7.60–7.14 (m, 7H, ArH), 5.41 (s, 2H,
NCH2); MS m/z (%): 379 ([M]2+, 12), 377 ([M]+, 17), 360 (25), 287 (19),
166 (23), 104 (21), 63 (27), 51 (100); Anal. calcd for C19H12ClN5O2: C,
60.41; H, 3.20; N, 18.54; found: C, 60.53; H, 3.18; N, 18.71%.
1-Benzyl-1H-indeno[2,1-g]pteridine-2,4,6(3H)-trione (13): Yield
77%; m.p. > 300°C; IR (νmax, cm–1) KBr: 3168 (NH), 3024 (CH Ar),
1
2843 (CH aliph.), 1692, 1601 (C=O), 1563 (C=C); H NMR (400
MHz, DMSO-d6) δ 10.21 (s, 1H, NH), 7.95–7.93 (d, 1H, ArH),
7.84–7.80 (m, 2H, ArH), 7.73–7.69 (m, 1H, ArH), 7.50–7.48 (d, 2H,
ArH), 7.34–7.25 (m, 3H, ArH), 5.44 (s, 2H, NCH2); MS m/z (%): 356
([M]+, 18), 284 (11), 232 (13), 230 (17), 132 (10), 91 (100); Anal. calcd
for C20H12N4O3: C, 67.41; H, 3.39; N, 15.72; found: C, 67.59; H, 3.45; N,
15.81%.
1-(2-Chlorophenyl)methyl-2H-indeno[2,1-g]pteridine-2,4,6-
(1H,3H)-trione (14): Yield 83%; m.p. > 300 °C; IR (νmax, cm–1)
KBr: 3303, 3175 (NH), 3059 (CH Ar), 2842 (CH aliph.), 1695, 1659
(C=O), 1557 (C=C), 751 (o-substituted phenyl); 1H NMR (400 MHz,
DMSO-d6) δ 10.52 (s, 1H, NH), 7.82–7.69 (m, 2H, ArH), 7.53–7.50
(m, 1H, ArH), 7.34–7.22 (m, 5H, ArH), 5.49 (s, 2H, NCH2); MS m/z
(%): 392 ([M]2+, 1.63), 390 ([M]+, 0.62), 355 (100), 318 (9), 284 (11),
154 (11), 125 (94), 102 (20), 76 (21); Anal. calcd for C20H11ClN4O3: C,
61.47; H, 2.84; N, 14.34; found: C, 61.61; H, 2.83; N, 14.51%.
1-Benzyl-1,10-dihydro-2H-indolo[3,2-g]pteridine-2,3(3H)-dione
(19): Yield 65% (Method A); m.p. > 300 °C; IR (νmax, cm–1) KBr:
3270, 3136 (NH), 3029 (CH Ar), 2920 (CH aliph.), 1712, 1642 (C=O),
1
1533 (C=C); H NMR (400 MHz, DMSO-d6) δ 12.63 (s, 1H, NH),
11.89 (s, 1H, NH), 8.21–8.20 (d, 1H, ArH), 7.63–7.59 (m, 1H, ArH),
7.54–7.52 (d, 1H, ArH), 7.40–7.21 (m, 6H, ArH), 5.43 (s, 2H, NCH2);
MS m/z (%): 343 ([M]+, 8), 271 (10), 181 (15), 125 (12), 102 (12), 91
(100); Anal. calcd for C19H13N5O2: C, 66.47; H, 3.82; N, 20.40; found:
C, 66.63; H, 3.79; N, 20.62%.
1,10-Dihydro-1-methyl-2H-indolo[3,2-g]pteridine-2,3(3H)-dione (20):
Yield 82% (Method B); m.p. > 300 °C; IR (νmax, cm–1) KBr: 3292, 3140
(NH), 3098 (CH Ar), 2942 (CH aliph.), 1694, 1623 (C=O), 1570 (C=C);
1H NMR (400 MHz, DMSO-d6) δ 11.89 (s, 1H, NH), 11.54 (s, 1H, NH),
8.18–7.35 (m, 4H, ArH), 3.55 (s, 3H, CH3); MS m/z (%): 267 ([M]+, 99),
196 (100), 181 (24), 156 (73), 102 (11), 58 (47); Anal. calcd for C13H9N5O2:
C, 58.43; H, 3.39; N, 26.21; found: C, 58.70; H, 3.43; N, 26.35%.
6-Amino-1-substituted-5-{[(3Z)-2-oxo-1,2-dihydro-3H-indol-3-
ylidene]amino}pyrimidine-2,4(1H,3H)-diones (15–17); general
procedure
A mixture of 5,6-diamino-1-substituted-uracils (3) (1.00 mmol) and
isatin (1.00 mmol) in ethanol (20 mL) in the presence of TEA as a
base was heated under reflux for 4–5 h. The resulting precipitate was
filtered hot, washed with ethanol and crystallised from DMF/ethanol
(1:3) to afford the desired compounds 15–17.
Biological investigation
Evaluation of the antitumour activity
6-Amino-1-methyl-5-{[(3Z)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]
amino}pyrimidine-2,4(1H, 3H)-dione (15): Yield 85%; m.p. > 300 °C;
IR (νmax, cm–1) KBr: 3349, 3309, 3260, 3146 (NH2 and NH), 3054 (CH
Ar), 2846 (CH aliph.), 1693, 1643 (C=O), 1575 (C=C); 1H NMR (400
MHz, DMSO-d6) δ 11.78 (s, 1H, NH), 10.54 (s, 1H, NH), 8.22–8.20 (d,
1H, ArH), 7.63–7.56 (m, 2H, ArH), 7.42–7.38 (m, 1H, ArH), 6.11 (s,
2H, NH2), 3.56 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ 28.7,
96.1, 120.2, 128.1, 136.5, 143.2, 146.0, 149.3, 151.2, 158.0, 159.4, 160.8,
171.4; MS m/z (%) 285 ([M]+,1.13), 156 (100), 139 (46), 98 (15), 84
(21), 57 (51); Anal. calcd for C13H11N5O3: C, 54.74; H, 3.89; N, 24.55;
found: C, 54.91; H, 3.94; N, 24.67%.
6-Amino-5-{[(3Z)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]amino}
pyrimidine-2,4(1H,3H)-dione (16): Yield: 88%; m.p > 300 °C; IR
(νmax, cm–1) KBr: 3459, 3407, 3360, 3194 (NH2 and NH), 3093 (CH
Ar), 2842 (CH aliph.), 1704, 1647 (C=O), 1564 (C=C); 1H NMR (400
MHz, DMSO-d6) δ 12.37 (s, 1H, NH), 10.90 (s, 1H, NH), 10.26 (s, 1H,
NH), 8.22–8.20 (d, 1H, ArH), 7.69–7.35 (m, 3H, ArH), 5.54 (s, 2H,
NH2); MS m/z (%): 271 ([M]+, 2.77) 266 (20), 142 (100), 97 (67), 71
(32), 43 (50); Anal. calcd for C12H9N5O3: C, 53.14; H, 3.34; N, 25.82;
found: C, 53.42; H, 3.23; N, 26.07%.
Mammalian cell lines: The cell line used in this study was
human hepatocellular carcinoma cell line (HepG2 cells)
obtained from the tissue culture Unit, VACSERA, Cairo, Egypt.
The mammalian cells were propagated in Dulbecco’s
modified Eagle’s37 medium (DMEM) or RPMI-1640 depending
on the type of cell line, supplemented with 10% heat-inactivated
fetal bovine serum, 1% L-glutamine, HEPES buffer and
50μg/mL gentamycin. All cells were maintained at 37 ºC in
a humidified atmosphere with 5% CO2 and were subcultured
twice a week.
Antitumour activity evaluation using viability assay
Antitumour activity assay was carried out according to the
method described previously.38 All the experiments concerning
the cytotoxicity evaluation were performed and analysed by
tissue culture unit at the Regional Centre for Mycology and
Biotechnology RCMB, Al-Azhar University, Cairo, Egypt.
The tumor cell lines were seeded in a sterile 96-well plate
in 100μl of growth medium at a cell concentration of 1 × 104
cells per well. After 24 h of seeding, the monolayers were
then washed with sterile phosphate buffered saline (0.01 M,
pH 7.2) and simultaneously the cells were treated with 100 μl
from different dilutions of the test sample in fresh maintenance
medium and incubated at 37 °C. Different two-fold dilutions
of the tested compound (100, 50, 25, 12.5, 6.25, 3.125, 1.56
and 0.78 μg mL–1) were added to confluent cell monolayers
(3Z) -3 - [( 6 - A m i n o -1- m ethyl- 4 - oxo -2- thioxo -1, 2 , 3, 4 -
tetrahydropyrimidin-5-yl)imino]-1,3-dihydro-2H-indol-2-one (17):
Yield 87%; m.p. > 300 °C; IR (νmax, cm–1) KBr: 3346, 3307, 3257, 3141
(NH2 & NH), 3026 (CH Ar), 2846 (CH aliph.), 1685, 1634 (C=O),
1
1572 (C=C); H NMR (400 MHz, DMSO-d6) δ 11.89 (s, 1H, NH),
10.20 (s, 1H, NH), 8.30–8.28 (d, 1H, ArH), 7.78–7.37 (m, 3H, ArH),
6.19 (s, 2H, NH2), 3.73 (s, 3H, CH3); MS m/z (%): 301 ([M]+, 2.24),
239 (9), 185 (16), 147 (67), 132 (16), 129 (29), 119 (100), 118 (23), 112

JOURNAL OF CHEMICAL RESEARCH 2016 777
9
U.K. Singh, S.N. Pandeya, A. Singh, B.K. Srivastava and M. Pandey, Int. J.
Pharm. Sci. Drug Res., 2010, 2, 151.
dispensed into a 96-well, flat-bottomed microtitre plates
(Falcon, NJ, USA) using a multichannel pipette. The microtitre
plates were incubated at 37 ºC in a humidified incubator with
5% CO2 for a period of 24 h. Untreated cells were served as
controls. Three independent experiments were performed each
containing six replicates for each concentration of the tested
samples. The cytotoxic effects of the tested compounds were
then measured using crystal violet staining viability assay.
Briefly, after 24 h of treatment, the medium was removed,
100 μL of 0.5% of crystal violet in 50% methanol was added
to each well and incubated for 20 minutes at room temperature
and subsequently excess dye was washed out gently by distilled
water. The plate was allowed to dry, then the viable crystal
violet-stained cells were lysed using 33% glacial acetic acid
solution. Absorbance at 570 nm was then measured in each
well using a microplate reader (SunRise, TECAN, Inc, USA).
5-Fluorouracil was used as a positive control. The absorbance
is proportional to the number of surviving cells in the culture
plate. Thus, using this colorimetric procedure, the tested
compounds-mediated cell lysis and the cytotoxic effect of 5-FU
(used as a positive control) were measured and compared to the
viability of untreated cells.
10 P. Selvam, N. Murgesh, M. Chandramohan, E. De Clercq, E. Keyaerts,
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17 J. Courant, D. Leblois, M. Tandon, S. Robert-Piessard, G. Le Baut, M. Juge,
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Because the stock solutions to prepare the different
concentrations of the tested compounds were dissolved in
DMSO, controls with DMSO alone were performed in parallel
for each concentration.
23 N.V. Lakshmi, P.M. Sivakumar, D. Muralidharan, M. Doble and P.T.
Perumal, RSC Adv., 2013, 3, 496.
24 M.R. Mohammadizadeh, J. Azizian, F. Teimouri, A.A. Mohammadi, A.R.
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Acknowledgements
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The author thanks Dr Mahmoud Elaasser for carrying out
the biological activity determinations at Regional Center for
Mycology and Biotechnology at Al-Azhar University.
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Received 6 October 2016; accepted 8 November 2016
Paper 1604362 doi: 10.3184/174751916X14798125870610
Published online: 5 December 2016
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