Synthesis and evaluation of anticancer and PDE 5 inhibitory activity of spiro-substituted quinazolin-4-ones

Article abstract of DOI:10.1007/s00706-017-1961-5

A series of novel spiro-substituted 2,3-dihydroquinazolin-4(1H)-ones was synthesized and structurally confirmed by spectral analysis, screened for their anticancer activity at a concentration of 10 μΜ against a panel of 56 cell lines derived from nine different types of cancers, including leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers. The synthesized compounds screened for their PDE 5 inhibitory activity and it showed encouraged activity compared to sildenafil. Graphical abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-017-1961-5

Monatsh Chem
DOI 10.1007/s00706-017-1961-5
ORIGINAL PAPER
Synthesis and evaluation of anticancer and PDE 5 inhibitory
activity of spiro-substituted quinazolin-4-ones
Mohamed A. Ameen1 Essam Kh. Ahmed1 Mohamed Ramadan2
•
•
•
Hisham A. Abd El-Naby1 Asmaa A. Abdel-Haseeb1
•
Received: 22 December 2016 / Accepted: 19 March 2017
Ó Springer-Verlag Wien 2017
Abstract A series of novel spiro-substituted 2,3-dihydro-
quinazolin-4(1H)-ones was synthesized and structurally
confirmed by spectral analysis, screened for their anti-
cancer activity at a concentration of 10 lL against a panel
of 56 cell lines derived from nine different types of can-
cers, including leukemia, melanoma, lung, colon, CNS,
ovarian, renal, prostate, and breast cancers. The synthe-
sized compounds screened for their PDE 5 inhibitory
activity and it showed encouraged activity compared to
sildenafil.
Introduction
The rational use of click approach for the discovery of new
medicinal agents takes advantages of the structurally
unique compounds provided by the rapid increase of
molecular complexity [1]. Quinazoline compounds teth-
ered to biomolecules via triazole linker produced by the
combination of an unusual quinazolinone scaffold with the
possibility of functionalization at N-atoms were not
reported. Quinazoline and its derivatives are versatile
nitrogen heterocyclic compounds, which known as a
promising class of biologically active compounds. Quina-
Graphical abstract
zolin-4-one derivatives exhibited
a wide range of
pharmacological properties such as CNS depressant,
sedative, hypnotic, antidepressant, anesthetic, tranquilizer,
anticonvulsant, muscle relaxant, antiviral, antibacterial,
antifungal, analgesic, anti-inflammatory, antipyretic,
antiulcer, antihypertensive, spore germination inhibition in
Drechslera rostrata and Fusarium oxysporum, antihis-
taminic, hypoglycemic, MAO inhibitor, insecticidal,
radioprotective, spasmolytic, contraceptive, and H2-antag-
onist [2]. They are also useful as cytotoxic agents [3], PDE
5 inhibitors [4, 5] antitubercular [6], antipsychotic [7],
dihydrofolate reductase (DHFR) inhibition [8].
Keywords Spiro compounds Á Cycloadditions Á
Antitumor agents Á Structure elucidation
Male erectile dysfunction (MED) is a common medical
problem affecting many of men worldwide. Phosphodi-
esterase 5 (PDE 5) inhibitors are very attractive therapeutic
agents for the treatment of MED [9]. Although Sildenafil
(ViagraÒ, IC50 = 3 nM; Fig. 1) was the lead compound
and the most famous PDE 5 inhibitor, but due to its low
selectivity against other PDEs, mainly towards PDE 6
which found in the retina, and its inhibition may cause
visual disturbances, perception of bluish haze, and
increased light sensitivity [10]. In the last few decades, the
focus on the design of more selective PDE 5 inhibitors
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-017-1961-5) contains supplementary
material, which is available to authorized users.
& Mohamed Ramadan
elbashamohammed@yahoo.com
1
Chemistry Department, Faculty of Science, Minia University,
El Minya, Egypt
2
Organic Chemistry Department, Faculty of Pharmacy,
Al-Azhar University, Assuit, Egypt
123

M. A. Ameen et al.
expanded. PDE 5 belongs to a superfamily of enzymes that
catalyzes the hydrolysis of cyclic nucleotides cAMP and
cGMP to the corresponding 5-nucleoside monophosphate.
Right now, 11 different isoforms of phosphodiesterases
(PDE 1–11) and their subtypes are known, distinguished by
substrate specificities and tissue concentration. The phos-
phodiesterase plays a vital role in the cellular signaling
process [11, 12].
Synthetic transformations based on the click approach
expand the medicinal potential of different heterocyclic
classes. So we illustrated the potential of acetylenic
quinazoline derivatives as a structural scaffold in the
design of therapeutic agents. The synthesized compounds
were screened for their anticancer and PDE 5 inhibitory
activity.
Fig. 1 Sildenafil
Scheme 1
Results and discussion
In continuation of our studies on the development of new
routes in heterocyclic synthesis [13–16] substituted
derivatives of spiro-2,3-dihydroquinazolin-4(1H)-one were
prepared as outlined in Scheme 1. Since the spirohexyl
Table 1 Reaction time and
yield percentage of compounds
3a–3g
Method A
Method B
Entry Ketone
A
Product 3
Yield/%
Time/h Yield/% Time/h
80
3
5
45
43
20
24
B
77
C
D
74
82
6
6
40
51
24
28
E
F
69
68
5
6
42
45
30
30
G
66
4
44
24
Method A: the reaction performed with catalyst at room temperature
Method B: the reaction performed without catalyst under reflux
123

Synthesis and evaluation of anticancer and PDE 5 inhibitory activity of spiro-substituted…
type was found to be favorable for high affinity and good
pharmacokinetic profiles [17]. It was found that the reac-
tion of equimolar amounts of 2-aminobenzamide and
cyclohexanone or N-substituted 4-piperidone derivatives in
the presence of 5% ammonium chloride as a catalyst in
ethanol at room temperature yielded the target products
3a–3g in good yields as shown in Table 1. When the
reactions were performed without a catalyst under reflux,
the products were obtained in lower yields and with longer
reaction times. So the treatment of aminobenzamide with
ketones to afford the spiro systems was best achieved by
running the reaction in the presence of catalytic quantities
13C NMR spectra and mass spectrometry of compounds
3a–3g clearly confirmed the formation of spiro-substituted
2,3-dihydroquinazolin-4(1H)-ones.
A plausible mechanism for this reaction is suggested in
Scheme 2. It is reasonable to assume that the functional-
ized spiroquinazoline 3a–3g could be obtained from the
initial condensation of amino group in 2-aminobenzamide
and carbonyl group of the ketone, followed by the attack of
the lone pair of N-atom of amide on the positively charged
carbon of imine. Finally, N-anion of ketenimine could
protonated to form the targeted compounds 3a–3g.
To generate the alkyne core 4, we examined the reaction
of NH-hydrochloride compound 3d with propargyl
1
of ammonium chloride at room temperature. The H and
Scheme 2
Scheme 3
(i) HCl, CH2Cl2, r.t.; (ii) propargyl bromide, DMF, K2CO3, r.t.; (iii) cat.
CuSO4∙5H2O, Na-ascorbate, H2O/THF, r.t.; (iv) CH3ONa, CH3OH, R = b
123

M. A. Ameen et al.
bromide in alkaline medium. The mass spectra for the
reaction product revealed the formation of 1:2 adduct.
Three possible nucleophilic centers are possible cites to be
attacked with alkyne electrophile, the spiroquinazoline N1,
N3 as well as the piperidine NH. Quinazoline nitrogen
were excluded realized in high inactivity [18] as a result of
either steric hindrance or tautomerization. Interestingly, the
piperidinium salt with attaching two propargyl groups was
formed. Accordingly, structure 4 was established for the
7 showed the presence of the broad band at 3407–3365 cm-1
1
corresponding to OH groups. H NMR spectrum of com-
pound 7 presents a signal at d = 3.74 ppm characteristic for
4 OH groups and disappearance of acetyl groups.
We designed the following scheme to optimize the
direct formation of compound 14, proceeding to the
desired target through 1,3-dipolar azide-alkyne cycload-
dition reaction (Scheme 4). The treatment of
aminobenzamide 1 with chloroacetyl chloride to afford
the acetylated product 8 was best achieved using an
excess amount of chloroacetyl chloride in chloroform
[20]. The subsequent cyclization step of compound 8 to
afford 2-(chloromethyl)quinazoline 9 was sluggish when
conducted in sodium ethoxide affording other products.
Refluxing compound 8 in an equivalent sodium ethoxide
afforded the pentacyclic ring system 10. In addition, we
investigated the effect of the excess amount of sodium
ethoxide at room temperature and then under reflux but
the two cases led to the formation of ethoxy methylene
product 11. In contrast when carrying out the reaction in
toluene in the presence of catalytic amount of TsOH
afforded 2-(chloromethyl)quinazoline 9. Hence, the
azido product 13 was obtained by treating the corre-
sponding compound 9 with sodium azide.
1
reaction product. H and 13C NMR was in support of the
proposed structure.
The alkyne product 4 and azide 5 coupling partners were
reacted together, under our previously developed CuAAC
click conditions [14] to deliver the new library of hybrid
triazole-bio ligand 6 in good yield (Scheme 3).
According to what has been previously reported [19] a
reasonable mechanism for the formation of clicked prod-
ucts 6 and 14 involves deprotonation of the triple bond
forming Cu-acetylide followed by 1,3-dipolar cycloaddi-
tion of azide.
Furthermore, we report here the chemical synthesis of
azido-alkyne functionalized glucose dimer 7 by hydrolysis
of 6 in a diluted sodium methoxide. The structure of 7 was
confirmed by spectroscopic data. IR spectrum of compound
Scheme 4
(i) ClCH2COCl, CH2Cl2, r.t.; (ii) TsOH, dry toluene, reflux; (iii) EtONa (1:1),
EtOH, reflux; (iv) EtONa (2:1), EtOH, reflux; (v) NaN3, acetonitrile, reflux; (vi)
EtONa (1:4), EtOH, r.t.; (vii) THF:H2O (1:1), sodium ascorbate,
cat.CuSO4∙5H2O, r.t.
123

Synthesis and evaluation of anticancer and PDE 5 inhibitory activity of spiro-substituted…
Table 2 56 cell line in vitro anticancer screening data of compounds 3c, 3d, 6, and 7
Subpanel tumor cell lines
Growth/%
3c NSC:793274
3d NSC:793275
6 NSC:793276
7 NSC:793277
Leukemia
CCRF-CEM
HL-60(TB)
MOLT-4
97.68
89.47
88.81
98.83
94.87
95.62
84.74
86.92
94.88
93.17
86.64
85.46
93.73
94.27
88.20
SR
100.27
Non-small cell lung cancer
A549/ATCC
EKVX
65.54
108.72
90.22
69.06
109.31
96.57
65.56
107.12
97.04
78.55
109.69
97.43
HOP-62
HOP-92
106.41
98.01
107.21
97.67
108.93
99.56
104.48
97.96
NCI-H226
NCI-H23
101.11
108.56
107.23
84.49
100.49
108.77
110.30
97.52
104.48
104.45
107.37
92.40
103.08
101.84
104.80
98.06
NCI-H322 M
NCI-H460
NCI-H522
Colon cancer
HCC-2998
HCT-116
HCT-15
104.12
103.16
113.68
93.23
103.23
100.40
112.99
98.18
101.62
97.63
104.04
96.30
116.45
98.02
111.49
100.22
102.97
98.26
HT29
KM12
106.62
96.69
105.19
102.34
102.81
100.39
SW-620
CNS cancer
SF-268
106.94
105.34
106.41
97.80
105.81
107.34
114.20
96.24
101.84
105.46
106.43
96.83
104.10
109.91
105.44
98.31
SF-295
SF-539
SNB-19
SNB-75
91.11
82.77
87.85
88.61
U251
85.25
91.18
91.14
93.28
Melanoma
LOX IMVI
MALME-3M
M14
101.06
105.62
101.63
106.46
100.08
119.11
107.37
58.33
101.89
117.27
100.04
103.74
98.06
103.24
102.11
102.47
105.27
108.31
114.48
109.09
62.90
97.95
98.84
102.65
107.40
101.82
113.55
104.60
83.07
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
116.41
113.99
85.66
95.59
96.12
96.23
100.01
106.42
114.43
110.84
111.66
82.31
113.16
108.09
115.93
114.89
84.38
109.29
108.25
113.55
112.57
76.12
106.74
109.43
119.53
107.17
88.65
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
109.36
96.11
108.66
103.48
107.66
79.33
108.21
87.49
123

M. A. Ameen et al.
Table 2 continued
Subpanel tumor cell lines
Growth/%
3c NSC:793274
3d NSC:793275
6 NSC:793276
7 NSC:793277
Renal cancer
786-0
103.02
98.98
107.40
95.47
110.38
111.25
99.14
98.26
89.44
109.68
96.51
108.59
119.71
97.06
94.92
90.37
106.67
106.19
108.66
102.48
97.57
A498
ACHN
113.82
107.50
101.86
94.65
RXF 393
SN12C
TK-10
101.84
84.14
UO-31
85.71
Prostate cancer
PC-3
94.78
88.54
92.77
82.27
DU-145
112.69
108.42
112.04
101.90
Breast cancer
MCF7
96.46
108.36
95.49
107.36
119.57
101.78
108.15
109.13
98.76
103.62
106.83
102.42
113.88
102.49
110.27
100.46
37.56
97.02
104.19
109.19
108.48
109.11
105.70
100.85
22.30
MDA-MB-231/ATCC
HS 578T
BT-549
114.28
96.61
T-47D
MDA-MB-468
Mean
112.08
100.32
41.99
102.03
32.97
Delta
Range
60.78
50.51
56.81
40.98
Range = highest growth percent - lowest growth percent. Delta = mean growth percent - lowest growth percent
An alternative reaction condition was devised using
sodium azide to convert the acetylated product 8 into azido
product 12 which was then subjected to cyclization in
sodium ethoxide to give compound 13. The copper(I)-
catalyzed 1,2,3-triazole-forming reaction between azide 13
and terminal alkyne 4 afforded the interesting two triazolo
linkers quinazoline trimer 14 (Scheme 4). The presence of
triazolo moieties in 14 was confirmed by the disappearance
of the characteristic bands in the IR spectrum for azide
The prepared compounds were added at single concen-
tration 10 lM and the culture was incubated for 48 h. End
point determinations were made with a protein binding dye,
sulforhodamine B (SRB). All the selected compounds
evaluated toward the panel of 56 cancer cell lines. The
human tumor cell lines were derived from nine different
cancer types: leukemia, melanoma, lung, renal, prostate,
colon, CNS, ovarian and breast cancers. Results for each
compound were reported as the growth percent of the
treated cells which are evaluated spectrophotometrically
and compared to that of the untreated control cells
(Table 2).
1
group. H NMR spectrum also introduced aromatic proton
signals for triazole rings at 8.08, 8.12 ppm. 13C NMR
showed signals of 2CHtriazoles and carbonyl groups at
135.0, 161.8, and 163.4 ppm, respectively.
The results listed in Table 2 revealed that compounds
3c, 3d, 6, and 7 possess a weak to moderate cytotoxic and/
or growth inhibitory effect toward different cell lines.
Screening of anticancer activity
Among the synthesized compounds, compound 3c, 3d, 6,
and 7 were selected by National Cancer Institute (NCI)
based on the protocol of the Drug Evaluation Branch of the
National Cancer Institute, Bethesda, USA, for in vitro
anticancer screening. The methodology of the NCI anti-
cancer screening has been illustrated in detail elsewhere
(http://www.dtp.nci.nih.gov).
Screening of PDE 5 inhibition activity
The synthesized compounds were screened for their ability
to inhibit PDE 5 compared to sildenafil as a standard ref-
erence drug. Most of the synthesized quinazoline-4-one
derivatives were found to have PDE 5 inhibitory activity at
a concentration of 3 nM (as IC50 of sildenafil) (Table 3).
123

Synthesis and evaluation of anticancer and PDE 5 inhibitory activity of spiro-substituted…
General procedure for synthesis of 3a–3g
Table 3 PDE 5 inhibition of compounds 3a–3d, 4, 6, and 7 at conc.
3 nM
Ammonium chloride (1 mmol) was added to a mixture of
2-aminobenzamide (1 mmol) and appropriate ketone
(1 mmol) in ethanol. Stirring was continued at room
temperature till the disappearance of the starting material,
followed by TLC. The precipitate was filtered off, washed
with ethanol to afford the title compounds 3a–3g.
Compound
PDE 5 inhibition/%
Sildenafil
100
100
100
34
3a
3b
3c
3d
4
10H-Spiro[cyclohexane-1,20-quinazolin]-40(30H)-one
47
(3a)
Yield: 0.17 g (80%); white solid; m.p.: 215–216 °C (Ref.
[21] 217–219 °C).
100
100
95
6
7
Ethyl 40-oxo-30,40-dihydro-10H-spiro[piperidine-4,20-quina-
zoline]-1-carboxylate (3b, C15H19N3O3)
Yield: 0.22 g (77%); white solid; m.p.: 188–190 °C
(EtOH); 1H NMR (300 MHz, DMSO-d6): d = 1.18 (t,
J = 7.2 Hz, 3H, CO2CH2CH3), 1.71 (m, 4H, 2CH2),
3.36–3.57 (m, 4H, 2CH2), 4.08 (q, J = 7.1 Hz, 2H,
CO2CH2CH3), 6.62 (t, J = 7.5 Hz, 1H, Ar–H), 6.72 (d,
J = 8.1 Hz, 1H, Ar–H), 7.19 (t, J = 7.5 Hz, 1H, Ar–H),
7.21 (s, 1H, NH), 7.56 (d, J = 8.0 Hz, 1H, Ar–H), 8.12 (s,
1H, NH) ppm; 13C NMR (75 MHz, DMSO-d6): d = 14.7
(CH3), 36.6 (C-3\, C-5\), 39.1 (C-2\, C-6\), 60.9 (CO2CH2-
CH3), 66.6 (C-4\), 114.4 (C-4a), 114.8 (C-8), 117.1 (C-6),
127.3 (C-5), 133.5 (C-7), 146.5 (C-8a), 154.7 (C=O, ester),
164.3 (C=O) ppm; MS: m/z = 289 (M?).
Conclusion
We designed the first synthesis of quaternary piperidinium
salt of quinazoline with two acetylenic groups and their
Cu-catalyzed alkyne-azide cycloaddition reaction. The
possibility of combining the acetylenic scaffolds with the
flexibility of structural ‘‘click’’ modifications could open
opportunities for the rational design of spiroquinazoline-
based therapeutic agents. The newly synthesized com-
pounds were screened for their anticancer activity and it
revealed moderate to weak anticancer activity. Encour-
agingly, most of the synthesized compounds possess
potent PDE 5 inhibitory activity. Despite the fact that this
assay is less sensitive compared to radioisotope PDE 5
assays and likely to underestimate the relative inhibitory
potencies of the test compounds and further studies
needed.
Tert-butyl 40-oxo-30,40-dihydro-10H-spiro[piperidine-4,20-
quinazoline]-1-carboxylate (3c, C17H23N3O3)
Yield: 0.24 g (74%); white solid; m.p.: 206–208 °C
(EtOH); 1H NMR (300 MHz, DMSO-d6): d = 1.39 (s,
9H, BOC), 1.71 (m, 4H, 2CH2), 3.32–3.52 (m, 4H,
2CH2), 6.64 (m, 1H, Ar–H), 6.77 (m, 1H, Ar–H), 7.22 (m,
2H, Ar–H), 7.56 (s, 1H, NH), 8.11 (s, 1H, NH) ppm; 13C
NMR (75 MHz, DMSO-d6): d = 28.1 (3CH3), 36.6 (C-3\,
C-5\), 56.8 (C-2\, C-6\), 66.6 (C-4\), 78.9 (C(CH3)3), 114.4
(C-4a), 114.7 (C-8), 117.0 (C-6), 127.3 (C-5), 133.4 (C-
7), 146.5 (C-8a), 154.0 (C=O, ester), 163.2 (C=O) ppm;
MS (ESI): m/z calcd. C17H24N3O3 ([M ? H]?) 318.17,
found 318.1.
Experimental
All reagents were purchased from Alfa Aesar and Fluka
companies and were used without further purification. 2-(2-
Chloroacetamido)benzamide (8) was synthesized accord-
ing to the literature [20]. Melting points were measured
with a Gallenkamp apparatus and are uncorrected. The
reactions and compounds purity were monitored by TLC,
on aluminum plates coated with silica gel with fluorescent
indicator (Merck, 60 F254). The NMR spectra were per-
10-H-Spiro[piperidine-4,20-quinazolin]-40(30H)-one
hydrochloride (3d, C12H16ClN3O)
Yield: 0.21 g (82%); white solid; m.p.: 269–271 °C
ꢀ
(MeOH); IR (KBr): m = 3190 (CH-arom.), 3060 (CH-aliph.),
1655 (C=O) cm-1 1H NMR (300 MHz, DMSO-d6):
;
formed on
(300 MHz for H NMR, 75 MHz for 13C NMR) and a
Bruker Avance (400 MHz for H, and 100 MHz for 13C)
a JHA-LAA 400 WB-FT spectrometer
1
d = 1.71 (m, 4H, 2CH2), 3.32 (m, 4H, 2CH2), 6.65 (t,
J = 7.5 Hz, 1H, Ar–H), 6.90 (d, J = 8.1 Hz, 1H, Ar–H),
7.22 (bs, 1H, NH.HCl), 7.23 (t, J = 7.5 Hz, 1H, Ar–H),
7.57 (d, J = 8.1 Hz, 1H, Ar–H), 7.96 (s, 1H, NH), 8.33 (s,
1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): d = 33.9
(C-3\, C-5\), 40.1 (C-2\, C-6\), 65.4 (C-4\), 114.8 (C-4a),
115.4 (C-8), 117.7 (C-6), 127.7 (C-5), 133.8 (C-7), 146.7
(C-8a), 163.5 (C=O) ppm; MS: m/z = 253 (M?).
1
with either deuterated DMSO-d6 or CDCl3 as a solvent.
Chemical shifts are expressed in d using TMS as a refer-
ence. The mass spectra were recorded on a Trace GC
2000/Finnegan Mat SSQ 7000 and a Shimadzu GCMS-QP-
1000EX mass spectrometer at 70 eV. Elemental analyses
were ascertained with a Euro EA analyzer.
123

M. A. Ameen et al.
2,2,6,6-Tetramethyl-1-(2,2,2-trifluoroacetyl)-10H-
spiro[piperidine-4,20-quinazolin]-40(30H)-one
(3e, C18H22F3N3O2)
2H, CH2-alkyne), 6.74 (t, J = 7.5 Hz, 1H, Ar–H), 7.07 (d,
1H, J = 8.1 Hz, Ar–H), 7.31 (t, 1H, J = 7.5 Hz, Ar–H),
7.53 (s, 1H, NH), 7.63 (d, 1H, J = 8.1 Hz, Ar–H), 7.83 (s,
1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): d = 31.6
(C-3\, C-5\), 49.3 (CH2-alkyne), 51.2 (CH2-alkyne), 54.6 (C-2\,
C-6\), 65.2 (C-4\), 71.4, 71.6 (2CH-alkyne), 84.1, 84.3 (2Cq-
alkyne), 114.4 (C-4a), 115.5 (C-8), 118.1 (C-6), 127.7 (C-5),
134.0 (C-7), 146.5 (C-8a), 163.3 (C=O) ppm; MS (ESI):
m/z calcd. C18H20BrN3O ([M-Br]?) 294.16, found 294.1.
Yield: 0.23 g (69%); white solid; m.p.: 196–198 °C
1
(MeOH); H NMR (300 MHz, DMSO-d6): d = 1.78 (s,
12H, 4CH3), 2.19 (m, 4H, 2CH2), 6.67 (t, J = 7.5 Hz, 1H,
Ar–H), 6.71 (d, J = 8.1 Hz, 1H, Ar–H), 7.28 (t,
J = 7.5 Hz, 1H, Ar–H), 7.57 (d, J = 8.1 Hz, 1H, Ar–H),
7.96 (bs, 1H, NH), 8.63 (s, 1H, NH) ppm; 13C NMR
(100 MHz, DMSO-d6): d = 25.1 (4CH3), 36.6 (C-3\, C-5\),
50.2 (C-2\, C-6\), 66.6 (C-4\), 114.6 (C-4a), 115.7 (C-8),
118.1 (C-6), 128.1 (C-5), 133.1 (C-7), 145.8 (C-8a), 154.3
(C=O, ester), 162.9 (C=O) ppm; MS: m/z = 369 (M?).
1,1-Bis[(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl-1H-
1,2,3-triazol-4-yl)methyl]-40-oxo-30,40-dihydro-10H-spiro-
[piperidine-4,20-quinazolin]-1-ium bromide
(6, C46H58BrN9O19)
1-Tosyl-10H-spiro[piperidine-4,20-quinazolin]-40(30H)-one
(3f, C19H21N3O3S)
Compound 4 (1 mmol) was added to glucose azide 5
(2 mmol) in THF/H2O (1:1), then sodium ascorbate
(0.4 mmol) and CuSO4Á5H2O (0.2 mmol) were added.
The reaction mixture was stirred at room temperature
overnight. The reaction progress was monitored by TLC.
After completion of the reaction, the reaction mixture was
poured into 50 cm3 of ice water and then extracted with
DCM. The solvent was removed under reduced pressure to
yield the title compounds. Yield: 0.68 g (65%); m.p.: 190–
Yield: 0.25 g (68%); white solid; m.p.: 288–290 °C
1
(EtOH); H NMR (300 MHz, DMSO-d6): d = 2.43 (m,
4H, 2CH2), 3.07 (m, 4H, 2CH2), 3.32 (s, 3H, CH3),
6.64–6.73 (m, 3H, Ar–H), 7.19 (s, 1H, Ar–H), 7.45–7.66
(m, 5H, Ar–H, NH), 8.12 (s, 1H, NH) ppm; 13C NMR
(100 MHz, DMSO-d6): d = 21.5 (CH3), 36.8 (C-3\, C-5\),
42.1 (C-2\, C-6\), 66.1 (C-4\), 114.9 (C-4a), 115.2, 117.4,
127.6, 127.8, 130.0 133.7 (8CH-Ar), 134.2 (CH3C), 143.8
(SO2C), 146.8 (C-8a), 163.6 (C=O) ppm; MS: m/z = 371
(M?).
ꢀ
192 °C (MeOH); IR (KBr): m = 1753 (C=O), 1661 (C=O)
1
cm-1; H NMR (300 MHz, DMSO-d6): d = 1.80, 1.84,
1.97, 2.01, 2.02, 2.08 (s, 24H, 8COCH3), 2.37 (m, 4H,
2CH2-spiro), 3.57 (m, 4H, 2CH2-spiro), 4.11–4.19 (m, 6H,
2C5H-CH2-OCO, 2C5H-CH2-OCO), 4.45 (m, 2H, C3H),
4.59 (s, 2H, CH2-Ctriazole), 4.66 (s, 2H, CH2-Ctriazole), 5.26
(m, 2H, 2C1H-Ntriazole), 5.71 (m, 4H, 2C2H, 2C4H,), 6.40
(t, J = 7.5 Hz, 1H, Ar–H), 6.77 (d, J = 8.0 Hz, 1H, Ar–
H), 6.90 (bs, 1H, NH), 7.35 (t, J = 7.5 Hz, 1H, Ar–H),
7.62 (d, J = 8.0 Hz, 1H, Ar–H), 8.09 (bs, 1H, NH), 8.86 (s,
1H, CHtriazole), 8.89 (s, 1H, CHtriazole) ppm; 13C NMR
(75 MHz, DMSO-d6): d = 19.97, 20.0, 20.2, 20.3, 20.5
(8CH3), 31.0 (C-3\, C-5\), 36.6 (2NCH2), 53.7 (C-2\, C-6\),
61.7 (2OCH2), 66.6 (C-4\), 67.4 (2C4H), 70.7 (2C2H), 71.5
(2C3H), 73.8 (2C5H), 84.3 (2C1H-Ntriazole), 114.1(C-4a),
114.9, 117.9, 127.3, 128.0 (4CH-Ar), 133.8 (2CH-triazole),
135.3 (C-triazole), 145.9 (C-8a), 162.9 (C=O), 169.1, 169.3,
169.4 169.9 (8C=O) ppm.
1-Benzyl-10H-spiro[piperidine-4,20-quinazolin]-40(30H)-
one (3g, C19H21N3O)
Yield: 0.2 g (66%); white solid; m.p.: 282–284 °C (EtOH);
1H NMR (300 MHz, DMSO-d6): d = 2.08 (s, 4H, 2CH2),
2.50 (s, 4H, 2CH2), 4.26 (s, 2H, CH2Ph), 6.70 (t,
J = 7.5 Hz, 1H, Ar–H), 6.74 (d, J = 8.0 Hz, 1H, Ar–H),
6.99 (t, J = 7.5 Hz, 1H, Ar–H), 7.27 (d, J = 8.1 Hz, 1H,
Ar–H), 7.46 (m, 3H, 2Ar–H, NH), 7.58 (m, 3H, Ar–H),
8.26 (s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6):
d = 33.9 (C-3\, C-5\), 47.1 (C-2\, C-6\), 58.6 (CH2), 65.5
(C-4\), 115.7 (C-4a), 117.8, 127.6, 128.1, 128.7, 131.8
(9CH-Ar), 133.6 (CH2C), 149.7 (C-8a), 163.5 (C=O) ppm;
MS: m/z = 307 (M?).
40-Oxo-1,1-di(prop-2-yn-1-yl)-30,40-dihydro-10H-
spiro[piperidine-4,20-quinazolin]-1-ium bromide
1,1-Bis[(b-D-glucopyranosyl-1H-1,2,3-triazol-4-yl)me-
thyl]-40-oxo-30,40-dihydro-10H-spiro[piperidine-4,20-
quinazolin]-1-ium bromide (7, C30H42BrN9O11)
(4, C18H20BrN3O)
Propargyl bromide (0.9 g, 7 mmol) was added portionwise
to a well-stirred mixture of 0.5 g of compound 3d
(2 mmol) and anhydrous potassium carbonate (6 mmol)
in 10 cm3 of dry DMF. Stirring was continued overnight at
room temperature, the reaction mixture was poured into
50 cm3 of ice water. The obtained white precipitate was
filtered off and dried well. Yield: 0.34 g (58%); m.p.:
236–237 °C (EtOH); 1H NMR (300 MHz, DMSO-d6):
d = 2.31 (m, 4H, 2CH2-spiro), 3.72 (m, 2H, 2CH-alkyne),
4.10 (m, 4H, 2CH2-spiro), 4.54 (s, 2H, CH2-alkyne), 4.68 (s,
Dry freshly prepared solution of NaOMe-MeOH (0.1
equiv) was added to a stirred solution of the glycoside
tetraacetate 6 (1.0 equiv) in 20 cm3 of MeOH at r.t. Stirring
was continued for 30 min (monitored by TLC). Neutralize
by addition of DOWEX 50 9 8 ion-exchange resin (pH 6),
followed by filtration. The filtrate was evaporated to
dryness to get the pure unprotected product as colorless
crystals in 0.45 g yield (57%). M.p.: 202–204 °C (EtOH);
-1
;
ꢀ
IR (KBr): m = 3407 (OH), 2925 (CH), 1648 (C=O) cm
123

Synthesis and evaluation of anticancer and PDE 5 inhibitory activity of spiro-substituted…
1H NMR (300 MHz, DMSO-d6): d = 2.39 (m, 4H, 2CH2-
spiro), 3.74 (s, 4H, 4OH), 3.79 (m, 4H, 2CH2-spiro),
4.05–4.19 (m, 6H, 2C5H-CH2-OCO, 2C5H-CH2-OCO),
4.61 (s, 2H, NCH2), 4.68 (s, 2H, NCH2), 5.17 (m, 2H,
C3H), 5.31 (m, 2H, C2H), 5.42 (m, 2H, C4H), 5.68 (m, 2H,
C1H-Ntriazole), 6.74 (t, J = 7.5 Hz, 1H, Ar–H), 6.92 (d, 1H,
J = 8.2 Hz, Ar–H), 7.08 (s, 1H, NH), 7.32 (t, J = 7.5 Hz,
1H, Ar–H), 7.61 (d, 1H, J = 8.1 Hz, Ar–H), 8.14 (s, 1H,
NH), 8.82 (s, 2H, CHar-triazole) ppm.
added. The reaction mixture was refluxed for 15 h then
cooled to room temperature. The reaction mixture was
poured into 20 cm3 of ice water and then extracted with
DCM. The organic layer was dried over anhydrous
Na2SO4, filtered and the solvent was evaporated under
vacuum. Yield: 0.204 g (93%); m.p.: 120–122 °C (EtOH);
IR (KBr): mꢀ = 3361 (NH2), 3186 (CH-aromatic), 2114 (N3),
1676 (C=O) cm-1 1H NMR (300 MHz, DMSO-d6):
;
d = 4.20 (s, 2H, CH2N3), 7.13 (t, J = 7.5 Hz, 1H, Ar–
H), 7.49 (d, 1H, J = 7.7 Hz, Ar–H), 7.65 (bs, 2H, NH2),
8.09 (t, J = 7.5 Hz, 1H, Ar–H), 8.29 (d, 1H, J = 8.1 Hz,
Ar–H), 12.07 (s, 1H, NH) ppm; MS: m/z = 219 (M?).
2-(Chloromethyl)quinazolin-4(3H)-one (9)
2-(Chloroacetamido)benzamide (8, 0.65 g, 3 mmol) was
suspended in 20 cm3 of toluene with 165 mg of toluene-
sulfonic acid monohydrate (30 mol %). The suspension
was heated under reflux using a Dean-Stark trap equipped
with a condenser for 16 h. The mixture was allowed to cool
then diluted with 40 cm3 of ethyl acetate, washed with
2-(Azidomethyl)quinazolin-4(3H)-one (13, C9H7N5O)
To a freshly prepared solution of sodium ethoxide (4 mmol
in 10 cm3 of dry ethanol), compound 12 (1 mmol) was
added. The reaction mixture was stirred at room temper-
ature overnight. The progress of the reaction was
monitored by TLC. After completion of the reaction, the
mixture was poured into 50 cm3 of ice water and then
extracted with DCM. The organic layer was dried using
anhydrous Na2SO4. The solvent was evaporated under
vacuum. Yield: 0.08 g (40%); m.p.: 220–222 °C (EtOH);
1H NMR (300 MHz, DMSO-d6): d = 4.38 (s, 2H, CH2N3),
7.48 (t, J = 7.5 Hz, 1H, Ar–H), 7.73 (d, 1H, J = 8.1 Hz,
Ar–H), 8.05 (t, J = 7.5 Hz, 1H, Ar–H), 8.16 (d, 1H,
J = 8.1 Hz, Ar–H), 12.35 (bs, 1H, NH) ppm; 13C NMR
(100 MHz, DMSO-d6): d = 51.5 (CH2), 121.6 (C-4a),
126.3, 127.3, 134.9 (4CH-Ar), 148.7 (C-8a), 152.7 (C-2),
161.8 (C=O) ppm; MS: m/z = 201 (M?).
saturated
aqueous
sodium
hydrogen
carbonate
(3 9 20 cm3), 50 cm3 of brine, dried over MgSO4, then
evaporated to dryness to give compound 9 in 65% yield.
M.p.: 246–247 °C (Ref. [20] 247 °C).
Pyrazino[2,1-b:5,4-b0]diquinazoline-8,16(6H,14H)-dione
(10, C18H12N4O2)
Sodium ethoxide solution (0.023 g, 1 mmol) in 5 cm3 of abs.
ethanol was added to 0.21 g of compound 8 (1 mmol). The
reaction mixture was refluxed for 8 h. The formed solid
product was collected by filtration to give compound 10.
1
Yield: 0.17 g (52%); m.p.: 240–242 °C (EtOH); H NMR
(300 MHz, DMSO-d6): d = 4.55 (s, 4H, 2CH2), 7.52–7.58
(m, 2H, Ar–H), 7.66–7.69 (m, 2H, Ar–H), 7.81–7.86 (m, 2H,
Ar–H), 8.11–8.14 (m, 2H, Ar–H) ppm; 13C NMR (100 MHz,
DMSO-d6): d = 43.7 (2CH2), 121.7 (C-8a, C-16a), 126.3,
127.7, 135.1 (8CH-Ar), 148.6 (C-4a, C-12a), 152.8 (C-5a,
C-13a), 161.9 (C=O) ppm; MS: m/z = 316 (M?).
40-Oxo-1,1-bis[[1-[(4-oxo-3,4-dihydroquinazolin-2-yl)me-
thyl]-1H-1,2,3-triazol-4-yl]methyl]-30,40-dihydro-10H-
spiro[piperidine-4,20-quinazolin]-1-ium bromide
(14, C36H34BrN13O3)
Compound 13 (2 mmol) was added to 1 mmol of 4 in THF/
H2O (1:1), then sodium ascorbate (0.4 mmol) and CuSO4-
5H2O (0.2 mmol) were added. The reaction mixture was
stirred for 18 h at room temperature then poured into
50 cm3 of ice water. The precipitate that formed was
filtered and dried well. Yield: 0.43 g (55%); m.p.:
248–250 °C (EtOH); 1H NMR (300 MHz, DMSO-d6):
d = 2.41 (m, 4H, 2CH2-spiro), 3.21 (m, 4H, CH2-spiro), 4.68
(s, 2H, CH2N-pip), 4.76 (s, 2H, CH2N-pip), 5.75 (s, 4H,
2CH2N-triaz), 6.74–6.79 (m, 3H, Ar–H), 7.32 (bs, 1H, NH),
7.44–7.50 (m, 3H, Ar–H), 7.62 (m, 3H, Ar–H), 7.74 (m,
3H, Ar-H), 8.02 (s, 1H, CH-triazole), 8.12 (s, 1H, CH-triazole),
8.82 (bs, 1H, NH), 12.66 (bs, 2H, 2 NH) ppm; 13C NMR
(100 MHz, DMSO-d6): d = 31.7 (C-3\, C-5\), 51.9
(2CH2N-pip), 54.2 (C-2\, C-6\), 59.7 (2CH2N-triazole), 65.2
(C-4\), 114.1(C-4a), 114.7, 115.5, 118.2, 126.4, 127.5,
127.6, 127.8, 131.2, 134.2 (12CH-Ar), 135.0 (2CH-triazole),
139.6 (2C-triazole), 148.4 (C-8a), 151.4 (C-2), 161.8
(2C=O), 163.4 (C=O) ppm.
2-(Ethoxymethyl)quinazolin-4(3H)-one
(11, C11H12N2O2)
Sodium ethoxide solution (0.05 g, 2 mmol) in 10 cm3 of abs.
ethanol was added to 2-(2-chloroacetamido)benzamide (8,
1 mmol). The reaction mixture was stirred at room temper-
ature overnight. The solvent was evaporated; the residue was
dissolved in water. HCl (1 M) was added to reach pH 4. The
solid product was filtered off, washed with water and
recrystallized from ethanol to give compound 11. Yield:
0.1 g (48%); m.p.: 140–142 °C (MeOH); 1H NMR
(300 MHz, DMSO-d6): d = 1.14 (t, J = 7.2 Hz, 3H, CH2-
CH3), 3.60(q, J = 6.9 Hz, 2H, CH2CH3), 4.36(s, 2H, CH2O),
7.48 (t, J = 7.5 Hz, 1H, Ar–H), 7.63 (d, 1H, J = 8.0 Hz, Ar–
H), 7.78 (t, J = 7.5 Hz, 1H, Ar–H), 8.09 (d, 1H, J = 8.1 Hz,
Ar–H), 12.14 (bs, 1H, NH) ppm; MS: m/z = 204 (M?).
2-(2-Azidoacetamido)benzamide (12, C9H9N5O2)
To a solution of 2-(2-chloroacetamido)benzamide (8,
1 mmol) in acetonitrile, sodium azide (1.5 mmol) was
123

M. A. Ameen et al.
Anticancer screening
assay or stored at -20 °C. A blank with protein, denatu-
rated by boiling water bath for 3 min, with and without
substrate was performed. Both incubation time and enzyme
concentration were adjusted so that no more than 25% of
the substrate was hydrolyzed under the assay conditions.
All assays were done in duplicate.
The synthesized compounds were screened for their anti-
cancer activity according to NCI protocol. Applying the
seven absorbance measurements [time zero (Tz), control
growth (C), and test growth in the presence of the tested
compound at the five concentration levels (Ti)], the per-
centage growth is calculated at each of the tested
compound concentrations levels. Percentage growth inhi-
bition is calculated as: [(Ti - Tz)/(C - Tz)] 9 100 for
concentrations for which Ti C Tz [(Ti - Tz)/Tz] 9 100 for
concentrations for which Ti \ Tz. Each compound is
exposed to 56 human tumor cell lines, including lung,
ovarian, breast, colon, melanoma, prostate, and kidney
cancers at five different doses for 48 h. Compounds tested
initially at a single high dose 10 lM in the full NCI 56 cell
panel. The data expressed as a mean graph of the percent
growth inhibition of treated cells [22–24]. Mean graph
midpoint (MG-MID) (differential activity patterns, bar
scale) were created for each cell line to facilitate visual
scanning of data for potential NCI patterns of selectivity,
with bars depicting the deviation of the individual tumor
cell lines from the overall mean value for all the cells
tested. In the mean graph, the center point is the mean of all
growth inhibition (GI) percentages over all cell lines.
Bars pointed to the right are (positive values) which
represent resistance where the inhibition is greater than the
average, while bars pointed to the left (negative values),
represent sensitivity to the selected cell line where the
inhibition is less than the average. The negative numbers
indicate cancer cell death.
The HPLC system was Agilent Technologies 1200
Series, G1315D DAD. The column used was Zorbax
Eclipse AAA rapid resolution 4.6 9 150 mm 3.5 lM
particle size. The mobile phase employed for the separation
(isocratic elution) consisted in 200 mM ammonium acetate
pH 6.0 with 2% acetonitrile (v/v). The flow rate was
1.5 cm3/min; the detector DAD at 254 nm. The injection
volume was 30 mm3. Peak identities were confirmed by
co-elution with standards. Quantitative measurements were
carried out by comparison, using standard solutions of
known concentrations.
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Inhibition of human PDE5 A1 was determined by the
reported protocol [25]. Human cGMP and phosphodi-
esterase (PDE 5A1) were purchased from Sigma-Aldrich.
All other reagents were of analytical grade from Merck.
The enzymatic reaction was carried out in 0.1 M Tris–
HCl buffer, pH 8.3, 10 mM MgCl2, 0.1 M KCl at 37 °C.
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by the addition of cGMP in a final concentration of
0.5 9 10-4 M. Enzyme and time of incubation may be
altered and if are not indicated in the specific experimental
procedure it will be 60 min. The reaction was finished by
transferring the reaction mixture tubes in boiling water bath
for 3 min. The sample was then centrifuged and filtered
through a nylon-66 filter, 0.2 mm (Rainin corporation).
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