Synthesis and antitumor screening of new series of pyrimido-[4,5-b] quinolines and [1,2,4]triazolo[2′,3′:3,4]pyrimido[6,5-b]quinolines

Article abstract of DOI:10.1007/s00044-012-0272-y

New series of pyrimido[4,5-b]quinolines and [1,2,4]triazolo[2′, 3′:3,4]pyrimido[6,5-b]quinolines have been synthesized. Compounds 4a, 4e, 4f, 4h, 5b, 5d, 6a, 6d, 6e, 8c, 8d, 10c-e, 10h, 11a, 11b, and 12a were tested for in vitro antitumor activity against

Full text of DOI:10.1007/s00044-012-0272-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2724–2736
DOI 10.1007/s00044-012-0272-y
ORIGINAL RESEARCH
Synthesis and antitumor screening of new series of pyrimido-[4,5-
b]quinolines and [1,2,4]triazolo[20,30:3,4]pyrimido[6,5-b]quinolines
•
M. B. El-Ashmawy M. A. El-Sherbeny
•
N. S. El-Gohary
Received: 20 July 2012 / Accepted: 5 October 2012 / Published online: 17 October 2012
Ó Springer Science+Business Media New York 2012
Abstract New series of pyrimido[4,5-b]quinolines and
[1,2,4]triazolo[20,30:3,4]pyrimido[6,5-b]quinolines have
been synthesized. Compounds 4a, 4e, 4f, 4h, 5b, 5d, 6a,
6d, 6e, 8c, 8d, 10c–e, 10h, 11a, 11b, and 12a were tested
for in vitro antitumor activity against human breast
carcinoma (MCF-7) cell line, where compound 8d was
found to be the most active member with IC50 value of
3.62 lM. The DNA-binding affinity for the same com-
pounds showed that compounds 8d and 10d exhibited the
highest affinity to DNA. The detailed synthesis, spec-
troscopic, and biological data are reported.
properties (El-Gazzar et al., 2008; Kidwai and Negi, 1997;
El-Gazzar et al., 2009a, b) as well as anti-inflammatory
activity (El-Gazzar et al., 2008, 2009a, b; Abd El-Salam
et al., 2009). Considering these published data and our
previous interests in the field of fused pyrimidines as
antitumor agents (El-Sayed et al., 2011; El-Ashmawy
et al., 2010; Mahran et al., 1998), it appeared worthwhile
to prepare certain derivatives of pyrimido[4,5-b]quinoline
and
[1,2,4]triazolo[20,30:3,4]pyrimido[6,5-b]quinoline
skeletons whose rigidity might contribute to a potential
DNA-binding affinity and antitumor activity. Herein, we
report their synthesis, preliminary DNA-binding assay and
antineoplastic evaluation against human breast carcinoma
(MCF-7) cell line.
Keywords Pyrimido[4,5-b]quinolines ꢀ
[1,2,4]Triazolo[2030:3,4]pyrimido[6,5-b]quinolines ꢀ
In vitro antitumor screening
Results and discussion
Introduction
Chemistry
Quinoline and pyrimidoquinoline derivatives continue to
attract the interest of medicinal chemists due to the wide
range of biological properties. Some of them showed
antitumor activity (Al-Said et al., 2011; Abbas et al., 2011;
Ghorab et al., 2010; Alqasoumi et al., 2010; Ghorab et al.,
2009; Ali et al., 2008; Shrestha et al., 2008; Abouzid and
Shouman, 2008; Ali et al., 2007; Wilson et al., 2007; Rhee
et al., 2007), while other derivatives have been used as
antimicrobial agents (El-Gazzar et al., 2008; Suresh et al.,
2003; Selvi et al., 2006). Also, some of these derivatives
showed antimalarial activity (Joshi et al., 2005), analgesic
2-Amino-1,4,5,6,7,8-hexahydro-4-(substituted)phenylquinoline-
3-carbonitriles (2a–d) were prepared (Elkholy, 2007), via
the reaction of cyclohexanone, ammonium acetate, and the
appropriate benzylidenemalononitrile 1a–d (Bigi et al.,
2000). The 1H NMR spectra of these compounds revealed
the presence of a singlet at d 5.51–6.55 ppm significant for
C4–H. This excluded structures 3a–d and supported the
formation of the adducts 2a–d. Compounds 2a–d, typical
b-enaminonitrile derivatives, were used as precursors for
the synthesis of pyrimidoquinoline derivatives. Refluxing
2a–d with aliphatic acids, namely formic and acetic acids in
the presence of catalytic amount of concentrated hydro-
chloric acid yielded 6,7,8,9-tetrahydro-2-(unsubstituted
or methyl)-5-(substituted)phenylpyrimido[4,5-b]quinolin-
4(3H, 5H, 10H)-ones (4a–h) (Scheme 1). The reaction
M. B. El-Ashmawy ꢀ M. A. El-Sherbeny ꢀ N. S. El-Gohary (&)
Medicinal Chemistry Department, Faculty of Pharmacy,
Mansoura University, Mansoura 35516, Egypt
e-mail: dr.nadiaelgohary@yahoo.com
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Med Chem Res (2013) 22:2724–2736
2725
Scheme 1 Synthesis of
compounds 2-7
R
O
R
CN
CN
x
+
CN
N
NH2
1a-d
3a-d
NH4OAc
Ethanol
R
R
R
S
O
N
CN
R1COOH
HCl
CS2
NH
NH
R1
Pyridine
S
N
H
N
H
N
NH2
N
H
H
5a-d
2a-d
4a-h
H
C
R1= H for 4a-d; CH3 for 4e-h
(
S
O
C
C
2
N
e
n
H
5
)
i
R
d
i
r
3
y
R
P
R
NH
R1
S
CN
CHOC2H5
N
N
N
H
N
H
N
H
6a-h
7a-d
R1= C6H5 for 6a-d; n-C4H9 for 6e-h
R = a) H; b) 3-Br; c) 4-Cl; d) 3,4-(OCH3)2
proceeds via initial acid hydrolysis of the nitrile group to
give the corresponding carboxamide derivatives, which
then condensed with formic or acetic acid with loss of two
molecules of water to yield the desired products 4a–h. The
the disappearance of the (C:N) absorption band con-
firming the formation of the desired cyclic structures. The
mass spectrum of compound 5b showed peaks at m/z 408
(M??2), 407 (M??1), 406 (M?) and a base peak at m/z
1
1
spectral data (IR, MS and H NMR) of these compounds
105. Regarding the H NMR spectrum of the same com-
were in agreement with the assigned structures. The IR
spectrum for compound 4d showed absorption band at
1,663 cm-1 for (C=O), also it showed the disappearance of
the (C:N) absorption band. Regarding the 1H NMR
spectra of compounds 4e–h, the protons of the (CH3) group
of the pyrimidine ring appeared as a singlet at d 2.11 and
1.96 ppm for the two tested compounds 4f and 4h,
respectively. The remaining protons were observed at the
expected regions. The mass spectrum of compound 4e
showed a molecular ion peak of m/z 293 and a base peak of
m/z 216.
pound, two singlets at d 6.57 and 8.60 ppm indicating
(C5–H) and (NH), respectively, were clearly identified.
Also, it showed two triplets at d 2.19 and 2.70 ppm for 2-
(CH2) groups, in addition to a multiplet at d 1.59–1.72 ppm
that represents the four protons of the other 2-(CH2)
groups. Aromatic protons appeared at the expected regions.
Upon heating compound 2a–d with n-butyl or phenyl
isothiocyanate in pyridine, 3,4,5,6,7,8,9,10-octahydro-
4-imino-3-(n-butyl or phenyl)-5-(substituted)phenylpyrim-
ido[4,5-b]quinoline-2(1H)-thiones (6a–h) were obtained
(Scheme 1). The structures of these compounds were con-
firmed by elemental analyses, IR and 1H NMR spectra. The
IR spectra of the tested compounds displayed absorption
band at the range 3,399–3,425 cm-1 for (3NH), also it
showed the absence of the (C:N) absorption band con-
firming the formation of the desired cyclic structures. The
1H NMR spectrum of 6h exhibited a significant multiplet at
d 0.88–1.38 ppm corresponding to the butyl protons, in
On the other hand, the ortho aminonitriles 2a–d were
further utilized for another cyclocondensation reaction
using carbon disulfide in pyridine to afford the 5,6,7,8,9,
10-hexahydro-5-(substituted)phenyl-pyrimido[4,5-b]quino-
line-2,4(1H, 3H)-dithiones (5a–d) (Scheme 1). The struc-
tural assignments of compounds 5a–d were based on
microanalytical and spectral data. The IR spectra showed
123

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Med Chem Res (2013) 22:2724–2736
addition to a singlet at d 3.41 ppm for (2NH) groups.
Moreover, the signals indicating (4CH2), (C5–H) and aro-
matic protons were observed at the expected regions.
Condensation of compound 2a–d with triethyl ortho-
formate yielded the corresponding ethyl N-[3-cyano-
1,4,5,6,7,8-hexahydro-4-(substituted)phenylquinolin-2-
yl]formimidates (7a–d) (Scheme 1). The structural
assignments of compounds 7a–d were based on analytical
and spectral data. The IR spectra displayed an absorption
Reaction of compound 7a–d with 35 % ammonia solu-
tion in ethanol produced the corresponding pyrimdoquin-
oline derivatives 8a–d rather than the opened analogs as
evidenced from IR data (Scheme 2). The structures of these
compounds were established based on their elemental
analyses and spectral data. The IR spectra revealed the
absence of the (C:N) absorption band, confirming the for-
1
mation of the desired cyclic structures. In addition, the H
NMR spectra of these compounds showed the disappear-
ance of the quartet-triplet pattern of the ethyl ester group
and the appearance of a significant singlet at d 3.01–
3.03 ppm in all derivatives corresponding to the (NH2)
group. Treatment of compounds 7a–d with hydrazine
hydrate in ethanol yielded 3-amino-3,4,6,7,8,9-hexahydro-
4-imino-5-(substituted)phenyl-5H, 10H-pyrimido[4,5-b]
quinolines (9a–d) (Scheme 2). The IR spectrum of com-
pound 9c showed three absorption bands at 3427, 3353, and
1
band at 2,208–2,213 cm-1 for (C:N). H NMR spectra
showed the typical quartet-triplet pattern characteristic for
1
the ethyl ester group. The H NMR spectrum of 7a dis-
played a quartet signal at d 4.34–4.38 ppm for (2H,
OCH2CH3), while the triplet signal was observed at d
1.34 ppm for (3H, OCH2CH3). Furthermore, the signals
characteristic for 4-(CH2), (NH), (N=CH), (C4–H) and the
aromatic protons appeared at the expected regions.
Scheme 2 Synthesis of
compounds 8-12
R
R
NH2
CN
CHOC2H5
N
N
NH3
Ethanol
N
H
N
H
N
7a-d
8a-d
NH2NH2.H2O
Ethanol
R
R
NH
NH
R1
R1
N
N
CHO
C
H
N
NH2
N
N
N
H
N
Piperidine/dioxane
H
10a-h
9a-d
R1= 4-Cl for 10a-d; 3,4-(OCH3)2 for 10e-h
R
2
5
3
N
R
Cl
N
N
CO
OC
2H5/
Benzene
N
H
N
NCOOC2H5
X
11a-d
N
NHCOOC2H5
N
H
N
R
12a-d
O
N
N
COOC2H5
N
N
H
N
13a-d
R = a) H; b) 3-Br; c) 4-Cl; d) 3,4-(OCH3)2
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Med Chem Res (2013) 22:2724–2736
2727
3201 cm-1 for (NH2) and 2-(NH) groups, respectively.
It is worth mentioning that reaction of compound 9a–d
with ethyl chloroformate in benzene afforded the unexpected
opened structures 12a–d rather than the desired cyclic
compounds 13a–d as evidenced by analytical and spectro-
scopic data (Scheme 2). The IR spectra showed an intense
carbonyl ester absorption band at 1,710–1,729 cm-1 in all
derivatives. The IR spectrum of 12b as an example displayed
absorption bands at 3,332 and 1,729 cm-1 indicating (2NH)
and(C=O)groups, respectively. Thenotablefeature inthe 1H
NMR spectra is the presence of two multiplets at d 1.26–1.42
and 4.21–4.31 ppm corresponding to (2COOCH2CH3) and
(2COOCH2CH3), respectively, confirming the formation of
compounds 12a–d.
Also, it showed the disappearance of the (C:N) absorp-
tion band. The H NMR spectrum of the same compound
1
revealed the presence of three singlets at d 5.46, 5.83, and
7.76 ppm indicating (NH2), (C5–H) and 2-(NH) groups,
respectively. In addition, the remaining protons appeared at
the expected regions.
The preparation of 3,4,6,7,8,9-hexahydro-4-imino-
3-(substituted benzylidene)amino-5-(substituted)-phenyl-
5H, 10H-pyrimido[4,5-b]quinolines (10a–h) has been
accomplished by reacting equimolar amounts of compound
9a–d with the appropriate benzaldehyde in dioxane in the
presence of piperidine as a basic catalyst (Scheme 2). The
elemental analyses and spectral data (IR, MS, and 1H
NMR) of compounds 10a–h were in agreement with the
assigned structures. The mass spectrum of compound 10c
showed a molecular ion peak of m/z 450 and a base peak of
m/z 283. The 1H NMR spectrum of compound 10a
exhibited three singlets at d 3.25, 3.70, and 5.24 ppm
indicating (NH), (NH), and (C5–H), respectively. Also, it
showed a multiplet at d 7.24–7.52 ppm characteristic for
(Ar–H, N=CH).
Biological investigation
The in vitro antitumor activity against breast cancer
(MCF-7) cell line
Potential antitumor activities of compounds 4a, 4e, 4f, 4h,
5b, 5d, 6a, 6d, 6e, 8c, 8d, 10c–e, 10h, 11a, 11b, and 12a were
screened at the National Cancer Institute (NCI), Cancer
Biology Department, Pharmacology Unit, Cairo, Egypt,
adopting the sulforhodamine B (SRB) assay, method of
(Skehan et al., 1990) using doxorubicin as a reference anti-
tumor agent.
Condensation of compound 9a–d with triethyl ortho-
formate afforded 7,8,9,10,11,12-hexahydro-12-(substituted)
phenyl-[1,2,4]triazolo[20,30:3,4]pyrimido[6,5-b]quinolines
(11a–d) (Scheme 2). The structures of compounds 11a–d
1
were ascertained through elemental analyses and H NMR
1
spectra. The notable feature in the H NMR spectra is the
The results of in vitro antitumor activity of the tested
compounds indicated that compound 8d exhibited the
highest cytotoxic activity against human breast cancer cells
(MCF-7) with IC50 value of 3.62 lM. Compounds 4e, 4f,
5b, 6a, 6d, 6e, 8c, 10d, 10e, 10h, and 12a showed lower
activity with IC50 values of 4.97, 4.03, 4.50, 6.98, 5.30,
5.70, 3.89, 3.76, 5.37, 6.17, and 5.44 lM, respectively. On
the other hand, compounds 4a, 4h, 5d, 10c, 11a, and 11b
are the least active members with IC50 values greater than
10 lM (Table 1).
presence of four singlets characteristic for (C2–H), (C5–H),
(NH), and (C12–H). For example, the 1H NMR spectrum of
11b showed four singlets at d 5.08, 8.20, 8.41, and
9.40 ppm corresponding to (C12–H), (NH), (C5–H), and
(C2–H), respectively. In addition, the signals representing
the (4CH2) groups and the aromatic protons appeared at the
expected regions. The mass spectrum of compound 11d
showed a molecular ion peak of m/z 364 and a base peak of
m/z 309.
Table 1 In vitro antitumor
Comp. no.
Antitumor activity
IC50 (lM)a, b
Comp. no.
Antitumor activity
IC50 (lM)a, b
activity of the selected
compounds and doxorubicin
against human breast cancer
cells (MCF-7)
4a
4e
4f
[10.00
4.97
8d
3.62
[10.00
3.76
10c
4.03
10d
4h
5b
5d
6a
6d
6e
8c
[10.00
4.50
10e
5.37
10h
6.17
[10.00
6.98
11a
[10.00
[10.00
5.44
11b
a
IC50, compound concentration
required to inhibit tumor cell
proliferation by 50 %
5.30
12a
5.70
Doxorubicin
0.70
b
Values are means of three
experiments
3.89
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Med Chem Res (2013) 22:2724–2736
Table 2 DNA-binding affinity
of the selected compounds and
bleomycin using methyl green/
DNA displacement assay
Comp. no. Methyl green/
DNA IC50 (lMa)
Comp. no. Methyl green/DNA Comp. no.
IC50 (lM)
Methyl green/DNA
IC50 (lM)
4a
4e
4f
88
22
32
25
72
76
38
25
2
2
2
2
1
1
2
2
6c
55
40
72
88
25
12
88
70
2
2
1
2
2
1
2
2
10d
10e
10h
11a
11b
12a
14
32
38
88
90
40
2
2
2
2
2
2
2
6d
6e
a
Values represent the
concentration (mean SD,
n = 3–5 separate
4h
5a
5b
5d
6a
6g
8c
8d
10a
10c
determinations) required for
50 % decrease in the initial
absorbance of DNA/methyl
green solution
Bleomycin 10
DNA-binding assay
of methyl green from DNA by compounds with ability to
bind DNA. The degree of displacement was determined
spectrophotometrically by measuring the change in initial
absorbance of methyl green/DNA solution in the presence
of reference compound.
The mechanism by which several known antitumor agents
produce their effects involves interaction with DNA.
Alkylating agents and intercalating agents represent two
major classes of antitumor drugs that act by direct inter-
action with DNA. Based on this fact, some short-term
procedures have been designed to be applied for the dis-
covery and evaluation of both naturally-occurring and
synthetic compounds that function by this mechanism.
DNA-binding assay (Pezzuto et al., 1983, 1991) and
methyl green/DNA displacement assay (Burres et al.,
1992) were applied for determination of the interaction of
small molecular weight compounds with DNA.
The obtained data indicated that compounds 8d and 10d
are the most active members with IC50 values of 12 and
14 lM, while compounds 4e, 4f, 4h, 5d, 6a, 6d, 8c, 10e,
10h and 12a showed moderate affinity with IC50 values of
22, 32, 25, 38, 25, 40, 25, 32, 38, and 40 lM, respectively
(Table 2).
Some structural features were found to be beneficial to
the antitumor activity of such compounds. In particular, the
pyrimido[4,5-b]quinoline derivatives were more active
than those possessing the [1,2,4]triazolo[20,30:3,4]pyrimi-
do[6,5-b]quinoline skeleton. Also, the presence of 3,
4-dimethoxyphenyl moiety at the 5-position of the pyrim-
idoquinoline ring system may contribute in the DNA-
binding affinity as in compounds 4h, 5d, 6d, 8d, 10d, and
10h. This indicates that better affinity could be attained by
the presence of 5-phenyl moiety substituted with electron-
donating groups. In addition, introduction of electron-
donating groups such as methyl or amino at the 2- or
4-position, respectively, of the pyrimidine ring favored the
activity as in compounds 4e, 4f, 4h, and 8c. On the other
hand, incorporation of the triazole ring into the pyrimido-
quinoline system completely abolished the activity as in
compounds 11a and 11b.
DNA-binding assay on TLC plates
It was demonstrated that when DNA was applied to RP-18
TLC plates, migration was observed when MeOH/H2O
(8:2) was used as the elution solvent. However, when DNA
was mixed with compounds with which it interacts (e.g.,
ethidium bromide), the complex was retained at the origin
with the same elution solvent. On the other hand, com-
pounds with no affinity did not retain the DNA at the origin
(Pezzuto et al., 1983, 1991).
Results from DNA-binding assay revealed that com-
pounds 8d and 10d displayed the highest DNA-binding
affinity which was demonstrated by retaining the complex at
the origin or by its migration for a very short distance.
Compounds 4e, 4f, 4h, 5d, 6a, 6d, 8c, 10e, 10h, and 12a
showed moderate affinity, while compounds 5a, 5b, 6c, 6e,
and 10c exhibited weak affinity. On the other hand, com-
pounds 4a, 6g, 10a, 11a, and 11b were proved to be inactive.
In summary, the present results suggest that the syn-
thesized compounds possess moderate antitumor activity
comparable to the activity of the commonly used antican-
cer drug, doxorubicin.
Methyl green/DNA displacement assay
Experimental
Methyl green reversibly binds polymerized DNA forming a
stable complex at neutral pH. The maximum absorption for
methyl green/DNA complex is 642.5–645 nm. This assay
(Burres et al., 1992) was used to measure the displacement
Chemistry
All melting points (°C) were recorded on Fisher-Johns
melting point apparatus and are uncorrected. The infrared
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Med Chem Res (2013) 22:2724–2736
2729
spectra were recorded in KBr disk using a Unicam SP 1000
IR spectrometer (v in cm-1) at Faculty of Science, Manso-
ura University. Nuclear magnetic resonance (1H NMR)
spectra were obtained on 300 MHz FT-NMR spectrometer
at Faculty of Science, Cairo University. The chemical
shifts are expressed in d ppm using tetramethylsilane
(TMS) as internal reference and DMSO-d6 or CDCl3 as
solvents. Mass spectra were recorded on JEOL JMS-600H
spectrometer using electron impact technique at 70 eV at
Microanalytical Unit, Cairo University. Microanalyses
(C, H, N) were performed at Micro-analytical Unit, Cairo
University, and were in agreement with the proposed
structures within 0.4 of the calculated values. Reaction
times were monitored using TLC plates, Silica gel 60 F254
precoated (E. Merck), and the spots were visualized by UV
(366 nm).
C16H16BrN3 (330.22): C 58.19, H 4.88, N 12.72. Found: C
58.40, H 5.18, N 12.53.
2-Amino-4-(4-chlorophenyl)-1,4,5,6,7,8-hexahydroquinoline-
3-carbonitrile (2c) Yield 85 %, m.p. 252–253 °C. 1H
NMR spectrum: (DMSO-d6, d ppm): 1.59–1.74 (m, 4H,
2CH2), 2.20 (t, 2H, CH2), 2.71 (t, 2H, CH2), CH2), 6.61 (s,
1H, C4–H), 7.34–7.59 (m, 7H, Ar–H, NH2, NH). MS m/z
(%): 288 (0.17, M??2), 287 (0.77, M??1), 283 (100.00,
M?-3). Anal.Calcd. for C16H16ClN3 (285.77): C 67.25, H
5.64, N 14.70. Found: C 67.00, H 5.80, N 14.53.
2-Amino-1,4,5,6,7,8-hexahydro-4-(3,4-dimethoxyphenyl)
quinoline-3-carbonitrile (2d) Yield 80 %, m.p. 254–
255 °C. IR spectrum (KBr, v, cm-1): 3429, 3303 (NH2);
3146 (NH); 2936, 2837 (CH); 2208 (C:N). 1H NMR
spectrum: (DMSO-d6, d ppm): 1.69–1.85 (m, 4H, 2CH2),
2.39 (t, 2H, CH2), 2.87 (t, 2H, CH2), 3.87 (s, 3H, OCH3),
3.94 (s, 3H, OCH3), 5.51 (s, 1H, C4–H), 6.76–6.99 (m, 6H,
Ar–H, NH, NH2). MS m/z (%): 313 (0.02, M??2), 312
(0.31, M??1), 311 (3.27, M?), 309 (100.00, M?-2). Anal.
Calcd. for C18H21N3O2 (311.38): C 69.43, H 6.80, N 13.49.
Found: C 69.17, H 6.58, N 13.23.
Compounds 1a–d were prepared according to the
reported procedure (Bigi et al., 2000) compounds, 2a–d
were prepared adopting the described procedure (Elkholy,
2007). The following materials were used in the biological
screening: Breast cancer (MCF-7) cell line (American
Type Culture Collection, Rockville, MD, USA). TLC
plates (RP-18F254, 0.25 mm, Merck). DNA and Anisalde-
hyde (Sigma-Aldrich Co., USA). Methyl green/DNA
(Sigma, St. Louis, MO, USA).
General procedure for the preparation of 6,7,8,9-
tetrahydro-2-(unsubstituted or methyl)-5-
(substituted)phenylpyrimido[4,5-b]quinolin-4(3H,
5H, 10H)-ones (4a–h)
General procedure for the preparation of 2-amino-
1,4,5,6,7,8-hexahydro-4-(substituted)phenyl-
quinoline-3-carbonitriles (2a–b)
A mixture of compound 2a–d (0.005 mol), formic acid or
glacial acetic acid (10 mL) and a catalytic amount of
concentrated hydrochloric acid (2 mL) was heated at reflux
temperature for 48 h. The reaction mixture was allowed to
cool to room temperature, poured onto crushed ice and
neutralized with 1N sodium hydroxide solution. The pre-
cipitated solid was collected by filtration, dried, and crys-
tallized from the appropriate solvent.
A mixture of cyclohexanone (4.9 g, 0.05 mol), ammonium
acetate (5.78 g, 0.075 mol) and the appropriate benzylid-
enemalononitrile 1a–d (0.05 mol) in absolute ethanol
(50 mL) was heated at reflux temperature for 6 h. The
precipitated solid was collected by filtration, dried and
crystallized from ethanol to yield the desired hexahydro-
quinoline derivatives 2a–d.
2-Amino-1,4,5,6,7,8-hexahydro-4-phenylquinoline-3-carboni-
trile (2a) Yield 75 %, m.p. 225–227 °C. IR spectrum
(KBr, v, cm-1): 3419, 3306 (NH2); 3150 (NH); 2928, 2861
(CH), 2211 (C:N). 1H NMR spectrum: (DMSO-d6, d
ppm): 1.57–1.76 (m, 4H, 2CH2), 2.21 (t, 2H, CH2), 2.71 (t,
2H, CH2), 6.55 (s, 1H, C4–H), 7.28–7.53 (m, 8H, Ar–H,
NH2, NH). MS m/z (%): 250 (6.30, M?-1), 249 (43.80,
M?-2), 51 (100.00). Anal. Calcd. for C16H17N3 (251.33):
C 76.46, H 6.82, N 16.72. Found: C 76.70, H 6.65, N 16.43.
6,7,8,9-Tetrahydro-5-phenylpyrimido[4,5-b]quinolin-4(3H, 5H,
10H)-one (4a) Crystallized from DMF, yield 76 %, m.p.
198–200 °C. IR spectrum (KBr, v, cm-1): 3,420 (2NH);
2975, 2899 (CH); 1,654 (C=O). 1H NMR spectrum:
(DMSO-d6, d ppm): 1.56–1.70 (m, 4H, 2CH2), 2.17–2.68
(m, 4H, 2CH2), 5.44 (s, 1H, C5–H), 6.49 (s, 1H, NH), 7.26–
7.46 (m, 7H, Ar–H, NH). Anal.Calcd. for C17H17N3O
(279.34): C 73.10, H 6.13, N 15.04. Found: C 72.85, H
6.31, N 15.23.
5-(3-Bromophenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-
4(3H, 5H, 10H)-one (4b) Crystallized from DMF, yield
82 %, m.p.117–119 °C. IR spectrum (KBr, v, cm-1): 3426,
3313 (2NH); 2955, 2875 (CH); 1667 (C=O). 1H NMR
spectrum: (DMSO-d6, d ppm): 1.63–1.82 (m, 4H, 2CH2),
2-Amino-4-(3-bromophenyl)-1,4,5,6,7,8-hexahydroquinoline-3-
carbonitrile (2b) Yield 70 %, m.p. 232–234 °C. IR spec-
trum (KBr, v, cm-1): 3420, 3302 (NH2); 3147 (NH); 2943,
2863 (CH), 2213 (C:N). MS m/z (%): 331 (3.30, M??1),
330 (20.18, M?), 329 (100.00, M?-1). Anal. Calcd. for
123

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6,7,8,9-Tetrahydro-5-(3,4-dimethoxyphenyl)-2-methylpy-
rimido[4,5-b]quinolin-4(3H, 5H, 10H)-one (4h) Crystal-
2.23–2.88 (m, 4H, 2CH2), 5.54 (s, 1H, C5–H), 6.67 (s, 1H,
NH), 7.45–7.73 (m, 6H, Ar–H, NH). MS m/z (%): 356
(5.70, M?-2), 327 (100.00). Anal. Calcd. for
C17H16BrN3O (358.23): C 57.00, H 4.50, N 11.73. Found:
C 56.75, H 4.21, N 11.53.
1
lized from DMF, yield 80 %, m.p. 210–212 °C. H NMR
spectrum: (DMSO-d6, d ppm): 1.68–1.91 (m, 4H, 2CH2),
1.96 (s, 3H, CH3), 2.41 (t, 2H, CH2), 2.90 (t, 2H, CH2),
3.91 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 5.61 (s, 1H, C5–
H), 6.77–7.00 (m, 4H, Ar–H, NH), 8.64 (s, 1H, NH). Anal.
Calcd. for C20H23N3O3 (353.41): C 67.97, H 6.56, N 11.89.
Found: C 67.70, H 6.21, N 11.63.
5-(4-Chlorophenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-
4(3H, 5H, 10H)-one (4c) Crystallized from DMF, yield
67 %, m.p. decomp. 220 °C. MS m/z (%): 315 (0.40,
M??1), 314 (0.70, M?), 60 (100.00). Anal. Calcd. for
C17H16ClN3O (313.78): C 65.07, H 5.14, N 13.39. Found:
C 65.32, H 4.91, N 13.61.
General procedure for the preparation of 5,6,7,8,9,10-
hexahydro-5-(substituted)phenylpyrimido[4,5-b]
quinoline-2,4(1H, 3H)-dithiones (5a–d)
6,7,8,9-Tetrahydro-5-(3,4-dimethoxyphenyl)pyrimido[4,5-b]
quinolin-4(3H, 5H, 10H)-one (4d) Crystallized from
DMF, yield 78 %, m.p. 257–259 °C. IR spectrum (KBr, v,
cm-1): 3336, 3164 (2NH); 2936, 2858 (CH); 1663 (C=O).
1H NMR spectrum: (DMSO-d6, d ppm): 1.52–1.72 (m, 4H,
2CH2), 2.17–2.65 (m, 4H, 2CH2), 3.89 (s, 3H, OCH3), 3.92
(s, 3H, OCH3), 5.44 (s, 1H, C5–H), 6.62–7.27 (m, 6H, Ar–
H, 2NH). MS m/z (%): 341 (0.10, M??2), 340 (0.70,
M??1), 339 (2.50, M?), 216 (100.00). Anal. Calcd. for
C19H21N3O3 (339.39): C 67.24, H 6.24, N 12.38. Found: C
67.46, H 6.21, N 12.53.
A mixture of compound 2a–d (0.005 mol) and carbon
disulfide (3.8 g, 0.05 mol) in pyridine (5 mL) was heated
at reflux temperature on a water bath (80 °C) for 18 h. The
reaction mixture was allowed to cool to 0 °C. Ethanol
(20 mL) was added to the reaction mixture and the pre-
cipitated solid was filtered, washed with ethanol, dried, and
crystallized from acetone.
5,6,7,8,9,10-Hexahydro-5-phenylpyrimido[4,5-b]quinoline-
2,4(1H, 3H)-dithione (5a) Yield 70 %, m.p. 228–230 °C.1H
NMR spectrum: (DMSO-d6, d ppm): 1.58–1.71 (m, 4H,
2CH2), 2.19 (t, 2H, CH2), 2.70 (t, 2H, CH2), 5.48 (s, 1H,
NH), 6.56 (s, 1H, C5–H), 7.19–7.58 (m, 7H, Ar–H, 2NH).
Anal. Calcd. for C17H17N3S2 (327.47): C 62.35, H 5.23, N
12.83. Found: C 62.50, H 5.41, N 12.65.
6,7,8,9-Tetrahydro-2-methyl-5-phenylpyrimido[4,5-b]quino-
lin-4(3H, 5H, 10H)-one (4e) Crystallized from DMF,
yield 80 %, m.p.200–202 °C. IR spectrum (KBr, v, cm-1):
3338, 3167 (2NH); 2934, 2859 (CH); 1664 (C=O). 1H
NMR spectrum: (DMSO-d6, d ppm): 1.57–1.76 (m, 4H,
2CH2), 2.05 (s, 3H, CH3), 2.19–2.72 (m, 4H, 2CH2), 5.52
(s, 1H, C5–H), 6.50 (s, 1H, NH), 7.13–7.54 (m, 6H, Ar–H,
NH). MS m/z (%): 293 (61.3, M?), 216 (100.00). Anal.
Calcd. for C18H19N3O (293.36): C 73.69, H 6.53, N 14.32.
Found: C 73.95, H 6.27, N 14.63.
5-(3-Bromophenyl)-5,6,7,8,9,10-hexahydropyrimido[4,5-b]
quinoline-2,4(1H, 3H)-dithione (5b) Yield 65 %, m.p.
235–237 °C. 1H NMR spectrum: (DMSO-d6, d ppm):
1.59–1.72 (m, 4H, 2CH2), 2.19 (t, 2H, CH2), 2.70 (t, 2H,
CH2), 6.57 (s, 1H, C5–H), 7.29–7.68 (m, 6H, Ar–H, 2NH),
8.64 (s, 1H, NH). MS m/z (%): 408 (14.00, M??2), 407
(14.00, M??1), 406 (14.00, M?), 105 (100.00). Anal.
Calcd. for C17H16BrN3S2 (406.36): C 50.25, H 3.97, N
10.34. Found: C 50.48, H 4.21, N 10.53.
5-(3-Bromophenyl)-6,7,8,9-tetrahydro-2-methylpyrimido
[4,5-b]quinolin-4(3H, 5H, 10H)-one (4f) Crystallized
from ethanol/DMF(3:2), yield 85 %, m.p. 170–172 °C. IR
spectrum (KBr, v, cm-1): 3338, 3152 (2NH); 2928, 2858
(CH); 1654 (C=O).1H NMR spectrum: (DMSO-d6, d ppm):
1.52–1.72 (m, 4H, 2CH2), 2.11 (s, 3H, CH3), 2.19–2.69 (m,
4H, 2CH2), 5.46 (s, 1H, C5–H), 6.50 (s, 1H, NH),
7.07–7.47 (m, 5H, Ar–H, NH). Anal. Calcd. for
C18H18BrN3O (372.26): C 58.08, H 4.87, N 11.29. Found:
C 58.34, H 4.62, N 11.15.
5-(4-Chlorophenyl)-5,6,7,8,9,10-hexahydropyrimido[4,5-b]
quinoline-2,4(1H, 3H)-dithione (5c) Yield 80 %, m.p.
250–252 °C. IR spectrum (KBr, v, cm-1): 3,396 (3NH);
1
2952, 2866 (CH). H NMR spectrum: (DMSO-d6, d ppm):
1.57–1.63 (m, 4H, 2CH2), 2.19 (t, 2H, CH2), 2.68 (t, 2H,
CH2), 6.56 (s,1H, C5–H), 7.32–7.58 (m, 6H, Ar–H, 2NH),
8.62 (s, 1H, NH). Anal. Calcd. for C17H16ClN3S2 (361.91):
C 56.42, H 4.46, N 11.61. Found: C 56.10, H 4.21, N 11.83.
5-(4-Chlorophenyl)-6,7,8,9-tetrahydro-2-methylpyrimido
[4,5-b]quinolin-4(3H, 5H, 10H)-one (4g) Crystallized
from ethanol/DMF (3:2), yield 72 %, m.p. 223–225 °C.
MS m/z (%): 328 (66.70, M?), 327 (55.60, M?-1), 283
(100.00). Anal. Calcd. for C18H18ClN3O (327.81): C 65.95,
H 5.53, N 12.82. Found: C 65.61, H 5.70, N 12.67.
5,6,7,8,9,10-Hexahydro-5-(3,4-dimethoxyphenyl)pyrimido
[4,5-b]quinoline-2,4(1H, 3H)-dithione (5d) Yield 80 %,
1
m.p. 239–240 °C. H NMR spectrum: (CDCl3, d ppm):
1.58–1.74 (m, 4H, 2CH2), 2.25 (t, 2H, CH2), 2.69 (t, 2H,
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Med Chem Res (2013) 22:2724–2736
2731
1
CH2), 3.76 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 5.58 (s, 1H,
C5–H), 6.66–7.07 (m, 5H, Ar–H, 2NH), 9.20 (s, 1H, NH).
Anal. Calcd. for C19H21N3O2S2 (387.52): C 58.89, H 5.46,
N 10.84. Found: C 58.60, H 5.21, N 10.75.
200–202 °C. H NMR spectrum: (CDCl3, d ppm): 0.91–
1.69 (m, 13H, CH3, 5CH2), 2.88–2.96 (m, 4H, 2CH2), 3.35
(s, 2H, 2NH), 5.20 (s, 1H, C5–H), 7.27–7.70 (m, 5H, Ar–
H), 8.03 (s, 1H, NH). Anal. NH). Anal. Calcd. for
C21H26N4S (366.52): C 68.82, H 7.15, N 15.29. Found: C
69.15, H 7.21, N 15.03.
General procedure for the preparation of 3,4,5,6,7,
8,9,10-octahydro-4-imino-3-(n-butyl or phenyl)-5-
(substituted)phenylpyrimido[4,5-b]quinoline-2(1H)-
thiones (6a–h)
5-(3-Bromophenyl)-3-n-butyl-3,4,5,6,7,8,9,10-octahydro-4-imi-
nopyrimido[4,5-b]quinoline-2(1H)-thione (6f) Yield 70 %,
m.p. [300 °C. IR spectrum (KBr, v, cm-1): 3,398 (3NH),
1
A mixture of compound 2a–d (0.005 mol) and the appro-
priate isothiocyanate (0.005 mol) in pyridine (5 mL) was
heated at reflux temperature for 12–16 h. The reaction
mixture was allowed to cool to 0 °C and poured onto
ethanol (20 mL). The precipitated solid was collected by
filtration, washed with ethanol, dried and crystallized from
ethanol/DMF (3:2).
3,024 (CH). H NMR spectrum: (CDCl3, d ppm): 0.83–
1.68 (m, 13H, CH3, 5CH2), 2.89–2.96 (m, 4H, 2CH2), 3.32
(s, 2H, 2NH), 5.40 (s, 1H, C5–H), 7.46–7.71 (m, 4H, Ar–
H), 8.03 (s, 1H, NH). Anal. Calcd. for C21H25BrN4S
(445.42): C 56.63, H 5.66, N 12.58. Found: C 56.87, H
5.80, N 12.43.
3-n-Butyl-5-(4-chlorophenyl)-3,4,5,6,7,8,9,10-octahydro-
(6g)
3,4,5,6,7,8,9,10-Octahydro-4-imino-3,5-diphenylpyrimido
[4,5-b]quinoline-2(1H)-thione (6a) Yield 70 %, m.p.
4-iminopyrimido[4,5-b]quinoline-2(1H)-thione
Yield 65 %, m.p. [300 °C. IR spectrum (KBr, v, cm-1):
3,425 (3NH), 3064, 2955, 2929 (CH). H NMR spectrum:
1
1
238–240 °C. H NMR spectrum: (CDCl3, d ppm): 1.77–
1.83 (m, 4H, 2CH2), 2.89–2.96 (m, 4H, 2CH2), 6.92–7.69
(m, 13H, Ar–H, C5–H, 2NH), 8.64 (s, 1H, NH). Anal.
Calcd. for C23H22N4S (386.51): C 71.47, H 5.74, N 14.50.
Found: C 71.25, H 5.97, N 14.33.
(CDCl3, d ppm): 0.93–1.86 (m, 13H, CH3, 5CH2), 2.35 (t,
2H, CH2), 2.84 (t, 2H, CH2), 3.41 (s, 2H, 2NH), 5.10 (s,
1H, C5–H), 7.25–7.57 (m, 4H, Ar–H), 8.63 (s, 1H, NH).
Anal. Calcd. for C21H25ClN4S (400.97): C 62.90, H 6.28, N
13.97. Found: C 63.15, H 6.45, N 13.83.
5-(3-Bromophenyl)-3,4,5,6,7,8,9,10-octahydro-4-imino-3-
phenylpyrimido[4,5-b]quinoline-2(1H)-thione (6b) Yield
3-n-Butyl-3,4,5,6,7,8,9,10-octahydro-4-imino-5-(3,4-dime-
thoxyphenyl)pyrimido[4,5-b]quinoline-2(1H)-thione (6h)
1
65 %, m.p. 165–167 °C. H NMR spectrum: (CDCl3, d
1
ppm): 1.88–1.92 (m, 4H, 2CH2), 2.39–2.43 (m, 4H, 2CH2),
6.87–7.94 (m, 12H, Ar–H, C5-H, 2NH), 9.15 (s, 1H, NH).
Anal. Calcd. for C23H21BrN4S (465.41): C 59.36, H 4.55,
N 12.04. Found: C 59.65, H 4.81, N 11.93.
Yield 50 %, m.p. [300 °C. H NMR spectrum: (CDCl3, d
ppm): 0.88–1.38 (m, 9H, CH3, 3CH2), 1.68–1.84 (m, 4H,
2CH2), 2.39 (t, 2H, CH2), 2.83 (t, 2H, CH2), 3.41 (s, 2H,
2NH), 5.06 (s, 1H, C5–H), 6.76–7.05 (m, 4H, Ar–H, NH).
Anal. Calcd. for C23H30N4O2S (426.57): C 64.76, H 7.09,
N 13.13. Found: C 65.15, H 7.21, N 12.93.
5-(4-Chlorophenyl)-3,4,5,6,7,8,9,10-octahydro-4-imino-3-
phenylpyrimido[4,5-b]quinoline-2(1H)-thione
(6c)
Yield 75 %, m.p. 218–220 °C. IR spectrum (KBr, v,
cm-1): 3,399 (3NH), 3,055 (CH). 1H NMR spectrum:
(DMSO-d6, d ppm): 1.82–1.89 (m, 4H, 2CH2), 2.41–2.47
(m, 4H, 2CH2), 6.77–7.57 (m, 12H, Ar–H, C5–H, 2NH),
8.66 (s, 1H, NH). Anal. Calcd. for C23H21ClN4S (420.96):
C 65.62, H 5.03, N 13.31. Found: C 65.99, H 5.21, N 13.53.
General procedure for the preparation of ethyl N-[3-
cyano-1,4,5,6,7,8-hexahydro-4-(substituted)-
phenylquinolin-2-yl]formimidates (7a–d)
A mixture of compound 2a–d (0.01 mol) and triethyl
orthoformate (10 mL) was heated at reflux temperature for
10–12 h. Excess reagent was evaporated under reduced
pressure, and the solid obtained was triturated with ice-
water, filtered, washed with water, dried and crystallized
from ethanol.
3,4,5,6,7,8,9,10-Octahydro-4-imino-5-(3,4-dimethoxyphenyl)-
3-phenylpyrimido[4,5-b]quinoline-2(1H)-thione (6d) Yield
1
80 %, m.p. 200–202 °C. H NMR spectrum: (CDCl3, d
ppm): 1.27–2.40 (m, 8H, 4CH2), 3.89 (s, 6H, 2OCH3), 5.89
(s, 1H, C5–H), 6.40–7.27 (m, 11H, Ar–H, 3NH). Anal.
Calcd. for C25H26N4O2S (446.56): C 67.24, H 5.87, N
12.55. Found: C 67.50, H 6.11, N 12.33.
Ethyl N-[3-cyano-1,4,5,6,7,8-hexahydro-4-phenylquinolin-
2-yl]formimidate (7a) Yield 70 %, m.p. 101–103 °C. IR
spectrum (KBr, v, cm-1): 3,421 (NH); 2940, 2873 (CH);
1
2,210 (C:N). H NMR spectrum: (DMSO-d6, d ppm):
1.34 (t, 3H, OCH2CH3), 1.66–1.78 (m, 4H, 2CH2), 2.36–
3-n-Butyl-3,4,5,6,7,8,9,10-octahydro-4-imino-5-phenylpyr-
imido[4,5-b]quinoline-2(1H)-thione (6e) Yield 72 %, m.p.
2.88 (m, 4H, 2CH2), 4.34–4.38 (q, 2H, OCH2CH3), 6.54
123

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Med Chem Res (2013) 22:2724–2736
(s, 1H, C4–H), 7.37–7.51 (m, 6H, Ar–H, N=CH), 8.55
(s,1H, NH). Anal. Calcd. for C19H21N3O (307.39): C 74.24,
H 6.89, N 13.67. Found: C 73.95, H 6.61, N 13.83.
2.21–2.71 (m, 4H, 2CH2), 3.03 (s, 2H, NH2), 6.55 (s, 1H,
C5–H), 7.29–7.62 (m, 6H, Ar–H), 8.45 (s, 1H, NH). Anal.
Calcd. for C17H18N4 (278.35): C 73.35, H 6.52, N 20.13.
Found: C 73.10, H 6.31, N 20.43.
Ethyl N-[4-(3-bromophenyl)-3-cyano-1,4,5,6,7,8-hexahy-
droquinolin-2-yl]formimidate (7b) Yield 72 %, m.p.
110–112 °C. IR spectrum (KBr, v, cm-1): 3,425 (NH);
2927, 2862 (CH); 2,237 (C:N). 1H NMR spectrum:
(DMSO-d6, d ppm): 1.39 (t, 3H, OCH2CH3), 1.84–1.87 (m,
4H, 2CH2), 2.43–2.94 (m, 4H, 2CH2), 4.34–4.37 (q, 2H,
OCH2CH3), 6.59 (s, 1H, C4–H), 7.35–7.63 (m, 5H, Ar–H,
N=CH), 8.51 (s, 1H, NH). Anal. Calcd. for C19H20BrN3O
(386.29): C 59.08, H 5.22, N 10.88. Found: C 59.30, H
5.36, N 11.13.
4-Amino-5-(3-bromophenyl)-5,6,7,8,9,10-hexahydropyrim-
ido[4,5-b]quinoline (8b) Yield 65 %, m.p. 185–187 °C.
IR spectrum (KBr, v, cm-1): 3,396 (NH2, NH); 3055,
1
2933, 2827 (CH). H NMR spectrum: (DMSO-d6, d ppm):
1.72–1.85 (m, 4H, 2CH2), 2.19–2.71 (m, 4H, 2CH2), 3.02
(s, 2H, NH2), 6.59 (s, 1H, C5–H), 7.32–7.72 (m, 5H, Ar–
H), 8.46 (s,1H, NH). Anal. Calcd. for C17H17BrN4
(357.25): C 57.15, H 4.80, N 15.68. Found: C 56.96, H
4.68, N 15.83.
Ethyl N-[4-(4-chlorophenyl)-3-cyano-1,4,5,6,7,8-hexahy-
droquinolin-2-yl]formimidate (7c) Yield 75 %, m.p.
125–127 °C. IR spectrum (KBr, v, cm-1): 3,420 (NH),
2932, 2882 (CH), 2213 (C:N). 1H NMR spectrum:
(DMSO-d6, d ppm): 1.34 (t, 3H, OCH2CH3), 1.67–1.77 (m,
4H, 2CH2), 2.36–2.88 (m, 4H, 2CH2), 4.44–4.47 (q, 2H,
OCH2CH3), 5.08 (s, 1H, C4–H), 7.19–7.61 (m, 5H, Ar–H,
N=CH), 8.42 (s, 1H, NH). Anal. Calcd. for C19H20ClN3O
(341.83): C 66.76, H 5.90, N 12.29. Found: C 66.45, H
5.71, N 12.46.
4-Amino-5-(4-chlorophenyl)-5,6,7,8,9,10-hexahydropyrim-
ido[4,5-b]quinoline (8c) Yield 75 %, m.p. 228–229 °C.
IR spectrum (KBr, v, cm-1): 3,399 (NH2, NH); 3055, 2924,
1
2887 (CH). H NMR spectrum: (DMSO-d6, d ppm): 1.71–
1.80 (m, 4H, 2CH2), 2.19–2.70 (m, 4H, 2CH2), 3.02 (s, 2H,
NH2), 6.59 (s, 1H, C5–H), 7.32–7.72 (m, 5H, Ar–H), 8.45
(s, 1H, NH). MS m/z (%): 314 (6.10, M??1), 313 (4.40,
M?), 117 (100.00). Anal. Calcd. for C17H17ClN4(312.80):
C 65.28, H 5.48, N 17.91. Found: C 65.50, H 5.62, N 17.75.
4-Amino-5,6,7,8,9,10-hexahydro-5-(3,4-dimethoxyphenyl)py-
rimido[4,5-b]quinoline (8d) Yield 68 %, m.p. 197–199 °C.
IR spectrum (KBr, v, cm-1): 3,405 (NH2, NH); 3055, 2929
Ethyl N-[3-cyano-1,4,5,6,7,8-hexahydro-4-(3,4-dimethoxy-
phenyl)quinolin-2-yl]formimidate (7d) Yield 70 %, m.p.
174–175 °C. IR spectrum (KBr, v, cm-1): 3,429 (NH);
2936, 2837 (CH); 2,208 (C:N). 1H NMR spectrum:
(DMSO-d6, d ppm): 1.34 (t, 3H, OCH2CH3), 1.66–1.77 (m,
4H, 2CH2), 2.51 (t, 2H, CH2), 2.87 (t, 2H, CH2), 3.77 (s,
3H, OCH3), 3.83 (s, 3H, OCH3), 4.32–4.39 (q, 2H,
OCH2CH3), 6.46 (s, 1H, C4–H), 6.86–7.11 (m, 4H, Ar–H,
N=CH), 8.48 (s, 1H, NH). MS m/z (%): 367 (5.70, M?),
366 (26.30, M?-1), 365 (100.00). Anal. Calcd. for
C21H25N3O3 (367.44): C 68.64, H 6.86, N 11.44. Found: C
68.36, H 6.71, N 11.38.
1
(CH). H NMR spectrum: (DMSO-d6, d ppm): 1.71–1.88
(m, 4H, 2CH2), 2.27–2.69 (m, 4H, 2CH2), 3.01 (s, 2H,
NH2), 3.81 (s, 6H, 2OCH3), 6.49 (s, 1H, C5–H), 6.88–7.16
(m, 4H, Ar–H), 8.43 (s, 1H, NH). Anal.Calcd. for
C19H22N4O2 (338.40): C 67.44, H 6.55, N 16.56. Found: C
67.25, H 6.82, N 16.73.
General procedure for the preparation of 3-amino-
3,4,6,7,8,9-hexahydro-4-imino-5-(substituted)-phenyl
-5H, 10H-pyrimido[4,5-b]quinolines (9a–d)
A mixture of compound 7a–d (0.005 mol) and hydra-
zine hydrate 98 % (2.5 g, 0.05 mol) in absolute ethanol
(15 mL) was heated at reflux temperature for 12 h. The
reaction mixture was cooled and the precipitated solid was
collected by filtration, washed with water, dried and crys-
tallized from dioxane.
General procedure for the preparation of 4-amino-
5,6,7,8,9,10-hexahydro-5-(substituted)phenyl-
pyrimido[4,5-b]quinolines (8a–d)
A mixture of compound 7a–d (0.005 mol) and ammonia
solution 35 % (10 mL) in absolute ethanol (15 mL) was
heated at reflux temperature for 12 h. The solvent was
evaporated under reduced pressure and the solid obtained
was triturated with ice-water, filtered, washed with water,
dried and crystallized from ethanol.
3-Amino-3,4,6,7,8,9-hexahydro-4-imino-5-phenyl-5H, 10H-
pyrimido[4,5-b]quinoline (9a) Yield 85 %, m.p. 266-
267 °C. 1H NMR spectrum: (DMSO-d6, d ppm): 1.58–1.70
(m, 4H, 2CH2), 2.10–2.64 (m, 4H, 2CH2), 5.45 (s, 2H,
NH2), 6.45 (s,1H, C5–H), 7.19–7.47 (m, 6H, Ar–H), 8.15
(s, 2H, 2NH). Anal. Calcd. for C17H19N5 (293.37): C
69.60, H 6.53, N 23.87. Found: C 69.30, H 6.81, N 23.68.
4-Amino-5,6,7,8,9,10-hexahydro-5-phenylpyrimido[4,5-b]
1
quinoline (8a) Yield 70 %, m.p. 262–263 °C. H NMR
spectrum: (DMSO-d6, d ppm): 1.59–1.72 (m, 4H, 2CH2),
123

Med Chem Res (2013) 22:2724–2736
2733
493 (4.73, M?-2), 125 (100.00). Anal. Calcd. for
C24H21BrClN5 (494.81): C 58.26, H 4.28, N 14.15. Found:
C 58.40, H 4.45, N 14.33.
3-Amino-5-(3-bromophenyl)-3,4,6,7,8,9-hexahydro-4-
imino-5H, 10H-pyrimido[4,5-b]quinoline (9b) Yield
86 %, m.p. 260–261 °C. 1H NMR spectrum: (DMSO-d6, d
ppm): 1.65–1.76 (m, 4H, 2CH2), 2.13–2.86 (m, 4H, 2CH2),
5.52 (s, 2H, NH2), 6.11 (s, 1H, C5–H), 7.32–7.64 (m, 5H,
Ar–H), 7.76 (s, 2H, 2NH). MS m/z (%): 374 (9.80, M??2),
373 (22.10, M??1), 372 (14.60, M?), 216 (100.00). Anal.
Calcd. for C17H18BrN5 (372.26): C 54.85, H 4.87, N 18.81.
Found: C 55.12, H 4.68, N 18.63.
3-(4-Chlorobenzylideneamino)-5-(4-chlorophenyl)-3,4,6,7,8,9-
hexahydro-4-imino-5H, 10H-pyrimido[4,5-b]quinoline (10c)
Yield 65 %, m.p. 250–251 °C. MS m/z (%): 451 (0.03,
M??1), 450 (0.05, M?), 283 (100.00). Anal.Calcd. for
C24H21Cl2N5 (450.36): C 64.01, H 4.70, N 15.55. Found: C
64.30, H 4.46, N 15.63.
3-Amino-5-(4-chlorophenyl)-3,4,6,7,8,9-hexahydro-4-imino-
5H, 10H-pyrimido[4,5-b]quinoline (9c). Yield 89 %, m.p.
IR spectrum (KBr, v, cm-1): 3427, 3353 (NH2); 3201 (2NH);
3-(4-Chlorobenzylideneamino)-3,4,6,7,8,9-hexahydro-4-
imino-5-(3,4-dimethoxyphenyl)-5H, 10H-pyrimido
[4,5-b]quinoline (10d) Yield 50 %, m.p. 251–253 °C.
IR spectrum (KBr, v, cm-1): 3,426 (2NH); 3024, 2922 (CH).
1H NMR spectrum: (CDCl3, d ppm): 1.67–1.87 (m, 4H,
2CH2), 2.39 (t, 2H, CH2), 2.84 (t, 2H, CH2), 3.71 (s, 2NH),
3.90 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 5.18 (s, 1H, C5–H),
6.76–6.99 (m, 8H, Ar–H), 7.27 (s, 1H, N=CH). MS m/z (%):
478 (0.83, M??2), 477 (2.07, M??1), 476 (2.26, M?), 84
(100.00). Anal. Calcd. for C26H26ClN5O2 (475.97): C 65.61,
H 5.51, N 14.71. Found: C 65.90, H 5.21, N 14.53.
1
2932, 2860 (CH). H NMR spectrum: (DMSO-d6, d ppm):
1.58–1.70 (m, 4H, 2CH2), 2.09–2.87 (m, 4H, 2CH2), 5.46 (s,
2H, NH2), 5.83 (s, 1H, C5–H), 7.21–7.57 (m, 5H, Ar–H), 8.23
(s, 2H, 2NH). Anal. Calcd. for C17H18ClN5 (327.81): C 62.29,
H 5.53, N 21.36. Found: C 62.50, H 5.31, N 21.43.
3-Amino-3,4,6,7,8,9-hexahydro-4-imino-5-(3,4-dimethoxy-
phenyl)-5H, 10H-pyrimido[4,5-b]quinoline (9d) Yield
70 %, m.p. 254–255 °C. IR spectrum (KBr, v, cm-1): 3428,
3354 (NH2); 3,203 (2NH); 2932, 2860 (CH). 1H NMR
spectrum: (DMSO-d6, d ppm): 1.58–1.70 (m, 4H, 2CH2),
2.22–2.66 (m, 4H, 2CH2), 3.74 (s, 6H, 2OCH3), 5.46 (s, 2H,
NH2), 5.83 (s,1H, C5–H), 6.32–6.83 (m, 4H, Ar–H), 8.12 (s,
2H, 2NH). Anal. Calcd. for C19H23N5O2 (353.42): C 64.57,
H 6.56, N 19.82. Found: C 64.70, H 6.31, N 19.64.
3,4,6,7,8,9-Hexahydro-4-imino-3-(3,4-dimethoxybenzylideneamino)
-5-phenyl-5H, 10H-pyrimido[4,5-b]quinoline (10e) Yield
65 %, m.p. 227–229 °C. 1H NMR spectrum: (DMSO-d6, d
ppm): 1.76–1.88 (m, 4H, 2CH2), 2.43 (t, 2H, CH2), 2.92 (t,
2H, CH2), 3.58 (s, 2NH), 3.89 (s, 3H, OCH3), 3.96 (s, 3H,
OCH3), 5.44 (s, 1H, C5–H), 6.73–7.40 (m, 9H, Ar–H,
N=CH). MS m/z (%): 441 (1.94, M?), 84 (100.00). Anal.
Calcd. for C26H27N5O2 (441.52): C 70.73, H 6.16, N 15.86.
Found: C 70.90, H 6.31, N 15.73.
General procedure for the preparation of 3,4,6,7,8,9-
hexahydro-4-imino-3-(substituted benzylidene)-amino-5-
(substituted)phenyl-5H, 10H-pyrimido[4,5-b]quinolines
(10a–h)
5-(3-Bromophenyl)-3,4,6,7,8,9-hexahydro-4-imino-3-(3,4-
10H-pyrimido[4,5-
A mixture of compound 9a–d (0.005 mol), the appropriate
benzaldehyde (0.005 mol) and a catalytic amount of
piperidine (0.5 mL) in dioxane (10 mL) was heated at
reflux temperature for 24 h. The precipitated solid was
collected by filtration, washed several times with cold
ethanol, dried and crystallized from acetone.
dimethoxybenzylideneamino)-5H,
1
b]quinoline (10f) Yield 65 %, m.p. 217–219 °C. H NMR
spectrum: (CDCl3, d ppm): 1.75–1.92 (m, 4H, 2CH2), 2.41 (t,
2H, CH2), 2.87 (t, 2H, CH2), 3.64 (s, 2NH), 3.86 (s, 3H, OCH3),
3.97 (s, 3H, OCH3), 5.36 (s, 1H, C5–H), 6.68–7.35 (m, 8H, Ar–
H, N=CH). MS m/z (%): 519 (0.25, M?-1), 518 (0.24, M?
-2), 329 (100.00). Anal. Calcd. for C26H26BrN5O2 (520.42): C
60.00, H 5.04, N 13.46. Found: C 60.27, H 4.98, N 13.71.
3-(4-Chlorobenzylideneamino)-3,4,6,7,8,9-hexahydro-4-imino-
5-phenyl-5H, 10H-pyrimido[4,5-b]quinoline (10a) Yield
1
72 %, m.p. 215–217 °C. H NMR spectrum: (CDCl3, d
5-(4-Chlorophenyl)-3,4,6,7,8,9-hexahydro-4-imino-3-(3,4-
dimethoxybenzylideneamino)-5H,
ppm): 1.64–1.89 (m, 4H, 2CH2), 2.35 (t, 2H, CH2), 2.86 (t,
2H, CH2), 3.25 (s,1H, NH), 3.70 (s,1H, NH), 5.24 (s, 1H,
C5–H), 7.24–7.52 (m, 11H, Ar–H, N=CH). Anal. Calcd. for
C24H22ClN5 (415.92): C 69.31, H 5.33, N 16.84. Found: C
69.65, H 5.21, N 16.73.
10H-pyrimido[4,5-
b]quinoline(10g) Yield 70 %, m.p. 240–242 °C. MS m/z
(%): 476 (2.10, M?), 475 (3.30, M?-1), 283 (100.00).
Anal. Calcd. for C26H26ClN5O2 (475.97): C 65.61, H 5.51,
N 14.71. Found: C 65.97, H 5.85, N 14.50.
5-(3-Bromophenyl)-3-(4-chlorobenzylideneamino)-3,4,6,7,
8,9-hexahydro-4-imino-5H, 10H-pyrimido-[4,5-b]quino-
line (10b) Yield 60 %, m.p. 158–159 °C. MS m/z (%):
3,4,6,7,8,9-Hexahydro-4-imino-3-(3,4-dimethoxybenzylide
neamino)-5-(3,4-dimethoxyphenyl)-5H,10H-pyrimido[4,5-b]
quinoline (10h) Yield 55 %, m.p. 217–218 °C. MS m/z
123

2734
Med Chem Res (2013) 22:2724–2736
(%): 503 (0.31, M??1), 258 (100.00). Anal.Calcd. for
C28H31N5O4 (501.58): C 67.05, H 6.23, N 13.95. Found: C
67.20, H 6.29, N 13.73.
General procedure for the preparation of 3-
(ethoxycarbonylamino)-4-(ethoxycarbonylimino)-
3,4,6,7,8,9-hexahydro-5-(substituted)phenyl-5H, 10H-
pyrimido[4,5-b]quinolines (12a–d)
General procedure for the preparation of 7,8,9,10,11,12-
hexahydro-12-(substituted)phenyl[1,2,4]-
A mixture of compound 9a–d (0.005 mol) and ethyl
chloroformate (1.08 g, 0.01 mol) in benzene (15 mL) was
heated at reflux temperature for 24 h. The reaction mixture
was concentrated, the precipitated solid was collected by
filtration, dried and crystallized from ethanol.
triazolo[20,30:3,4]pyrimido[6,5-b]quinolines (11a–d)
A mixture of compound 9a–d (0.005 mol) and triethyl
orthoformate (5 mL) was heated at reflux temperature for
8–10 h. The reaction mixture was cooled and diluted with
ethanol (5 mL). The precipitated solid was collected by
filtration, dried and crystallized from ethanol.
3-(Ethoxycarbonylamino)-4-(ethoxycarbonylimino)-3,4,6,7,8,9-
hexahydro-5-phenyl-5H, 10H-pyrimido[4,5-b]quinoline (12a)
Yield 60 %, m.p.110–112 °C. IR spectrum (KBr, v, cm-1):
3313, 3208 (2NH), 2933, 2861 (CH), 1710 (2C=O). MS m/
z (%): 438 (0.09, M??1), 391 (0.10, M?–C2H5OH), 249
(100.00). Anal. Calcd. for C23H27N5O4 (437.49): C 63.14,
H 6.22, N 16.01. Found: C 63.40, H 5.97, N 16.23.
7,8,9,10,11,12-Hexahydro-12-phenyl-[1,2,4]triazolo[20,30:3,4]
pyrimido[6,5-b]quinoline (11a) Yield 50 %, m.p. 110–
1
112 °C. H NMR spectrum: (CDCl3, d ppm): 1.22–3.26
(m, 8H, 4CH2), 5.15 (s, 1H, C12–H), 7.12–7.55 (m, 5H,
Ar–H), 8.18 (s, 1H, C5–H), 9.38 (s, 1H, C2–H), 10.90 (s,
1H, NH). Anal. Calcd. for C18H17N5 (303.36): C 71.27,
H 5.65, N 23.09. Found: C 71.50, H 5.41, N 23.33.
5-(3-Bromophenyl)-3-(ethoxycarbonylamino)-4-(ethoxycar-
bonylimino)-3,4,6,7,8,9-hexahydro-5H, 10H-pyrimido[4,5-
b]quinoline (12b) Yield 60 %, m.p.104–105 °C. IR
spectrum (KBr, v, cm-1): 3,332 (2NH); 2933, 2863 (CH);
1729 (2C=O).1H NMR spectrum: (CDCl3, d ppm): 1.27–
1.42 (m, 6H, 2COOCH2CH3), 1.73–1.88 (m, 4H, 2CH2),
2.29–2.99 (m, 4H, 2CH2), 4.21–4.31 (m, 4H,
2COOCH2CH3), 6.50 (s, 1H, C5–H), 7.21–7.70 (m, 7H,
C2–H, Ar–H, 2NH). Anal. Calcd. for C23H26BrN5O4
(516.39): C 53.50, H 5.07, N 13.56. Found: C 53.12, H
4.92, N 13.29.
12-(3-Bromophenyl)-7,8,9,10,11,12-hexahydro-[1,2,4]triaz-
olo[20,30:3,4]pyrimido[6,5-b]quinoline (11b) Yield 55 %,
m.p.120–122 °C.1H NMR spectrum: (CDCl3, d ppm):
1.26–2.96 (m, 8H, 4CH2), 5.08 (s, 1H, C12–H), 7.21–7.58
(m, 4H, Ar–H), 8.20 (s, 1H, NH), 8.41 (s, 1H, C5–H),
9.40 (s, 1H, C2–H). Anal. Calcd. for C18H16BrN5
(382.26): C 56.56, H 4.22, N 18.32. Found: C 56.70, H
4.28, N 18.49.
5-(4-Chlorophenyl)-3-(ethoxycarbonylamino)-4-(ethoxy-
carbonylimino)-3,4,6,7,8,9-hexahydro-5H,
12-(4-Chlorophenyl)-7,8,9,10,11,12-hexahydro-[1,2,4]triazol-
o[20,30:3,4]pyrimido[6,5-b]quinoline (11c) Yield 62 %,
10H-pyrimido
[4,5-b]quinoline (12c) Yield 55 %, m.p. 112–113 °C. IR
m.p. 125–127 °C. 1H NMR spectrum: (CDCl3,
d
spectrum (KBr, v, cm-1): 3380, 3297 (2NH), 2935, 2863
1
ppm):1.30–2.79 (m, 8H, 4CH2), 5.15 (s, 1H, C12–H), 7.26–
7.46 (m, 4H, Ar–H), 8.04 (s, 1H, NH), 8.41 (s, 1H, C5–H),
9.55 (s,1H, C2–H). Anal. Calcd. for C18H16ClN5 (337.81):
C 64.00, H 4.77, N 20.73. Found: C 64.23, H 4.91, N 20.57.
(CH), 1,725 (2C=O). H NMR spectrum: (CDCl3, d ppm):
1.28–1.41 (m, 6H, 2COOCH2CH3), 1.76–1.95 (m, 4H,
2CH2), 2.32–2.96 (m, 4H, 2CH2), 4.19–4.33 (m, 4H,
2COOCH2CH3), 6.65 (s, 1H, C5–H), 7.32–7.70 (m, 7H,
C2–H, Ar–H, 2NH). MS m/z (%): 426 (0.84, M?–
C2H5OH), 283 (100.00). Anal. Calcd. for C23H26ClN5O4
(471.94): C 58.53, H 5.55, N 14.84. Found: C 58.73, H
5.31, N 14.67.
7,8,9,10,11,12-Hexahydro-12-(3,4-dimethoxyphenyl)-[1,2,4]triaz-
olo[20,30:3,4]pyrimido[6,5-b]quinoline (11d) Yield 55 %,
m.p.175–176 °C. 1H NMR spectrum: (DMSO-d6, d ppm):
1.30–2.79 (m, 8H, 4CH2), 3.87 (s, 3H, OCH3), 3.91 (s,
3H, OCH3), 5.15 (s, 1H, C12–H), 6.73–7.15 (m, 3H, Ar–
H), 8.22 (s, 1H, NH), 8.63 (s, 1H, C5–H), 9.47 (s,1H, C2–
H). MS m/z (%): 364 (4.93, M??1), 309 (100.00). Anal.
Calcd. for C20H21N5O2 (363.41): C 66.10, H 5.82, N
19.27. Found: C 65.96, H 5.71, N 19.46.
3-(Ethoxycarbonylamino)-4-(ethoxycarbonylimino)-3,4,6,
7,8,9-hexahydro-5-(3,4-dimethoxyphenyl)-5H,10H-pyrimi-
do[4,5-b]quinoline (12d) Yield 65 %, m.p. 155–156 °C.
IR spectrum (KBr, v, cm-1): 3,334 (2NH), 2935, 2863
(CH), 1,724 (2C=O).1H NMR spectrum: (CDCl3, d ppm):
123

Med Chem Res (2013) 22:2724–2736
2735
1.26–1.36 (m, 6H, 2COOCH2CH3), 1.74–1.91 (m, 4H,
2CH2), 2.50 (t, 2H, CH2), 2.99 (t, 2H, CH2), 3.90 (s, 3H,
OCH3), 3.94 (s, 3H, OCH3), 4.23–4.30 (m, 4H,
2COOCH2CH3), 6.76–6.99 (m, 6H, C5–H, Ar–H, 2NH),
7.36 (s, 1H, C2–H). Anal. Calcd. for C25H31N5O6 (497.54):
C 60.35, H 6.28, N 14.08. Found: C 60.53, H 5.99, N 14.13.
each tube. Samples were incubated in the dark at ambient
temperature. After 24 h, the final absorbance was deter-
mined. Readings were corrected for initial absorbance and
normalized as a % of the unreacted DNA/methyl green
absorbance value (Burres et al., 1992). Bleomycin was
used as a reference antitumor agent. The activity of com-
pounds 4e, 4f, 4 h, 5d, 6a, 6d, 8c, 8d, 10d, 10e, 10h, and
12a which showed DNA-binding affinity have been
determined and expressed as IC50 (concentration required
for 50 % decrease in the initial absorbance of the methyl
green/DNA solution) (Table 2).
Biological screening
The in vitro antitumor activity against breast cancer
(MCF-7) cell line
Cells were placed in 96-multiwell microtiter plate (104 cell/
well) for 24 h before treatment with the compounds to
allow attachment of the cells to the wall of the plate.
Different concentrations of the tested compounds (0, 1, 2.5,
5 and 10 lM) were added to the cell monolayer. Triplicate
wells were prepared for each individual dose. Monolayer
cells were incubated with the compounds for 48 h at 37 °C
and in atmosphere of 5 % CO2. After 48 h, cells were
fixed, washed and stained with SRB stain. Unbound dye
was removed by four washes with 1 % acetic acid and
attached stain was recovered with Tris EDTA buffer. Color
intensity was measured in an ELISA reader. The relation
between surviving fraction and drug concentration is
plotted to get the survival curve for breast tumor cell line
after the specified time (Skehan et al., 1990). The con-
centration required for 50 % inhibition of cell viability
(IC50) was calculated for each compound, and the results
are given in Table 1.
Conclusion
New series of pyrimido[4,5-b]quinolines and [1,2,4]triaz-
olo[20,30:3,4]pyrimido[6,5-b]quinolines have been synthe-
sized and evaluated for in vitro antitumor activity against
human breast carcinoma (MCF-7) cell line and DNA-
binding affinity. The results of in vitro antitumor testing
against MCF-7 breast cancer cells and DNA-binding assay
indicated that compounds, 4f, 6d, 8c, 8d, 10d, 10e, 10h,
and 12a exhibited better activities among compounds
tested.
Acknowledgments The authors would like to express their sincere
thanks to Prof. Dr. Farid A. Badria, Pharmacognosy Department, Fac-
ulty of Pharmacy, Mansoura University, Egypt, for carrying out the
DNA-binding assay, and to all members of Pharmacology Unit at
the National Cancer Institute (NCI), Cairo University, for performing
the cytotoxicity testing.
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