Synthesis and biological evaluation of some novel tetrahydroquinolines as anticancer and antimicrobial agents

Article abstract of DOI:10.3109/14756366.2013.787421

This study reports the synthesis of a series of new 2-amino-3-cyano-8- methyl-4-substituted-5,6,7,8-tetrahydroquinolines along with some derived fused-ring systems. Ten compounds have shown remarkable cytotoxic activity against human colon carcinoma HT29, hepatocellular carcinoma HepG2 and Caucasian breast adenocarcinoma MCF7 cell lines. Six compounds showed considerable broad-spectrum cytotoxic activity among which two proved to be the most active derivatives. Likewise, seven compounds from the series were found to exhibit significant antimicrobial activity and three of them proved to be the most active candidates. Two alkylthio-pyrimido quinolines are suggested as possible antimicrobial and anticancer candidates in the present series.

Full text of DOI:10.3109/14756366.2013.787421

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J Enzyme Inhib Med Chem, Early Online: 1–12
2013 Informa UK Ltd. DOI: 10.3109/14756366.2013.787421
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ORIGINAL ARTICLE
Synthesis and biological evaluation of some novel tetrahydroquinolines
as anticancer and antimicrobial agents
Hassan M. Faidallah1, Alaa A. Saqer1, Khalid A. Alamry1, Khalid A. Khan1, and Abdullah M. Asiri1,2
2
1Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia and Center of Excellence for Advanced Materials
Research, King Abdulaziz University, Jeddah, Saudi Arabia
Abstract
Keywords
This study reports the synthesis of a series of new 2-amino-3-cyano-8-methyl-4-substituted-
5,6,7,8-tetrahydroquinolines along with some derived fused-ring systems. Ten compounds have
shown remarkable cytotoxic activity against human colon carcinoma HT29, hepatocellular
carcinoma HepG2 and Caucasian breast adenocarcinoma MCF7 cell lines. Six compounds
showed considerable broad-spectrum cytotoxic activity among which two proved to be the
most active derivatives. Likewise, seven compounds from the series were found to exhibit
significant antimicrobial activity and three of them proved to be the most active candidates.
Two alkylthio-pyrimido quinolines are suggested as possible antimicrobial and anticancer
candidates in the present series.
2-Amino-3-cyano-8-methyl-4-substituted-
5,6,7,8-tetrahydroquinolines,
3H-pyrimido[4,5-b]quinolin-4-ones,
antimicrobial agents, cytotoxic agents
History
Received 27 November 2012
Revised 8 March 2013
Accepted 15 March 2013
Published online 22 April 2013
Introduction
with diverse chemotherapeutic activities, much concern has been
raised on the antimicrobial and antitumor potentials of some
pyridine derivatives14–17. The obtained results prompted further
structure modification of the disubstituted-2(1H)-pyridinone
scaffold by fusing it with a cyclohexane ring leading to the
synthesis of new tetrahydroquinoline analogs. The substitution
profile of the main tetrahydroquinoline ring was attempted to
comprise some counterparts that would confer different elec-
tronic, lipophilic and steric environment, which would influence
the targeted biological activities. It was, therefore, considered
worthwhile to synthesize some tetrahydroquinoline-derived
fused-ring systems as interesting structural variation to improve
the anticipated chemotherapeutic profile.
The ongoing efforts of research on the treatment of malignancy
are focused on the discovery of novel products interacting with
novel biological targets. Among the wide variety of heterocycles
that have been explored for developing pharmaceutically import-
ant molecules, pyridines and fused pyridine ring systems have
received much attention since they are proved to be biologically
versatile compounds possessing a variety of activities. A wide
range of chemotherapeutic activities have been ascribed to
pyridine derivatives including antimicrobial1–3, antiamoebic1,
antiparasitic4 and antiviral5,6 activities. As far as the antineoplas-
tic potential is concerned, pyridine derivatives were reported to
contribute to a variety of anticancer activity including cyto-
toxic7,8, antiproliferative9 and CDK kinase inhibitory activity10.
Cyanopyridylureas have been claimed for their properties
Experimental
in treating hyperproliferative and angiogenesis disorders. The Chemistry
3-cyano-2,6-dihydropyridine is a potent inhibitor of dihydrouracil
Melting points were determined on a Gallenkamp melting point
dehydrogenase and its co-administration with 1-ethoxymethyl-5-
fluorouracil enhances the antitumor effect11. Furthermore,
Piritrexim (2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyr-
ido[2,3-d]pyrimidine), a second-generation antineoplastic drug,
having a pyridine ring among their structures was reported to
inhibit the enzyme dihydrofolate reductase. Recently, it has been
reported that some fused pyridines emerged as potential inhibitors
of protein kinase B12 and HIF 1-a prolyl hydroxylase13, which
play a major role in cancer cell division. During our ongoing
studies aimed at the discovery of new structure leads endowed
apparatus and are uncorrected. The microwave reactor used was
CEM Discover 300 W. The infrared (IR) spectra were recorded on
Shimadzu FT-IR 8400S IR spectrophotometer using the KBr
1
pellet technique. H and 13C NMR spectra were recorded on a
Bruker DPX-400 FT NMR spectrometer using tetramethylsilane
as the internal standard and DMSO-d6 as a solvent (chemical
shifts in ꢀ, ppm). Splitting patterns were designated as follows:
s, singlet; d, doublet; m, multiplet and q, quartet. Elemental
analyses were performed on a 2400 Perkin Elmer Series 2
analyzer and the found values were within ꢀ0.4% of the
theoretical values. Follow up of the reactions and checking the
homogeneity of the compounds were made by TLC on silica gel
pre-coated aluminum sheets (Type 60 F254, Merck) and the spots
were detected by exposure to UV-lamp at ꢁ 254. The analysis data
are reported in Tables 1 and 2.
Address for correspondence: Prof. Hassan M. Faidallah, PhD, Department
of Chemistry, Faculty of Science, King Abdulaziz University, P O Box
80203, Jeddah-21589, Saudi Arabia. Tel: +00-966-567743180. E-mail:
hfaidallahm@hotmail.com

2
H. M. Faidallah et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Table 1. Physicochemical and analytical data of compounds 1–28.
Analysis %
ꢁ
Compound no.
R
M.p., C
Yield %
67
Mol. formula (mol. weight)
C17H17N3 (263.34)
Calcd.
Found
1
C6H5
162–164
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
77.54
6.51
77.58
6.67
15.96
59.66
4.71
12.28
73.70
6.53
14.32
77.95
6.90
15.15
66.88
5.61
15.60
72.15
6.81
21.04
74.24
6.89
13.67
58.39
4.36
11.35
71.01
5.96
13.07
74.73
6.27
13.76
64.62
5.08
14.13
59.39
4.72
10.94
71.62
6.31
12.53
75.21
6.63
13.16
65.57
5.50
13.49
70.11
6.54
18.17
72.33
5.56
16.12
59.75
4.83
12.21
73.42
6.71
14.09
78.08
6.72
15.40
66.97
5.36
15.44
72.32
6.95
20.88
74.32
6.97
13.81
58.42
4.45
11.44
71.32
5.73
12.91
74.51
6.47
13.53
64.71
4.89
14.34
59.42
4.89
10.85
71.54
6.42
12.70
75.41
6.47
13.25
65.63
5.39
13.21
69.92
6.69
18.03
72.19
5.66
2
3
4-BrC6H4
190–192
177–179
210–212
186–188
150–152
160–162
150–152
186–188
137–139
166–168
168–169
122–124
135–137
182–184
4300
54
68
73
81
50
59
62
72
47
63
42
41
52
63
36
82
82
78
76
73
59
85
C17H16BrN3 (342.23)
C18H19N3O (293.37)
C18H19N3 (277.37)
4-CH3OC6H4
4-CH3C6H4
2-Thienyl
4
5
C
C
15H15N3S (269.37)
16H18N4 (266.35)
6
1-Methyl-pyrrol-2-yl
C6H5
7
C18H17N3O (307.39)
C18H16BrN3O (370.24)
C19H19N3O2 (321.38)
C19H19N3O (305.38)
8
4-BrC6H4
9
4-CH3OC6H4
4-CH3C6H4
2-Thienyl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
C
16H15N3OS (297.38)
4-BrC6H4
C19H18BrN3O (384.27)
C20H21N3O2 (335.40)
C20H21N3O (319.40)
4-CH3OC6H4
4-CH3C6H4
2-Thienyl
C
C
17H17N3OS (311.41)
18H20N4O (308.39)
1-Methyl-pyrrol-2-yl
C6H5
140–142
140–142
134–136
134–136
184–186
136–138
126–128
C24H22N4S (398.53)
C24H21BrN4S (477.42)
C25H24N4OS (428.55)
C25H24N4S (412.56)
14.06
60.38
4.43
11.74
70.07
5.64
13.07
72.78
5.86
13.58
65.32
4.98
13.85
68.80
5.77
17.44
70.40
5.20
14.14
60.46
4.61
11.65
69.98
5.66
13.15
72.62
5.66
13.71
65.09
5.17
13.56
68.93
5.56
17.24
70.51
5.09
4-BrC6H4
4-CH3OC6H4
4-CH3-C6H4
2-Thienyl
C
C
22H20N4O2 (404.55)
23H23N5S (401.54)
1-Methyl-pyrrol-2-yl
C6H5
C25H22N4OS (426.54)
12.72
12.86
(continued )

DOI: 10.3109/14756366.2013.787421
Table 1. Continued
Novel tetrahydroquinolines as anticancer and antimicrobial agents
3
Analysis %
ꢁ
Compound no.
R
M.p., C
Yield %
85
Mol. formula (mol. weight)
C25H21BrN4OS (505.43)
Calcd.
Found
24
25
26
27
28
4-BrC6H4
126–128
176–178
102–103
194–196
152–154
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
59.41
4.19
11.08
68.40
5.30
12.27
70.88
5.49
12.72
63.86
4.66
59.34
4.09
11.15
68.58
5.17
12.09
71.02
5.31
12.45
63.64
4.83
4-CH3OC6H4
4-CH3-C6H4
83
79
73
52
C26H24N4O2S (456.57)
C26H24N4OS (440.57)
2-Thienyl
C
C
24H20N4OS2 (432.56)
24H23N5OS (429.54)
12.95
67.11
5.40
13.07
66.92
5.73
1-Methyl-pyrrol-2-yl
16.30
16.18
Table 2. Physicochemical and analytical data of the scheme 2 compounds 29–51.
Analysis %
Compound no.
R
Y or R1
S
M.p., ꢁC
120–122
Yield, %
67
Mol. formula (mol. weight)
C18H18N4S (322.43)
Calcd.
Found
29
C6H5
C
H
N
C
H
N
C
H
N
S
67.05
5.63
17.38
53.87
4.27
13.96
64.75
5.72
15.90
9.10
66.89
5.78
17.21
53.69
4.19
13.78
64.88
5.59
16.07
8.89
30
31
4-BrC6H4
S
S
143–145
207–209
52
73
C18H17BrN4S (401.32)
C19H20N4OS (352.46)
4-OCH3–C6H4
32
33
4-CH3C6H4
2-Thienyl
S
S
143–145
178–180
52
71
C19H20N4S (336.46)
C
H
N
S
C
H
N
S
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
S
67.83
5.99
16.65
9.53
58.51
4.91
17.06
19.52
70.57
5.92
18.29
67.84
5.99
16.66
61.52
5.16
17.93
68.54
6.33
15.99
70.88
5.49
67.02
6.14
16.38
9.61
58.66
4.73
16.87
19.69
70.71
5.69
18.03
68.06
6.14
16.49
61.41
5.37
18.07
68.79
6.14
16.07
71.06
5.58
C16H16N4S2 (328.46))
34
35
36
37
38
39
C6H5
O
222–224
234–236
250–252
146–148
138–139
250–252
69
71
75
67
74
75
C18H18N4O (306.36)
C19H20N4O2 (336.39)
4-CH3OC6H4
2-Thienyl
C6H5
O
O
C
16H16N4OS (312.39)
C2H5
C20H22N4S (350.49)
C26H24N4OS (440.57)
C19H19BrN4S (415.35)
C6H5
CH2COC6H5
CH3
12.72
59.94
4.61
13.49
7.72
12.44
60.02
4.50
13.52
7.64
4-BrC6H4
40
4-CH3OC6H4
CH3
146–148
67
C20H22N4OS (366.48)
C
H
N
S
65.55
6.05
15.29
8.75
65.59
6.14
15.17
8.86
41
42
4-OCH3–C6H4
4-CH3OC6H4
CH2COC6H5
C2H5
140–142
176–178
76
59
C27H26N4O2S (470.60)
C21H24N4OS (380.52)
C
H
N
C
H
N
68.91
5.57
11.91
66.29
6.36
68.67
5.73
12.14
66.12
6.53
14.72
14.62
(continued )

4
H. M. Faidallah et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Table 2. Continued
Analysis %
Compound no.
R
Y or R1
CH3
M.p., ꢁC
183–185
Yield, %
66
Mol. formula (mol. weight)
C20H22N4S (350.48)
Calcd.
Found
43
4-CH3C6H4
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
C
H
N
S
68.54
6.33
15.99
73.21
6.14
13.13
59.62
5.30
16.36
60.64
5.65
15.72
66.00
5.30
13.39
74.46
6.25
19.29
58.55
4.64
15.17
7.52
68.41
6.50
13.48
73.41
5.87
13.34
59.55
5.27
16.34
60.95
5.71
15.84
66.34
5.15
13.17
74.72
6.07
19.18
58.43
4.53
15.21
7.42
44
45
46
47
48
49
4-CH3C6H4
2-Thienyl
2-Thienyl
2-Thienyl
C6H5
CH2C6H5
CH3
240–242
240–242
167–169
4300
61
61
52
63
81
61
C26H26N4S (426.59)
C17H18N4S2 (342.48)
C18H20N4S2 (356.51)
C23H22N4S2 (418.58)
C18H18N4 (290.37)
C18H17BrN4 (369.26)
C2H5
CH2C6H5
139–141
240–242
4-BrC6H4
50
51
4-OCH3–C6H4
4-CH3–C6H4
121–123
200–201
83
78
C19H20N4O (320.40)
C19H20N4 (304.40)
C
H
N
C
H
N
71.23
6.29
17.49
74.97
6.62
71.12
6.40
17.60
75.13
6.49
18.41
18.56
Compounds 29–33 and 34–36 were also prepared using microwave-assisted synthesis. The yields were about 20–25% much better than the conventional
method.
2-Amino-3-cyano-8-methyl-4-substituted-5,6,7,8-
tetrahydroquinolines (1–6)
After cooling, the reaction mixture was poured onto ice-cold
water; the precipitated solid was filtered, washed with water
and crystallized from ethanol. IR (cmꢂ1): 3258–3150 (NH),
1695–1715 (C¼O). 13CNMR (ꢀ ppm) for 7 (R ¼C6H5): 21.2
(CH3), 21.4, 27.2, 31.0, 36.4 (cyclohexyl C), 119.6, 126.8, 129.0,
129.1, 135.4, 138.4, 152.2, 162.1, 163.4, 171.5 (ArC), 170.2
(CO). 8 (R ¼ 4-BrC6H4): 20.8 (CH3), 21.6, 27.5, 31.2, 36.8
(cyclohexyl C), 118.6, 126.4, 129.2, 129.3, 136.4, 138.2, 153.2,
162.6, 163.0, 170.3 (ArC), 169.2 (CO). 9 (R ¼ 4-CH3OC6H4):
20.7 (CH3), 21.7, 27.4, 31.8, 39.0 (cyclohexyl C), 56.1 (OCH3),
115.2, 127.4, 131.5, 135.2, 138.5, 152.6, 162.7, 163.0, 171.0 (Ar
C), 170.5 (CO). 10 (R ¼ 4-CH3C6H4): 21.0 (CH3), 22.1 (CH3),
21.6, 27.3, 30.4, 36.8 (cyclohexyl C), 118.4, 126.5, 129.1, 129.2,
135.6, 138.6, 152.7, 162.4, 163.2, 171.6 (ArC), 169.7 (CO).
11 (R ¼ 2-thienyl): 21.3 (CH3), 22.2, 27.8, 31.4, 37.6 (cyclohexyl
C), 120.3, 122.4, 125.3, 127.4, 135.8, 142.4, 148.4, 162.2, 163.0,
171.7 (Ar C), 170.1 (CO).
A mixture of the 2-methylcyclohexanone (1.12 g, 0.01 mol), the
appropriate aldehyde (0.01 mol), malononitrile (0.66 g, 0.01 mol)
and anhydrous ammonium acetate (6.2 g, 0.08 mol) in absolute
ethanol (50 mL) was refluxed for 3–6 h. The reaction mixture
was cooled and the resulting precipitate was filtered, washed
with water, dried and crystallized with the appropriate solvent.
IR (cmꢂ1): 3210–3450 (NH2), 2214–2226 (CN). 13C NMR
(ꢀ ppm) for 1 (R ¼C6H5): 20.8 (CH3), 21.3, 27.12, 30.5, 36.6
(cyclohexyl C), 117.0 (CN), 91.8, 122.4, 152.9, 157.4, 166.1
(pyridine C), 123.9, 129.6, 132.4, 135.8 (Ar C). 2 (R ¼ 4-
BrC6H4): 20.2 (CH3), 21.1, 27.02, 30.9, 36.5 (cyclohexyl C),
116.8 (CN), 89.7, 120.3, 153.1, 157.2, 165.8 (pyridine C), 123.2,
129.8, 132.0, 135.3, (Ar C). 3 (R ¼ 4-CH3OC6H4): 20.6 (CH3),
22.1, 27.6, 31.2, 38.4 (cyclohexyl C), 56.2 (OCH3), 116.8 (CN),
89.7, 121.3, 152.8, 157.7, 165.3 (pyridine C), 114.2, 127.8, 131.5,
162.4 (Ar C). 4 (R ¼ 4-CH3C6H4): 20.2(CH3), 21.2 (CH3), 21.4,
27.1, 30.7, 36.5 (cyclohexyl C), 116.9 (CN), 90.1, 120.6, 154.6,
157.2, 165.3 (pyridine C), 128.0, 129.3, 133.4, 138.6 (Ar C).
5 (R ¼ 2-Thienyl): 20.3 (CH3), 21.5, 28.2, 31.2, 37.81 (cyclohexyl
C), 116.8 (CN), 92.3, 122.3, 148.8, 158.5, 165.3 (pyridine C),
127.4, 127.6, 128.8, 136.9, (thiophene ring C). 6 (R ¼ 1-Methyl-
pyrrol-2-yl): 21.9 (CH3), 32.4 (NCH3), 21.9, 27.28, 31.5, 37.6
(cyclohexyl C), 117.3 (CN), 90.5, 121.8, 153.9, 156.6, 164.8
(pyridine C), 110.9, 112.2, 122.9, 124.1 (pyrrole C).
2,9-Dimethyl-5-substituted-6,7,8,9-tetrahydro-3H-
pyrimido[4,5-b]quinolin-4-ones (12–16)
A mixture of the appropriate compound 2–6 (0.01 mol), acetic
anhydride (5 mL) and concentrated H2SO4 (0.5 mL) was heated in
a boiling water bath for 10 min then cooled, poured into ice-cold
water and treated with 20% NaOH solution until the pH is alkaline
(pH 11). The crude solid product was filtered and crystallized
from ethanol. IR (cmꢂ1): 3226–3431 (NH), 1707–1715 (C¼O).
13CNMR (ꢀ ppm) for 12 (R ¼ 4-BrC6H4): 19.9 (CH3), 20.9 (CH3),
22.3, 27.4, 31.2, 37.4 (cyclohexyl C), 119.8, 123.5, 129.1, 132.3,
135.2, 137.2, 152.4, 162.4, 164.0, 170.6 (ArC), 168.9 (CO).
9-Methyl-5-substituted-6,7,8,9-tetrahydro-3H-
pyrimido[4,5-b]quinolin-4-ones (7–11)
A mixture of the appropriate compound 1–5 (0.01 mol) and 13 (R ¼ 4-CH3OC6H4): 19.8 (CH3), 20.7 (CH3), 21.7, 27.4, 31.8,
formic acid (5 mL) was heated in a boiling water bath for 30 min. 39.0 (cyclohexyl C), 56.2 (OCH3), 114.2, 127.7, 130.5, 135.2,

DOI: 10.3109/14756366.2013.787421
Novel tetrahydroquinolines as anticancer and antimicrobial agents
5
138.5, 152.6, 162.0, 162.4, 163.9, 171.6 (ArC), 170.2 (CO). General methods for the preparation of 4-amino-9-
14 (R ¼ 4-CH3C6H4): 19.5 (CH3), 20.1 (CH3), 21.4 (CH3), 22.6, methyl-5-substituted-6,7,8,9-tetrahydro-1H-pyrimido
27.6, 30.5, 37.0 (cyclohexyl C), 119.4, 126.3, 129.2, 129.4, [4,5-b]quinoline-2-thiones (29–33) and 4-amino-9-methyl-
135.5, 138.2, 152.6, 162.3, 163.6, 170.6 (ArC), 169.8 (CO). 5-substituted-6,7,8,9-tetrahydro-1H-pyrimido[4,5-b]
16 (1-Methyl-pyrrol-2-yl): 20.0 (CH3), 21.3 (CH3), 32.4 quinoline-2-ones (34–36)
(NCH3), 23.2, 28.1, 31.9, 37.4 (cyclohexyl C), 113.1, 119.6,
120.2, 122.2, 135.6, 147.2, 153.4, 161.4, 164.4, 171.6 (ArC),
170.7 (CO).
Method A
A solution of the appropriate thioureido derivative 23–27
(0.02 mol) in a mixture of 1N sodium hydroxide (5.5 mL) and
EtOH (10 mL) was heated on a boiling water bath for 30 min.
The reaction mixture was filtered, if necessary, allowed to cool
and rendered acidic with 10% acetic acid. The product was
filtered, washed thoroughly with H2O, dried and crystallized from
DMF containing few drops of water. IR (cmꢂ1): 3339–3445
(NH2), 1158–1172 (C¼S).
4-Imino-9-methyl-3-phenyl-5-substituted-3,4,6,7,8,9-
hexahydro-1H-pyrimido[4,5-b]-quinoline-2-thiones
(17–22)
A mixture of the appropriate compound 1–6 (0.01 mol), phenyl
isothiocyanate (1.35 g, 0.01 mol) in pyridine (15 mL) was refluxed
for 2 h. After cooling, the solid was filtered, washed thoroughly
with water, dried and recrystallized from acetic acid. IR (cmꢂ1):
3180–3327 (NH), 1629–1615 (C¼N), 1195–1210 (C¼S).
13CNMR (ꢀ ppm) for 17 (R ¼C6H5): 22.2 (CH3), 25.4, 31.2,
32.5, 39.4 (cyclohexyl C), 109.8, 124.5, 124.7, 125.5, 126.8,
128.8, 129.1, 138.3, 139.2, 149.6, 156.8, 163.4 (ArC), 166.0
(C¼NH), 179.8 (CS). 18 (R ¼ 4-BrC6H4): 21.9 (CH3), 25.3, 31.4,
32.3, 39.2 (cyclohexyl C), 109.5, 123.2, 124.6, 125.3, 126.8,
128.8, 129.3, 132.3, 137.3, 139.4, 148.9, 156.7, 163.4 (ArC),
165.4 (C¼NH), 180.5 (CS). 19 (R ¼ 4-CH3OC6H4): 22.0 (CH3),
26.0, 31.8, 32.5, 39.4 (cyclohexyl C), 56.6 (OCH3), 109.8, 114.4,
124.3, 125.2, 127.6, 128.6, 130.5, 139.7, 148.8, 157.1, 162.3,
163.2 (ArC), 164.7 (C¼NH), 178.4 (CS). 20 (R ¼ 4-CH3C6H4):
20.8 (CH3), 22.3 (CH3), 25.6, 30.8, 32.5, 38.8 (cyclohexyl C),
109.8, 124.1, 124.5, 125.4, 126.6, 128.6, 129.5, 135.4,
139.5, 148.8, 157.1, 163.2 (ArC), 164.9 (C¼NH), 179.0 (CS).
22 (1-Methyl-pyrrol-2-yl): 20.6 (CH3), 32.4 (NCH3), 25.2, 31.4,
32.1, 38.5 (cyclohexyl C), 108.1, 110.5, 112.2, 124.6, 125.2,
128.8, 129.8, 133.6, 139.6, 145.2, 157.4, 163.4 (ArC), 164.4
(C¼NH), 179.6 (CS).
Method B
A mixture of the appropriate derivative 1–5 (0.001 mol) and
thiourea (0.4 g, 0.005 mol) or urea (0.3 g, 0.005 mol) was fused
at 260–300 ꢁC using sand bath for 1 h. The reaction mixture was
allowed to attain room temperature. The resulting solid product
was treated with water, then rubbed with ethanol, filtered and
crystallized from DMF containing few drops of water.
Method C
A mixture of the appropriate derivative 1–5 (0.001 mol) and
thiourea (0.4 g, 0.005 mol) or urea (0.3 g, 0.005 mol) was heated
in a microwave reactor at 250 ꢁC for 15 min. Working up of the
reaction was carried out exactly as described under method B.
IR (cmꢂ1) for compounds: 3320–3432 (NH), 1695–1708 (C¼O).
13C NMR (ꢀ ppm) for 29 (R ¼C6H5): 21.9 (CH3), 25.3, 31.4,
32.6, 39.2 (cyclohexyl C), 109.9, 124.3, 126.9, 129.1, 138.2,
149.4, 157.1, 163.2, 164.7 (ArC), 182.4 (CS). 30 (R ¼ 4-BrC6H4):
22.1 (CH3), 25.4, 31.2, 32.3, 37.6 (cyclohexyl C), 109.5, 123.4,
124.7, 129.2, 132.3, 137.1, 148.9, 157.3, 163.2, 164.4 (ArC),
183.2 (CS). 31 (R ¼ 4-CH3OC6H4): 21.8 (CH3), 25.3, 31.3, 32.4,
38.7 (cyclohexyl C), 56.0 (OCH3), 109.7, 114.4, 124.6, 127.8,
130.3, 149.6, 156.9, 162.5, 163.4, 165.3, (ArC), 182.8 (CS).
32 (R ¼ 4-CH3C6H4): 20.8 (CH3), 22.2 (CH3) 25.4, 31.2, 32.3,
38.2 (cyclohexyl C), 109.9, 124.7, 126.4, 129.8, 135.2, 138.2,
149.5, 157.1, 162.9, 163.7 (ArC), 180.2 (CS). 33 (R ¼ 2-thienyl):
21.9 (CH3), 25.2, 31.3, 32.3, 38.7 (cyclohexyl C), 110.4, 122.2,
125.3, 126.4, 127.5, 142.5, 145.3, 157.2, 163.4, 164 (ArC), 181.3
(CS). 34 (R ¼C6H5): 21.8 (CH3), 25.1, 30.8, 31.9, 37.9 (cyclo-
hexyl C), 109.5, 124.4, 126.9, 129.0, 138.2, 149.3, 157.2, 163.3,
164.4 (ArC), 163.2 (CO).
1-Benzoyl-3-(3-cyano-8-methyl-4-substituted-5,6,7,8-
tetrahydroquinolin-2-yl)-thioureas (23–28)
To a solution of the appropriate derivative 1–6 (0.02 mol) in dry
acetone (20 mL), a solution of benzoyl isothiocyanate (3.3 g,
0.02 mol) in dry acetone (10 mL) was added. The resultant
solution was heated under reflux for 3 h. The reaction mixture was
left overnight, concentrated and allowed to cool. The separated
crystalline product was filtered, washed with Et2O and recrys-
tallized from ethanol. IR (cmꢂ1): 3345–3450 (NH), 2210–2220
(CN), 1710–1700 (C¼O), 1185–1168 (C¼S). 13C NMR (ꢀ ppm)
for 23 (R ¼C6H5): 21.7 (CH3), 25.0, 31.3, 32.4, 39.1 (cyclohexyl
C), 91.6, 125.3, 126.8, 127.4, 128.4, 129.1, 129.2, 131.7, 133.5,
138.2, 156.7, 163.2, 166.2 (ArC), 117.6 (CN), 179.5 (CS), 169.9
(CO). 24 (R ¼ 4-BrC6H4): 22.1 (CH3), 25.7, 31.3, 32.4, 37.9
(cyclohexyl C), 91.4, 123.3, 126.8, 127.4, 128.4, 129.1, 131.2,
2-Alkylthio-4-amino-9-methyl-5-substituted-6,7,8,9-
tetrahydro-1H-pyrimido[4,5-b]quinolines (37–47)
132.7, 133.5, 137.2, 156.7, 163.2, 166.2 (ArC), 117.2 (CN), 179.1 To a stirred solution of the proper thione 29–33 (0.02 mol) in a
(CS), 168.7 (CO). 25 (R ¼ 4-CH3OC6H4): 21.8 (CH3), 25.4, 31.5, mixture of 1N NaOH (5 mL) and ethanol (2 mL), the selected
32.2, 39.1 (cyclohexyl C), 56.1 (OCH3), 91.3, 114.6, 125.2, 127.3, alkyl or aralkyl halide (0.022 mol) was added. The reaction
128.6, 130.4, 131.8, 133.5, 156.7, 162.5, 163.2, 166.1 (ArC), mixture was stirred at room temperature for 2–3 h and the
118.0 (CN), 179.8 (CS), 169.5 (CO). 26 (R ¼ 4-CH3C6H4): 20.9 precipitated product was filtered, washed with aqueous ethanol,
(CH3), 22.4 (CH3) 25.6, 31.5, 32.3, 38.0 (cyclohexyl C), 91.7, dried and crystallized from ethanol. IR (cmꢂ1): 3440–3150 (NH).
125.3, 126.8, 127.3, 128.7, 129.7, 131.8, 133.5, 135.2, 138.3, IR (cmꢂ1) for compounds 38 and 41: 1680–1685 (ketone C¼O).
156.6, 163.4, 165.7 (ArC), 117.5 (CN), 179.2 (CS), 169.4 (CO). 13C NMR (ꢀ ppm) for 37 (R ¼C6H5; R0 ¼C2H5): 15.8 (CH3), 21.5
27 (R ¼ 2-thienyl): 21.8 (CH3), 24.9, 31.1, 32.5, 37.9 (cyclohexyl (CH3), 28.1 (CH2), 25.2, 31.3, 32.4, 38.4 (cyclohexyl C), 102.8,
C), 92.4, 122.7, 125.3, 125.8, 127.2, 127.4, 128.5, 131.8, 133.5, 126.9, 127.1, 129.0, 135.2, 138.2, 150.0, 157.9, 162.6, 167.3,
142.6, 152.4, 163.4, 166.2 (ArC), 118.1 (CN), 180.0 (CS), 168.8 167.6 (ArC). 38 (R ¼C6H5; R0 ¼CH2COC6H5): 22.3 (CH3), 44.2
(CO). 28 (1-Methyl-pyrrol-2-yl): 20.6 (CH3), 32.4 (NCH3), 25.2, (CH2) 24.9, 31.5, 32.4, 38.3 (cyclohexyl C), 102.2, 126.4, 126.7,
31.4, 32.1, 38.5 (cyclohexyl C), 92.4, 110.2, 123.1, 125.8, 127.3, 128.4, 128.7, 135.3, 129.2, 132.8, 135.3, 137.1, 138.4, 148.9,
128.5, 131.9, 133.4, 152.2, 163.1, 165.8 (ArC), 117.7 (CN), 179.6 158.3, 162.2, 162.8 (ArC), 196.2 (CO). 39 (R ¼ 4-BrC6H4;
(CS), 169.7 (CO).
R0 ¼CH3): 18.8 (CH3), 21.7 (CH3), 25.1, 31.2, 32.3, 38.8

6
H. M. Faidallah et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Table 3. 1H NMR data of compounds 1–51.
a
NH2 (s)
Compound
Cyclohexyl H (3 m,7H)
CH3 (d, J ¼ 10.0 Hz)
ArH (m)
Others
1
2
3
4
5
6
7
8
9
1.56, 2.36, 2.93
1.48, 2.42, 2.85
1.68, 2.39, 2.88
1.48, 2.42, 2.85
1.65, 2.52, 2.97
1.50, 2.45, 2.90
1.62, 2.35, 3.07
1.52, 2.28, 3.12
1.52, 2.28, 3.12
1.39
1.37
1.36
1.37
1.38
1.35
1.37
1.38
1.38
5.03
5.06
5.09
5.06
5.08
5.05
6.98–7.21 (5H)
7.32–7.49 (4H)
6.99–7.26 (4H)
7.12–7.41 (4H)
6.80–7.27 (3H)b
6.46–6.68 (3H)c
7.22–7.42 (5H)
7.37–7.49 (4H)
6.97–7.26 (4H)
3.85 (s, 3H, OCH3)
2.33 (s, 3H, CH3)
3.52 (s, 3H, NCH3)
7.89 (s, 1H, NH)
7.91 (s, 1H, NH)
3.91 (s, 3H, OCH3)
7.91 (s, 1H, NH)
2.37 (s, 3H, CH3)
7.89 (s, 1H, NH)
8.12 (s, 1H, NH)
2.31 (s, 3H, CH3)
7.95 (s, 1H, NH)
2.35 (s, 3H, CH3)
3.87 (s, 3H, OCH3)
8.12 (s, 1H, NH)
2.37 (s, 3H, CH3)
2.32 (s, 3H, CH3)
8.14 (s, 1H, NH)
2.31 (s, 3H, CH3)
8.16 (s, 1H, NH)
3.5 (s, 3H, NCH3)
7.98 (s, 1H, NH)
5.2 (s, 1H, NH)
10
1.62, 2.35, 3.07
1.34
6.84–7.32 (4H)
11
12
1.62, 2.26, 2.89
1.52, 2.20, 2.95
1.39
1.36
6.83–7.25 (3H)
7.37–7.45 (4H)
13
14
1.62, 2.22, 2.89
1.39
1.36
6.83–7.25 (4H)
7.01–7.59 (4H)
1.63–2.58 (6H)
3.08 (1H)
15
16
1.68–2.43 (6H)
3.10 (1H)
1.52, 2.35, 2.85
1.37
1.35
6.76–7.26 (3H)
6.48–6.72 (3H)
17
18
19
1.62, 2.70, 3.02
1.67, 2.62, 3.22
1.55, 2.65, 3.18
1.35
1.35
1.38
6.92–7.60 (11H)d
7.31–7.45 (10H)d
6.87–7.67 (10H)d
5.14 (s, 1H, NH)
3.95 (s, 3H, OCH3)
5.14 (s, 1H, NH)
3.29 (s, 3H, CH3)
5.07 (s, 1H, NH)
5.20 (s, 1H, NH)
20
21
22
23
24
25
1.67, 2.52, 3.02
1.36
1.40
1.37
1.36
1.39
1.38
7.07–7.52 (10H)d
6.65–7.20 (9H)d
6.42–7.19 (9H)d
7.10–7.79 (10H)
7.12–7.68 (9H)
6.97–7.26 (9H)
1.59–2.49 (6H)
3.20 (1H)
1.68–2.59 (6H)
3.10 (1H)
1.72, 2.78, 3.22
3.46 (s, 3H, NCH3)
5.15 (s, 1H, NH)
5.04 (s, 1H, NHCS)
7.95 (s, 1H, NHCO)
5.10 (s, 1H, NHCS)
8.01 (s, 1H, NHCO)
3.89 (s, 3H, OCH3)
5.14 (s, 1H, NHCS)
8.16 (s, 1H, NHCO)
3.68 (s, 3H, CH3)
5.14 (s, 1H, NHCS)
8.16 (s, 1H, NHCO)
5.18 (s, 1H, NHCS)
7.89 (s, 1H, NHCO)
3.49 (s, 3H, NCH3)
5.09 (s, 1H, NHCS)
8.03 (s, 1H, NHCO)
4.21 (s, 1H, NH)
1.57, 2.60, 3.12
1.48, 2.28, 2.48
26
1.68, 2.29, 3.25
1.37
6.84–7.25 (9H)
27
28
1.69–2.68 (6H),
3.20 (1H)
1.52, 2.39, 2.92
1.35
1.37
6.85–7.76 (9H)
6.52–7.64 (9H)
29
30
31
1.66, 2.51, 2.90
1.56, 2.36, 2.93
1.59, 2.49, 3.20
1.36
1.39
1.40
5.28
5.26
5.29
7.14–7.42 (5H)
6.98–7.68 (4H)
6.90–7.30 (4H)
4.26 (s, 1H, NH)
3.84 (s, 3H, OCH3)
4.19 (s, 1H, NH)
32
1.67, 2.52, 3.02
1.38
5.72
7.07–7.57 (4H)
3.29 (s, 3H, CH3)
4.18 (s, 1H, NH)
4.30 (s, 1H, NH)
8.02 (s, 1H, NH)
3.84 (s, 3H, OCH3)
7.96 (s, 1H, NH)
33
34
35
1.56, 2.44, 3.24
1.53, 2.64, 3.05
1.57, 2.62, 3.14
1.41
1.41
1.39
5.32
5.24
5.35
6.88–7.30 (3H)
6.97–7.54 (5H)
7.12–7.68 (4H)
36
37
1.54, 2.26, 3.10
1.52, 2.18, 3.12
1.38
1.38
5.44
5.22
6.95–7.28 (3H)
6.94–7.48 (5H)
8.10 (s, 1H, NH)
3.22 (q, 2H, CH2)e
1.23 (t, 3H, CH3)e
4.05 (s, 2H, CH2)
2.46 (s, 3H, CH3)
2.51 (s, 3H, CH3)
3.88 (s, 3H, OCH3)
4.17 (s, 2H, CH2)
3.85 (s, 3H, OCH3)
1.22 (t, 3H, CH3)e
3.25 (q, 2H, CH2)e
3.88 (s, 3H, OCH3)
38
39
40
1.68–2.33, 3.20
1.57, 2.48, 3.01
1.48–2.30, 3.23
1.32
1.34
1.39
5.32
5.44
5.25
6.99–7.61 (10H)
7.37–7.46 (4H)
6.99–7.61 (4H)
41
42
1.52, 2.55, 3.04
1.66, 2.51, 2.98
1.34
1.36
5.34
5.28
6.99–7.61 (9H)
7.01–7.52 (4H)
(continued )

DOI: 10.3109/14756366.2013.787421
Table 3. Continued
Novel tetrahydroquinolines as anticancer and antimicrobial agents
7
a
NH2 (s)
Compound
Cyclohexyl H (3 m,7H)
CH3 (d, J ¼ 10.0 Hz)
1.37
ArH (m)
Others
43
1.65, 2.54, 3.12
1.70, 2.62, 3.09
5.65
7.10–7.55 (4H)
2.46 (s, 3H, CH3)
3.29 (s, 3H, CH3)
2.39 (s, 3H, CH3)
4.17 (s, 2H, CH2)
2.51 (s, 3H, CH3)
1.26 (t, 3H, CH3)e
3.28 (q, 2H, CH2)e
4.19 (s, 2H, CH2)
8.40 (s, 1H, H-2)
8.38 (s, 1H, H-2)
44
1.33
5.43
7.17–7.78 (9H)
45
46
1.52, 2.24, 3.03
1.55, 2.26, 3.14
1.32
1.33
5.28
5.27
7.05–7.26 (3H)
6.93–7.28 (3H)
47
48
49
50
1.62, 2.54, 2.94
1.64, 2.55, 2.99
1.59, 2.53, 2.96
1.65, 2.63, 3.02
1.37
1.34
1.36
1.35
5.47
5.66
5.49
5.78
7.03–7.42 (7H)
7.22–7.49 (5H)
7.36–7.51 (4H)
6.83–7.36 (4H)
3.74 (s, 3H, OCH3)
8.39 (s, 1H, H-2)
2.35 (s, 3H, CH3)
8.36 (s, 1H, H-2)
51
1.63, 2.55, 2.94
1.34
5.62
7.12–7.37 (4H)
b
c
d
e
aD2O exchangeable protons; Thiophene ring protons; Pyrrole ring protons; ArH þ HN ¼ ; J ¼ 8.0 Hz.
(cyclohexyl C), 102.1, 123.7, 129.1, 132.4, 135.3, 137.1, 148.7,
Table 4. Cytotoxic effects (LC50; mM)a of the active compounds on some
human tumor cell lines using the MTT assay.
158.4, 162.4, 167.3, 167.7 (ArC). 40 (R ¼ 4-CH3OC6H4;
R0 ¼CH3): 18.9 (CH3), 21.9 (CH3), 25.2, 31.4, 32.2, 38.9
(cyclohexyl C), 56.2 (OCH3), 102.2, 114.7, 127.8, 130.5, 135.3,
150.1, 158.4, 162.5, 162.6, 167.3 (ArC). 41 (R ¼ 4-CH3OC6H4;
R0 ¼CH2COC6H5): 21.8 (CH3), 44.1 (CH2), 25.4, 31.1, 32.2, 38.6
(cyclohexyl C), 56.1 (OCH3) 102.1, 114.6, 127.8, 128.4, 128.7,
130.1, 132.8, 135.4, 137.3, 149.5, 158.2, 162.3, 162.6, 167.3,
167.8 (ArC), 196.4 (CO). 42 (R ¼ 4-CH3OC6H4; R0 ¼C2H5): 15.5
(CH3), 21.6 (CH3), 28.2 (CH2), 25.4, 31.1, 32.2, 38.6 (cyclohexyl
C), 56.1 (OCH3), 102.3, 114.4, 127.9, 130.4, 135.4, 149.9,
158.0, 162.2, 162.5, 167.2, 167.6 (ArC). 44 (R ¼ 4-CH3C6H4;
R0 ¼CH2C6H5): 20.9 (CH3), 22.2 (CH3), 25.0, 31.5, 32.3,
38.1 (cyclohexyl C), 40.7 (CH2), 102.5, 124.3, 126.8, 126.9,
127.6, 128.4, 129.7, 135.2, 135.3, 141.2, 149.9, 158.3, 167.4,
167.7 (ArC).
Compound
no.
Human colon
carcinoma HT29
Human hepatocellular Human breast
carcinoma HepG2
cancer MCF 7
24
25
28
30
33
39
40
42
23.6
28.4
30.3
45.6
50.4
7.9
10.1
15.4
11.5
18.2
12.1
50.3
b
49.2
–
–
–
–
43.2
47.7
20.2
24.6
37.2
30.4
42.6
1.69
–
40.4
2.0
2.8
8.7
3.8
9.4
2.14
45
46
Doxorubicinc
aLC50: Lethal concentration of the compound that causes death of 50% of
cells in 24 h (mM).
4-Amino-9-methyl-5-substituted-6,7,8,9-tetrahydro-
pyrimido[4,5-b]quinolines(48–51)
bTotally inactive against this cell line.
cPositive control cytotoxic agent.
A mixture of the appropriate intermediate 1–4 (0.01 mol) and
formamide (10 ml) was heated under reflux for 2–3 h. The
reaction mixture was cooled and the precipitated solid was
collected, washed with cold ethanol and crystallized from acetic
acid containing few drops of water. IR (cmꢂ1): 3330–3265 (NH2).
48 (R ¼C6H5): 22.0 (CH3), 24.7, 31.3, 32.0, 38.4 (cyclohexyl C),
106.8, 126.9, 129.1, 135.3, 138.2, 149.9, 157.3, 158.5, 162.4,
167.7 (ArC). 49 (R ¼ 4-BrC6H5): 21.7 (CH3), 25.3, 31.4, 32.2,
38.6 (cyclohexyl C), 106.1, 123.7, 129.0, 132.3, 135.2, 137.3,
149.7, 157.4, 158.1, 162.4, 167.7 (ArC). 50 (R ¼ 4-CH3OC6H5):
21.9 (CH3), 25.6, 31.4, 32.3, 38.7 (cyclohexyl C), 56.0 (OCH3),
106.7, 114.4, 127.9, 130.3, 135.4, 149.5, 157.2, 158.3, 162.4,
167.4 (ArC).
(Sheldon, TC2323, Cornelius, OR). Media was aspirated, fresh
medium (without serum) was added and cells were incubated
either alone (negative control) or with eight different concentra-
tions of the test compounds (100, 50, 25, 12.5, 6.25, 3.125, 1.56
and 0.78 mg/mL). DMSO was employed as a vehicle for dissol-
ution of the tested compounds and its final concentration on the
cells was less than 0.2%. Cells were suspended in RPMI 1640
medium (for HepG2 and HT29 cell lines) and DMEM (for MCF 7
cell line), 1% antibiotic-antimycotic mixture (10 000 IU/mL
penicillin potassium, 10 000 mg/mL streptomycin sulphate and
25 mg/mL amphotericin B), and 1% L-glutamine in 96-well flat
bottom microplate at 37 ꢁC under 5% CO2.
Biological activity
After 24 h of incubation, the medium was aspirated, 40 mL of
MTT salt (2.5 mg/mL) was added to each well and incubated for
further 4 h at 37 ꢁC under 5% CO2. To stop the reaction and
In vitro MTT cytotoxicity assay
The synthesized compounds were investigated for their in vitro dissolve the formed crystals, 200 mL of 10% sodium dodecyl
cytotoxic effect via the standard 3-(4,5-dimethylthiazol-2-yl)-2,5- sulphate in deionized water was added to each well and incubated
diphenyltetrazolium bromide (MTT) method18,19 against a panel overnight at 37 ꢁC. The absorbance was then measured using
of three human tumor cell lines: Caucasian breast adenocarcin- a microplate multiwell reader (Bio-Rad Laboratories Inc., model
oma MCF7, hepatocellular carcinoma HepG2 and colon carcin- 3350, Hercules, CA) at 595 nm and a reference wavelength of
oma HT29. The procedures were done in a sterile area using a 620 nm. A statistical significance was tested between samples and
laminar flow cabinet biosafety class II level (Baker, SG403INT, negative control (cells with vehicle) using independent t-test
Stanford, ME). Cells were batch cultured for 10 d, then seeded at by SPSS 11 program. Doxorubicin is used as positive control
concentration of 10 ꢃ 103 cells/well in fresh complete growth cytotoxic agent. The results of LC50 (mM), which is the lethal
medium in 96-well microtiter plastic plates at 37 ꢁC for 24 h under concentration of the compound causing death of 50% of the cells
5% CO2 using a water-jacketed carbon dioxide incubator in 24 h, are presented in Table 4.

8
H. M. Faidallah et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Table 5. Minimal inhibitory concentrations [MIC, mg/mL (mM)] of the tested compounds.
Compound no.
S. aureus
B. subtilis
M. luteus
E. coli
P. aeruginosa
K. pneumoniae
C. albicans
a
2
3
4
5
6
12
13
18
19
21
22
24
25
28
30
31
33
39
40
41
42
44
45
46
50 (146.1)
50 (170.4)
100 (360.5)
50 (185.6)
25 (93.8)
100 (260.2)
100 (298.1)
25 (52.3)
50 (146.1)
100 (340.8)
100 (360.5)
100 (371.2)
100 (375.5)
100 (260.2)
200 (596.3)
50 (104.7)
100 (233.3)
100 (247.1)
100 (249.0)
100 (197.8)
100 (219.0)
100 (232.8)
50 (124.5)
100 (283.6)
100 (304.4)
25 (80.0)
–
50 (146.1)
100 (340.8)
100 (360.5)
200 (742.4)
50 (187.7)
25 (65.0)
100 (298.1)
25 (52.3)
25 (58.3)
100 (292.3)
–
–
–
–
–
–
–
–
–
–
–
–
200 (721.0)
100 (371.2)
–
100 (260.2)
–
–
200 (742.4)
100 (375.5)
100 (260.2)
100 (298.1)
50 (104.7)
50 (116.6)
100 (247.1)
50 (124.5)
100 (197.8)
50 (109.5)
200 (405.6)
25 (62.2)
100 (283.6)
200 (608.8)
12.5 (40.0)
25 (71.2)
50 (106.2)
25 (65.7)
100 (234.4)
100 (292.2)
50 (140.0)
12.5 (36)
–
–
–
200 (596.3)
100 (209.4)
200 (466.6)
–
100 (209.4)
100 (233.3)
–
50 (104.7)
–
25 (58.3)
50 (123.5)
100 (249.0)
50 (98.9)
–
–
100 (247.1)
50 (124.5)
50 (98.9)
–
–
50 (98.9)
100 (219.0)
50 (116.4)
100 (249.0)
200 (567.2)
100 (304.4)
50 (160.0)
–
–
–
–
100 (197.8)
200 (438.0)
50 (116.4)
100 (249.0)
50 (141.8)
–
25 (80.0)
100 (284.8)
200 (424.8)
100 (263.1)
–
50 (109.5)
100 (232.8)
50 (124.5)
100 (283.6)
50 (152.2)
6.25 (20.0)
12.5 (35.6)
25 (53.1)
12.5 (32.8)
50 (117.2)
25 (73.1)
12.5 (35.0)
6.25 (18)
100 (219.0)
100 (232.8)
50 (124.5)
50 (141.8)
50 (152.2)
6.25 (20.0)
6.25 (17.8)
25 (53.1)
12.5 (32.8)
100 (234.4)
12.5 (36.5)
12.5 (35.0)
6.25 (18)
50 (124.5)
100 (283.6)
50 (152.2)
12.5 (40.0)
25 (71.2)
100 (212.4)
25 (65.7)
–
25 (71.2)
100 (284.8)
–
100 (212.4)
50 (131.4)
100 (234.4)
50 (146.1)
50 (140.0)
12.5 (36)
–
–
–
50 (146.1)
100 (280.0)
12.5 (36)
–
50 (117.2)
12.5 (35.0)
–
–
12.5 (36)
–
Ampicillin
Clotrimazole
–
–
–
6.25 (18)
aMIC4200 (600) mg/mL (mM).
In vitro antibacterial and antifungal activities
The key intermediates in this scheme are the 2-amino-3-cyano-
8-methyl-4-substituted-5,6,7,8-tetrahydroquinolines 1–6. These
compounds were easily synthesized via one-pot multicomponent
reaction by treating an appropriate aromatic aldehyde and
2-methylcyclohexanone, with an excess of ammonium acetate
and malononitrile in boiling ethanol21,22. This reaction is of
considerable importance since it is easy to perform, gives high
yields, and is less time consuming compared with the traditional
two-step procedure that involves the formation of the chalcone via
Claisen-Schmidt condensation followed by cyclocondensation
All of the newly synthesized compounds were evaluated for their
in vitro antibacterial activity against Staphylococcus aureus
(ATCC 6538), Bacillus subtilis (ATCC 6633) and Micrococcus
luteus (ATCC 21881) as examples of Gram-positive bacteria and
Escherichia coli (ATCC 25922), Pseudomonas aeruginosa
(ATCC 27853) and Klebsiella pneumonia (clinical isolate) as
examples of Gram-negative bacteria. They were also evaluated for
their in vitro antifungal potential against Candida albicans
(ATCC 10231) and Aspergillus niger (recultured) fungal strains.
Each 100 mL of sterile molten agar (at 45 ꢁC) received 1 mL of
6 h broth culture and then the seeded agar was poured into
sterile Petri dishes. Cups (8 mm in diameter) were cut in the
agar. Each cup received 0.1 mL of the 1 mg/mL solution of the
test compounds. The plates were then incubated at 37 ꢁC for 24 h
or, in case of C. albicans, for 48 h. A control using DMSO without
the test compound was included for each organism. Ampicillin
trihydrate and clotrimazole were used as reference drugs. The
results were recorded for each tested compound as the average
diameter of inhibition zones of bacterial or fungal growth around
the discs in mm. The minimal inhibitory concentrations (MIC) of
the most active compounds were measured using the twofold
serial broth dilution method20. The test organisms were grown in
their suitable broth: 24 h for bacteria and 48 h for fungi at 37 ꢁC.
Twofold serial dilutions of solutions of the test compounds were
prepared using 200, 100, 50, 25, 12.5 and 6.25 mg/mL. The tubes
were then inoculated with the test organisms; each 5 mL received
0.1 mL of the above inoculums and were incubated at 37 ꢁC for
48 h. Then, the tubes were observed for the presence or absence of
microbial growth. The MIC values in mg/mL of the prepared
compounds are listed in Table 5.
with malononitrile and ammonium acetate23,24
.
The IR spectra of these compounds revealed absorption bands
at 3210–3450 characteristic for the NH2 and at 2214–2225 cmꢂ1
attributed to the CN group. 1H-NMR of this series showed beside
the aromatic protons at ꢀ 7.11–7.42 a doublet of three protons
intensity at ꢀ 1.13–1.26 ppm (J ¼ 10.0 Hz) for the CH3 group,
a D2O exchangeable proton singlet of two proton intensity at ꢀ
5.00–5.42 due to the NH2 group, as well as three multiplets at ꢀ
1.53–1.70, 2.29–2.38 and 2.73–2.84 ppm for the cyclohexyl
moiety. The structures were further supported from their 13C
NMR spectral data that showed the expected number of aliphatic
and aromatic carbons.
Reaction of the 2-aminohydroquinoline derivatives 1–5 with
formic acid afforded the targeted 9-methyl-5-substituted-6,7,8,9-
tetrahydro-3H-pyrimido[4,5-b]quinolin-4-ones (7–11). The IR
spectra of these compounds were characterized by the absence
of the CN group absorption and the presence of new sharp
absorption bands at 1695–1715 cmꢂ1 due to the new C¼O groups.
Moreover, the 1H-NMR spectra (Table 3) showed beside the
aromatic protons a doublet of three protons intensity in the
range ꢀ 1.31–1.38 ppm for the CH3 group as well as three
multiplets at ꢀ 1.53–1.70, 2.29–2.38 and 2.83–2.84 ppm corres-
ponding to the protons of the cyclohexyl moiety. Further
confirmation of the structure is supported by their 13C NMR
spectral data (see Experimental section) that exhibited beside
the expected number of aliphatic and aromatic carbons, signals
Results and discussion
Chemistry
The synthetic strategies adopted for the preparation of the at ꢀ 169.2–170.5 ppm for the CO. Treatment the 2-aminohydro-
intermediate and target compounds are described in Scheme 1. quinoline derivatives 2–6 with acetic anhydride in presence of

DOI: 10.3109/14756366.2013.787421
Novel tetrahydroquinolines as anticancer and antimicrobial agents
9
Scheme 1. Reagents and reaction conditions: (i) 2-methylcyclohexanone (0.01 mol), approp. aldehyde (0.01 mol) malononitrile (0.01 mol), NH4OAc
(0.08 mol) in Abs. EtOH (50 mL), reflux 3–6 h; (ii) approp. starting compd. (0.01 mol), formic acid (5 mL), heated in boiling water bath 30 min;
(iii) approp. starting compd. (0.01 mol), Ac2O (5 mL), conc. H2SO4 (0.5 mL), heated in boiling water bath 10 min; (iv) approp. starting compd.
(0.01 mol), PhNCS (0.01 mol) in pyridine (15 mL), reflux 2 h; (v) approp. starting compd. (0.02 mol), PhCONCS (0.02 mol), dry acetone (10 mL),
reflux 3 h; (vi) approp. thioureido deriv. (0.02 mol), 1N NaOH (5.5 mL), EtOH (10 mL), heated in boiling water bath 30 min; (vii) approp. starting
compd. (0.001 mol), urea or thiourea (0.005 mol), fused at 260–300 ꢁC, sand bath 1 h; (viii) approp. starting compd. (0.001 mol), urea or thiourea
(0.005 mol), heated in microwave reactor at 250 ꢁC, 15 min; (ix) approp. thione (0.02 mol), 1N NaOH (5 mL), EtOH (2 mL), alkyl or aralkyl halide
(0.022 mol), stirred at r.t., 2–3 h and (x) approp. starting compd. (0.01 mol), HCONH2 (10 mL), reflux, 2–3 h.
few drops of concentrated sulphuric acid gave the corresponding isothiocyanate in the presence of pyridine gave 4-imino-9-methyl-
2,9-dimethyl-5-substituted-6,7,8,9-tetrahydro-3H-pyrimido[4,5-b] 3-phenyl-5-substituted-3,4,6,7,8,9-hexahydro-1H-pyrimido[4,5-b]
quinolin-4-ones 12–16. The IR spectra of these pyrimido- quinoline-2-thiones 17–22. Their IR spectra revealed two
quinolin-4-one derivatives lacked the CN bands that were present characteristic absorption bands at 1615–1629 cmꢂ1 and 1195–
in the starting quinolines and instead exhibited a carbonyl 1210 cmꢂ1 due to the (C¼N) and (C¼S) groups, respectively. The
absorption bands at 1707–1715 cmꢂ1. The 1H-NMR spectra 1H-NMR spectra showed along with multiplets of the cyclohexyl
showed new singlets at ꢀ 2.45–2.31 ppm due to the new CH3 moiety a doublet of three protons at ꢀ 1.34–1.42 ppm for the CH3
group introduced as well as the three multiplets for cyclohexyl group. The structures of the above pyrimido[4,5-b]quinoline-2-
moiety at ꢀ 1.51–1.75, 2.24–2.36 and 2.72–2.88 ppm. Likewise, thiones 17–22 were further substantiated by their 13C NMR
their 13C NMR spectral data exhibited beside the expected spectra that exhibited two characteristic signals at 178.0–180.0
number of aliphatic and aromatic carbons, a new CH3 singlet at ꢀ and ꢀ 164.4–166.0 ppm for the CS and C¼NH groups, respect-
19.5–20.2 ppm in addition to a CO signal at ꢀ 168.9–170.7. ively, in addition to the expected number of aliphatic (cyclohexyl)
Reacting the starting aminohydroquinolines 1–6 with the phenyl and aromatic carbons. On the other hand, when compounds 1–6

10 H. M. Faidallah et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
Figure 1. 1-Benzoyl-3-[3-cyano-8-methyl-4-
(1-methyl-1H-pyrrol-2-yl)-5,6,7,8-tetrahy-
droquinolin-2-yl]thiourea.
were reacted with benzoyl isothiocyanate in dry acetone, the intensity at ꢀ 1.30–1.35 ppm for the CH3 group, three multi-
corresponding substituted thioureido derivatives 23–28 were plets for the cyclohexyl moiety as well as a broad singlet at ꢀ
formed in good yields. The IR spectra of these compounds 7.89–8.12 for the NH2 group, in addition the methylthio
maintained the characteristic CN absorptions 2210–2220 cmꢂ1 in derivatives 39, 40, 43 and 45 that exhibited a singlet at ꢀ
addition to the two new bands at 1700–1710 and 1168–1185 cmꢂ1 2.45–2.48 for the S-CH3 group while the ethylthio derivatives 37,
attributed to the C¼O and C¼S groups, respectively. The 13C 42 and 46 afforded a triplet of three proton intensity at
NMR spectra of these compounds showed three signals at ꢀ ꢀ 1.23–1.26 (J ¼ 8.0 Hz) and a quartet of two proton intensity
117.2–118.1, 179.1–180.3 and 168.7–169.9 ppm for the CN, at ꢀ 2.96–3.21 (J ¼ 8.0 Hz) due to the CH3 and CH2 groups,
CS and C¼NH groups, respectively. Moreover, the structure respectively. Further confirmation for the structure was achieved
of the quinoline derivative 28 was also supported by by their 13C NMR spectral data that lacked the CS signals
X-ray crystallography25 (Figure 1). Cyclization of compounds that existed in the original compounds and exhibited instead an
23–27 was done by heating them with sodium hydroxide S-CH3 signal at ꢀ 18.5–19.2 in case of the S-CH3 derivatives
to form 4-amino-9-methyl-5-substituted-6,7,8,9-tetrahydro-1H- and two signals at ꢀ 15.5–15.8 and ꢀ 27.8–28, in case of the
pyrimido[4,5-b]quinoline-2-thiones 29–33. It is worth mentioning S-ethyl compounds. Furthermore, heating the key intermediates
that direct condensation of 1–5 either with thiourea or urea 1–4 with formamide resulted in the formation of the targeted
was attempted as another efficient procedure for a one-step 4-amino-9-methyl-5-substituted-6,7,8,9-tetrahydro-pyrimido[4,5-
synthesis of the target compounds 29–33 and 34–36, respectively. b]quinolines 48–51. The IR spectra of these pyrimido[4,5-b]
The IR spectra of these derivatives (29–33) were characterized quinolines derivatives were characterized by the disappearance
by the disappearance of the cyano and the carbonyl absorptions of the cyano groups absorptions and the appearance of broad
and the appearance of broad absorption bands at 3339–3445 cmꢂ1 absorption bands at 3265–3330 cmꢂ1 due to the amino group.
1
due to amino group as well as a CS absorption at 1158– Their H-NMR spectra showed beside the aromatic protons at ꢀ
1172 cmꢂ1. The 1H-NMR spectra showed beside the aromatic 6.77–7.45, a distinct doublet for three protons at ꢀ 1.33–1.37 ppm
protons at ꢀ 6.78–7.42, a doublet of three protons at intensity for the CH3 group along with three multiplets at of the cyclohexyl
of ꢀ 1.33–1.36 ppm for the CH3 group. Further confirmation moiety and a broad singlet at ꢀ 7.92–8.22 for the NH2 group.
for the structure was done from their 13C NMR spectral data Further structural assignments were done by their 13C NMR
that exhibited beside the expected number of aliphatic and spectral data.
aromatic carbons, a signal at ꢀ 162.3–163.5 or at ꢀ 182.4–184.6
for the CO (in compounds 29–33) and CS (in compounds 34–36),
respectively. Thioakylation of the 2-thione derivatives 29–33
In vitro MTT cytotoxicity assay
could be affected by treating these compounds with a variety Twenty-one analogs (1, 4, 5, 7, 8, 11, 12, 15, 16, 24, 25, 28, 30,
of alkyl or aralkyl halides in the presence of sodium hydroxide 33, 39, 40, 42, 45, 46, 48 and 49) were selected to be evaluated for
to produce the alkylthio derivatives 37–47. Their IR spectra their in vitro cytotoxic effect via the standard MTT method
exhibited broad absorption bands at 3262–3338 cmꢂ1 due to against a panel of three human tumor cell lines: Caucasian breast
the amino group. The 1H-NMR spectra showed beside the adenocarcinoma MCF7, hepatocellular carcinoma HepG2 and
aromatic protons at ꢀ 6.84–7.62, a doublet of three protons with colon carcinoma HT29. The results of LC50 (mM), which is the

DOI: 10.3109/14756366.2013.787421
Novel tetrahydroquinolines as anticancer and antimicrobial agents 11
lethal concentration of the compound causing death of 50% of the
cells in 24 h, are presented in Table 4.
Among the tested Gram-positive bacterial strains, two organ-
isms, namely S. aureus and B. subtilis, showed relatively high
The obtained data revealed that the three tested human tumor sensitivity towards the tested compounds. In this view, compound
cell lines exhibited variable degree of sensitivity profiles towards 39 was almost equipotent to ampicillin (MIC 6.25 mg/mL) against
ten of the tested compounds: 24, 25, 28, 30, 33, 39, 40, 42, 45 and S. aureus, whereas the analogs 40, 42 and 46 (MIC 12.5 mg/mL)
46. Among these, compounds 39, 40 and 45 showed strong were 50% less active than ampicillin. Compounds 39 and 40
activity against the human colon carcinoma HT29 cell line with showed half the activity of ampicillin (MIC 25 mg/mL) against
LC50 values of 7.9, 10.1 and 11.5 mM, respectively. Moreover, B. subtilis. However, these two compounds were able to produce
a remarkable cytotoxic potential was displayed by compounds 42 a distinctive growth inhibitory profile against E. coli that was
and 46 against the same cell line (15.4 and 18.2 mM). Compounds equipotent to ampicillin (MIC 6.25 mg/mL), whereas compounds
24, 25 and 28 revealed almost similar cytotoxicity profile against 42, 45 and 46 showed 50% less activity than ampicillin (MIC
colon carcinoma HT29 with LC50 values of 23.6, 28.4 and 12.5 mg/mL) against the same organism. Meanwhile, the tested
30.3 mM, respectively. However, compounds 30 and 33 were able P. aeruginosa strain showed moderate sensitivity towards
to exhibit moderate activity against the same cell line with LC50 most of the active compounds, particularly compound 39 (MIC
value range of 45.6 and 50.4 mM. Furthermore, the growth of the 12.5 mg/mL) which was equipotent to ampicillin.
human hepatocellular carcinoma HepG2 cell line was found to be
Eleven compounds 18, 19, 30, 31, 33, 39, 40, 41, 42, 45 and 46
moderately inhibited by eight of the active compounds 24, 30, 33, have displayed a significant growth inhibitory potential against
39, 40, 42, 45 and 46 with LC50 values ranging from 20.2 to C. albicans, among which compounds 39 and 46 (MIC
50.3 mM. Among these, the highest cytotoxic activity was 12.5 mg/mL) were the most active members when compared
displayed by compounds 39 and 40 (LC50 values of 20.2 and with clotrimazole (MIC 6.25 mg/mL). A close examination of the
24.6 mM, respectively). On the other hand, human breast cancer structures of the active compounds revealed that their antimicro-
MCF 7 was proved to be the least sensitive among the cell lines bial activity is strongly related to the nature of the substituents
tested as it was affected by only seven of the test compounds. at positions C-2 and C-4, together with the nature of the rings
However, an outstanding growth inhibition potential was shown fused with the quinoline system. In general, it could be seen
by compounds 39, 40, 42, 45 and 46 as evidenced from their LC50 that potential antibacterial activity was connected with the
values (2.0, 2.8, 8.7, 3.8 and 9.4 mM, respectively). The remaining electron-withdrawing group (R ¼ 4-BrC6H4) at C-4, whereas
two active compounds, namely 24 and 33, showed moderate moderate activity was displayed by the electron-donating
to mild activity against the same cell line with LC50 values of (R ¼ 4-CH3OC6H4) group at the same carbon. In this context,
49.2 and 40.4 mM, respectively. Further interpretation of the hexahydroquinoline, the key precursors 2, 3, 4, 5 and 6 showed
results revealed that compounds 39, 40, 42, 45 and 46 showed moderate antimicrobial potential, with particular effectiveness
considerable broad spectrum of cytotoxic activity against the against S. aureus and E. coli. Tetrahydropyrimido[4,5-b]quinolin-
three tested human tumor cell lines. In particular, compound 39 4(3H)-ones 7–11 obtained as a result of formic acid-induced
proved to be the most active member in this study with a broad cyclization led to the complete abolishment of the activity.
spectrum of activity against the tested cell lines, with special However, acetic anhydride-derived cyclized products introduced
effectiveness against the human colon carcinoma HT29 and a methyl group at position 2. In these series, two compounds,
human breast cancer MCF 7 cell lines (LC50 values 7.9 and 12 and 13, were found to retain antimicrobial activity where
2.0 mM, respectively). A close examination of the structure of the substituent R at position 4 is either 4-bromophenyl or
the active compounds showed that the 4-bromophenyl counterpart 4-methoxyphenyl, although these two compounds are noticeably
at position 4 of the tetrahydroquinoline skeleton is the most less active than the parent compounds 2 and 3. On the other hand,
favourable substituent when compared with other analogs. phenylisothiocyanate-derived cyclization gave rise to four active
Moreover, the thioalkyl substituent at position 2 as in compounds compounds 18, 19, 21 and 22 among which the bromo-derivative
39, 40, 42, 45 and 46 was responsible for the high activity 18 was the most active one. Furthermore, reaction of the key
displayed by these analogs. Thiomethyl analogs, 39 and 40, intermediates, hexaquinoline derivatives with benzoylisothiocya-
displayed better profile when compared with a thioethyl deriva- nate, produced a series of compounds, out of which 24 and 25
tive as in case of compound 42. Although compound 30 showed showed slightly improved antimicrobial profiles when compared
some activity because it carries a 4-bromophenyl substituent, but with 2 and 3. The cyclization of the thiourea derivatives, 23–27,
it lacks optimal activity owing to the presence of 2-thione and/or to the corresponding 2-thiones, 29–33, however, did not change
the absence of a thioalkyl substituent at position 2. Substitution at the biological profile significantly. Moreover, alkylation of the
position 2 with a thiourea moiety or cyclization of these thiourea quinoline-2-thiones 29–33 resulted in an obvious enhancement
derivatives resulted in moderate to weakly active compounds 24, of the antimicrobial activity as well as the spectrum as can be seen
25, 28, 30 and 33 whereas cyclization of the intermediate in 39, 40, 42, 45 and 46. It could be clearly recognized that the
quinoline derivatives (1–6) with formic acid, acetic anhydride activity of these analogs is connected with the aryl substituent
and formamide led to complete abolishment of the cytotoxic in position 4 as well as to the substituent linked to position 2.
activity.
Within this series, compound 39 (R ¼ 4-Br–C6H4) was the
most active member as it displayed a fourfold increase in the
antimicrobial activity against S. aureus, E. coli and P. aeruginosa
(MIC 6.25, 6.25 and 12.5 mg/mL, respectively), together with
In vitro antibacterial and antifungal activities
As revealed from MIC data recorded in Table 5 [MIC expressed a remarkable antifungal activity (12.5 mg/mL), when compared
in both concentration units mg/mL (mM)], 24 of the 51 newly with the parent compound 2.
synthesized compounds displayed variable inhibitory effects on
the growth of the tested Gram-positive and Gram-negative
Conclusion
microorganisms with pronounced activity against S. aureus
and E. coli bacterial strains. In addition, some members The present paper describes the synthesis of 2-amino-3-cyano-
exhibited moderate antifungal activity against C. albicans, 8-methyl-4-substituted-5,6,7,8-tetrahydroquinolines as well as
whereas, all the tested compounds lacked antifungal activity derived fused-ring systems. Ten compounds showed remarkable
against Aspergillus niger.
cytotoxic efficiency against human colon carcinoma HT29,

12 H. M. Faidallah et al.
J Enzyme Inhib Med Chem, Early Online: 1–12
thiosemicarbazone Sn(IV) complexes. Eur J Med Chem 2005;40:
467–72.
hepatocellular carcinoma Hep-G2 and Caucasian breast adeno-
carcinoma MCF7 cell lines. Compounds 24, 39, 40, 42, 45 and 46
showed considerable broad-spectrum cytotoxic activity among
which 39 and 40 proved to be the most active derivatives. A close
examination of the structure of the active compounds showed that
the 4-bromophenyl counterpart of the tetrahydroquinoline skel-
eton is the most favourable substituent when compared with other
analogs. Moreover, the thioalkyl substituent at position 2 as in
compounds 39, 40, 42 and 45 was responsible for the high activity
displayed by these analogs. However, the cyclization of the
intermediate quinoline derivatives (1–6) with formic acid, acetic
anhydride and formamide led to complete abolishment of activity.
Likewise compounds 18, 19, 39, 40, 42, 45 and 46 were found to
exhibit moderate antimicrobial activity and compounds 39, 40 and
46 proved to be the most active candidates. Compounds 39 and 40
could be considered as possible antimicrobial and anticancer
candidates that deserve further investigation and derivatization in
order to explore the scope and limitation of their biological
activities.
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some 3-cyano-1,4,6-trisubstituted -2-(1H)-pyridinones and their
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Acknowledgements
The authors are deeply thankful to the authorities of the Bioassay-Cell
Culture Laboratory, Drug Discovery Unit, National Research Centre,
Cairo, Egypt, for their efforts in performing the MTT cytotoxicity assay.
16. Faidallah HM, Rostom SAF, Asiri AM, et al. 3-Cyano-8-methyl-2-
oxo-1,4-disubstituted-1,2,5,6,7,8-hexahydroquinolines:
synthesis
and biological evaluation as antimicrobial and cytotoxic agent.
J Enzyme Inhibition Med Chem 2013; 28:123–30.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
17. Faidallah HM, Rostom SAF, Khan KA, Basaif SA. Synthesis and
characterization of some hydroxypyridone derivatives and their
evaluation as antimicrobial agents. J Enzyme Inhibition Med Chem
2013; 28:495–508.
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