Antitumor studies. Part 5: Synthesis, antitumor activity, and molecular docking study of 5-(monosubstituted amino)-2-deoxo-2-phenyl-5-deazaflavins

Article abstract of DOI:10.1016/j.bmc.2008.09.022

Various novel 5-(monosubstituted amino)-2-deoxo-2-phenyl-5-deazaflavins derivatives have been synthesized by direct coupling of 5-deazaflavins and N-alkyl or aryl amines. The antitumor activities against human tumor cell lines CCRF-HSB-2 and KB cells have been investigated in vitro and many compounds showed promising potential antitumor activities with less cytotoxicities. AutoDock molecular docking into PTK (PDB code: 1t46) has been done for lead optimization of these compounds as potential PTK inhibitors. Some of the synthesized 5-(monosubstituted amino)-2-deoxo-2-phenyl-5-deazaflavins at the 5-position exhibited reasonable binding affinities into PTK with the hydrogen bond through their C₅-NH moiety.

Full text of DOI:10.1016/j.bmc.2008.09.022

Bioorganic & Medicinal Chemistry 16 (2008) 9161–9170
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antitumor studies. Part 5: Synthesis, antitumor activity, and molecular
docking study of 5-(monosubstituted amino)-2-deoxo-2-phenyl-5-deazaflavins
Ajaya R. Shrestha a, Hamed I. Ali a, Noriyuki Ashida b, Tomohisa Nagamatsu a,
*
a Division of Pharmaceutical Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama Univesity 1-1-1, Tsushima-naka, Okayama 700-8530, Japan
b Biology Laboratory, Research and Development Division, Yamasa Shoyu Co. Choshi, Chiba 288-0056, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 July 2008
Revised 6 September 2008
Accepted 9 September 2008
Available online 12 September 2008
Various novel 5-(monosubstituted amino)-2-deoxo-2-phenyl-5-deazaflavins derivatives have been syn-
thesized by direct coupling of 5-deazaflavins and N-alkyl or aryl amines. The antitumor activities against
human tumor cell lines CCRF-HSB-2 and KB cells have been investigated in vitro and many compounds
showed promising potential antitumor activities with less cytotoxicities. AutoDock molecular docking
into PTK (PDB code: 1t46) has been done for lead optimization of these compounds as potential PTK
inhibitors. Some of the synthesized 5-(monosubstituted amino)-2-deoxo-2-phenyl-5-deazaflavins at
the 5-position exhibited reasonable binding affinities into PTK with the hydrogen bond through their
C5-NH moiety.
Keywords:
5-Deazaflavin
Antitumor activity
AutoDock
Ó 2008 Elsevier Ltd. All rights reserved.
Protein tyrosine kinase
1. Introduction
Design, synthesis, and biological evaluation of 5-deazaflavins
and flavin analogs have been greatly attracted our interest.1–8
Our previous study on the selective protein kinase C (PKC) inhibi-
tory activities of 5-deazaflavins, 10-substituted 2-deoxo-2-phenyl-
5-deazaflavins
{2-phenylpyrimido[4,5-b]quinolin-4(10H)-ones},
and flavin-5-oxides have revealed that they can exhibit significant
potential antitumor activities against NCI-H 460, HCT 116, A 431,
CCRF-HSB-2, and KB cells.2 The interest on 5-deazaflavins is by rea-
son of their first synthesis as potential riboflavin antagonists or fla-
vin models,9 and the discovery that they can serve as the bio-
reductive cofactors for several flavin-catalyzed reactions.10
(Scheme 1).
Scheme 1. Flavin and its analogs.
Moreover, it was found that the redox active coenzyme factor
420 (F420), which has the 5-deazaflavin skeleton, was isolated from
the methanogenic bacteria.11 5-Deazaflavins {5-deazaisoalloxa-
zines, pyrimido[4,5-b]quinoline-2,4(3H,10H)-diones} also have
the potent broad-spectrum activity against coccidiosis.12 5-Deazaf-
lavins have long been known for its strong oxidizing power which
can oxidize alcohols and amines to the corresponding carbonyl
compounds, and they behave as autorecycling turnover catalysts
in the redox reaction.13 The oxidizing power of 5-deazaflavin is
due to the presence of high electron deficiency at the 5-position.
The oxidizing property of 5-deazaflavin is effective especially in
the case of the structure without a substituent at the 5-position,
whereas the compound with a substituent cannot perform as the
redox catalyst. The 5-substituted 5-deazaflavins are, however, re-
ported to have appreciable bioactivity and can act as potential anti-
tumor agents. Namely, 5-amino-5-deazaflavins have been reported
to have antiproliferative activity against L1210 and KB cells.14 Also,
10-aryl-5-deazaflavin derivatives have been reported as inhibitors
of the E3 activity of HMD2 in tumors that retain wild-type p53.15
Molecular modeling is a powerful tool in drug design which
generously assists in investigating, interpreting, explaining and
identification of molecular properties using three-dimensional
structures. Many programs, including AutoDock,16 LigandFit/Ceri-
us2,17 ICM,18 FlexX,19 GOLD,20 Glide,21 and DOCK,22 are now avail-
able to quantify possible interactions between small molecules and
proteins, in silico. The role of tyrosine kinase in the control of cel-
* Corresponding author. Tel.: +81 86 251 7931; fax: +81 86 251 7926.
E-mail address: nagamatsu@pheasant.pharm.okayama-u.ac.jp (T. Nagamatsu).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.022

9162
A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 9161–9170
lular growth and differentiation is central to all organisms and the
tyrosine kinase has been found to participate in human neoplastic
diseases. Tyrosine kinase inhibitors and their potentials in the clin-
ical applications are well documented by dramatic examples such
as Gleevec, Iressa, and Herceptin. Several tyrosine kinase inhibitors
are undergoing human trials and several are in the pipeline of drug
discovery.23 Molecular docking has been a focus of attention for
many years. Generally speaking, today’s flexible docking programs
such as AutoDock can predict protein-ligand complex structures
with reasonable accuracy and speed.24 One of the most reliable,
robust and popular energy-based docking packages is AutoGrid/
AutoDock (Morris et al.)16 because it allows a very efficient docking
of flexible ligands (e.g., substrates, drug candidates, inhibitors,
peptides, etc.) onto receptors (e.g., enzymes, antibodies, nucleic
acids, etc.).25
In this study, our main objective is the development of potential
inhibitors synthesized newly 5-(monosubstituted amino)-
5-deazaflavins and their analogs as antitumor agents, based on
molecular modeling and the investigation of SAR between new
inhibitors and protein tyrosine kinase (PTK). As we described in
our previous paper,2 many of the synthesized, 2-deoxo-2-phenyl-
5-deazaflavin derivatives showed promising antitumor activities
against CRF-HSB-2 and KB cells as well as in vivo investigation
against A 431human adenocarcinomacells transplanted subcutane-
ously into nude mouse exhibited effective antitumor activity. More-
over, molecular docking study revealed that the compounds
possessed higher binding affinities into differentPTKs. In the present
paper, we describe the preparation of 5-(monosubstituted amino)-
2-deoxo-2-phenyl-5-deazaflavins, expectingtogetbetter antitumor
activities with lesser cytotoxicities. Also the AutoDock study for new
inhibitors docked into the active sites of PTK has been carried out.
imidin-4(3H)-ones (2a–e) were prepared by direct fusion of
6-chloro-2-phenylpyrimidin-4(3H)-one (1) with appropriate
N-monoalkylanilines at 180–200 °C for 0.5–5 h according to the
known procedure26 as shown in Scheme 2.
The N-monomethylated aniline derivatives were synthesized
from appropriate aniline derivatives by refluxing aniline deriva-
tives with ethyl formate for 15 h to get the corresponding N-aryl-
formamides, followed by reduction with LiAlH4 in THF to afford
the respective N-monomethylated anilines.27 Similarly, the N-
monoethylated aniline derivatives were synthesized by refluxing
appropriate aniline derivatives with ethyl orthoformate in pres-
ence of concd sulfuric acid to get N-ethyl-formanilide derivatives
and followed by refluxing with dilute hydrochloric acid and then
aqueous potassium hydroxide.28 The synthesized products were
identified by comparing the IR spectra with authentic samples.
The 10-alkyl-2-deoxo-2-phenyl-5-deazaflavins (3a–e) were pre-
pared by following known procedure,29 refluxing 6-(N-monoalky-
lanilino)-2-phenylpyrimidin-4(3H)-ones (2a–e) with Vilsmeier
reagent (N,N-dimethylformamide-phosphoryl chloride) at 90 °C
for 2–4 h. The target compounds, 5-(monosubstituted amino)-
10-alkyl-2-deoxo-2-phenyl-5-deazaflavins (4a–r and 5a–c) were
then synthesized by treating the appropriate amines with 3a–e
and refluxing at 90–110 °C for 2–6 h. Particularly, alkyl amines
were found to be easier to undergo the reaction. In case of aryl
amines, more strenuous conditions were required and the reac-
tions were carried out in the sealed glass tube at 120–135 °C for
10–15 h. This may be due to the steric hindrance of bulky aromatic
ring in case of aryl amines. The reactions in general were carried
out by direct fusion of the reactants, but in case of aryl amines,
n-butanol as the reaction medium and triethylamine as a catalyst
were used.
UV–vis, IR, NMR spectra, and elemental analyses were used for
the identification and confirmation of the newly assigned struc-
tures. The structures of the products were confirmed in particu-
larly by the disappearance of characteristic C5-H in 1H NMR
spectra. In case of 2-deoxo-2-phenyl-5-deazaflavin derivatives
showed a characteristic singlet signal at the lower field at 9.2–
9.4 ppm due to the proton at the 5-position, implying that the
2. Results and discussion
2.1. Chemistry
The requisite precursors for the preparation of 2-deoxo-2-phe-
nyl-5-deazaflavins (3a–e), 6-(N-monoalkylanilino)-2-phenylpyr-
Scheme 2. General method of the preparation of 6-(N-monoalkylanilino)-2-phenylpyrimidin-4(3 H)-ones (2a–e), 2-deoxo-2-phenyl-5-deazaflavins (3a–e) and 5-
(monosubstituted amino)-2-deoxo-2-phenyl-5-deazaflavins (4a–r, 5a–c). Reagents and conditions: (i) fusion at 180–200 °C, 0.5–5 h; (ii) Vilsmeier reagents (DMF-POCl3),
90 °C, 2–4 h; (iii) Et3 N, 90–110 °C, 2–6 h; (iv) Et3 N, n-BuOH, sealed glass tube, 135 °C, 10–15 h.

A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 9161–9170
9163
5-position of these compounds is the most electron-deficient
methine and very reactive towards nucleophiles.2 This explains
the nucleophilic substitution of the C5 proton by alkyl or aryl
amines, producing 5-(monosubstituted amino)-2-deoxo-2-phe-
nyl-5-deazaflavins (4a–r and 5a–c). Hence, the disappearance of
signal at the 9.2–9.4 ppm in 1H NMR spectra confirmed the substi-
tution of amines at 5-position. In 13C NMR, the C5 carbon appeared
at more deshielded region around 153–156 ppm due to the pres-
ence of the more electronegative nitrogen atom. UV absorption
spectra of the 5-(monoalkylamino)-2-deoxo-2-phenyl-5-deazaf-
lavins (4a–r) showed two absorption maxima at 270–277 and
342–351 nm together with three absorption maxima as shoulders
at 229–238, 251–260, and 380–410 nm. Comparing these data
with those of the corresponding 2-deoxo-2-phenyl-5-deazaflavin,2
which showed four absorption maxima at 283–297, 336–355, 414–
431, and 433–456 nm together with an absorption maximum as
shoulder at 262–288 nm, it has been revealed the hypsochromic
shift at 283–297 nm with loss of two absorption maxima at 414–
431 and 433–456 nm and addition of two shoulders at 229–238
and 380–410 nm. In contrast with the aforementioned UV absorp-
tion spectra, the compound with aryl amino substituent at the
5-position (5a and b) exhibited hypsochromic shift in the region
of longer wavelength with two absorption maxima at 231 and
282–283 nm along with an absorption maximum as shoulder at
329–337 nm. In case of compound 5c, the rather different pattern
of UV absorption spectra was gained with four absorption maxima
at 229, 299, 337, and 452 nm along with one absorption maximum
as shoulder at 434 nm. This may due to the presence of ester moi-
ety in the aryl group at the 5-position. In general, due to the pres-
ence of absorption maximum at 452 nm, compound 5c was
reddish in color and other compounds showed yellow or colorless
crystals due to the absence of absorption maxima in the region of
longer wavelength.
the antitumor agent, cytosine arabinoside (Ara-C), was used as po-
sitive control in the study. The IC50 values [the concentration
(lg/mL) required for 50% inhibition of cell growth] for these com-
pounds are shown in Table 1.
As can be seen in Table 1, many 5-(monosubstituted amino)-2-
deoxo-2-phenyl-5-deazaflavins (4a–k, n–p, and 5a–c) have been
found to show fairly good antitumor activities. Although the tested
compounds exhibited less antitumor activities than that of Ara-C
(IC50: 0.017
the 4.7–8.9
l
l
g/mL) as the control, most of the compounds showed
g/mL potential growth inhibitory activities against
CCRF-HSB-2 cell line appreciably revealing their promising poten-
tial inhibitory activities and less cytotoxicities. It was also note-
worthy that these compounds exhibited less potential growth
inhibitory activities than the corresponding 2-deoxo-2-phenyl-5-
deazaflavin,2 also supporting the fact that substitution at 5-posi-
tion leads to reduce the cytotoxicities of the compounds. Among
them, the compounds 5a, 5b, and 4j were found to be better inhib-
itors with less than 2.5 lg/mL (IC50). Similarly, in case of KB cell
line, the compounds 4j, 4o, 5a, and 5b exhibited good growth
inhibitory activities of about one-fifth antitumor potency of Ara-
C (IC50: 0.42
1.6, and 1.8
l
g/mL), that is, the IC50 values of them were 2.7, 2.0,
lg/mL, respectively. From this study, it has been gen-
eralized that the arylamino substituents at the 5-position in-
creased the potency in the case of 5a and 5b, while the longer
chained alkylamino substituents reduced the potency in the case
of 4c, 4g, and 4k comparing with that of 4b, 4f, and 4j. Substituents
at the 7-position such as the cases of 4j, 4k, and 4o were found to
improve the potential antitumor activities of the compounds than
their corresponding 7-unsubstituted derivatives.
2.3. Molecular docking study
Both pharmaceutical companies and university laboratories
have been active to develop compounds which can inhibit tyrosine
kinase activity in the expectation that the potent and selective
inhibitors would represent a new class of therapeutics for cancers
as well as other proliferative diseases. Therefore, PTK inhibitors can
be applied aptly as a new mode of cancer therapy. Depending on
the above mentioned idea, herein, we investigated the AutoDock
binding affinities of the synthesized 5-(monosubstituted amino)-
2-deoxo-2-phenyl-5-deazaflavins into PTK. Towards optimization
of the aforementioned lead compounds of the promising antitumor
activities, the advanced docking program AutoDock 3.0516 was
2.2. In vitro antitumor activities of 5-(monosubstituted
amino)-2-deoxo-2-phenyl-5-deazaflavins against human tumor
cell lines
The in vitro tests of the synthesized compounds were carried
out for the growth inhibitory activities against various cultured tu-
mor cell lines. Two human tumor cell lines viz, human T-cell acute
lymphoblastoid leukemia cell line (CCRF-HSB-2) and human oral
epidermoid carcinoma cell line (KB) were used for the test and
Table 1
Growth inhibitory activities of 5-(monosubstituted amino)-2-deoxo-2-phenyl-5-deazaflavins (4a–k, n–p, and 5a–c) against human tumor cell lines of CCRF-HSB-2 and KB cells
Compound
R1
R2
R3
Inhibitory activity against tumor cell lines
[IC50 g/mL)]
(
l
CCRF-HSB-2
KB
18.00
6.60
32.90
66.90
22.00
7.60
4a
4b
4c
4tJ
4e
4f
Me
Me
Me
Me
Me
Et
H
H
H
H
H
H
n-Pr
n-Bu
n-C8H17
CH2C6H5
CH2CH2C6H5
n-Bu
>100.00
4.70
>100.00
>100.00
17.70
8.30
4g
4h
4i
Et
Et
Et
Me
Me
Et
Et
Et
H
H
H
Me
Me
Me
Me
Me
H
n-C8H17
CH2C6H5
CH2CH2C6H5
n-Bu
n-C8H17
n-Bu
n-C8H17
CH2C6H5
3,4-Me2-C6H3
4-Me-C6H4
2-COOMe-C6H4
20.40
6.10
63.30
2.10
8.60
8.90
5.70
6.90
0.75
1.10
12.00
4.70
53.40
2.70
4.70
8.60
2.00
17.20
1.60
1.80
>100.00
0.42
4j
4k
4n
4o
4p
5a
5b
5c
AraC
Me
Me
Me
H
H
>100.00
0.02

9164
A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 9161–9170
used to evaluate the binding free energies as potential inhibitors
into the target PTK macromolecule.
1t46) with its bound ligand STI exhibited hydrogen bonds via their
C5-NH group as illustrated for compounds 4b and 4g in Figure 1
and for compounds 4a and 5b in Figure 2.
2.3.1. Validation of the accuracy and performance of AutoDock
According to the method of validation cited in literature,23
where if the RMSD (root mean square deviation) of the best docked
conformation is 62.0 Å from the experimental one, the used scor-
ing function is successful. The obtained success rates of AutoDock
(Morris et al)16 was highly excellent as illustrated in Table 2 for the
native ligand STI ligand (ImatinibÒ or GleevecÒ), 4-(4-methylpi-
perazin-1-ylmethyl)-n-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-
ylamino)phenyl]-benzamide, which was docked into its c-Kit
receptor PTK (PDB code: 1t46). The RMSD of the docked ligand
was 0.25 Å. The validation results were discussed in details in
our previous publication.3 The results indicated the high accuracy
of the AutoDock simulation in comparison with the biological
methods.30
The correlation between the growth inhibitory activities (IC50
g/mL) of the synthesized 5-(monosubstituted amino)-2-deoxo-
,
l
2-phenyl-5-deazaflavins against tumor cells and the binding affin-
ities predicted by AutoDock was highly good for some compounds.
Considering the growth inhibition against CCRF-HSB-2 cells, it was
noticed that the correlation between IC50 of 4b–g, i, n, and 5a–c
and their AutoDock binding free energies revealed an excellent
correlation coefficient (R2) of 0.7871 as represented in Figure 3.
Moreover, the growth inhibition against KB cells revealed a bet-
ter correlation with AutoDock binding free energies for compounds
4b–g, i, n and 5a–c of correlation coefficient (R2) value of 0.7753 as
shown in Figure 4.
3. Conclusions
2.3.2. AutoDock binding affinities of the synthesized and
designed compounds into PTK
The binding affinity was evaluated by the binding free energies
(DGb, kcal/mol), inhibition constants (Ki), hydrogen bonding, and
In this study, various 5-alkylamino and 5-arylamino deriva-
tives (4a–r and 5a–c) of 2-deoxo-2-phenyl-5-deazaflavin have
been synthesized by direct coupling of 2-deoxo-2-phenyl-5-
deazaflavins and appropriate N-alkyl or aryl amines to investigate
their potential antitumor activities. In vitro growth inhibitory
activities of the compounds 4a–k, n–p, and 5a–c against two hu-
man tumor cell lines viz, human T-cell acute lymphoblastoid leu-
kemia cell line (CCRF-HSB-2) and human oral epidermoid
carcinoma cell line (KB) have been investigated. From the study,
it has been revealed that the alkylamino substituents at the 5-po-
sition decreased the antitumor activities, whereas arylamino sub-
stituents at the 5-position increased it. Especially, compounds 5a
and 5b were found to exhibit better antitumor activities with
RMSD values. The compounds which revealed the highest binding
affinities, that is, lowest binding free energies, within PTK and
the hydrogen bond interactions into the target macromolecule
are represented in Table 2. These compounds include 5-alkyl-
amino-2-deoxo-10-methyl-5-deazaflavins (4a–e and 4j–l), 5-alkyl-
amino-2-deoxo-10-ethyl-5-deazaflavins (4f–i, and 4n–p), and
5-arylamino-2-deoxo-10-methyl-5-deazaflavins (5a–c). Many of
these derivatives exhibited one or two hydrogen bonds between
C5-NH of the docked 5-deazaflavins and different amino acids of
the target PTK including Leu 595, Ala 636, Glu 640, Thr 670, Ilu
789, His 790, and Asp 810 as cited in Table 2.
IC50 values less than 2.0 lg/mL. Also, it has been generalized that
the substitution of 7-H by a methyl group tends to increase the
antitumor activities. The overall antitumor activities of com-
pounds 4a–k, n–p, and 5a–c were found to be lesser than corre-
The molecular docking study revealed that the majority of the
compounds docked into the c-Kit receptor of PTK (PDB code:
Table 2
The best docking results based on the binding free energies (DGb) and inhibition constants (Ki) of compounds docked into PTK, the distances and angles of hydrogen bonds
between compounds and amino acids involved in PTK, and RMSD from the co-crystallized STI ligand
a
Compound
D
Gb (kcal/mol)
Kib
Hydrogen bonds between atoms of compounds and amino acids
RMSDc(A)
2 24
Atom of compound
Amino acid
Distance (A)
Angle (°)
4a
ꢀ10.28
2.90Eꢀ08
N1-N
HN of Asp 810
OH of Thr 670
CO of Leu 595
CO of His 790
O of His 790
O of Asp 810
CO of IIe 789
Co of Glu 640
O of Asp 810
O of Asp 810
—
1.68
2.11
2.26
1.94
2.33
1.71
2.10
2.44
1.94
2.06
—
158.5
143.6
137.4
177.1
149.4
130.0
124.5
113.7
114.4
125.5
C5-NH
C5-NH
C5-NH
C5-NH
C5-NH
C5-NH
C5-NH
C5-NH
C5-NH
—
C5-NH
C5-NH
C5-NH
C5-NH
C5-NH
C5-NH
C5-NH
C5-NH
C5-NH
N3-N
4b
4c
4d
4e
4f
4g
4h
4i
ꢀ8.74
ꢀ8.17
ꢀ7.68
ꢀ8.49
ꢀ9.07
ꢀ9.18
ꢀ7.84
ꢀ7.82
ꢀ7.34
ꢀ10.39
ꢀ7.95
ꢀ8.90
3.91Eꢀ07
1.02Eꢀ06
2.33Eꢀ06
5.96Eꢀ07
2.26Eꢀ07
1.88Eꢀ07
1.80Eꢀ06
1.86Eꢀ06
4.18Eꢀ06
2.43Eꢀ08
1.49Eꢀ06
2.99Eꢀ07
7.32
8.49
9.33
8.80
7.61
4.98
9.06
9.01
7.85
7.98
8.76
7.09
d
4j
4k
4l
—
CO of Leu 595
O of Ala 636
O of His 790
O of IIe 789
O of Ala 636
NH of Glu 640
NH of Glu 640
O of Ala 636
O of Ala 636
HN of Cys 673
OH of Thr 670
HN of Asp 810
1.85
2.08
2.45
2.37
2.13
2.18
2.08
2.45
2.17
1.66
1.86
2.01
145.7
113.4
163.5
106.7
146.7
163.4
168.2
110.1
133.0
161.0
147.2
133.3
4n
4o
5a
5b
ꢀ7.94
ꢀ9.20
ꢀ9.35
1.52Eꢀ06
1.79 Eꢀ07
1.39 Eꢀ07
8.88
8.75
8.09
5c
ꢀ7.85
1.75 Eꢀ06
3.08Eꢀ14
8.91
0.25
STIe
ꢀ18.43
NH (H79)
O29
a
Binding free energy.
Inhibition constant.
Root mean square deviation.
No hydrogen bond.
The native co-crystallized bound ligand (STI-571) of PTK (PDB code: 1t46).
b
c
d
e

A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 9161–9170
9165
Figure 1. Compound 4b (colored by element, ball and stick) is bound into PTK receptor site via hydrophobic interactions with Leu 595, Tyr 672, Leu 799, and Phe 811 in
addition to hydrophilic binding by hydrogen bond between its C5-NH and C@O of Leu 595. While 4g (blue, stick) is extended through the whole groove of the pocket forming
one hydrogen bond with Glu 640. The binding pocket is shown in the solid surface with labeled amino acids and the STI ligand is shown as yellow stick.
Figure 2. Differential binding affinities of 2-deoxo-10-methyl-2-phenyl-5-(n-propylamino)-5-deazaflavins (4a; colored by element, ball and stick) and 2-deoxo-10-methyl-
2-phenyl-5-(p-toluidino)-5-deazaflavins (5b: yellow, stick) into PTK. Each of the docked compounds exhibited two hydrogen bonds shown as green lines. The binding pocket
of PTK is shown in the wire mesh surface with labeled amino acids, the PTK protein structure is shown in solid ribbon view, and the STI ligand is shown as a blue line.
sponding 2-deoxo-2-phenyl-5-deazaflavins, which reveals the
potential antitumor activities of the compounds with lesser
cytotoxicities.
The AutoDock investigation of the synthesized 5-(monosubsti-
tuted amino)-2-deoxo-2-phenyl-5-deazaflavins (4a–l, n, o, and
5a–c) was carried out by docking into the c-kit of protein tyrosine
kinase (PDB code: 1t46). The correlation between the growth
inhibitory activities (IC50, lg/mL) of the synthesized 5-(monosub-
stituted amino)-2-deoxo-2-phenyl-5-deazaflavins (4b–g, i, n, and
5a–c) against tumor cells namely CCRF-HSB-2 and KB cell lines
and the binding affinities predicted by AutoDock was highly good,
being of the correlation coefficients (R2) of 0.7871 and 0.7753,
respectively. The docked analogs exhibited preferential binding
affinities into PTK with one or two hydrogen bonds and reasonably
low binding free energies. Thus, the studies of the 5-(monosubsti-
tuted amino)-2-deoxo-2-phenyl-5-deazaflavins by synthesis, bio-
logical investigation and in silico study revealed the less
cytotoxicities with reasonably low binding free energies and prom-
ising potential antitumor activities. Therefore, these compounds
Figure 3. Correlation between the binding free energy (DGb) and antitumor activity
(IC50) of 5-(monosubstituted amino)-2-deoxo-2-phenyl-5-deazaflavins (4 b–g, i, n
and 5a–c) against human T-cell acute lymphoblastoid leukemia cell line (CCRF-
HSB-2).

9166
A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 9161–9170
(1H, s, 5-H). Anal. Calcd for C20H17N3O2ꢁ0.66H2O: C, 71.20; H,
5.28; N, 12.46. Found: C, 71.33; H, 5.52; N, 12.76.
4.2. General procedure for the preparation of 5-alkyl amino-2-
deoxo-5-deazaflavins (4a–r)
A mixture of 10-alkyl-2-deoxo-2-phenyl-5-deazaflavins (3a–e)2
(1.5 g) and N-alkylamines (15 equiv.) was refluxed at 90–110 °C for
2–6 h. After cooling, the solution was diluted with diethyl ether to
get the precipitate, which was filtered off, washed with diethyl
ether, dried and recrystallized from ethanol to afford the corre-
sponding products as colorless or pale yellow needles.
4.2.1. 10-Methyl-5-(n-propylamino)-2-phenylpyrimido[4,5-b]-
quinolin-4(10H)-one (4a)
Yield, (1.1 g, 61%); mp 233–235 °C; UV (EtOH): kmax/nm
e
/dm3 molꢀ1cmꢀ1): 231 (4.09), 251 (4.21), 271 (4.28), 346
Figure 4. Correlation between the binding free energy (DGb) and antitumor activity
(IC50) of 5-(monosubstituted amino)-2-deoxo-2-phenyl-5-deazaflavins (4 b–g, i, n
and 5a–c) against human oral epidermoid carcinoma cell line (KB).
(log
(4.02), 380 (3.76); IR (
m
max/cmꢀ1): 1648 (C@O), 3140 (NH); 1H
NMR (CDCl3): dY 1.17 (3H, t, J = 7.5 Hz, 5-CH2CH3), 1.95 (2H, m,
5-CH2CH2 CH3), 3.93 (2H, m, 5-NCH2CH2CH3), 4.14 (3H, s, 10-
NCH3), 7.34 (1H, m, 7-H), 7.43–7.46 (3H, m, 2-Ph-m, pH), 7.63
(1H, d, J = 9.0 Hz, 9-H), 7.78 (1H, m, 8-H), 8.27 (1H, d, J = 8.7 Hz,
6-H), 8.57–8.60 (2H, m, 2-Ph-oH); 13C NMR (CDCl3): dC 11.51
(5-NCH2CH2CH3), 23.75 (5-NCH2CH2CH3), 31.62 (10-NCH3), 50.45
(5-NCH2CH2CH3), 97.59 (C4a), 115.51 (C7), 116.22 (C9), 121.85 (C8),
may be considered as promising candidates for further antitumor
investigations.
0
0
0
0
0
4. Experimental
4.1. Chemistry
127.94 (C3 and C5 ), 128.27 (C6), 128.97 (C2 and C6 ), 130.78 (C4 ),
0
134.03 (C1 ), 138.25 (C5a), 141.90 (C9a), 155.73 (C5), 158.72 (C10a),
166.30 (C2), 175.62 (C@O). Anal. Calcd for C21H20N4Oꢁ0.16H2O: C,
72.60; H, 5.90; N, 16.13. Found: C, 72.84; H, 6.17; N, 15.73.
Mps were obtained on a Yanagimoto micro melting point appa-
ratus and were uncorrected. Microanalyses were measured by
Yanaco CHN corder MT-5 apparatus. IR spectra were recorded on
a JASCO FT/IR-200 spectrophotometer as Nujol mulls. 1H and 13C
NMR spectra were obtained using a Varian VXR 300 and 75 MHz
spectrophotometer, respectively, and chemical shift values are ex-
pressed in d values (ppm) relative to tetramethylsilane (TMS) as
internal standard. Coupling constants are given in Hz and signals
are quoted as follows: s, singlet; d, doublet; t, triplet; q, quartet;
quin, quintet; sex, sextet; sept, septet; br, broad; m, multiplet.
UV spectra were measured in absolute EtOH using Beckman DU-
68S UV spectrophotometers and absorption values in italics refer
to wave lengths at which shoulders or inflexions occur in the
absorption. All reagents were of commercial quality and were used
without further purification. Organic solvents were dried in the
presence of appropriate drying agents and were stored over suit-
able molecular sieves. Reaction progress was monitored by analyt-
ical thin-layer chromatography (TLC) on pre-coated glass plates
(Silica gel 60 F254-plate-Merck) and the products were visualized
by UV light.
4.2.2. 5-(n-Butylamino)-10-methyl-2-phenylpyrimido[4,5-b]-
quinolin-4(10H)-one (4b)
Yield, (1.05 g, 56%); mp 249–251 °C; UV (EtOH): kmax/nm
(log
e
/dm3 molꢀ1cmꢀ1): 229 (4.38), 252 (4.45), 272 (4.56), 346
(4.26), 384 (3.99); IR (
m
max/cmꢀ1): 1651 (C@O), 3140 (NH); 1H
NMR (CDCl3): dY 1.10 [3H, t, J = 7.2 Hz, 5-N(CH2)3CH3], 1.61 [2H,
m, 5-N(CH2)2CH2CH3], 1.92 [2H, m, 5-NCH2CH2CH2CH3], 3.97 [2H,
m, 5-NCH2CH2CH2CH3], 4.15 (3H, s, 10-NCH3), 7.33 (1H, m,
7-H),7.45–7.47 (3H, m, 2-Ph-m, pH), 7.66 (1H, d, J = 8.7 Hz, 9-H),
7.79 (1H, m, 8-H), 8.33 (1H, d, J = 8.4 Hz, 6-H), 8.57–8.61 (2H, m,
2-Ph-oH); 13C NMR (CDCl3): dC 13.74 (5-NCH2CH2CH2CH3), 20.08
(5-NCH2CH2CH2CH3), 31.61 (10-NCH3), 32.38 (5-NCH2CH2CH2CH3),
48.47 (5-NCH2CH2CH2CH3), 97.61 (C4a), 115.52 (C7), 116.21 (C9),
0
0
0
0
121.84 (C8), 127.95 (C3 and C5 ), 128.27 (C6), 128.99 (C2 and C6 ),
0
0
130.78 (C4 ), 134.02 (C1 ), 138.28 (C5a), 141.92 (C9a), 155.75 (C5),
158.70 (C10a), 166.33 (C2), 175.62 (C@O); Anal. Calcd for
C22H22N4Oꢁ0.16H2O: C, 73.11; H, 6.23; N, 15.50. Found: C, 73.11;
H, 5.99; N, 15.37.
4.2.3. 10-Methyl-5-(n-octylamino)-2-phenylpyrimido[4,5-b]-
quinolin-4(10H)-one (4c)
4.1.1. Preparation of 10-ethyl-7-methoxy-2-phenylpyrimido-
[4,5-b]quinolin-4(10H)-one (3e)
Yield, (0.97 g, 45%); mp 205–207 °C; UV (EtOH): kmax/nm
A mixture of 6-(N-ethylanisidino)-2-phenylpyrimidin-4(3H)-
one (2e)2 (14.5 g, 0.05 mol) and phosphoryl chloride (77 g,
0.5 mol) in N,N-dimethylformamide (100 mL) was heated under
stirring at 90 °C for 3 h. Then, the reaction mixture was poured
onto ice and neutralized (pH 7) with aqueous ammonia. The sepa-
rated yellow crystals were filtered off, washed with water, dried,
and recrystallized from ethanol to get yellow needles.
(log
e
/dm3 molꢀ1 cmꢀ1): 230 (4.51), 252 (4.66), 271 (4.72), 345
(4.51), 383 (4.25); IR (
m
max/cmꢀ1): 1680 (C@O), 3180 (NH); 1H
NMR (CDCl3): dY 0.8 8 [3Y, t, J = 6.9 Hz, 5-NCH2(CH2)6CH3], 1.22–
1.41 [8H, m, 5-NCH2CH2CH2(CH2)4CH3], 1.50–1.61 [2H, m, 5-
NCH2CH2CH2(CH2)4CH3], 1.87–1.98 [2H, m, 5-NCH2CH2(CH2)5CH3],
3.96 [2H, m, 5-NCH2(CH2)6CH3], 4.15 (3H, s, 10-NCH3), 7.32 (1H, m,
7-H), 7.44–7.47 (2H, m, 2-Ph-m, pH), 7.63 (1H, d, J = 8.7 Hz, 9-H),
7.78 (1H, m, 8-H), 8.30 (1H, d, J = 8.7 Hz, 6-H), 8.57–8.60 (2H, m,
2-Ph-oH); 13C NMR (CDCl3): dC 14.25 [5-NCH2(CH2)6CH3], 22.80
[5-NCH2(CH2)5CH2CH3], 27.07 [5-NCH2CH2CH2(CH2)4CH3], 29.41
[5-NCH2(CH2)3CH2(CH2)2CH3], 30.56 [5-NCH2(CH2)2CH2(CH2)3CH3],
31.08 [5-NCH2(CH2)4CH2CH2CH3], 31.75 (10-NCH3), 31.95 [5-
NCH2CH2(CH2)5CH3], 48.93 [5-NCH2(CH2)6CH3], 97.74 (C4a), 115.67
Yield, 15.1 g (94%); mp 238–240 °C; UV (EtOH): kmax/nm (log
dm3 molꢀ1 cmꢀ1): 235 (3.99), 293 (4.04), 318 (3.72), 445 (3.36),
470 (3.39); IR (
max/cmꢀ1): 1621 (C@O); 1H NMR (CDCl3): dY 1.63
e/
m
(3H, t, J = 7.2 Hz, 10-NCH2CH3), 3.96 (3H, s, 7-OCH3), 5.17 (2H, q,
J = 7.2 Hz,, 10-NCH2CH3), 7.34 (1H, d, J6,8 = 3.0 Hz, 6-H), 7.44–7.53
(3H, m, Ph-m, pH), 7.64 (1H, dd, J6,8 = 3.0 Hz, J8,9 = 9.6Hz, 8-H),
7.84 (1H, d, J8,9 = 9.6 Hz, 9-H), 8.63–8.66 (2H, m, Ph-oH), 9.28
0
0
(C7), 116.35 (C9), 121.97 (C8), 128.09 (C3 and C5 ), 128.43 (C6),

A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 9161–9170
9167
0
0
0
0
129.14 (C2 and C6 ), 130.92 (C4 ), 134.16 (C1 ), 138.45 (C5a), 142.06
(C9a), 155.90 (C5), 158.80 (C10a), 166.47 (C2), 175.78 (C@O). Anal.
Calcd for C26H30N4Oꢁ0.12H2O: C, 74.92; H, 7.32; N, 13.44. Found:
C, 74.94; H, 7.04; N, 13.26.
NMR (CDCl3): dY 0.87 [3Y, t, J = 6.9 Hz, 5-NCH2(CH2)6CH3], 1.22–
1.26 (10H, m, NCH2CH2(CH2)5CH3), 1.40–1.51 [2H, m, 5-
NCH2CH2(CH2)5CH3], 1.56 (3H, t, J = 7.2 Hz, 10-NCH2CH3), 2.70
[2H, m, 5-NCH2CH2(CH2)5CH3], 4.91 (2H, br s, 10-NCH2CH3),
7.38–7.56 (4H, m, 7-H, 2-Ph-m, pH), 7.70 (1H, d, J = 7.8 Hz, 9-H),
7.84–7.89 (1H, m, 8-H), 8.52–8.61 (3H, m, 6-H, 2-Ph-oH); 13C
NMR (CDCl3): dC 12.89 [5-NCH2(CH2)6CH3], 14.26 (10-NCH2CH3),
22.80 [5-N(CH2)6CH2CH3], 27.08 [5-N(CH2)2CH2(CH2)4CH3], 29.40
[5-N(CH2)4CH2(CH2)2CH3], 30.63 [5-N(CH2)3CH2(CH2)3CH3], 31.09
[5-N(CH2)5CH2CH2CH3], 31.95 [5-NCH2CH2(CH2)5CH3], 39.71 [5-
NCH2CH2(CH2)5CH3], 48.98 [10-NCH2CH3], 97.81 (C4a), 115.96
4.2.4. 5-Benzylamino-10-methyl-2-phenylpyrimidio[4,5-b]-
quinolin-4(10H)-one (4d)
Yield, (1.12 g, 55%); mp 238–240 °C (lit.,26 228 °C); UV (EtOH):
kmax/nm (log
(4.11), 351 (3.92), 381 (3.64); IR (
e
/dm3 molꢀ1 cmꢀ1): 229 (3.92), 252 (4.04), 271
m
max/cmꢀ1): 1670 (C@O), 3120
(NH); 1H NMR (CDCl3): dY 4.18 (3 H, s, 10-NCH3), 5.18 (2H, d,
J = 6.0 Hz, 5-NHCH2Ph), 7.25–7.30 (1H, m, 7-H), 7.34–7.49 (9H, m,
5-Ph-H, 2-Ph-m, pH), 7.65 (1H, d, J = 8.1 Hz, 9-H), 7.78 (1H, m, 8-
H), 8.23 (1H, d, J = 8.4 Hz, 6-H), 8.57–8.59 (2H, m, 2-Ph-oH); 13C
NMR (CDCl3): dC 31.81 (10-NCH3), 51.78 (5-NCH2Ph), 98.19 (C4a),
0
0
(C7), 116.17 (C9), 121.74 (C8), 128.12 (C3 and C5 ), 128.73 (C6),
0
0
0
0
129.22 (C2 and C6 ), 130.93 (C4 ), 134.13 (C1 ), 138.65 (C5a),
141.15 (C9a), 156.37 (C5), 158.95 (C10a), 166.77 (C2), 175.85
(C@O). Anal. Calcd for C27H32N4O: C, 75.67; H, 7.53; N, 13.07.
Found: C, 75.32; H, 7.24; N, 12.90.
00
00
115.21 (C7), 116.35 (C9), 122.10 (C8), 126.93 (C3 and C5 ), 128.06
0
0
00
00
0
0
(C3 , C5 and C6), 129.04 (C2 and C6 ), 129.18 (C2 and C6 ), 130.96
0
0
00
(C4 ), 134.30 (C1 ), 136.41 (C1 ), 138.25 (C5a), 142.01 (C9a), 155.98
(C5), 159.6 (C10a), 166.78 (C2), 175.71 (C@O). Anal. Calcd for
C25H20N4Oꢁ0.60H2O: C, 74.09; H, 5.77; N, 13.82. Found: C, 73.97;
H, 6.00; N, 13.69.
4.2.8. 5-Benzylamino-10-ethyl-2-phenylpyrimido[4,5-
b]quinolin-4(10H)-one (4h)
Yield, (0.88 g, 44%); mp 171–173 °C; UV (EtOH): kmax/nm
(log
e
/dm3 molꢀ1 cmꢀ1): 229 (3.89), 252 (3.99), 271 (4.09), 340
(3.79), 372 (3.55), 381 (3.57); IR (
m
max/cmꢀ1): 1670 (C@O), 3180
(NH); 1H NMR (CDCl3): dY 1.54 (3H, t, J = 6.9 Hz, 10-NCH2CH3),
4.90 (2H, br s, 10-NCH2CH3), 5.20 (2H, d, J = 4.5 Hz, 5-NHCH2Ph),
7.27–7.40 (6H, m, 7-H, 5-Ph-H), 7.42–7.49 (3H, m, 2-Ph-m, pH),
7.70 (1H, d, J = 9.0 Hz, 9-H), 7.79 (1H, m, 8-H), 8.26 (1H, d,
J = 8.7 Hz, 6-H), 8.56–8.58 (2H, m, 2-Ph-oH)); 13C NMR (CDCl3):
dC 13.08 (10-NCH2CH3), 39.42 (10-NCH2CH3), 55.14 (5-NCH2Ph),
4.2.5. 10-Methyl-5-phenethylamino-2-phenylpyrimido[4,5-b]-
quinolin-4(10H)-one (4e)
Yield, (1.22 g, 57%); mp 271–273 °C; UV (EtOH): kmax/nm
(log
e
/dm3 molꢀ1cmꢀ1): 229 (4.18), 253 (4.30), 271 (4.38), 346
(4.15), 383 (3.92); IR (
m
max/cmꢀ1): 1650 (C@O), 3120 (NH); 1H
NMR (CDCl3): dY 3.2 2.2Y, t, J = 7.2 Hz, 5-NCH2CH2Ph), 4.19 (3 H,
s, 10-NCH3), 4.23 (2H, m, 5-NCH2CH2Ph), 7.25–7.40 (6H, m, 5-Ph-
H and 7-H), 7.44 (3H, m, 2-Ph-m, pH), 7.61 (1H, d, J = 9.0 Hz,
9-H), 7.75 (1H, m, 8-H), 8.24 (1H, d, J = 8.4 Hz, 6-H), 8.57–8.60
(2H, m, 2-Ph-oH); 13C NMR (CDCl3): dC 31.63 (10-NCH3), 36.86
(5-NCH2CH2Ph), 50.62 (5-NCH2CH2Ph), 97.71 (C4a), 115.46 (C7),
00
95.82 (C4a), 114.94 (C7), 117.11 (C9), 123.37 (C8), 125.37 (C4 ),
0
00
0
00
0
00
0
128.42 (C3 , C3 , C5 and C5 ), 128.54 (C6), 128.64 (C2 , C2 , C5 and
00
0
0
00
C5 ), 131.17 (C4 ), 131.78 (C1 ), 135.20 (C1 ), 138.65 (C5a), 139.32
(C9a), 156.07 (C5), 158.57 (C10a), 166.43 (C2), 174.87 (C@O). Anal.
Calcd for C26H24N4O2ꢁH2O: C, 73.56; H, 5.70; N, 13.20. Found: C,
73.78; H, 5.73; N, 12.96.
00
0
0
116.26 (C9), 121.94 (C8), 126.99 (C4 ), 127.97 (C3 and C5 ), 128.18
00
00
00
00
0
(C6), 128.83 (C3 and C5 ), 129.02 (C2 and C6 ), 129.06 (C2 and
0
0
0
00
C6 ), 130.86 (C4 ), 134.09 (C1 ), 137.72 (C1 ), 138.19 (C5a), 141.86
(C9a), 155.72 (C5), 158.78 (C10a), 166.41 (C2), 175.61 (C@O). Anal.
Calcd for C26H22N4Oꢁ0.33H2O: C, 75.71; H, 5.54; N, 13.58. Found:
C, 75.69; H, 5.89; N, 13.35.
4.2.9. 10-Ethyl-5-phenethylamino-2-phenylpyrimido[4,5-
b]quinolin-4(10H)-one (4i)
Yield, (0.80 g, 38%); mp 258–260 °C; UV (EtOH): kmax/nm
(log
e
/dm3 molꢀ1 cmꢀ1): 233 (4.02), 254 (4.13), 273 (4.21), 350
(3.97), 381 (3.71); IR (
m
max/cmꢀ1): 1650 (C@O), 3150 (NH); 1H
NMR (CDCl3): dY 1.51 (3H, t, J = 7.2 Hz, 10-NCH2CH3), 3.23 (2H, t,
J = 7.2 Hz, 5-NCH2CH2Ph), 4.23 (2H, m, 5-NCH2CH2Ph), 4.91 (2H,
br s, 10-NCH2CH3), 7.27–7.39 (6H, m, 5-Ph-H), 7.43–7.56 (3 H, m,
2-Ph-m, pH), 7.65 (1H, d, J = 9.0 Hz, 9-H), 7.77 (1H, m, 8-H), 8.28
(1H, d, J = 8.4 Hz, 6-H), 8.58–8.63 (2H, m, 2-Ph-oH); 13C NMR
(CDCl3): dC 12.76 (10-NCH2CH3), 36.24 (5-NCH2CH2Ph), 39.42 (10-
NCH2CH3), 49.90 (5-NCH2CH2Ph), 97.08 (C4a), 115.29 (C7), 116.65
4.2.6. 5-(n-Butylamino)-10-ethyl-2-phenylpyrimido[4,5-b]-
quinolin-4(10H)-one (4f)
Yield, (1.04 g, 56%); mp 186–188 °C; UV (EtOH): kmax/nm
(log
e
/dm3 molꢀ1 cmꢀ1): 229 (4.47), 253 (4.58), 271 (4.38), 346
(4.15), 382 (4.18); IR (
m
max/cmꢀ1): 1650 (C@O), 3150 (NH); 1H
NMR (CDCl3): dY 1.01 [3H, t, J = 7.5 Hz, 5-N(CH2)3CH3], 1.52 [3H, t,
J = 7.2 Hz, 10-NCH2CH3], 1.56–1.64 (2H, m, 5-NCH2CH2CH2CH3),
1.83–1.96 (2H, m, 5-NCH2CH2CH2CH3), 3.95–4.02 (2H, m,
5-NCH2CH2CH2CH3), 4.85 (2H, br s, 10-NCH2CH3), 7.30–7.36 (1H,
m, 7-H), 7.44–7.48 (3H, m, 2-Ph-m, pH), 7.65 (1H, d, J = 8.7 Hz,
9-H), 7.57–7.81 (1H, m, 8-H), 8.31 (1H, d, J = 8.4 Hz, 6-H),
8.58–8.61 (3H, m, 2-Ph-oH); 13C NMR (CDCl3): dC 12.73 [5-
N(CH2)3CH3 ], 13.72 (10-NCH2CH3), 20.07 (5-NCH2CH2CH2CH3),
32.44 (5-NCH2CH2CH2CH3), 39.54 (5-NCH2CH2CH2CH3), 48.51
(10-NCH2CH3), 97.66 (C4a), 115.79 (C7), 116.02 (C9), 121.61 (C8),
00
00
00
0
(C9), 122.34 (C8), 126.66 (C4 ), 128.12 (C3 and C5 ), 128.51 (C3 ,
0
00
00
0
0
0
C5 and C6), 129.07 (C2 and C6 ), 129.90 (C2 and C6 ), 130.89 (C4 ),
0
00
134.69 (C1 ), 138.06 (C1 ), 138.21 (C5a), 140.48 (C9a), 154.84 (C5),
158.61 (C10a), 165.27 (C2), 174.39 (C@O). Anal. Calcd for
C27H24N4Oꢁ0.50H2O: C, 75.50; H, 5.87; N, 13.04. Found: C, 75.76;
H, 6.06; N, 12.94.
4.2.10. 5-(n-Butylamino)-7,10-dimethyl-2-phenylpyrimido[4,5-
b]quinolin-4(10H)-one (4j)
0
0
0
0
0
127.97 (C3 and C5 ), 128.56 (C6), 129.05 (C2 and C6 ), 130.77 (C4 ),
0
133.99 (C1 ), 138.47 (C5a), 140.97 (C9a), 155.20 (C5a), 158.82
Yield, (1.15 g, 60%); mp 220–222 °C; UV (EtOH): kmax/nm
e
/dm3 molꢀ1 cmꢀ1): 236 (4.26), 255 (4.42), 271 (4.49), 347
(C10a), 166.60 (C2), 175.68 (C@O). Anal. Calcd for C23H24N4O: C,
74.17; H, 6.49; N, 15.04. Found: C, 74.26; H, 6.44; N, 14.91.
(log
(4.19), 368 (3.96), 390 (3.90); IR (
m
max/cmꢀ1): 1634 (C@O), 3185
(NH); 1H NMR (CDCl3): dY 1.01 [3H, t, J = 7.5 Hz, 5-N(CH2)3CH3],
1.56–1.65 (2H, sex, J = 7.5 Hz, 5-NCH2CH2CH2 CH3), 1.91 (2H, quin,
J = 7.5 Hz, 5-NCH2CH2CH2CH3), 2.46 (3H, s, 7-CH3), 3.95 (2H, m, 5-
NCH2CH2CH2CH3), 4.09 (3 H, s, 10-NCH3), 7.41–7.48 (3H, m, 2-Ph-
m, pH), 7.49 (1H, d, J = 8.7 Hz, 9-H), 7.56 (1H, d, J = 8.7 Hz, 8-H),
8.01 (1H, s, 6-H), 8.53–8.57 (2H, m, 2-Ph-oH); 13C NMR (CDCl3):
4.2.7. 10-Ethyl-5-(n-octylamino)-2-phenylpyrimido[4,5-
b]quinolin-4(10H)-one (4g)
Yield, (1.05 g, 49%); mp 163–165 °C; UV (EtOH): kmax/nm
(log
e
/dm3 molꢀ1 cmꢀ1): 229 (4.15), 254 (4.21), 270 (4.27), 346
(4.04), 384 (3.79); IR (
m
max/cmꢀ1): 1680 (C@O), 3120 (NH); 1H

9168
A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 9161–9170
dC 13.76 [5-N(CH2)3CH3], 20.08 (5-NCH2CH2CH2CH3), 21.16 (7-
NCH3), 31.56 (5-NCH2CH2CH2CH3), 32.38 (10-NCH3), 48.39 (5-
NCH2CH2CH2CH3), 97.61 (C4a), 115.48 (C8), 116.12 (C9), 127.72
4.2.14. 5-(n-Butylamino)-10-ethyl-7-methyl-2-phenylpyri-
mido[4,5-b]quinolin-4(10H)-one (4n)
Yield, (0.90 g, 49%); mp 231–233 °C; UV (EtOH): kmax/nm
(C6), 127.93 (C3 and C5 ), 128.96 (C2 and C6 ), 130.71 (C4 ), 131.55
(log
e
/dm3 molꢀ1 cmꢀ1): 259 (4.50), 271 (4.55), 308 (4.09), 346
0
0
0
0
0
(C7), 135.46 (C1 ), 138.36 (C5a), 139.96 (C9a), 155.46 (C5), 158.56
(4.31), 372 (4.06), 390 (4.04); IR (m
max/cmꢀ1): 1637 (C@O), 3150
0
(C10a), 166.15 (C2), 175.65 (C@O). Anal. Calcd for C23H24N4Oꢁ
(NH); 1H NMR (CDCl3): dY 1.03 [3H, t, J = 7.2 Hz, 5-N(CH2)3CH3],
1.45 (3H, t, J = 7.2 Hz, 10-NCH2CH3), 1.58 (2H, m, 5-NCH2CH2
CH2CH3), 1.85 (2H, m, 5-NCH2CH2CH2CH3), 2.48 (3H, s, 7-CH3),
4.04 (2H, m, 5-NCH2CH2CH2CH3), 4.81 (2H, br s, 10-NCH2CH3),
7.42–7.45 (3H, 2-Ph-m, pH), 7.65 (1H, d, J = 8.1 Hz, 9-H), 7.78
(1H, d, J = 9.0 Hz, 8-H), 8.16 (1H, s, 6-H), 8.40–8.42 (2H, m,
2-Ph-oH); 13C NMR (CDCl3): dC 12.86 [5-N(CH2)3CH3 ], 13.86
(10-NCH2CH3), 19.82 (5-NCH2CH2CH2CH3), 20.85 (7-CH3), 32.22
(5-NCH2CH2CH2CH3), 39.42 (5-NCH2CH2CH2CH3), 47.92 (10-
0.66H2O:C,71.85;H,6.64;N,14.57.Found:C,71.88;H,6.68;N,14.45.
4.2.11. 7,10-Dimethyl-5-(n-octylamino)-2-phenylpyrimido[4,5-
b]quinolin-4(10H)-one (4k)
Yield, (1.32 g, 62%); mp 192–194 °C; UV (EtOH): kmax/nm
(log
e
/dm3 molꢀ 1 cmꢀ1): 232 (4.32), 257 (4.46), 271 (4.51), 346
(4.21), 371 (3.97), 392 (3.90); IR (
m
max/cmꢀ1): 1630 (C@O), 3170
(NH); 1H NMR (CDCl3): dY 0.88 [3H, t, J = 6.9 Hz, 5-NCH2(CH2)6CH3],
1.25–1.28 (10H, m, NCH2CH2(CH2)5CH3), 1.40–1.46 [2H, m, 5-NCH2
CH2(CH2)5CH3], 2.47 (3H, s, 7-CH3), 2.69 [2H, t, J = 6.9 Hz, 5-NCH2
CH2(CH2)5CH3], 4.18 (3H, s, 10-NCH3), 7.48–7.51 (3H, m, 2-Ph-m,
pH), 7.56 (1H, d, J = 9.0 Hz, 9-H), 7.64 (1H, d, J = 9.0 Hz, 8-H), 8.3
(1H, s, 6-H), 8.54–8.57 (2H, m, 2-Ph-oH); 13C NMR (CDCl3): dC
14.11 [5-NCH2(CH2)6CH3], 21.16 (7-CH3), 22.66 [5-NCH2(CH2)5
CH2CH3], 26.95 [5-N(CH2)2CH2(CH2)4CH3], 29.31 [5-N(CH2)4
CH2(CH2)2CH3], 30.44 [5-N(CH2)3CH2(CH2)3CH3], 30.94 [5-N(CH2)5
CH2CH2CH3], 31.54 (10-NCH3), 31.81 [5-NCH2CH2(CH2)5CH3], 48.69
[5-NCH2(CH2)6CH3], 97.55 (C4a), 115.47 (C8), 116.10 (C9), 127.70
0
0
NCH2CH3), 97.16 (C4a), 115.36 (C8), 116.51 (C9), 128.04 (C3 , C5
0
0
0
0
and C6), 128.57 (C2 and C6 ), 130.71 (C4 ), 131.54 (C1 ), 136.01
(C7), 138.58 (C5a), 138.67 (C9a), 154.65 (C5a), 158.39 (C10a), 165.41
(C2), 174.84 (C@O). Anal. Calcd for C24H26N4Oꢁ0.33H2O: C, 73.44;
H, 6.85; N, 14.27. Found: C, 73.55; H, 7.06; N, 13.95.
4.2.15. 10-Ethyl-7-methyl-5-(n-octylamino)-2-phenylpyri-
mido[4,5-b]quinolin-4(10H)-one (4o)
Yield, (0.98 g, 47%); mp 179–181 °C; UV (EtOH): kmax/nm (log
dm3 molꢀ1 cmꢀ1): 238 (3.82), 258 (4.02), 270 (4.05), 283 (3.98), 345
(3.72), 370 (3.52), 390 (3.41); IR (
max/cmꢀ1): 1634 (C@O), 3150
e/
0
0
0
0
0
(C6), 127.91 (C3 and C5 ), 128.96 (C2 and C6 ), 130.68 (C4 ), 131.55
m
0
(C7), 135.44 (C1 ), 138.37 (C5a), 139.92 (C9a), 155.42 (C5), 158.45
(NH); 1H NMR (CDCl3): dY 0.87 [3H, t, J = 6.6 Hz, 5-NCH2(CH2)6CH3],
1.22–1.38 (10 H, m, NCH2CH2(CH2)5CH3), 1.49 (3H, t, J = 7.2 Hz, 10-
NCH2CH3), 1.50–1.57 [2H, m, 5-N(CH2)6CH2CH3], 1.86–1.96 [2H, m,
5-NCH2CH2(CH2)5CH3], 2.48 (3H, s, 7-CH3), 3.95 [2H, m, 5-
NCH2CH2(CH2)5CH3], 4.87 (2H, br s, 10-NCH2CH3), 7.44–7.47 (3H,
m, 2-Ph-m, pH), 7.51 (1H, d, J = 7.8 Hz, 9-H), 7.59 (1H, d,
J = 7.8 Hz, 8-H), 8.06 (1H, s, 6-H), 8.58–8.61 (2H, m, 2-Ph-oH); 13C
NMR (CDCl3): dC 12.89 [5-NCH2(CH2)6CH3], 14.26 (10-NCH2CH3),
(C10a), 166.10 (C2), 175.63 (C@O). Anal. Calcd for C27H32N4Oꢁ
0.33H2O: C, 74.62; H, 7.58; N, 12.89. Found: C, 74.50; H, 7.58; N,
12.89.
4.2.12. 7,10-Dimethyl-5-phenethylamino-2-phenylpyrimido-
[4,5-b]quinolin-4(10H)-one (4l)
Yield, (1.35 g, 65%); mp 138–140 °C; UV (EtOH): kmax/nm
(log
e
/dm3 molꢀ1 cmꢀ1): 233 (4.03), 260 (4.15), 270 (4.17), 345
21.14
(7-CH3),
22.64
[5-N(CH2)6CH2CH3],
26.93
[5-
(3.74), 394 (3.43); IR (
m
max/cmꢀ1): 1634 (C@O), 3150 (NH); 1H
N(CH2)2CH2(CH2)4CH3], 29.15 [5-N(CH2)4CH2(CH2)2CH3], 29.29
[5-N(CH2)3CH2(CH2)3CH3], 30.50 [5-N(CH2)5CH2CH2CH3], 31.80
[5-NCH2CH2(CH2)5CH3], 39.44 [5-NCH2CH2(CH2)5CH3], 48.75 [10-
NMR (CDCl3): dY 2.43 (3H, s, 7-CH3), 3.22 (2H, t, J = 6.9 Hz,
5-NCH2CH2Ph), 4.08 (3H, s, 10-NCH3), 4.20 (2H, m, 5-NCH2CH2Ph),
7.32–7.41 (5H, m, 5-Ph-H), 7.43–7.46 (3H, m, 2-Ph-m, pH), 7.51
(1H, d, J = 9.0 Hz, 8-H), 7.57 (1H, d, J = 9.0 Hz, 9-H), 7.98 (1H, s,
6-H), 8.55–8.58 (2H, m, 2-Ph-oH); 13C NMR (CDCl3): dC 21.15
(7-CH3), 31.55 (10-NCH3), 36.88 (5-NCH2CH2Ph), 50.41
(5-NCH2CH2Ph), 97.66 (C4a), 115.37 (C8), 116.16 (C9), 126.97
0
NCH2CH3], 97.66 (C4a), 115.75 (C8), 115.92 (C9), 127.93 (C3 and
0
0
0
0
0
C5 ), 128.40 (C6), 129.02 (C2 and C6 ), 130.68 (C4 ), 135.44 (C1 ),
136.69 (C7), 138.54 (C5), 138.98 (C9a), 154.90 (C5), 158.60 (C10a),
166.41 (C2), 175.71 (C@O). Anal. Calcd for C28H34N4Oꢁ0.33H2O: C,
74.97; H, 7.79; N, 12.49. Found: C, 75.09; H, 7.56; N, 12.43.
00
00
00
0
0
(C4 ), 127.44 (C6), 127.92 (C3 and C5 ), 128.82 (C3 , C5 and C6),
00
00
0
0
0
128.97 (C2 and C6 ), 129.11 (C2 and C6 ), 130.75 (C4 ), 131.71
4.2.16. 5-Benzylamino-10-ethyl-7-methyl-2-phenylpyri-
mido[4,5-b]quinolin-4(10H)-one (4p)
0
00
(C7), 135.57 (C1 ), 137.81 (C1 ), 138.22 (C5a), 139.84 (C9a), 155.31
(C5), 158.57 (C10a), 166.10 (C2), 175.58 (C@O). Anal. Calcd for
C27H24N4Oꢁ0.66H2O: C, 74.98; H, 5.90; N, 12.95. Found: C, 75.05;
H, 6.13; N, 12.63.
Yield, (0.85 g, 42%); mp 243–245 °C; UV (EtOH): kmax/nm
(log
e
/dm3 molꢀ1 cmꢀ1): 256 (3.96), 272 (3.99), 309 (3.48), 347
(3.71), 375 (3.45), 390 (3.42); IR (
m
max/cmꢀ1): 1638 (C@O), 3180
(NH); 1H NMR (CDCl3): dY 1.37 (3H, t, J = 6.9 Hz, 10-NCH2CH3),
2.40 (3H, s, 7-CH3), 4.82 (2H, br s, 10-NCH2CH3), 5.27 (2H, d,
J = 4.8 Hz, 5-NCH2Ph), 7.20–7.46 (5H, m, 5-Ph-H), 7.47–7.52 (3H,
m, 2-Ph-m, pH), 7.73 (1H, d, J = 8.7 Hz, 9-H), 7.85 (1H, d, J = 8.7 Hz,
8-H), 8.24 (1H, s, 6-H), 8.38–8.42 (2H, m, 2-Ph-oH); 13C NMR (CDCl3):
dC 12.79 (10-NCH2CH3), 20.54 (7-CH3), 39.39 (10-NCH2CH3), 51.38
4.2.13. 5-(iso-Butylamino)-7,10-dimethyl-2-phenylpyrimido-
[4,5-b]quinolin-4(10H)-one (4m)
Yield, (1.15 g, 62%); mp 236–238 °C; UV (EtOH): kmax/nm
(log
e
/dm3 molꢀ1 cmꢀ1): 258 (4.10), 273 (4.17), 344 (3.78), 370
(3.51), 392 (3.41); IR (
m
max/cmꢀ1): 1635 (C@O), 3120 (NH); 1H
NMR (CDCl3): dY 1.16 [6H, d, J = 6.6 Hz, 5-NCH2CH(CH3)2], 2.15-
2.24 [1H, sept, J = 6.6 Hz, 5-NCH2CH(CH3)2], 2.48 (3H, s, 7-CH3),
3.79 [2H, t, J = 6.6 Hz, 5-NCH2CH(CH3)2], 4.12 (3H, s, 10-NCH3),
7.44–7.46 (3H, m, 2-Ph-m, pH), 7.51–7.61 (2 H, m, 8 and 9-H),
8.02 (1 H, s, 6-H), 8.57–8.61 (2 H, m, 2-Ph-oH); 13C NMR (CDCl3):
dC 20.24 [5-NCH2CH(CH3)2], 21.18 (7-CH3), 29.69 [5-
NCH2CH(CH3)2], 31.53 (10-NCH3), 56.37 [5-NCH2CH(CH3)2], 97.55
00
(5-NCH2Ph), 97.30 (C4a), 115.03 (C7), 116.59 (C9), 126.67 (C4 ),
00
00
0
0
00
127.77 (C3 and C5 ), 127.97 (C6), 128.14 (C3 and C5 ), 128.41 (C2
00
0
0
0
0
and C5 ), 128.93 (C2 and C5 ), 130.82 (C4 ), 131.63 (C1 ), 136.11 (C7),
00
137.42 (C1 ), 138.45 (C5a), 139.09 (C9a), 154.60 (C5), 158.47 (C10a),
165.34 (C2), 174.71 (C@O). Anal. Calcd for C27H24N4O: C, 77.12; H,
5.75; N, 13.32. Found: C, 77.23; H, 5.97; N, 13.32.
0
0
(C4a), 115.45 (C8), 116.11 (C9), 127.73 (C6), 127.89 (C3 and C5 ),
0
0
0
0
4.2.17. 10-Ethyl-7-methyl-5-phenethylamino-2-phenylpyrimi-
do[4,5-b]quinolin-4(10H)-one (4q)
128.92 (C2 and C6 ), 130.67 (C4 ), 131.54 (C7), 135.48 (C1 ), 138.27
(C5a), 139.92 (C9a), 155.36 (C5), 158.67 (C10a), 166.02 (C2), 175.59
(C@O). Anal. Calcd for C23H24N4Oꢁ0.14H2O: C, 73.66; H, 6.53; N,
14.94. Found: C, 73.63; H, 6.28; N, 15.13.
Yield, (0.90 g, 44%); mp 215–217 °C; UV (EtOH): kmax/nm
(loge
/dm3 molꢀ1cmꢀ1): 233 (3.59), 272 (3.78), 282 (3.78), 351

A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 9161–9170
9169
(3.36), 373 (3.12), 390 (2.95); IR (
m
max/cmꢀ1): 1640 (C@O), 3150
4.3.2. 10-Methyl-2-phenyl-5-(p-toluidino)pyrimido[4,5-b]-
quinolin-4(10H)-one (5 b)
Yield, (1.20 g, 59%); mp 234-236 °C; UV (EtOH): kmax/nm (log
(NH); 1H NMR (CDCl3): dY 1.38 (3 H, t, J = 6.9 Hz, 10-NCH2CH3),
2.45 (3 H, s, 7-CH3), 3.10 (2H, t, J = 6.6 Hz, 5-NCH2CH2Ph), 4.31
(2H, m, 5-NCH2CH2Ph), 4.79 (2H, br s, 10-NCH2CH3), 7.20–7.42
(5H, m, 5-Ph-H), 7.47–7.49 (3H, m, 2-Ph-m, pH), 7.70 (1H, d,
J = 9.0 Hz, 9-H), 7.80 (1H, d, J = 9.0 Hz, 8-H), 8.19 (1H, s, 6-H),
8.39–8.43 (2H, m, 2-Ph-oH); 13C NMR (CDCl3): dC 12.90 (10-
NCH2CH3), 36.21 (5-NCH2CH2Ph), 39.42 (10-NCH2CH3), 49.69 (5-
e
/
/
dm3 molꢀ1 cmꢀ1): 231 (3.88), 283 (4.09), 329 (3.07); IR (mmax
cmꢀ1): 1649 (C@O), 3150 (NH); 1H NMR (CDCl3): dY 3.08 (3 H, s,
5-Ph-CH3), 3.83 (3H, s, 10-NCH3), 6.92 (2H, d, J = 7.5 Hz, 5-Ph-
mH), 6.91–6.98 (1H, m, 7-H), 7.10–7.18 (1H, m, 8-H), 7.14 (2H, d,
J = 7.5 Hz, 5-Ph-oH), 7.45–7.52 (4H, m, 9-H, 2-Ph-m, pH), 8.16–
8.19 (3H, m, 6-H and 2-Ph-oH); 13C NMR (CDCl3): dC 24.59 [5-Ar-
CH3], 30.67 (10-NCH3), 94.11 (C4a), 114.12 (C7), 121.83 (C9),
00
NCH2CH2Ph), 97.08 (C4a), 114.00 (C8), 116.79 (C9), 126.82 (C4 ),
00
00
0
0
00
128.52 (C3 and C5 ), 128.67 (C3 , C5 and C6), 128.86 (C2 and
00
0
0
0
0
00
00
0
0
0
C6 ), 129.29 (C2 and C6 ), 131.08 (C4 ), 131.84 (C1 ), 136.28 (C7),
122.69 (C8), 127.59 (C3 and C5 ), 127.84 (C3 and C5 ), 128.95 (C2
00
0
00
00
0
138.46 (C1 and C5a), 139.64 (C9a), 154.60 (C5), 158.52 (C10a),
and C6 ), 129.10 (C2 and C6 ), 131.82 (C4 ), 132.33 (C5a), 133.02
00
00
165.33 (C2), 174.81 (C@O). Anal. Calcd for C28H26N4Oꢁ0.5H2O: C,
(C4 ), 140.52 (C9a), 150.85 (C1 ), 153.16 (C5), 155.61 (C10a), 162.59
(C2), 175.58 (C@O). Anal. Calcd for C25H20N4Oꢁ0.66H2O: C, 74.24;
H, 5.32; N, 13.85. Found: C, 74.16; H, 5.60; N, 14.07.
75.82; H, 6.14; N, 12.63. Found: C, 75.88; H, 6.38; N, 12.87.
4.2.18. 10-Ethyl-7-methoxy-5-(n-octylamino)-2-phenylpyri-
mido[4,5-b]quinolin-4(10H)-one (4r)
4.3.3. 5-(2-Methoxycarbonylanilino)-10-methyl-2-phenyl-
Yield, (1.35 g, 65%); mp 144–146 °C; UV (EtOH): kmax/nm (log
dm3 molꢀ1cmꢀ1): 257 (4.44), 277 (4.48), 282 (4.47), 342 (4.12), 387
(3.94), 410 (3.84); IR (
max/cmꢀ1): 1635 (C@O), 3140 (NH); 1H NMR
e
/
pyrimido[4,5-b]quinolin-4(10H)-one (5c)
Yield, (1.02 g, 45%); mp >300 °C; UV (EtOH): kmax/nm (log
dm3 molꢀ1cmꢀ1): 229 (4.23), 299 (4.02), 337 (3.80), 434 (3.66),
452 (3.72); IR (
max/cmꢀ1): 1692, 1645 (C@O), 3180 (NH); 1H
e/
m
(CDCl3): dY 0.88 [3H, t, J = 7.2 Hz, 5-NCH2(CH2)6CH3], 1.22–1.32
(8H, m, 5-NCH2CH2CH2(CH2)4CH3), 1.50 (3H, t, J = 7.2 Hz,
10-NCH2CH3), 1.52–1.56 [2H, m, 5-NCH2CH2CH2(CH2)4CH3],
1.88–1.97 [2H, m, 5-NCH2CH2(CH2)5CH3], 3.91 (3H, s, 7-OCH3),
3.91–4.03 [2H, m, 5-NCH2CH2(CH2)5CH3], 4.91 (2H, br s, 10-
NCH2CH3), 7.38–7.56 (4H, m, 7-H, 2-Ph-m, pH), 7.60–7.75 (2H, m,
8 and 9-H), 8.52–8.61 (3H, m, 6-H, 2-Ph-oH); 13C NMR (CDCl3):
dC 12.89 [5-NCH2(CH2)6,CH3], 14.10 (10-NCH2CH3), 22.27 [5-
N(CH2)6CH2CH3], 26.42 [5-NCH2CH2CH2(CH2)4CH3], 28.83 [5-
N(CH2)3CH2CH2(CH2)2CH3], 30.13 [5-N(CH2)5CH2CH2CH3], 31.39
[5-NCH2CH2(CH2)5CH3], 39.71 [5-NCH2CH2(CH2)5CH3], 48.76 [10-
NCH2CH3], 54.85 (7-OCH3), 96.97 (C4a), 115.96 (C8), 118.24 (C9),
m
NMR (CDCl3): dY 3.74 (3H, s, 10-NCH3), 4.50 (3H, s, 5-COOCH3),
6.91–6.97 (3H, m, 40, 50 and 60-H of 5-Ph), 7.19–7.22 (1H, m, 30-H
of 5-Ph), 7.49–7.59 (4H, m, 7-H, 2-Ph-m, pH), 7.70 (1H, d,
J = 8.4 Hz, 9-H), 8.08 (1H, m, 8-H), 8.18 (1H, d, J = 9.0 Hz, 6-H),
8.49–8.51 (2H, m, 2-Ph-oH); 13C NMR (CDCl3): dC 30.56 (10-
NCH3), 51.80 (–COOCH3), 102.25 (C4a), 108.30 (C9), 115.42 (C7
00
0
00
00
0
and C6 ), 122.73 (C2 and C6), 124.88 (C4 ), 125.34 (C2 and C6 ),
0
0
0
00
00
128.56 (C3 , C5 and C8), 129.08 (C4 ), 130.81 (C3 ), 132.07 (C5 ),
0
00
136.10 (C1 ), 137.84 (C5a), 140.34 (C9a), 151.62 (C1 ), 157.18 (C5),
158.24 (C10a), 162.60 (C2), 167.89 (C@O). Anal. Calcd for
C26H20N4O3ꢁH2O: C, 68.71; H, 4.88; N, 12.33. Found: C, 68.63; H,
4.92; N, 12.29.
0
0
0
0
0
128.11 (C3 and C5 ), 128.42 (C2 and C6 ), 128.52 (C6), 130.73 (C4 ),
0
131.23 (C7), 134.46 (C1 ), 135.06 (C5a), 138.56 (C9a), 153.87 (C5),
158.05 (C10a), 166.41 (C2), 174.79 (C@O). Anal. Calcd for
C28H34N4O2ꢁ0.5H2O: C, 71.92; H, 7.54; N, 11.98. Found: C, 72.14;
H, 7.61; N, 11.68.
4.4. Growth inhibitory activities of test compounds against
human tumor cell lines
The procedure was carried out using the modified 3-(3,4-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
colorimetric assay31 to determine the inhibitory effects of test
compounds, namely, 5-(monosubstituted amino)-2-deoxo-2-phe-
nyl-5-deazaflavins (4 and 5), on cell growth in vitro as mentioned
in detail in our previous paper.2 Two human tumor cell lines CCRF-
HSB-2 (human T-cell acute lymphoblastoid leukemia) and KB (hu-
man oral epidermoid carcinoma) were used in this study. AraC was
used as a positive control, where the IC50 was determined from the
dose-response curve.
4.3. General procedure for the preparation of 5-anilino-2-
deoxo-2-phenyl-5-deazaflavins (5a–c)
A mixture of 10-methyl-2-deoxo-2-phenyl-5-deazaflavins (3a–
e) (1.5 g) and N-aryl-amines (15 equiv.) in n-butanol was refluxed
in sealed glass tube at 135 °C for 10–15 h. After cooling, the solu-
tion was diluted with diethyl ether to get the precipitate, which
was filtered off, washed with diethyl ether, dried, and recrystal-
lized from ethanol to afford the corresponding products as red
needles.
4.5. Experimental protocol of docking study
4.3.1. 10-Methyl-2-phenyl-5-(3,4-xylidino)pyrimido[4,5-b]-
quinolin-4(10H)-one (5a)
The automated docking studies were carried out using Auto-
Dock version 3.0.5. First, AutoGrid component of the program
pre-calculates a three-dimensional grid of interaction energies
based on the macromolecular target using the AMBER force field.
Yield, (0.98 g, 47%); mp 207–209 °C; UV (EtOH): kmax/nm
(log
max/cmꢀ1): 1646 (C@O), 3150 (NH); 1H NMR (CDCl3): dY 2.16
[6H, d, J = 8.4 Hz, 5-Ar(CH3)2], 3.61 (3H, s,10-NCH3), 6.46 (1H, d,
e
/dm3 molꢀ1 cmꢀ1): 231 (3.96), 282 (4.14), 337 (3.20); IR
(m
0
0
The cubic grid box of 60 ÅA size (x, y, z) with a spacing of 0.375 ÅA
and grid maps were created representing the catalytic active target
site region where the native ligand was embedded. Then auto-
mated docking studies were carried out to evaluate the binding
free energy of the inhibitors within the macromolecules. The GA-
LS search algorithm (genetic algorithm with local search) was cho-
sen to search for the best conformers. The parameters were set
using the software ADT (Auto-Dock Tool Kit) on PC which is asso-
ciated with Auto-Dock 3.0.5. For all docking parameters, default
values were used with 10 independent docking runs for each dock-
ing case. The AutoDock performs the task of the docking, where the
00
00
00
J = 7.2 Hz, C6 ), 6.52 (1H, s, C2 ), 6.91 (1H, d, J = 7.8 Hz, C5 ), 6.95–
7.03 (2H, m, 7 and 9-H), 7.16–7.25 (2H, m, 6 and 8-H), 7.54 (3H,
m, 2-Ar-m, p-H), 8.19–8.22 (2H, m, 2-Ar-oH); 13C NMR (CDCl3):
dC 24.51 [5-Ar-(CH3)2], 30.57 (10-NCH3), 94.02 (C4a), 114.02 (C7),
00
0
0
121.75 (C9), 122.57 (C8), 127.48 (C2 and C6), 127.75 (C3 and C5 ),
00
0
0
00
0
128.54 (C5 ), 128.83 (C2 and C6 ), 129.00 (C6 ), 131.71 (C4 ),
00
00
00
132.13 (C5a), 132.83 (C4 ), 140.46 (C9a and C3 ), 142.12 (C1 ),
154.50 (C5), 155.51 (C10a), 162.47 (C2), 169.79 (C@O). Anal. Calcd
for C26H22N4Oꢁ0.33H2O: C, 75.98; H, 5.52; N, 13.63. Found: C,
75.68; H, 5.50; N, 13.97.

9170
A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 9161–9170
ligand moves randomly in any one of six degrees of freedom, and
the energy of the new ligand ‘state’ is calculated. If the energy of
the new state is lower than that of the old state, the new one is
automatically accepted as the next step in docking.
Acknowledgments
The authors are indebted to the SC NMR Laboratory of Okayama
University for the NMR spectral measurements.
4.5.1. Preparation of ligands and target tyrosine kinase
References and notes
The compounds involved in this study as ligands including 4a–l,
n, o and 5a–c were studied for their binding activities into protein
tyrosine kinase. The three-dimensional structures of the aforemen-
tioned compounds were constructed using Chem 3D ultra 8.0 soft-
ware [Chemical Structure Drawing Standard; Cambridge Soft
corporation, USA (2003)], then they were energetically minimized
by using MOPAC with 100 iterations and minimum RMS gradient
of 0.10. The Gasteiger-Hückel charges of ligands were assigned.
Thecrystal structures of proteintyrosine kinase(1t46) complex with
(STI) were retrieved from the RCSB Protein Data Bank (http://
www.rcsb.org/pdb/home/home.do) (PDB code: 1t46). All bound
waters and ligands were eliminated from the protein and the polar
hydrogens and the Kollman-united charges were added to the pro-
teins. The amino acids of the ligand-target binding site were defined
using data in pdbsum (http://www.ebi.ac.uk/pdbsum/).
1. Part 4 Ali, H. I.; Ashida, N.; Nagamatsu, T. Biorg. Med. Chem. 2008, 16, 922.
2. Ali, H. I.; Tomita, K.; Akaho, E.; Kambara, H.; Miura, S.; Hayakawa, H.; Ashida,
N.; Kawashima, Y.; Yamagishi, T.; Ikeya, H.; Yoneda, F.; Nagamatsu, T. Biorg.
Med. Chem. 2007, 15, 242.
3. Ali, H. I.; Ashida, N.; Nagamatsu, T. Bioorg. Med. Chem. 2007, 15, 6336.
4. Sakuma, Y.; Nagamatsu, T.; Yoneda, F. J. Chem. Soc. Chem. Commun. 1975,
977.
5. Sakuma, Y.; Matsumoto, S.; Nagamatsu, T.; Yoneda, F. Chem. Pharm. Bull. 1976,
24, 338.
6. Nagamatsu, T.; Yamato, H.; Ono, M.; Takarada, S.; Yoneda, F. J. Chem. Soc. Perkin
Trans. 1 1992, 2101.
7. Nagamatsu, T.; Kuroda, K.; Mimura, N.; Yanada, R.; Yoneda, F. J. Chem. Soc.
Perkin Trans. 1 1994, 1125.
8. Nagamatsu, T.; Yamada, H.; Shiromoto, K. Heterocycles 2004, 63, 9.
9. O0Brien, D. E.; Weinstock, L. T.; Cheng, C. C. J. Heterocycl. Chem. 1970, 7, 99.
10. Hemmerich, P.; Massey, V.; Fenner, H. FEBS Lett. 1977, 84, 5.
11. Eirich, L. D.; Vogels, G. D.; Wolfe, R. S. Biochemistry 1978, 17, 4583.
12. Graham, D. W.; Brown, J. E.; Ashton, W. T.; Brown, R. D.; Rogers, E. F. Experientia
1977, 33, 1274.
13. Yoneda, F.; Sakuma, Y.; Hemmerich, P. J. Chem. Soc., Chem. Commun. 1977, 825.
14. Kimachi, T.; Yoneda, F.; Sasaki, T. J. Heterocycl. Chem. 1992, 29, 763.
15. Wilson, J. M.; Henderson, G.; Black, F.; Sutherland, A.; Ludwig, R. L.; Vousden, K.
H.; Robins, D. J. Bioorg. Med. Chem. 2007, 15, 77.
16. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
17. Venkatachalam, C. M.; Jiang, X.; Oldfield, T.; Waldman, M. J. Mol. Graph. Model.
2003, 21, 289.
18. Abagyan, R.; Totrov, M.; Kuznetsov, D. J. Comput. Chem. 1994, 15, 488.
19. Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. J. Mol. Biol. 1996, 261, 470.
20. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. J. Mol. Biol. 1997, 267,
727.
21. Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W.
T.; Banks, J. L. J. Med. Chem. 2004, 47, 1750.
22. Ewing, T. J. A.; Makino, S.; Skillman, A. G.; Kuntz, I. D. J. Comput. Aided Mol. Des.
2001, 15, 411.
23. Paul, M. K.; Mukhopadhyay, A. K. Int. J. Med. Sci. 2004, 1, 101.
24. Wang, R.; Lu, Y.; Wang, S. J. Med. Chem. 2003, 46, 2287.
25. Vaque, M.; Arola, A.; Aliagas, C.; Pujadas, G. Bioinformatics 2006, 22, 1803.
26. Yoneda, F.; Mori, K.; Ono, M.; Kadokawa, Y.; Nagao, E.; Yamaguchi, H. Chem.
Pharm. Bull. 1980, 28, 3514.
27. Coppola, G. M.; Schuster, H. F. J. Heterocycl. Chem 1989, 26, 957.
28. Roberts, R. M.; Vogt, P. J. Org. Synth. 1958, 38, 29.
29. Yoneda, F.; Sakuma, Y.; Mizumoto, S.; Ito, R. J. Chem. Soc. Perkin Trans. 1 1976,
1805.
4.5.2. Molecular modeling and analysis of the docked results
The predicted binding free energy that includes the intermolec-
ular energy and torsional free energy was used as the criterion for
ranking. Furthermore, the intermolecular hydrogen bonds, whose
effect has already been counted in the binding energy, were also
investigated in order to find useful information for drug design. A
comparison of the results suggests that the binding free energy is
more reliable as a criterion for the virtual screening via molecular
docking. Cluster analysis was performed on the docked results
0
using a root mean square (RMS) tolerance of 0.5 ÅA. Each of the clus-
ters that exhibited significant negative interaction energies was
examined by Accelrys, DS visualizer 2.0 [Accelrys Inc., San Diego
CA (2007)] to determine their binding orientations, molecular
modeling, evaluation of the hydrogen bonds. Being the native li-
gand (STI) and the docked inhibitors have different chemical struc-
tures, the RMSD values were measured as distance between the
centroids of the docked inhibitor and the native ligand. The mode
of interaction of the native ligand (STI) within the crystal structure
of PTK was used as a standard docked model as well as for RMSD
calculation. Regarding the hydrogen bond interaction, the more
linear hydrogen bond is likely to be stronger. Therefore, in our
modeling results we consider the hydrogen bond angle more than
100° to be of a reasonable strength.
30. Mol, C. D.; Dougan, D. R.; Schneider, T. R.; Skene, R. J.; Kraus, M. L.; Scheibe,
D. N.; Snell, G. P.; Zou, H.; Sang, B. C.; Wilson, K. P. J. Biol. Chem. 2004, 279,
31655.
31. Miura, S.; Yoshimura, Y.; Endo, M.; Machida, H.; Matsuda, A.; Tanaka, M.;
Sasaki, T. Cancer Lett. 1998, 129, 103.