Antitumor studies. Part 4: Design, synthesis, antitumor activity, and molecular docking study of novel 2-substituted 2-deoxoflavin-5-oxides, 2-deoxoalloxazine-5-oxides, and their 5-deaza analogs

Article abstract of DOI:10.1016/j.bmc.2007.10.014

Various novel 10-alkyl-2-deoxo-2-methylthioflavin-5-oxides and their 2-alkylamino derivatives were prepared by facile nitrosative cyclization of 6-(N-alkylanilino)-2-methylthiopyrimidin-4(3H)-ones followed by nucleophilic replacement of the 2-methylthio moiety by different amines, and acidic hydrolysis of the 2-methylthio moiety afforded the corresponding flavin derivatives. 2-Deoxo-2-methylthio-5-deazaalloxazines and 2-deoxo-2-methylthioalloxazine-5-oxides were also prepared by Vilsmeier reaction and by nitrosation of 6-anilino-2-methylthiopyrimidin-4(3H)-ones, respectively. Then, they were subjected to nucleophilic replacement with appropriate amines to produce the corresponding 2-alkylamino derivatives. Regiospecific N₃-alkylation of 2-deoxo-2-methylthioalloxazine-5-oxides was carried out with various alkylating agents in the usual way. The antitumor activities against CCRF-HSB-2 and KB tumor cells have been investigated in vitro, and many compounds showed promising antitumor activities. Furthermore, AutoDock molecular docking into PTK (PDB: 1t46) has been done for lead optimization of the aforementioned compounds as potential PTK inhibitors.

Full text of DOI:10.1016/j.bmc.2007.10.014

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 922–940
Antitumor studies. Part 4: Design, synthesis, antitumor activity,
and molecular docking study of novel 2-substituted 2-deoxoflavin-
5-oxides, 2-deoxoalloxazine-5-oxides, and their 5-deaza analogs
Hamed I. Ali,a Noriyuki Ashidab and Tomohisa Nagamatsua,*
aDepartment of Drug Discovery and Development, Division of Pharmaceutical Sciences, Graduate School of Medicine,
Dentistry and Pharmaceutical Sciences, Okayama University 1-1-1, Tsushima-Naka, Okayama 700-8530, Japan
bBiology Laboratory, Research and Development Division, Yamasa Shoyu Co., Choshi, Chiba 288-0056, Japan
Received 12 September 2007; revised 1 October 2007; accepted 4 October 2007
Available online 11 October 2007
Abstract—Various novel 10-alkyl-2-deoxo-2-methylthioflavin-5-oxides and their 2-alkylamino derivatives were prepared by facile
nitrosative cyclization of 6-(N-alkylanilino)-2-methylthiopyrimidin-4(3H)-ones followed by nucleophilic replacement of the 2-meth-
ylthio moiety by different amines, and acidic hydrolysis of the 2-methylthio moiety afforded the corresponding flavin derivatives.
2-Deoxo-2-methylthio-5-deazaalloxazines and 2-deoxo-2-methylthioalloxazine-5-oxides were also prepared by Vilsmeier reaction
and by nitrosation of 6-anilino-2-methylthiopyrimidin-4(3H)-ones, respectively. Then, they were subjected to nucleophilic replace-
ment with appropriate amines to produce the corresponding 2-alkylamino derivatives. Regiospecific N3-alkylation of 2-deoxo-2-
methylthioalloxazine-5-oxides was carried out with various alkylating agents in the usual way. The antitumor activities against
CCRF-HSB-2 and KB tumor cells have been investigated in vitro, and many compounds showed promising antitumor activities.
Furthermore, AutoDock molecular docking into PTK (PDB: 1t46) has been done for lead optimization of the aforementioned com-
pounds as potential PTK inhibitors.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
O
N
O
N
Cancer remains as a major threat to the public health. In
our previous publications,1–6 a series of synthesized 2-
phenylflavin-5-oxide and 2-phenyl-5-deazaflavin analogs
(Scheme 1) has been reported for their in vitro signifi-
cant antitumor activities against different tumor cell
lines. Compounds comprising flavin-N-oxides have been
recently used for treatments of solid tumors, non-solid
tumor masses, leukemia, and non-small cell lung cancers
involving in situ activator mixed with the flavin-N-oxide
for a period of time, resulting in damage to the DNA in
the cancer cells without substantial damage to the DNA
of normal cells.7 Considering their biological activities,
6-nitro-5-deazaflavin derivatives showed the most
potent antitumor activities among the nitro-5-deazafla-
vin derivatives.8 The 6- and 8-nitro-5-deazaflavin
derivatives generating stable one-electron reduction
X
N
N
HN
N
R
O
N
H
: 2-Deoxo-2-phenyl-5-deazaflavin
: 2-Deoxo-2-phenylflavin-5-oxide
X =CH
X =
Flavin
(Isoalloxazine)
N O
O
N
O
O
N
HN
MeS
HN
N
N
O
N
H
N
Alloxazine
2-Deoxo-2-methylthioalloxazine-5-oxide
Scheme 1. Structures of different flavin and flavin-5-oxide analogs.
product(s) showed marked selective cytotoxicities to-
ward hypoxic cells.9 Furthermore, it has been found that
the combination of conjugate molecules of 8-amino-5-
deazaflavin with the sialosylalkyl group has been found
Keywords: Antitumor activity; Flavin-5-oxide; Alloxazine-5-oxide;
AutoDock.
*
Corresponding author. Tel.: +81 86 251 7931; fax: +81 86 251
7926; e-mail: nagamatsu@pheasant.pharm.okayama-u.ac.jp
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.014

H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
923
to give rise to significant increase in their antitumor
activities.10 Nitro-5-deazaflavin-pyrrolecarboxamide(s)
hybrid molecules were evaluated in vitro as novel
DNA targeted bioreductive antitumor agents on KB
cells.11
were investigated for their in vitro antitumor activities.
In fact, many of the synthesized compounds showed
promising antitumor activities against CCRF-HSB-2
and KB tumor cells. The molecular docking study using
AutoDock was carried out to get the designed com-
pounds possessing higher binding affinities into PTK.
As the result, the correlation between the growth inhib-
itory activities (IC50, lg/mL) of the synthesized flavin
analogs against tumor cells and the AutoDock binding
free energies was investigated.
5-Amino-5-deazaflavins also revealed antiproliferative
activity against L1210 and KB tumor cells.12 The devel-
opment of a potent riboflavin antagonist is one of the
major interests for the fundamental studies for investi-
gation of possible chemotherapeutic activity. Those ana-
logs of riboflavin inhibit flavoenzymes or influence
riboflavin metabolism in a variety of biological systems
providing a reason to prepare a number of isoalloxa-
zines for evaluation of their therapeutic activity against
cancer.13 Therefore, 8-chloroalloxazine was one of an
early series of alloxazines and isoalloxazines prepared
for the investigation of the possible antitumor activity
of riboflavin inhibitors.14 And isoalloxazines with a b-
hydroxyethyl group at the 9 position as well as some
of their esters are also designed as riboflavin antago-
nists, sometimes as antitumor agents.15 Moreover,
6,7-dimethyl-9-formylmethyl-isoalloxazine and its thio-
semicarbazone analogs were found to be reversible ribo-
flavin antagonists and they showed antitumor activity
against the Murphy–Sturm lymphosarcoma in rats.13
The 10-aryl-5-deazaflavin derivatives were preliminarily
reported to act as inhibitors of E3 activity of HMD2 in
tumors that retain wild-type P53.16 Some heterocyclic
2. Results and discussion
2.1. Chemistry
The requisite starting material, 6-(N-alkylanilino)-2-
methylthiopyrimidin-4(3H)-ones (1a–j), was synthesized
by treatment of 6-chloro-2-methylthiopyrimidin-4(3H)-
one19,20 with appropriate N-alkyl anilines in n-butanol
under reflux for 12–72 h in 55–83% yields.18 The in-
tended
10-alkyl-2-deoxo-2-methylthioflavin-5-oxides
(2a–i) were prepared by nitrosative cyclization of 6-(N-
alkylanilino)-2-methylthiopyrimidin-4(3H)-ones (1a–j)
with excess NaNO2 in glacial acetic acid at 10–15 ꢁC
for 1–2 h to afford the red compounds in 65–89% yields
(Scheme 2). The preparation of such alkylamino deriva-
tives by reaction of alkylthio derivatives with appropri-
ate amines has long been utilized in heterocyclic
chemistry. Many of these reactions require rather stren-
uous conditions and are usually carried out in a stainless
steel vessel except when amines of high boiling point are
used.21 Nucleophilic replacement of the methylthio moi-
ety of 10-methyl-2-deoxo-2-methylthioflavin-5-oxides
(2a) by dimethylamine in steel sealed tube at 160–
165 ꢁC (15 kg/cm2 pressure) for 4 h exhibited the
replacement accompanied with the N10-demethylation
and N5-deoxygenation to give 2-deoxo-2-dimethylami-
noalloxazine (3), where the singlet signal of the N10-
compounds containing
a quinoline ring such as
5-deazaflavins are of importance owing to their miscella-
neous biological activities with higher activity toward
tumor cells.17
Despite the aforementioned potential antitumor activi-
ties of various flavin analogs and the numerous publica-
tions on chemistry of the related flavin compounds,
information of the 2-methylthio- and 2-alkylamino-fla-
vin analogs is none yet except our previous report.18
These circumstances led us to seek a convenient syn-
thetic route to the 2-methylthio- and 2-alkylamino-fla-
vin analogs to search their potential antitumor
activities and to develop potent antitumor agents show-
ing a higher selectivity toward tumor cells.
1
methyl at d 4.1 and 33.5 of H NMR and 13C NMR,
respectively, was lost. Therefore, to avoid the N10-
demethylation, this reaction should be carried out at
lower temperature for short time. Interestingly, facile ra-
pid nucleophilic substitution of 10-alkyl-2-deoxo-2-
methylthioflavin-5-oxides (2) by different amines was
carried out by heating the mixture in n-butanol under re-
flux to afford the yellow crystalline solid of 10-alkyl-2-
alkylamino-2-deoxoflavin-5-oxides (4a–e) in 62–89%
yields. The pyrimidine moieties of 2a–i undergo nucleo-
philic substitution on the carbon adjacent to the ring
nitrogen which is well authenticated and readily explica-
ble in view of the p-electron-deficient nature.15 There-
fore, various aminations were applied for the
replacement of 2-methylthio group. The nucleophilic
substitution of 10-alkyl-2-deoxo-2-methylthioflavin-5-
oxide analogs (2) involves short time reaction (15 min)
in comparison with the substitution reaction in case of
2-deoxo-2-methylthio-5-deazaflavin analogs, which
needs reaction time of 2–5 h.18 In contrast, acidic hydro-
lysis of 2 by heating in 5 N hydrochloric acid under re-
flux for 7–12 h gave the corresponding 10-alkylflavins
(5a and b) or 7-methoxy-10-methylflavin-5-oxide (6) as
yellow needles in 68–97% yields as shown in Scheme 2.
Thus, in the present paper, we describe a facile synthesis
of 10-alkyl-2-deoxo-2-methylthioflavin-5-oxides, which
were used as versatile intermediates for the synthesis
of various 2-alkylamino-2-deoxoflavin-5-oxides by
nucleophilic substitution involving different primary
and secondary alkylamines. Furthermore, they were de-
rived to 10-alkylflavins and 10-alkylflavin-5-oxide as a
novel synthetic route. 2-Deoxo-2-methylthio-5-deazaal-
loxazines and 2-methylthioalloxazine-5-oxides were also
prepared from 6-anilino-2-methylthiopyrimidin-4(3H)-
one. And the N3-alkyl analogs of 2-deoxo-2-methylthio-
alloxazin-5-oxides were prepared by alkylation using
various alkylating agents. Moreover, 2-deoxo-2-methyl-
thio-5-deazaalloxazines and 2-deoxo-2-methylthio-
alloxazine-5-oxides were subjected to nucleophilic
replacement with some amine derivatives to prepare 2-
alkylamino-2-deoxo-5-deazaalloxazines and 2-alkylami-
no-2-deoxoalloxazines, respectively. These compounds

924
H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
O
N
O
N
N
N
HN
MeS
HN
R2
Me
N
N
R1
Me
a
c
b
d
1a—i
3
O
N
O
N
O
N
O
N
O
N
R3
N
N
N
HN
R2
R2
N
MeS
N
O
N
R
R1
R1
2a—i
4a—e
5a,b
d
a: R1 = Me, R2 = piperidino, R3 = OMe
b: R1 = Et, R2 = (Me)2CHCH2NH, R3 = H
c: R1 = Me, R2 = cyclohexylamino, R3 = Me
d: R1 = Me, R2 = PhCH2NH, R3 = Cl
a: R1 = Me, R2 = H
b: R1 = Et, R2 = H
a: R = Me
b
: R = Et
c: R1 = Me, R2 = 7-Me
d: R1 = Me, R2 = 9-Me
e: R1 = Me, R2 = 7-OMe
f: R1 = Me, R2 = 8-OMe
g: R1 = Me, R2 = 7-Cl
h: R1 = Et, R2 = 7-Me
i: R1 = Me, R2 = 7,8-(Me)2
O
N
O
N
e: R1 = Me, R2 = HOCH2CH2NH, R3 = Me
OMe
HN
O
N
Me
6
Scheme 2. General methods for the preparation of 2-methylthio- and 2-alkylamino-10-alkyl-2-deoxoflavin-5-oxides (2a–i and 4a–e), 2-deoxo-2-
dimethylaminoalloxazine (3), flavins (5a and b), and 7-methoxy-10-methylflavin-5-oxide (6). Reagents and conditions: (a) NaNO2, AcOH, 10–15 ꢁC,
1–2 h; (b) 50% aq NH(Me)2, steel sealed tube, 160–165 ꢁC, 4 h; (c) appropriate amines, n-BuOH, reflux 15 min; (d) 5 N HCl, reflux, 7–12 h.
O
N
R
N
Me
N
H
N
b
c
O
N
O
N
8a,b
a
a: R = Me
b: R = n-C8H17
HN
MeS
HN
R
N
Me
N
N
Me
H
O
N
O
N
1a
7a,b
N
R
a: R = Me
b: R = n-C8H17
N
Me
N
H
9a,b
a: R = Me
b: R = n-C8H17
Scheme 3. General methods for the preparation of 2-alkylamino-2-deoxo-10-methyl-5-deazaflavins (8a and b), and 2-alkylamino-2-deoxo-10-
methylflavin-5-oxides (9a and b). Reagents and conditions: (a) 40% aq NH2Me, steel sealed tube, 160 ꢁC, 15 h for 7a; n-octylamine, 160 ꢁC, 10 h for
7b; (b) Vilsmeier reagent (DMF–POCl3), 90 ꢁC, 3 h; (c) NaNO2, AcOH, 10–15 ꢁC, 2–3 h.
Another route for synthesis of 10-alkyl-2-alkylamino-2-
deoxo-5-deazaflavins (8a and b) was established by the
nucleophilic substitution of 2-methylthio group of 6-
(N-methylanilino)-2-methylthiopyrimidin-4(3H)-one (1a)
by an appropriate amine before the cyclization of 1a
as shown in Scheme 3. This nucleophilic substitution

H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
925
was done either by fusion of 1a with n-octylamine of
high boiling point or by heating in steel sealed tube at
150–160 ꢁC with volatile methylamine. Then, the com-
pounds 7a, b were used as precursors for the preparation
of 2-alkylamino-2-deoxo-5-deazaflavins (8a and b) by
heating with Vilsmeier reagent at 90 ꢁC for 3 h, and
for the preparation of 2-alkylamino-2-deoxoflavin-5-
oxides (9a and b) by nitrosative cyclization involving ex-
cess NaNO2 in glacial acetic acid for 2–3 h.
SAR that the higher binding affinities were obtained
with the structure features on the flavin or 5-deazaflavin
skeleton; NH2 or Ph group at the C2-position, H (or
alloxazine conformation) or Ph group at the N10-posi-
tion. Herein, we tried to synthesize the designed analogs
based on AutoDock possessing higher binding affinities,
namely, 2-deoxo-2-methylthio-5-deazaalloxazines (12)
and
2-deoxo-2-methylthioalloxazine-5-oxides
(13).
These derivatives can be synthesized from the key inter-
mediate, 6-anilino-2-methylthiopyrimidin-4(3H)-ones
(11), by facile methods of interactions (Scheme 4). The
requisite key compounds11a–e cannot be prepared
directly from 6-chloro-2-methylthiopyrimidin-4(3H)-ones
by adapting the same procedure for the preparation of
Based on our previous structure based drug design
(SBDD) study and docking investigation of different fla-
vin analogs, and aiming to discovery of antitumor
agents,22 it was concluded from the results considering
O
O
O
a
X
HN
HN
HN
R
R
S
N
H
S
N
H
NH2
N
H
S
N
H
N
10a–e
a: R = Ph
b: R = 4-Me-C6H4
c: R = 2,4-(Me)2-C6H3
d: R = 4-MeO-C6H4
e: R = 4-HO-C6H4
X = CH or N O
b
O
O
O
N
O
N
c
d
HN
MeS
HN
HN
MeS
R
R
R
MeS
N
N
N
N
N
H
12a,b
11a–e
13a–e
a: R = H
b: R = 7,9-(Me)2
a: R = Ph
a: R = H
b: R = 4-Me-C6H4
c: R = 2,4-(Me)2-C6H3
d: R = 4-MeO-C6H4
e: R = 4-HO-C6H4
b: R = 7,9-(Me)2
c: R = 7-OMe
d: R = 7-Me
e: R = 7-OH
e
g
f
O
O
O
N
O
N
R1
N
HN
HN
N
R2
R2
R2
R1
R1
N
N
N
N
N
MeS
15a,b
16a–e
14a,b
a: R1 = morpholino, R2 = 7-OMe
b: R1 = (Me)2N, R2 = H
a: R1 = Me, R2 = H
a: R1 = morpholino, R2 = H
b: R1 = HOCH2CH2(Me)N,
R2 = 7,9-(Me)2
b: R1 = Me, R2 = 7,9-(Me)2
c: R1 = Et, R2 = 7,9-(Me)2
d: R1 = EtOOCCH2, R2 = H
e: R1 = Me, R2 = 7-Me
Scheme 4. General methods for the preparation of 2-deoxo-5-deazaalloxazines (12a, b and 14a, b), 2-deoxo-alloxazine-5-oxides (13a–e and 16a–f),
and 2-deoxoalloxazines (15a and b). Reagents and conditions: (a) ArNH2=ArNH3þClꢀ, 170 ꢁC, 12 h; (b) MeI, 2 N KOH, 0–5 ꢁC, 30 min; (c)
Vilsmeier reagent (DMF–POCl3), 90 ꢁC, 1 h; (d) NaNO2, AcOH, 10–15 ꢁC, 1–2 h; (e) appropriate amines, DMF or n–BuOH, reflux 12–24 h; (f)
morpholine, DMF, reflux, 6 h for 15a; 50% aq NH(Me)2, steel sealed tube, 135 ꢁC, 8 h for 15b; (g) R1I or R1Br, DMF, K2CO3, rt, 3–10 h.

926
H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
compounds 12 and 13 by alkylamines was carried out
50000
40000
30000
20000
10000
0
by heating the reaction mixture with appropriate amines
in DMF or n-butanol under reflux for high boiling point
amines (morpholine or N-hydroxyethyl-N-methylamine)
for 6–24 h or in sealing vessel for volatile one (dimethyl-
amine) for 8 h to afford the corresponding yellow
crystalline solid of the 2-alkylamino analogs (14a, b
and 15a, b) in 67–86% yields. This replacement was
accompanied by deoxygenation of 2-deoxo-2-methyl-
thioalloxazin-5-oxides (13) to produce the 2-alkylamino-2-
deoxoalloxazine analogs (15a and b). Interestingly, the
regiospecific N3-alkylation of 13a–e was carried out
using excess alkyl iodide or bromide in the presence of
anhydrous potassium carbonate in DMF at room tem-
perature to afford the 3-alkyl-2-deoxo-2-methylthioal-
loxazine-5-oxides (16a–e) in 71–82% yields. This
alkylation is 100% regioselective at the N3-position,
giving highly pure one-spot product without purification
by column chromatography as previously described
in methylation of 2-deoxo-2-phenyl-5-deazaflavins.6
The discrimination between the positional isomers,
3-methyl-2-deoxo-2-methylthioalloxazine-5-oxide (16a)
and 10-methyl-2-deoxo-2-methylthioflavin-5-oxide (2a),
was accurately proved by obvious different melting
points and IR spectra where the 4-oxo group of 3-
methyl analog(16a) was shown at higher frequency of
1700 cmꢀ1, whereas that of 10-methyl analog (2a) was
2b
4b
5b
13a
ε
200 250 300 350 400 450 500 550
λ
/nm
Figure 1. UV–vis spectra of 2-deoxo-10-ethyl-2-methylthioflavin-5-
oxide (2b), 2-deoxo-10-ethyl-2-isobutylaminoflavin-5-oxide (4b), 10-
ethylflavin (5b), and 2-deoxo-2-methylthioalloxazine-5-oxide (13a).
6-(N-alkylanilino)-2-methylthiopyrimidin-4(3H)-ones
(1a–j).18 Both of the 6-chloro and 2-methylthio groups
were replaced by the anilines. Therefore, the compounds
11a–e were prepared in a two-step reaction. The first
reaction by amine exchange route, namely, the reaction
of 6-amino-2-thiouracil with anilines in the presence of
their corresponding anilinium chloride salts at high tem-
perature to afford the corresponding 6-anilino-4-oxo-2-
thioxo-1,2,3,4-tetrahydropyrimidins (10a–e) in 67–83%
yields. Compounds 10a–e were prepared by modifica-
tion of known procedure23,24 to improve the yields and
to get more pure products. Hence, compound 10a was
obtained in higher yield of 83% (literatures23,24: 78%
and 68% yields). In our proposed procedure, the anilin-
ium chloride derivatives were used in more quantities of
1.5–2.0 equivalents and the crude product was further
purified by dissolving it in alkaline solution, then repre-
cipitation by 10% HCl to get more pure compounds.
The second-step reaction involves S-methylation by
MeI in alkaline solution in ice bath to afford the 2-meth-
ylthio analogs (11a–e) in excellent yields of 77–98%.
Synthesis of 2-thio-5-deazaalloxazines and 2-thioallox-
azine-5-oxides was not successful directly from the com-
pounds 10a–e. This may be attributed to oxidative
dimerization of the 2-thioxo derivatives. Therefore, the
2-thioxo moiety should be protected by methylation be-
fore the cyclization step to get the desired compounds 12
and 13. The 2-deoxo-2-methylthio-5-deazaalloxazines
(12a and b) were prepared from 11 by Vilsmeier reagent
at 90 ꢁC for 1 h as yellow needles in 72 and 63% yields,
respectively. 2-Deoxo-2-methylthioalloxazine-5-oxides
(13a–e) were also prepared from 11 by nitrosative cycli-
zation using excess NaNO2 in glacial acetic acid at 10–
15 ꢁC and then at room temperature for 1–2 h. After
adding NaNO2 to the cold reaction mixture, the formed
greenish yellow nitroso intermediates have to be dis-
solved in acetic acid by mild warming in water bath to
enhance the prompt cyclization to afford the red prod-
ucts in 66–89% yields (Scheme 4). Moreover, the nucle-
ophilic replacement of 2-methylthio group for these
1
shown at lower frequency of 1645 cmꢀ1. In H NMR
spectra, the N3-methyl of 16a as a singlet signal was
shown at 3.64 ppm, while the N10-methyl of 2a was
shown at lower field of 4.13 ppm.
1
UV–vis, IR, H NMR spectra, and elemental analyses
were used for determination and identification of the
newly assigned structures. The 2-deoxo-2-methylthiofla-
vin-5-oxides (2a–i) were differentiated from the non-cyc-
lized compounds (1a–i) by the absence of a singlet signal
1
proton at the 5-position of 1a–i in their H NMR spec-
tra and loss of one ortho-aromatic proton by the oxida-
tive cyclization. The compounds 2 in UV–vis spectra
revealed the characteristic bathochromic shift in the re-
gions of the longest two wavelengths compared with all
other flavin analogs, as shown in Figure 1 and experi-
mental part. Considering 1H NMR, the spectra of 10-al-
kyl-2-deoxo-2-alkylaminoflavin-5-oxides (4a–e) are
characterized by disappearance of the strong character-
istic singlet signal of 2-methylthio group, which was as-
signed for compounds 2a–i at 2.57–2.60 ppm.
Interestingly, the phenomenon of reversible interconver-
sion of two isomers at room temperature in case of the
2-monoalkylamino (secondary amino) derivatives (4b–
1
e, 8a, b, and 9a, b) was observed as tautomerism in H
NMR spectra as reported the similar phenomenon in
the previous paper.18 The twin overlapped spectra of
approximately 1:2 or 2:1 ratio of the 2-monoalkylamino
and 2-monoalkylimino tautomers in (CD3)2SO were ob-
tained for the two resonance species (tautomers). Espe-
cially, such duplicated spectra were observed in the
range of 4.0–9.0 ppm based on the tautomers of the gua-
nidine adjacent to the carbonyl group. At higher tem-
perature of 85–100 ꢁC, the coalescence of the
duplicated spectrum was observed to produce the single
spectrum. This phenomenon is mainly attributed to the

H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
927
Table 1. Growth inhibitory activities against CCRF-HSB-2 and KB
cells for flavin-5-oxides (2, 4, 6, and 9a), flavins (5), 5-deazaflavins (8),
5-deazaalloxazine (12a), alloxazine-5-oxides (13 and 16), and alloxa-
zines (15)
presence of a secondary amine at the 2-position, whereas
it does not take place in case of 2-substituted primary
and tertiary amines. On the other hand, 2-deoxo-2-
methylthio-5-deazaalloxazines (12a and b) showed a
characteristic singlet signal of the most electron-deficient
C5-methine proton in the lower field at 8.98 and
9.16 ppm, respectively. 2-Deoxo-2-methylthioallox-
azine-5-oxides (13a–e) can be easily differentiated from
the 2-deoxo-2-methylthioflavin-5-oxide analogs (2a–i)
by the presence of singlet signals of 3-NH proton at
12.69–12.79 ppm, whereas such signals of 2a–i were lost.
In contrast, the N10-alkyl signals of 2a–i at 4.07–4.19
and 4.77 ppm for methyl and methylene moieties were
observed, respectively, whereas such signals of 13a–e
did not exist. Both of them have a singlet signal identi-
fied to their 2-SMe moiety at 2.57–2.60 ppm. The 2-
alkylamino-2-deoxo-5-deazaalloxazines (14a and b)
and 2-alkylamino-2-deoxoalloxazines (15a and b) are
characterized by disappearance of the strong character-
istic singlet signals of the 2-methylthio groups of the cor-
responding precursors (12 and 13) at 2.44–2.83 ppm.
Compound
Inhibitorty activity against tumor
cell lines [IC50 (lg/mL)]
CCRF-HSB-2
KB
1.44
8.19
27.7
0.61
11.0
2a
2b
1.56
3.72
17.1
1.46
9.84
10.0
6.66
34.7
14.3
6.18
6.0
2c
2d
2e
2f
2g
12.5
10.8
46.3
30.0
27.6
8.1
2h
2i
4a
4b
4c
7.1
33.7
1.7
4e
5a
1.46
1.8
2.1
1.89
5b
6
1.57
5.1
6.2
14.5
7.9
8a
2.7
8.9
The UV–vis absorption spectra of the 2-deoxo-2-meth-
ylthioflavin analogs located at shorter wavelength (nm)
in comparison with the 2-deoxo-2-phenylflavin analogs6
which have more conjugated system. As shown in
Figure 1, the 2-deoxo-2-methylthioflavin-5-oxides (2a–
i) exhibited four absorption maxima, three in the UV re-
gion at 219–221, 265–289, and 347–370 nm, and one in
the visible region at 457–495 nm with bathochromic
shift (red shift) in the regions of the longest two wave-
lengths in comparison with the 2-deoxo-2-methylthio-
5-deazaflavin analogs.18 All compounds of 2a–i showed
red color owing to the presence of absorption maximum
at 457–495 nm in the longest wavelength. The acid
hydrolyzed products, 10-alkylflavins (5a and b), exhib-
ited hypsochromic shift mainly in the longest two wave-
lengths in comparison with the UV–vis spectra of 2a–i as
can be seen in Figure 1 and experimental part. The 2-
deoxoflavin-5-oxides (4b–e) substituted by the primary
amines at the 2-position exhibited four absorption max-
ima at 217–223, 266–274, 330–350, and 436–453 nm,
and an additional shoulder at 472–487 nm as shown in
Figure 1 for compound 4b. While, 2-deoxo-2-methyl-
thioalloxazine-5-oxides (13a–e) exhibited five absorption
maxima at 241–247, 270–275, 285–298, 351–361, and
443–457 nm.
8ba
9a
1.74
23.2
6.77
6.29
4.11
5.86
41.4
51.2
8.63
8.96
5.9
6.93
44.1
6.76
11.8
5.35
12a
13a
13b
13c
13d
15a
15b
16a
16b
16c
16d
16e
Ara-C
5.8
71.7
63.1
9.31
14.2
10.0
54.8
13.4
34.2
7.74
0.047
0.23
a Ref. 18.
2-deoxo-2-methylthioflavin-5-oxides (2), 2-deoxo-2-
alkylaminoflavin-5-oxides (4), flavin-5-oxides (6), and
2-deoxo-2-alkylamino-5-deazaflavins (8) have been
found to show significant antitumor activities, but
they were of inferior antitumor activities than that
of Ara-C against CCRF-HSB-2 cell line. The activities
for the compounds (2a, d, 4e, 5a, b, 8a, and 9a) were
the highest among their analogs (IC50: 1.56, 1.46, 1.46,
1.8, 1.57, 2.7, and 1.74 lg/mL, respectively). Further,
against KB cell line they exhibited good growth inhib-
itory activities of one-third to one-ninth against the
antitumor potency of Ara-C (IC50: 0.23 lg/mL), where
IC50 of compounds 2a, d, 4e, and 5a, b were of 1.44,
0.61, 1.7, 2.1, and 1.89 lg/mL, respectively. This may
reveal a fairly good and less toxic antitumor activity
of these flavin analogs in comparison with Ara-C.
Moreover, compounds 2b, g, 4b, c, and 6 exhibited
moderate potential growth inhibitory activities against
CCRF-HSB-2 cell line with IC50 of 3.72, 6.66, 6.0,
7.1, and 5.1 lg/mL, respectively. Also compounds 2b,
4b, and 6 exhibited prospective growth inhibitory
activities against KB cell line with IC50 being 8.19,
8.1, and 6.2 lg/mL, respectively.
2.2. In vitro antitumor activities of different 2–methyl-
thioflavin-5-oxides, alloxazin-5-oxides, 5-deazaalloxa-
zines, and their 2-alkylamino derivatives against human
tumor cell lines
The compounds (2, 4, 6, 8, 9, 12, 13, 15, and 16) syn-
thesized in this study were tested in vitro for their
growth inhibitory activities against two human cul-
tured tumor cell lines, namely, human T-cell acute
lymphoblastoid leukemia cell line (CCRF-HSB-2)
and human oral epidermoid carcinoma cell line
(KB), by using the modified MTT colorimetric as-
say.25 The antitumor agent, cytosine arabinoside
(Ara-C), was used as a positive control in this study.
As can be noticed in Table 1, some compounds of

928
H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
In comparison with the antitumor activities of the above
studied flavin-5-oxide, 2-alkylaminoflavin-5-oxide, and
5-deazaflavin derivatives (2, 4, 5, and 8), which revealed
the IC50 in the range of 1.46–2.7 and 0.61–2.1 lg/mL
against CCRF-HSB-2 and KB tumor cell lines, respec-
tively, and the activities of our previously reported potent
2-deoxo-2-phenylflavin analogs,6 which revealed the IC50
in the range of 0.15–0.68 lg/mL for CCRF-HSB-2 cells
and 0.16–0.72 lg/mL for KB cell. It can be explained that
the phenyl group at the C2-position provides better affin-
ity to the PTK enzyme on account of the force of electro-
static attraction between the planar phenyl and the target
site pocket of the PTK. Hence, the phenyl derivatives ex-
hibit a good fitting into the active site and better antitu-
mor activity. The results considering SAR revealed that
the highest antitumor activities (ca. 1.5 lg/mL) were ob-
tained with the structure features on the flavin-5-oxide,
5-deazaflavin, and alloxazine skeletons; SMe or HNMe
group at the C2-position, H (or alloxazine conformation)
or Me group at the N10-position, and unsubstituted qui-
noxaline nucleus or substituted by 7-Me, or 9-Me group.
The SAR revealing moderate antitumor activity (ca.
5.5 lg/mL) was obtained with the structure features:
HN-n-Bu or oxo at the C2-position, Me or Et group at
the N10-position, and unsubstituted quinoxaline nucleus
or substituted by 7-OMe group. Noteworthy, the N3-alkyl
substituted alloxazin-5-oxides exhibited lower antitumor
activities than their unsubstituted analogs as shown in
Table 1. And the N-5-oxide analogs revealed higher
potencies than 5-deazaflavins. These results confirm our
previously reported SAR study.22
intervention. The selective PTK inhibitors into the
receptor and non-receptor PTK represent a promising
class of antitumor agents. These agents are shown to in-
hibit multiple features of cancer cells, including prolifer-
ation, survival, invasion, and angiogenesis. Depending
on the above-mentioned idea, herein we investigated
the AutoDock binding affinities into PTK (PDB code,
1t46) for the synthesized flavin analogs (2–6, 8, 9, and
12–16). For the purpose of optimization of the afore-
mentioned lead compounds for the promising antitumor
activities, the advanced docking program AutoDock
3.0526 was used to evaluate the binding free energies of
these inhibitors into the target PTK macromolecule.
2.3.1. Validation of the docking performance and accu-
racy. The validation of the docking accuracy was done
by docking of the native co-crystallized STI-571 ligand
(Imatinib or Gleevec), 4-(4-methylpiperazin-1-ylmeth-
yl)-n-[4-methyl-3-(4-pyridin-3-ylpyrimidin-2-ylami-
no)phenyl]-benzamide, into its binding site of PTK. The
obtained success rates of AutoDock, as mentioned in de-
tail in our previous paper,18 was highly excellent in com-
parison with the biological method,27 where the docked
ligand was exactly superimposed on the native co-crys-
˚
tallized one with RMSD being 0.25 A and binding free
energies (DGb) of ꢀ18.43 kcal/mol. The hydrogen bonds
exhibited between the docked ligand and amino acids
were quite similar to those between the native ligand
and amino acids.
2.3.2. AutoDock binding affinities of the synthesized and
the designed compounds into PTK. The binding affinity
was evaluated by the binding free energies (DGb, kcal/
mol), inhibition constants (Ki), hydrogen bonds, and
RMSD values. The compounds, which revealed the
highest binding affinities (in other words, lowest binding
2.3. Molecular docking study
Due to their involvement in various forms of cancers,
PTKs have become prominent targets for therapeutic
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
c
RMSD (A)
Kib
Hydrogen bonds between atoms of compounds and amino acids
˚
˚
Compound
DGb (kcal/mol)
Atom of compound
Amino acid
Distance (A)
Angle (ꢁ)
3
4b
ꢀ13.73
ꢀ13.39
8.61Eꢀ11
1.54Eꢀ10
N3–H
C4-Oxo
N3–N
CO of Asp 810
HN of Asp 810
HN of Asp 810
HN of Cys 673
HN of Cys 673
CO of Asp 810
HM of Asp 810
OH of Glu 640
OH of Glu 640
HN of Asp 810
CO of Cys 673
OH of Thr 670
OH of The 670
CO of Asp 810
CO of Asp 810
HN of Cys 673
HN of Cys 673
OH of Thr 670
HN of Asp 810
2.06
2.03
1.52
1.82
1.82
2.39
2.19
1.94
2.25
2.23
2.21
1.94
2.44
2.05
2.19
2.06
1.66
1.86
2.01
136.7
133.3
136.0
124.8
142.0
144.8
132.7
146.3
132.5
131.0
137.0
131.0
173.1
137.7
145.2
132.7
161.0
147.2
133.3
3.55
1.66
4c
ꢀ14.47
1.35Eꢀ11
C4-Oxo
C5-Oxide
C2–NH
C2NCH2CH2O
C2–NCH2CH2OH
C2–NH
C2–NH
C2–NH
C2–NH
C2–NH
N3–H
7.32
4d
4e
ꢀ14.47
ꢀ13.76
2.47Eꢀ11
8.14Eꢀ11
4.63
4.33
8a
ꢀ13.01
2.93Eꢀ10
3.96
8b
9a
ꢀ13.41
ꢀ12.75
ꢀ12.97
ꢀ8.93
1.48Eꢀ10
4.53Eꢀ10
3.09Eꢀ10
2.87Eꢀ07
2.77Eꢀ07
1.39Eꢀ07
3.08Eꢀ14
9.07
6.40
6.11
3.92
3.98
5.48
9b
13c
13d
16b
STId
ꢀ8.95
N3–H
C4-Oxo
N3–N
ꢀ9.36
ꢀ18.43
NH (H79)
O29
0.25
a Binding free energy.
b Inhibition constant.
c Root mean square deviation.
d The native co-crystallized bound ligand (STI-571) of PTK (PDB code: 1t46).

H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
929
free energies) within PTK and the hydrogen bond
interactions into the target macromolecule, are represented
in Table 2. These compounds include 2-dimethyl-
amino-2-deoxoalloxazine (3), 10-alkyl-2-alkylamino-2-
deoxoflavin-5-oxides (4b–e), 2-alkylamino-10-methyl-5-
deazaflavins (8a and b), 2-alkylamino-2-deoxo-10-
methyl-flavin-5-oxides (9a and b), 2-methylthioallox-
azine-5-oxides (13c and d), and 3,7,9-trimethyl-2-
docking results of compounds 4e and 9a are highly cor-
related to their antitumor activities cited in Table 1.
Figure 4 illustrates the comparative docking modes of
8a and 13d into the c-Kit receptor PTK together with
the bound ligand STI. Compounds 8a (DGb:
ꢀ13.01 kcal/mol) and 13d (DGb: ꢀ8.95 kcal/mol) were
docked into same groove of the binding site along with
STI-ligand and exhibited two (with Glu 640 and Asp
810) and one (with Asp 810) hydrogen bonds,
respectively.
methylthioalloxazine-5-oxide
(16b).
Compounds
2-(2-hydroxyethylamino)-7,10-dimethyl-2-deoxoflavin-5-
oxide (4e) and 10-methyl-2-methylamino-2-deoxoflavin-
5-oxide (9a) as shown in Figures 2 and 3, respectively,
were deeply embedded into the catalytic and activation
loops of the ATP-binding cleft of the c-Kit receptor
tyrosine kinase domain with DGb: ꢀ13.76 for 4e and
ꢀ12.75 kcal/mol for 9a. Compound 4e showed two
hydrogen bonds with Asp 810 and Glu 640, whereas
9a showed one hydrogen bond with Thr 679. These
The docking for 2-deoxo-2-methylthioflavin-5-oxides
(2), flavins (5), and flavin-5-oxide (6) exhibited mostly
higher binding energies (lower binding affinities) being
ꢀ9.11 to ꢀ7.43 kcal/mol (DGb) due to less proper fitting
into the target site by hydrogen bond formation, in com-
parison with the binding energies of other derivatives (3,
O
O
N
Me
N
H
N
N
N
HOH2CH2C
I789
Me
4e
D810
STI
D810
V643
4e
E640
STI
L799
C673
C809
2.15Å
V668
Y672
V643
1.94Å
E640
V622
4e
Figure 2. Stereo view of compound 2-(2-hydroxyethylamino)-7,10-dimethyl-2-deoxoflavin-5-oxide (4e) (ball and stick, colored by element) deeply
embedded into the ATP-binding cleft of the c-Kit receptor PTK domain with the bound ligand STI-571. Hydrogen bonds are shown as green dashed
lines.
Figure 3. Docking of compound 2-methylamino-2-deoxo-10-methylflavin-5-oxide (9a) (ball and stick, colored by element) into the ATP-binding cleft
˚
of the c-Kit receptor PTK, exhibiting one hydrogen bond with amino acid Thr 670 (in green dashed lines) and RMSD being 6.40 A from the bound
ligand STI-571.

930
H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
Figure 4. AutoDock binding affinities of compound 2-deoxo-2-methylamino-5-deazaflavin (8a; colored by element, ball and stick) and 2-deoxo-7-
methyl-2-methylthioalloxazine-5-oxide (13d; red, stick) into PTK (1t46). They exhibit two and one hydrogen bonds, respectively, shown as green
dashed lines. The binding pocket of the PTK is shown in transparent solid surface with labeled amino acids and the STI ligand is shown as yellow
stick.
4, 8, and 9) (DGb: ꢀ14.83 to ꢀ12.75 kcal/mol). However,
-6
some compounds showed better antitumor activities in
spite of the lower binding affinities as cited in Table 1.
It appeared reasonable to conclude the reason for the
better antitumor activities as described below. That is,
the planar pyrimidoquinoxaline ring of compounds 2,
5, and 6 was involved in hydrophobic electrostatic sur-
face interaction, where it was sandwiched within dis-
2f
13b
2e
-8
-10
-12
-14
-16
16c
2g
16b
16e
9a
˚
4b
8b
tance of ca. 5.0 A between the phenyl moieties of Phe
4e
811 and Tyr 672 and the terminal hydrocarbon chain
of Leu 595. Also their 2-methylthio group interacts with
Leu 644, Val 654, Val 668, and Cys 809 by hydrophobic
y = 0.5364x–15.722
2
R = 0.680
˚
attraction within distance of ca. 3.62–5.16 A. This inter-
action keeps these derivatives (2, 5, and 6) in the binding
0
2
4
6
8
μ
10
12
14
16
IC ( g/mL)
50
˚
site, within RMSD values of 4.09–6.53 A.
Figure 6. Correlation between the binding free energy (DGb) and IC50
(lg/mL) of compounds 2e, f, g, 4b, e, 8b, 9a, 13b, and 16b, c, e against
KB tumor cells.
The overall correlation between the growth inhibitory
activities of the synthesized flavin analogs against tumor
cells and the AutoDock binding affinities was better for
some compounds. The growth inhibitory activities of
compounds 2c, h, 4a–c, e, 8a, b, 9a, 12a, and 16d against
CCRF-HSB-2 tumor cells were correlated to their Auto-
Dock binding free energies with a good correlation coef-
ficient (R2) of 0.759 as shown in Figure 5.
-6
12a
Whereas, the growth inhibition against KB tumor cells
revealed a reasonable correlation with AutoDock bind-
ing free nergies for compounds 2e, f, g, 4b, e, 8b, 9a, 13b,
and 16b, c, e of correlation coefficient (R2) of 0.68 as
shown in Figure 6.
2h
-8
-10
-12
-14
-16
2c
16d
9a
8a
4b
8b
3. Conclusions
y = 0.1952x–14.29
4c
4a
4e
2
R = 0.759
In this study, various novel 10-alkyl-2-deoxo-2-methyl-
thioflavin-5-oxides (2a–i) were synthesized from 6-(N-
alkylanilino)-2-methylthiopyrimidin-4(3H)-ones (1a–i)
by nitrosative cyclization. The 2-alkylamino derivatives
(4a–e) were synthesized by the facile replacement of
the C2-methylthio moiety of 2 by different amines. Fla-
vins (5a and b) and flavin-5-oxide (6) were prepared by
0
5
10
15
20
25
30
35
40
μg/mL)
(
IC
50
Figure 5. Correlation between the binding free energy (DGb) and IC50
(lg/mL) of compounds 2c, h, 4a–c, e, 8a, b, 9a, 12a, and 16d against
CCRF-HSB-2 tumor cells.

H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
931
acidic hydrolysis of 2 in excellent yields. Nucleophilic
substitution of 2-methylthio group of the non-cyclized
6-(N-methylanilino)-2-methylthiopyrimidin-4(3H)-one (1a)
by an appropriate amine was carried out, followed by
Vilsmeier reaction to afford the 2-alkylamino-2-deoxo-
5-deazaflavins (8a and b) or by nitrosative cyclization
to afford the 2-alkylaminoflavin-5-oxides (9a and b).
Compounds 2-deoxo-2-methylthio-5-deazaalloxazines
(12) and 2-deoxo-2-methylthioalloxazine-5-oxides (13)
were facilely synthesized from 6-anilino-2-methylthio-
pyrimidin-4(3H)-ones (11) by Vilsmeier reaction and
nitrosative cyclization, respectively. The nucleophilic
replacement of 2-methylthio group of compounds (12
and 13) was carried out by heating with alkylamines
to afford their 2-alkylamino analogs (14 and 15). Addi-
tionally, 3-alkyl-2-methylthioalloxazine-5-oxides (16a–
e) were prepared by the regiospecific N3-alkylation of
2-deoxo-2-methylthioalloxazine-5-oxides (13) using ex-
cess alkyl iodide or bromide.
were measured by Yanaco CHN Corder MT-5 appa-
ratus. IR spectra were recorded on a JASCO FT/IR-
200 spectro-photometer as Nujol mulls. 1H NMR
spectra were obtained using
a
Varian VXR
300 MHz spectrophotometer and chemical shift values
were expressed in d values (ppm) relative to tetra-
methylsilane (TMS) as internal standard. Coupling
constants are given in Hz. All NH and OH protons
were exchangeable with D2O. UV spectra were mea-
sured in absolute EtOH using Beckman DU-68S
UV spectrophotometer and absorption values fol-
lowed by sh refer to wavelengths 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 an appropriate drying agent and were
stored over suitable molecular sieves. Reaction pro-
gress was monitored by analytical thin layer chroma-
tography (TLC) on pre-coated glass plates (silica gel
60F254-plate-Merck) and the products were visualized
by UV light.
In vitro growth inhibitory activities of compounds (2,
4, 6, 8, 9, 12, 13, 15, and 16) against T-cell acute lym-
phoblastoid leukemia cell line (CCRF-HSB-2) and hu-
man oral epidermoid carcinoma cell line (KB) revealed
potential antitumor activities of many derivatives.
Among them, compounds 2a, 2d, 4e, 5a, 5b, 8a, and
9a exhibited the most significant antiproliferative
potencies with good IC50 of 1.46–2.70 and 0.61–
2.1 lg/mL against CCRF-HSB-2 and KB cells, respec-
tively. Some other derivatives, namely 2b, 4b, 6, 13a,
13c, and 13d, showed moderate IC50 of 3.72–6.77
and 5.35–8.19 lg/mL against CCRF-HSB-2 and KB
cells, respectively. These promising antitumor activities
provide these derivatives as new lead entities in cancer
therapy. Moreover, the structure–activity relationships
(SAR) revealed that the flavin-5-oxide, 5-deazaflavin,
and alloxazine skeletons with SMe or HNMe group
at the C2-position, alloxazine conformation or Me
group at the N10-position, and unsubstituted quinoxa-
line nucleus or substituted by 7-Me or 9-Me group
provide higher antitumor activities.
4.1.1. General procedure for the preparation of 10-alkyl-
2-deoxo-2-methylthioflavin-5-oxides {10-alkyl-2-methyl-
thio-4-oxo-4,10-dihydrobenzo[g]pteridine-5-oxides} (2a–
i). To a stirring solution of 6-(N-alkylanilino)-2-methyl-
thiopyrimidin-4(3H)-one (1, 0.01 mol) in acetic acid (5–
15 mL) at 10–15 ꢁC was added sodium nitrite (0.02–
0.04 mol) by portions, and the mixture was stirred for
1–2 h at room temperature. The solid deposited was col-
lected by suction filtration and washed with water. The
filtrate was evaporated in vacuo and diluted with excess
water to afford the second crop. The collected solids
were dried and crystallized from an appropriate solvent
to afford the corresponding products as red needles in
65–89% yields.
4.1.1.1. 10-Methyl-2-methylthio-4-oxo-4,10-dihydro-
benzo[g]pteridine-5-oxide (2a). Yield, (2.0 g, 73%); mp
215–217 ꢁC (decomp., from DMF); UV (EtOH):
k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 219 (4.53), 269 (4.39),
1
368 (4.00), 464 (4.09); IR (mmax/cmꢀ1): 1650 (C@O); H
NMR (CDCl3): d 2.59 (3H, s, 2-SMe), 4.13 (3H, s, 10-
Me), 7.60 (1H, dt, J6,7 = J7,8 = 8.7 Hz, J7,9 = 1.5 Hz, 7-
H), 7.75 (1H, dd, J7,9 = 1.5 Hz, J8,9 = 8.7 Hz, 9-H),
7.94 (1H, dt, J7,8 = J8,9 = 8.7 Hz, J6,8 = 1.5 Hz, 8-H),
8.57 (1H, dd, J6,7 = 8.7 Hz, J6,8 = 1.5 Hz, 6-H). Anal.
Calcd for C12H10N4O2S: C, 52.54; H, 3.67; N, 20.43.
Found: C, 52.61; H, 3.91; N, 20.59.
The AutoDock investigation of the synthesized deriva-
tives (2–6, 8, 9, and 12–16) was carried out for lead opti-
mization and they docked into c-kit protein–tyrosine
kinase. The correlation between the binding free ener-
gies (DGb, kcal/mol) predicted by AutoDock and the
growth inhibitory activities (IC50, lg/mL) against
CCRF-HSB-2 tumor cells for compounds 2c, h, 4a–c,
e, 8a, b, 9a, 12a, and 16d was good with a correlation
coefficient (R2) of 0.759. Also the correlation between
and the AutoDock binding free energies and IC50
against KB tumor cells for compounds 2e, f, g, 4b, e,
8b, 9a, 13b, and 16b, c, e was good with a correlation
coefficient (R2) of 0.68.
4.1.1.2. 10-Ethyl-2-methylthio-4-oxo-4,10-dihydro-
benzo[g]pteridine-5-oxide (2b). Yield, (2.57 g, 89%); mp
234–236 ꢁC (decomp., from DMF); UV (EtOH):
k
max/nm (log e/dm3 molꢀ1 cmꢀ1): 220 (4.53), 278 (4.38),
1
371 (4.06), 468 (4.16); IR (mmax/cmꢀ1): 1655 (C@O); H
NMR (CDCl3): d 1.53 (3H, t, J = 7.2 Hz, 10-CH2-
CH3), 2.59 (3H, s, 2-SMe), 4.77 (2H, q, J = 7.2 Hz, 10-
CH2–CH3), 7.60 (1H, dt, J6,7 = 8.7 Hz, J7,8 = 8.4 Hz,
J7,9 = 1.5 Hz, 7-H), 7.74 (1H, br d, J8,9 = 8.4 Hz, 9-H),
7.94 (1H, dt, J7,8 = J8,9 = 8.4 Hz, J6,8 = 1.5 Hz, 8-H),
8.58 (1H, dd, J6,7 = 8.7 Hz, J6,8 = 1.5 Hz, 6-H). Anal.
Calcd for C13H12N4O2S: C, 54.15; H, 4.19; N, 19.43.
Found: C, 54.31; H, 4.38; N, 19.16.
4. Experimental
4.1. Chemistry
Mps were obtained on a Yanagimoto micro melting
point apparatus and were uncorrected. Microanalyses

932
H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
4.1.1.3. 7,10-Dimethyl-2-methylthio-4-oxo-4,10-dihydro-
benzo[g]pteridine-5-oxide (2c). Yield, (2.02 g, 70%); mp
273 (4.31), 368 (3.87), 470 (3.93); IR (mmax/cmꢀ1): 1650
(C@O); H NMR (CDCl3): d 1.51 (3H, t, J = 7.2 Hz,
10-CH2–CH3), 2.57 (3H, s, 7-Me), 2.59 (3H, s, 2-SMe),
4.76 (2H, q, J = 7.2 Hz, 10-CH2–CH3), 7.68 (1H, d,
J8,9 = 9.0 Hz, 9-H), 7.75 (1H, dd, J8,9 = 9.0 Hz,
J6,8 = 1.5 Hz, 8-H), 8.37 (1H, br s, 6-H). Anal. Calcd
for C14H14N4O2S: C, 55.61; H, 4.67; N, 18.53. Found:
C, 55.33; H, 4.79; N, 18.16.
1
221–223 ꢁC (decomp., from DMF); UV (EtOH): kmax
/
nm (loge/dm3 molꢀ1 cmꢀ1): 221 (4.48), 279 (4.38), 369
(4.00), 472 (4.11); IR (mmax/cmꢀ1): 1650 (C@O); 1H
NMR (CDCl3): d 2.57 (3H, s, 7-Me), 2.59 (3H, s, 2-
SMe), 4.11 (3H, s, 10-Me), 7.63 (1H, d, J8,9 = 8.4 Hz, 9-
H), 7.75 (1H, dd, J8,9 = 8.4 Hz, J6,8 = 2.1 Hz, 8-H), 8.35
(1H, br s, 6-H). Anal. Calcd for C13H12N4O2S: C, 54.15;
H, 4.19; N, 19.43. Found: C, 54.15; H, 4.26; N, 19.12.
4.1.1.9. 7,8,10-Trimethyl-2-methylthio-4-oxo-4,10-
dihydrobenzo[g]pteridine-5-oxide (2i). Yield, (2.45 g,
81%); mp >300 ꢁC (decomp., from DMF–H2O); UV
(EtOH): kmax/nm (loge/dm3 molꢀ1 cmꢀ1): 222 (4.64),
280 (4.59), 378 (4.29), 474 (4.35); IR (mmax/cmꢀ1): 1650
4.1.1.4. 9,10-Dimethyl-2-methylthio-4-oxo-4,10-dihydro-
benzo[g]pteridine-5-oxide (2d). Yield, (2.05 g, 71%); mp
200–202 ꢁC (decomp., from DMF); UV (EtOH): kmax
/
nm (loge/dm3 molꢀ1 cmꢀ1): 219 (4.55), 268 (4.47), 377
(4.10), 466 (4.04); IR (mmax/cmꢀ1): 1650 (C@O); 1H
NMR (CDCl3): d 2.58 (3H, s, 2-SMe), 2.88 (3H, s, 9-
Me), 4.19 (3H, s, 10-Me), 7.47 (1H, t,
J6,7 = J7,8 = 8.1 Hz, 7-H), 7.69 (1H, d, J7,8 = 8.1 Hz, 8-
H), 8.42 (1H, d, J6,7 = 8.1 Hz, 6-H). Anal. Calcd for
C13H12N4O2S: C, 54.15; H, 4.19; N, 19.43. Found: C,
54.47; H, 4.49; N, 19.39.
(C@O); H NMR (CDCl3): d 2.52 (3H, s, 7-Me), 2.53
1
(3H, s, 8Me), 2.57 (3H, s, 2-SMe), 4.10 (3H, s, 10-Me),
7.49 (1H, s, 6-H), 8.21 (1H, s, 9-H). Anal. Calcd for
C14H14N4O2S: C, 55.61; H, 4.67; N, 18.53. Found: C,
55.85; H, 4.68; N, 18.38.
4.1.1.10. Preparation of 2-deoxo-2-dimethylaminoal-
loxazine {2-dimethylamino-4-oxo-3,4-dihydrobenzo[g]-
pteridine} (3). A mixture of 2-deoxo-10-methyl-2-meth-
ylthioflavin-5-oxide (2a, 1.4 g, 5.0 mmol) and 50% aque-
ous dimethylamine (50 mL) was heated in steel sealed
tube at 160–165 ꢁC (15 kg/cm2 pressure) for 4 h. After
the reaction was complete, the precipitated crystals were
collected by filtration, and mother liquor was evapo-
rated in vacuo to get the second crop. The product
was washed with water, dried, and recrystallized from
EtOH to afford the yellow crystals in 89% yield.
4.1.1.5. 7-Methoxy-10-methyl-2-methylthio-4-oxo-4,10-
dihydrobenzo[g]pteridine-5-oxide (2e). Yield, (2.37 g,
78%); mp >300 ꢁC (from DMF); UV (EtOH): kmax/nm
(loge/dm3 molꢀ1 cmꢀ1): 220 (4.66), 289 (4.61), 365
(4.14), and 495 (4.28); IR (mmax/cmꢀ1): 1640 (C@O);
1H NMR (CDCl3): d 2.59 (3H, s, 2-SMe), 3.98 (3H, s,
7-OMe), 4.14 (3H, s, 10-Me), 7.56 (1H, dd,
J8,9 = 9.3 Hz, J6,8 = 2.7 Hz, 8-H), 7.69 (1H, d,
J8,9 = 9.3 Hz, 9-H), 7.96 (1H, d, J6,8 = 2.7 Hz, 6-H).
Anal. Calcd for C13H12N4O3S: C, 51.31; H, 3.97; N,
18.41. Found: C, 51.19; H, 4.10; N, 18.32.
Yield, (1.07 g, 89%); mp >300 ꢁC (decomp., from
EtOH); UV (EtOH): kmax/nm (loge/dm3 molꢀ1 cmꢀ1):
222 (4.39), 268 (4.32), 288 (4.32), 335 (3.63), 376sh
(3.59), 398 (3.71), 429 (3.77); IR (mmax/cmꢀ1): 3190
4.1.1.6. 8-Methoxy-10-methyl-2-methylthio-4-oxo-4,10-
dihydrobenzo[g]pteridine-5-oxide (2f). Yield, (2.04 g,
67%); mp 214–217 ꢁC (decomp., from DMF); UV
(EtOH): kmax/nm (loge/dm3 molꢀ1 cmꢀ1): 228 (4.50),
265 (4.34), 379 (4.26), 457 (4.23); IR (mmax/cmꢀ1): 1630
1
(NH), 1700 (C@O); H NMR (CDCl3): d 3.42 (6H, s,
2-N–(CH3)2), 7.66 (1H, dt, J6,7 = J7,8 = 8.4 Hz,
J7,9 = 1.5 Hz, 7-H), 7.81 (1H, dt, J7,8 = J8,9 = 8.4 Hz,
J6,8 = 1.5 Hz, 8-H), 8.04 (1H, br d, J8,9 = 8.4 Hz, 9-H),
8.23 (1H, br d, J6,7 = 8.4 Hz, 6-H), 10.13 (1H, br s, 3-
NH, exchangeable with D2O). Anal. Calcd for
C12H11N5OÆ0.2H2O: C, 58.86; H, 4.69; N, 28.60. Found:
C, 58.78; H, 4.87; N, 28.58.
1
(C@O); H NMR (CDCl3): d 2.57 (3H, s, 2-SMe), 4.06
(3H, s, 8-OMe), 4.07 (3H, s, 10-Me), 6.96 (1H, d,
J7,9 = 2.4 Hz, 9-H), 7.14 (1H, dd, J6,7 = 9.6 Hz,
J7,9 = 2.4 Hz, 7-H), 8.45 (1H, d, J6,7 = 9.6 Hz, 6-H).
Anal. Calcd for C13H12N4O3S: C, 51.31; H, 3.97; N,
18.41. Found: C, 51.28; H, 4.40; N, 18.40.
4.1.2. General procedure for the preparation of 10-alkyl-
2-alkylamino-2-deoxoflavin-5-oxides {10-alkyl-2-alkyla-
mino-4-oxo-4,10-dihydrobenzo[g]pteridine-5-oxides} (4a–
e). A mixture of 10-alkyl-2-deoxo-2-methylthioflavin-5-
oxides (2, 5.0 mmol) and an appropriate alkylamine
(0.025–0.05 mol) in n-butanol (30 mL) was refluxed with
stirring for 15 min. After the clear yellow solution ob-
tained under reflux was cooled under refrigeration over-
night, the resulting yellow crystals were collected by
filtration to get the first crop. The filtrate was concen-
trated in vacuo and the residue was treated with ethanol
or water to get the second crop free from amines. The
collected solids were dried and recrystallized from an
appropriate solvent to give the corresponding products
as bright yellow-orange needles in 62–89% yields.
4.1.1.7. 7-Chloro-10-methyl-2-methylthio-4-oxo-4,10-
dihydrobenzo[g]pteridine-5-oxide (2g). Yield, (2.0 g,
65%); mp 303–305 ꢁC (decomp., from DMF); UV
(EtOH): kmax/nm (loge/dm3 molꢀ1 cmꢀ1): 220 (4.39),
268 (4.34), 347 (4.72), 469 (3.85); IR (mmax/cmꢀ1): 1660
1
(C@O); H NMR (CDCl3): d 2.60 (3H, s, 2-SMe), 4.10
(3H, s, 10-Me), 7.68 (1H, d, J8,9 = 9.6 Hz, 9-H), 7.87
(1H, dd, J8,9 = 9.6 Hz, J6,8 = 2.4 Hz, 8-H), 8.54 (1H, d,
J6,8 = 2.4 Hz, 6-H). Anal. Calcd for C12H9ClN4O2S: C,
46.68; H, 2.94; N, 18.15. Found: C, 46.89; H, 3.09; N,
17.88.
4.1.1.8. 10-Ethyl-7-methyl-2-methylthio-4-oxo-4,10-
dihydrobenzo[g]pteridine-5-oxide (2h). Yield, (2.6 g,
86%); mp 236–238 ꢁC (decomp., from DMF); UV
(EtOH): kmax/nm (loge/dm3 molꢀ1 cmꢀ1): 220 (4.45),
4.1.2.1. 7-Methoxy-10-methyl-2-piperidino-4-oxo-4,10-
dihydrobenzo[g]pteridine-5-oxide (4a). Yield, (1.06 g,

H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
933
62%); mp 276–278 ꢁC (decomp., from DMF); UV
(EtOH): kmax/nm (loge/dm3 molꢀ1 cmꢀ1): 223 (4.37),
266 (4.28), 284 (4.48), 350 (3.88), 444sh (3.95), 476
(4.17), 506 (4.14); IR (mmax/cmꢀ1): 1645 (C@O); 1H
NMR (CDCl3): d 1.66 (2H, br s, 40-CH2), 3.74–3.80
(4H, m, 30 and 50-CH2), 3.92 (3H, s, 7-OMe), 4.04–
4.12 (4H, m, 20 and 60-CH2), 4.09 (3H, s, 10-Me), 7.47
(1H, dd, J8,9 = 9.3 Hz, J6,8 = 2.7 Hz, 8-H), 7.56 (1H, d,
J8,9 = 9.3 Hz, 9-H), 7.75 (1H, d, J6,8 = 2.7 Hz, 6-H).
Anal. Calcd for C17H19N5O3: C, 59.81; H, 5.61; N,
20.52. Found: C, 59.58; H, 5.44; N, 20.75.
m, 2-NHCH2CH2OH), 3.97 (3H, s, 10-Me), 4.86 (1H,
br s, 2-NHCH2CH2OH, exchangeable with D2O), 7.42
(1H, br s, 2-NH, exchangeable with D2O), 7.70–7.81
(1H, m, 9-H), 7.85–7.97 (2H, m, 6 and 8-H). Anal. Calcd
for C14H15N5O3: C, 55.81; H, 5.02; N, 23.24. Found: C,
56.04; H, 4.95; N, 23.34.
4.1.3. General procedure for the preparation 10-alkylf-
lavins
{10-alkylbenzo[g]pteridine-2,4(3H,10H)-diones}
(5a, b) and 7-methoxy-10-methylflavin-5-oxide {7-meth-
oxy-10-methyl-2,4-dioxo-2,3,4,10-tetrahydro-benzo[g]pteri-
dine-5-oxide} (6). To 10-alkyl-2-deoxo-2-methylthioflavin-
5-oxides (2, 0.01 mol) was added 5 N hydrochloric acid
(250 mL). Then, the mixture was refluxed for 7–12 h,
and the resulting clear yellow solution was concentrated
in vacuo. The yellow residue was treated with water and
neutralized with aqueous ammonia (pH 7). The precipitate
powdery crystals were filtered off, washed well with water,
dried, and recrystallized from acetic acid to afford the cor-
responding products as yellow needles in 68–97% yields.
4.1.2.2. 10-Ethyl-2-isobutylamino-4-oxo-4,10-dihydro-
benzo[g]pteridin-5-oxide (4b). Yield, (1.30 g, 83%); mp
264–267 ꢁC (decomp., from EtOH-n-hexane); UV
(EtOH): kmax/nm (loge/dm3 molꢀ1 cmꢀ1): 219 (4.47),
271 (4.47), 343sh (3.96), 443 (3.12), 472sh (3.98); IR
(mmax/cmꢀ1): 3195 (NH), 1620 (C@O); 1H NMR
(CDCl3): d 0.99–1.02 (6H, m, NH–CH2–CH–(CH3)2),
1.46–1.56 (3H, m, 10-CH2–CH3) 1.92–2.08 (1H, m,
NH–CH2–CH–(CH3)2), 3.44–3.51 (2H, m, NH–CH2–
CH–(CH3)2), 4.75 (2H, q, J = 6.9 Hz, 10-CH2–CH3),
5.75 (1H, br s, 2-NH, exchangeable with D2O), 7.53–
7.65 (2H, m, 7 and 9-H), 7.76–7.88 (1H, m, 8-H),
7.32–7.40 (1H, m, 6-H). Anal. Calcd for C16H19N5O2:
C, 61.33; H, 6.11; N, 22.35. Found: C, 61.51; H, 6.44;
N, 22.43.
4.1.3.1. 10-Methylbenzo[g]pteridine-2,4(3H,10H)-
dione (5a). Yield, (2.05 g, 90%); mp >300 ꢁC (from
HOAc; lit.28 mp >360 ꢁC); UV (EtOH): kmax/nm (loge/
dm3 molꢀ1 cmꢀ1): 214 (4.31), 265 (4.39), 332 (3.74),
436 (3.81); IR (mmax/cmꢀ1): 3170 (NH), 1720, 1660
1
(C@O); H NMR [(CD3)2SO]: d 3.98 (3H, s, 10-Me),
7.62–7.69 (1H, m, 7-H), 7.93–7.98 (2H, m, 8 and 9-H),
8.13 (1H, br d, J6,7 = 7.8 Hz, 6-H), 11.10 (1H, br s,
3-NH, exchangeable with D2O). Anal. Calcd for
C11H8N4O2Æ0.6H2O: C, 55.28; H, 3.88; N, 23.44. Found:
C, 55.47; H, 3.70; N, 23.02.
4.1.2.3. 2-Cyclohexylamino-7,10-dimethyl-4-oxo-4,10-
dihydrobenzo[g]pteridine-5-oxide (4c). Yield, (1.05 g,
62%); mp 288–291 ꢁC (decomp., from EtOH); UV
(EtOH): kmax/nm (loge/dm3 molꢀ1 cmꢀ1): 220 (4.56),
271 (4.57), 341 (3.94), 436 (4.10), 483sh (3.83); IR
(mmax/cmꢀ1): 3270 (NH), 1665 (C@O); 1H NMR
[(CD3)2SO], 100 ꢁC: d 1.10–1.68 (6H, m, 30, 40, and 50-
CH2), 1.68–1.98 (4H, m, 20 and 60-CH2), 2.49 (3H, s,
7-Me), 3.95 (3H, s, 10-Me), 4.02–4.06 (1H, br m, 10-
CH), 7.37 (1H, br s, 2-NH, exchangeable with D2O),
7.67–7.78 (1H, m, 9-H), 7.87 (1H, br s, 6-H), 7.95 (1H,
br d, J8,9 = 9.0 Hz, 8-H). Anal. Calcd for C18H21N5O2:
C, 63.70; H, 6.24; N, 20.64. Found: C, 63.99; H, 6.34;
N, 20.25.
4.1.3.2. 10-Ethylbenzo[g]pteridine-2,4(3H,10H)-dione
(5b). Yield, (2.35 g, 97%); mp >300 ꢁC (from HOAc;
lit.28 mp 347 ꢁC); UV (EtOH):
kmax/nm (loge/
dm3 molꢀ1 cmꢀ1): 216 (4.53), 265 (4.65), 335 (3.98),
434 (4.13); IR (mmax/cmꢀ1): 3180 (NH), 1720, 1680
1
(C@O); H NMR (CDCl3): d 1.33 (3H, t, J = 7.2 Hz,
10-CH2–CH3), 4.65 (2H, q, J = 7.2 Hz, 10-CH2–CH3),
7.65 (1H, d t, J6,7 = J7,8 = 7.8 Hz, J7,9 = 1.2 Hz, 7-H),
7.92–8.03 (2H, m,
8 and 9-H), 8.14 (1H, dd,
J6,7 = 7.8 Hz, J6,8 = 1.2 Hz, 6-H), 11.39 (1H, br s, 3-
NH, exchangeable with D2O). Anal. Calcd for
C12H10N4O2: C, 59.50; H, 4.16; N, 23.13. Found: C,
59.10; H, 4.40; N, 23.06.
4.1.2.4. 2-Benzylamino-7-chloro-10-methyl-4-oxo-
4,10-dihydroenzo[g]pteridine-5-oxide (4d). Yield, (1.34 g,
73%); mp >300 ꢁC (from EtOH); UV (EtOH): kmax/nm
(loge/dm3 molꢀ1 cmꢀ1): 221 (4.85), 274 (4.90), 330
(4.24), 453 (4.38), 487sh (4.24); IR (mmax/cmꢀ1): 3190
4.1.3.3. 7-Methoxy-10-methyl-2,4-dioxo-2,3,4,10-tet-
rahydrobenzo[g]pteridine-5-oxide (6). Yield, (1.86 g,
1
(NH), 1655 (C@O); H NMR [(CD3)2SO], at 100 ꢁC: d
4.00 (3H, s, 10-Me), 4.60–4.80 (2H, m, 2-NH–CH2),
7.20–7.52 (5H, m, 2-CH2–C6H5), 7.82–8.02 (2H, m, 2-
NH and 9-H), 8.10–8.28 (2H, m, 6 and 8-H). Anal.
Calcd for C18H14ClN5O2Æ0.25H2O: C, 58.07; H, 3.93;
N, 18.81. Found: C, 57.89; H, 4.28; N, 18.42.
68%); mp >300 ꢁC (from HOAc); UV (EtOH): kmax
/
nm (loge/dm3 molꢀ1 cmꢀ1): 216 (4.57), 272 (4.63), 339
(4.04), 474 (4.18); IR (mmax/cmꢀ1): 3160 (NH), 1710,
1
1665 (C@O); H NMR [(CD3)2SO]: d 3.93 (3H, s, 7-
OMe), 3.99 (3H, s, 10-Me), 7.59 (1H, d, J6,8 = 3.0 Hz,
6-H), 7.63–7.65 (1H, m, 8-H), 7.91(1H, d,
J8,9 = 9.0 Hz, 9-H), 11.03 (1H, br s, 3-NH, exchangeable
with D2O). Anal. Calcd for C12H10N4O4: C, 52.56; H,
3.68; N, 20.43. Found: C, 52.87; H, 3.94; N, 20.13.
4.1.2.5. 2-(2-Hydroxyethylamino)-7,10-dimethyl-4-
oxo-4,10-dihydrobenzo[g]pteridine-5-oxide (4e). Yield,
(1.34 g, 89%); mp 266–268 ꢁC (decomp., from EtOH);
UV (EtOH): kmax/nm (loge/dm3 molꢀ1 cmꢀ1): 217
(4.62), 273 (4.64), 344 (4.12), 453 (4.24), 483sh (4.11);
IR (mmax/cmꢀ1): 3290 (OH), 3200 (NH), 1660 (C@O);
1H NMR [(CD3)2SO], at 100 ꢁC: d 2.49 (3H, s, 7-Me),
3.38–3.46 (2H, m, 2-NHCH2CH2OH), 3.48–3.60 (2H,
4.1.4. Preparation of 2-methylamino-6-(N-methylanili-
no)pyrimidin-4(3H)-one (7a). A mixture of 6-(N-methy-
lanilino)-2-methylthiopyrimidin-4(3H)-one (1a, 0.5 g,
2.0 mmol) and 40% aqueous methylamine (50 mL) in

934
H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
steel sealed tube was heated at 160 ꢁC (10 kg/cm2 pres-
sure) for 15 h. After the reaction was complete, the
resulting clear yellow solution was concentrated in va-
cuo. The yellow residue was treated with ethyl acetate
to get the solid, which was filtered off, washed with
water, dried, and crystallized from DMF to afford the
pure product as colorless needles.
(4.16), 321 (3.67), 410 (3.66); IR (mmax/cmꢀ1): 3290
(NH), 1705 (C@O); H NMR (CDCl3): d 3.34 (3H, s,
2-NMe),4.10 (3H, s, 10-Me), 7.46–7.58 (1H, m, 7-H),
7.67 (1H, br s, 2-NH, exchangeable with D2O), 7.82–
7.98 (2H, br m, 8 and 9-H), 8.11 (1H, br d,
J6,7 = 7.8 Hz, 6-H), 8.82 (1H, s, 5-H). Anal. Calcd for
C13H12N4OÆ0.7H2O: C, 61.75; H, 5.34; N, 22.16. Found:
C, 62.04; H, 5.74; N, 22.0.
1
Yield, (0.40 g, 87%); mp 219–221 ꢁC (from DMF); UV
(EtOH): kmax/nm (loge/dm3 molꢀ1 cmꢀ1): 229 (4.49),
258sh (4.15), 284 (4.21); IR (mmax/cmꢀ1): 3310, 3176
4.1.4.4. 10-Methyl-2-n-octylaminopyrimido[4,5-b]quin-
olin-4-(10H)-one (8b).18 Yield, (1.02 g, 60%); mp 187–
189 ꢁC (decomp., from EtOH); UV (EtOH): kmax/nm
(loge/dm3 molꢀ1 cmꢀ1): 220 (4.45), 269 (4.54), 275sh
(4.47), 320 (3.98), 406 (4.05); IR (mmax/cmꢀ1): 3220
1
(NH), 1625 (C@O); H NMR (CDCl3): d 2.88 (3H, s,
2-NMe), 3.44 (3H, s, 6-NMe), 4.67 (1H, s, 5-H), 5.52
(1H, br s, 2-NH, exchangeable with D2O), 7.01–7.44
(5H, m, Ph–H), 12.19 (1H, br s, 3-NH, exchangeable
with D2O). Anal. Calcd for C12H14N4O: C, 62.59; H,
6.13; N, 24.33. Found: C, 62.23; H, 6.07; N, 24.33.
1
(NH), 1720 (C@O); H NMR [(CD3)2SO], at 100 ꢁC: d
0.86 (3H, t, J = 7.2 Hz, 2-N(CH2)7–CH3), 1.20–1.42
(10H, m, 2-N(CH2)2–(CH2)5–CH3), 1.60 (2H, quint,
J = 7.2 Hz, 2-NCH2–CH2–(CH2)5–CH3), 3.35–3.51
(2H, m, 2-NCH2–(CH2)6–CH3), 4.07 (3H, s, 10-Me),
7.17 (1H, br s, 2-NH, exchangeable with D2O), 7.42–
7.53 (1H, m, 7-H), 7.75–7.93 (2H, m, 8 and 9-H), 8.07
(1H, d, J = 8.1 Hz, 6-H), 8.77 (1H, s, 5-H). Anal. Calcd
for C20H26N4OÆ0.1H2O: C, 70.60; H, 7.76; N, 16.47.
Found: C, 70.30; H, 7.59; N, 16.14.
4.1.4.1. Preparation of 6-(N-methylanilino)-2-n-octyla-
minopyrimidin-4(3H)-one (7b). A mixture of 6-(N-methy-
lanilino)-2-methylthiopyrimidin-4(3H)-one (1a, 1.24 g,
5.0 mmol) and n-octylamine (2.58 g, 0.02 mol) was
heated at 160 ꢁC for 10 h. After cooling, the resulting
mixture was triturated with water and acidified with ace-
tic acid to remove the excess n-octylamine as salt. The
product was extracted from the mixture with dichloro-
methane (3· 50 mL) and the extract was washed with
saturated brine (2· 20 mL). The combined organic ex-
tracts were dried over anhydrous magnesium sulfate, fil-
tered, and the solvent was removed under reduced
pressure to afford the product 7b as orange oil. The
product which was dried under vacuum was used for
the next step without further purification.
4.1.5. General procedure for the preparation of 2-alkyla-
mino-2-deoxo-10-methylflavin-5-oxides
{2-alkylamino-
10-methyl-4-oxo-4,10-dihydrobenzo[g]pteridine-5-oxides}
(9a,b). To a stirring solution of 2-alkylamino-6-(N-
methylanilino)pyrimidin-4(3H)-ones (7, 5.0 mmol) in
acetic acid (5–15 mL) at 10–15 ꢁC was added sodium ni-
trite (0.01–0.02 mol) by portions, and the mixture was
stirred at room temperature for 2–3 h. The solid depos-
ited was collected by suction filtration and washed well
with water. The filtrate was concentrated in vacuo and
diluted with excess water to afford the second crop.
The collected solids were washed with diluted ammonia
solution, dried, and crystallized from an appropriate sol-
vent to afford the corresponding products as orange nee-
dles in 76–88% yields.
Yield, (1.41 g, 86%); oily compound; UV (EtOH): kmax
/
nm (loge/dm3 molꢀ1 cmꢀ1): 230 (4.56), 259sh (4.28), 282
(4.29); IR (mmax/cmꢀ1): 3278, 3156 (NH), 1640 (C@O);
1H NMR (CDCl3): d 0.84–0.92 (3H, m, 2-N–(CH2)7–
CH3), 1.22–1.33 (10H, m, 2-N–(CH2)2–(CH2)5–CH3),
1.38–1.59 (2H, m, 2-N–CH2–CH2–(CH2)5–CH3), 3.27
(2H, q, J = 7.2 Hz, 2-N–CH2–(CH2)6–CH3), 3.41 (3H,
s, 6-NMe), 4.66 (1H, s, 5-H), 5.82 (1H, br s, 2-NH,
exchangeable with D2O), 7.19–7.28 (3H, m, Ph-o, pH),
7.34–7.42 (2H, m, Ph-mH), 11.57 (1H, br s, 3-NH,
exchangeable with D2O).
4.1.5.1. 10-Methyl-2-methylamino-4-oxo-4,10-dihydro-
benzo[g]pteridine-5-oxide (9a). Yield, (1.13 g, 88%); mp
>300 ꢁC (decomp., from DMF); UV (EtOH): kmax/nm
(loge/dm3 molꢀ1 cmꢀ1): 216 (4.27), 247sh (4.34), 274
(4.48), 348 (3.99), 455 (4.05); IR (mmax/cmꢀ1): 3290
1
4.1.4.2. General procedure for the preparation of 2-
alkylamino-2-deoxo-10-methyl-5-deazaflavins {2-alkyl-
amino-10-methylpyrimido[4,5-b]quinolin-4(10H)-ones}
(8a,b). A mixture of 2-alkylamino-6-(N-methylanilino)-
pyrimidin-4(3H)-ones (7, 5.0 mmol) and phosphoryl
chloride (3.83 g, 25 mmol) in anhydrous DMF (5 mL)
was heated under stirring at 90 ꢁC for 3 h. Then, the
reaction mixture was poured onto ice and treated with
aqueous ammonia (pH 8). The separated yellow crystals
were filtered off, washed with water, dried and recrystal-
lized from an appropriate solvent to afford the products
as yellow needles in 60–82% yields.
(NH), 1670 (C@O); H NMR [(CD3)2SO], at 85 ꢁC: d
2.89 (3H, s, 2-NMe), 3.93 (3H, s, 10-Me), 7.31 (1H, br
s, 2-NH, exchangeable with D2O), 7.43–7.62 (1H, m,
7-H), 7.78–8.0 (2H, m, 8 and 9-H), 8.34 (1H, d,
J = 8.1 Hz, 6-H). Anal. Calcd for C12H11N5O2Æ0.2H2O:
C, 55.25; H, 4.41; N, 26.85. Found: C, 55.09; H, 4.54;
N, 26.85.
4.1.5.2. 10-Methyl-2-n-octylamino-4-oxo-4,10-dihydro-
benzo[g]pteridine-5-oxide (9b). Yield, (1.35 g, 76%); mp
172–174 ꢁC (from EtOAc); UV (EtOH): kmax/nm (loge/
dm3 molꢀ1 cmꢀ1): 216 (4.40), 246sh (4.29), 273 (4.47),
347 (3.95), 444 (4.05); IR (mmax/cmꢀ1): 3270 (NH), 1655
1
4.1.4.3. 10-Methyl-2-methylaminopyrimido[4,5-b]quin-
olin-4-(10H)-one (8a). Yield, (0.99 g, 82%); mp 259–
251 ꢁC (decomp., from EtOH); UV (EtOH): kmax/nm
(loge/dm3 molꢀ1 cmꢀ1): 221 (5.07), 269 (4.20), 275sh
(C@O); H NMR [(CD3)2SO], at 100 ꢁC: d 0.89 (3H, t,
J = 6.6 Hz, 2-N(CH2)7–CH3), 1.22–1.45 (10H, m, 2-
N(CH2)2–(CH2)5–CH3), 1.61 (2H, quint, J = 6.6 Hz, 2-
NCH2–CH2–(CH2)5–CH3), 3.30–3.51 (2H, m, 2-NCH2–

H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
935
0
s, 5-H), 6.98 (2H, dd, J2 ,3 = J5 ,6 = 8.7 Hz, J2 ,6
0
0
0
0
0
(CH2)6–CH3), 3.96 (3H, s, 10-Me), 7.32 (1H, br s, 2-NH,
exchangeable with D2O), 7.43–7.65 (1H, m, 7-H), 7.75–
8.0 (2H, m, 8 and 9-H), 8.36 (1H, d, J = 8.4 Hz, 6-H).
Anal. Calcd for C19H25N5O2: C, 64.20; H, 7.09; N,
19.70. Found: C, 64.14; H, 6.83; N, 19.48.
=
2.1 Hz, Ar-oH), 7.17 (2H, dd, J2 ,3 = J5 ,6 = 8.7 Hz,
0
0
0
0
0
0
J3 ,5 = 2.1 Hz, Ar-mH), 7.92 (1H, s, 6-NH), 11.53 (1H,
br s, 1-NH), 11.83 (1H, br s, 3-NH). Anal. Calcd for
C11H11N3O2S: C, 53.00; H, 4.45; N, 16.86. Found: C,
52.87; H, 4.52; N, 16.70.
4.1.6. General procedure for the preparation of 6-(N-
anilino)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidines
(10a–e). A mixture of 6-amino-2-thiouracil (7.10 g,
0.05 mol), an appropriate aniline (0.1 mol) together with
anilinium chloride (0.075–0.10 mol) was heated at
170 ꢁC for 4–12 h. The cooled mixture was diluted with
65% ethanol (200 mL) to afford the solid product, which
was collected by filtration, dissolved in hot ca. 5%
NaOH solution, and reprecipitated by neutralization
with 10% HCl to get more pure product. The solid
deposited was collected by suction filtration, washed
with water, dried, and crystallized from a mixture of
DMF and H2O to afford the corresponding products
as colorless needles in 67–83% yields.
4.1.6.5. 6-(4-Hydroxyanilino)-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine (10e). Yield, (8.35 g, 71%); mp
>300 ꢁC (decomp., from DMF–H2O); UV (EtOH):
k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 208 (4.43), 279 (4.47);
IR (mmax/cmꢀ1): 3330 (OH), 3285, 3205, 3095 (NH),
1
1685 (C@O); H NMR [(CD3)2SO]: d 4.60 (1H, s, 5-
0
H), 6.79 (2H, dd, J2 ,3 = J5 ,6 = 8.7 Hz, J2 ,6 = 2.1 Hz,
0
0
0
0
0
0 0 0 0
J2 ,3 = J5 ,6 = 8.7 Hz,
Ar-oH),
7.05
(2H,
dd,
0
0
J3 ,5 = 2.1 Hz, Ar-mH), 7.81 (1H, s, 6-NH), 9.55 (1H,
s, OH), 11.51 (1H, br s, 1-NH), 11.81 (1H, br s, 3-
NH). Anal. Calcd for C10H9N3O2S: C, 51.05; H, 3.86;
N, 17.86. Found: C, 50.79; H, 4.19; N, 17.63.
4.1.7. General procedure for the preparation of 6-
anilino-2-methylthio-4-oxo-3,4-dihydropyrimidines (11a–
e). To a solution of 6-anilino-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidines (10, 0.01 mol) in 2 N KOH
(250 mL) at 0–5 ꢁC was added methyl iodide (2.84 g,
0.02 mol) and the solution was shaken vigorously un-
der chilling for 30 min. The solid precipitated was col-
lected by filtration and the filtrate was neutralized with
10% HCl to get the second crop. The collected solid
products were dissolved in hot ca. 5% NaOH solution
and reprecipitated by neutralization with 10% HCl to
get the more pure products. The solid products were
collected by suction filtration and washed with water,
dried, and crystallized from an appropriate solvent to
afford the corresponding silver colored crystals in 77–
98% yields.
4.1.6.1. 6-Anilino-4-oxo-2-thioxo-1,2,3,4-tetrahydro-
pyrimidine (10a). Yield, (9.10 g, 83%); mp 296–298 ꢁC
(decomp., from DMF–H2O); (lit.,23,24 287–288 and
281–283 ꢁC); UV (EtOH): kmax/nm (loge/dm3 molꢀ1
cmꢀ1): 205 (4.47), 277 (4.57); IR (mmax/cmꢀ1): 3335,
1
3220, 3105 (NH), 1670 (C@O); H NMR (CDCl3): d
5.19 (1H, s, 5-H), 7.15–7.23 (3H, m, Ph-o, pH), 7.34–
7.40 (2H, m, Ph-mH), 7.78 (1H, s, 6-NH), 11.28 (1H,
br s, 1-NH), 11.41 (1H, br s, 3-NH). Anal. Calcd for
C10H9N3OSÆ0.1H2O: C, 54.33; H, 4.19; N, 19.01.
Found: C, 54.27; H, 4.20; N, 18.80.
4.1.6.2. 6-(4-Methylanilino)-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine (10b). Yield, (7.82 g, 67%); mp
293–295 ꢁC (decomp., from DMF–H2O); UV (EtOH):
k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 205 (4.41), 255 (4.24);
IR (mmax/cmꢀ1): 3330, 3210, 3120 (NH), 1655 (C@O);
1H NMR [(CD3)2SO]: d 2.30 (3H, s, Me), 4.85 (1H, s,
5-H), 7.11 (2H, br d, JAB = 7.8 Hz, Ar-oH), 7.21 (2H,
br d, JAB = 7.8 Hz, Ar-mH), 8.06 (1H, s, 6-NH), 11.49
(1H, br s, 1-NH), 11.86 (1H, br s, 3-NH). Anal. Calcd
for C11H11N3OS: C, 56.63; H, 4.75; N, 18.01. Found:
C, 56.32; H, 5.08; N, 17.65.
4.1.7.1. 6-Anilino-2-methylthio-4-oxo-3,4-dihydropy-
rimidine (11a). Yield, (2.10 g, 90%); mp 294–295 ꢁC (de-
comp., from MeOH); UV (EtOH): kmax/nm (loge/
dm3 molꢀ1 cmꢀ1): 204 (4.46), 258 (4.41), 297 (4.16); IR
1
(mmax/cmꢀ1): 3250, 3140 (NH), 1640 (C@O); H NMR
[(CD3)2SO]: d 2.47 (3H, s, 2-SMe), 5.32 (1H, s, 5-H),
6.98 (1H, br t, J = 7.5 Hz, Ph-pH), 7.29 (2H, br t,
J = 7.5 Hz, Ph-mH), 7.42 (2H, br d, J = 7.5 Hz, Ph-
oH), 9.06 (1H, s, 6-NH), 11.84 (1H, br s, 3-NH). Anal.
Calcd for C11H11N3OS: C, 56.63; H, 4.75; N, 18.01.
Found: C, 56.32; H, 4.78; N, 17.66.
4.1.6.3. 6-(2,4-Dimethylanilino)-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine (10c). Yield, (9.77 g, 79%); mp 293–
295 ꢁC (decomp., from DMF–H2O); UV (EtOH): kmax
/
nm (loge/dm3 molꢀ1 cmꢀ1): 204 (4.45), 275 (4.41); IR
1
(mmax/cmꢀ1): 3350, 3190, 3095 (NH), 1650 (C@O); H
4.1.7.2. 6-(4-Methylanilino)-2-methylthio-4-oxo-3,4-
dihydropyrimidine (11b). Yield, (1.90 g, 77%); mp 252–
254 ꢁC (decomp., from DMF–H2O); UV (EtOH):
NMR [(CD3)2SO]: d 2.27 (6H, s, 20 and 40-CH3), 5.50
(1H, s, 5-H), 6.69 (1H, s, 6-NH), 7.09 (1H, s, Ar-30-H),
7.50–7.57 (1H, m, Ar-50-H), 8.14 (1H, d, J5 ,6 = 8.1 Hz,
Ar-60-H), 9.04 (1H, s, 1-NH), 11.98 (1H, br s, 3-NH).
Anal. Calcd for C12H13N3OS: C, 58.28; H, 5.30; N,
16.99. Found: C, 58.05; H, 5.39; N, 17.14.
k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 204 (4.39), 258 (4.38),
0
0
305 (4.09); IR (mmax/cmꢀ1): 3300, 3160 (NH), 1640
(C@O); 1H NMR (CDCl3): d 2.34 (3H, s, Ar-pMe),
2.54 (3H, s, 2-SMe), 5.51 (1H, s, 5-H), 6.47 (1H, s, 6-
NH), 7.12 (2H, d, JAB = 8.7 Hz, Ar-oH), 7.16 (2H, d,
JAB = 8.7 Hz, Ar-mH), 12.76 (1H, br s, 3-NH). Anal.
Calcd for C12H13N3OS: C, 58.28; H, 5.30; N, 16.99.
Found: C, 58.06; H, 5.41; N, 16.71.
4.1.6.4. 6-(4-Methoxyanilino)-4-oxo-2-thioxo-1,2,3,4-
tetrahydropyrimidine (10d). Yield, (9.60 g, 77%); mp
284–286 ꢁC (decomp., from DMF–H2O); UV (EtOH):
k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 204 (4.36), 278 (4.35);
IR (mmax/cmꢀ1): 3340, 3240, 3100 (NH), 1665 (C@O);
1H NMR [(CD3)2SO]: d 3.76 (3H, s, OMe), 4.66 (1H,
4.1.7.3. 6-(2,4-Dimethylanilino)-2-methylthio-4-oxo-
3,4-dihydropyrimidine (11c). Yield, (2.56 g, 98%); mp

936
H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
258–260 ꢁC (decomp., from DMF–H2O); UV (EtOH):
4.1.8.2. 7,9-Dimethyl-2-methylthiopyrimido[4,5-b]quino-
lin-4(3H)-ne (12b). Yield, (1.71 g, 63%), mp >300 ꢁC (de-
comp., from DMF); UV (EtOH): kmax/nm (loge/
dm3 molꢀ1 cmꢀ1): 230 (4.33), 244sh (4.38), 278sh
(4.55), 287 (4.62), 320sh (3.91), 369 (3.80), 393 (3.66);
IR (mmax/cmꢀ1): 3160 (NH), 1660 (C@O); 1H NMR
(CDCl3): d 2.47 (3H, s, 7-Me), 2.66 (3H, s, 9-Me), 2.70
(3H, s, 2-SMe), 7.61 (1H, s, 8-H), 7.77 (1H, s, 6-H),
8.98 (1H, s, 5-H), 12.69 (1H, br s, 3-NH, exchangeable
with D2O). Anal. Calcd for C14H13N3OSÆ0.4H2O: C,
60.37; H, 4.99; N, 15.09. Found: C, 60.15; H, 5.04; N,
14.88.
k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 208 (4.52), 245 (4.50),
286 (4.10); IR (mmax/cmꢀ1): 3390, 3140 (NH), 1640
1
(C@O); H NMR (CDCl3): d 2.22 (3H, s, 20-Me), 2.32
(3H, s, 40-Me), 2.53 (3H, s, 2-SMe), 5.04 (1H, s, 5-H),
6.16 (1H, s, 6-NH), 7.00 (1H, d, J5 ,6 = 7.8 Hz, Ar-50-
0
0
H), 7.06 (1H, s, Ar-30-H), 7.12 (1H, d, J5 ,6 = 7.8 Hz,
Ar-60-H), 12.26 (1H, br s, 3-NH). Anal. Calcd for
C13H15N3OS: C, 59.74; H, 5.79; N, 16.08. Found: C,
59.44; H, 5.85; N, 15.89.
0
0
4.1.7.4. 6-(4-Methoxyanilino)-2-methylthio-4-oxo-3,4-
dihydropyrimidine (11d). Yield, (2.24 g, 85%); mp 229–
231 ꢁC (decomp., from DMF–H2O); UV (EtOH):
4.1.9. General procedure for the preparation of 2-deoxo-2-
methylthioalloxazine-5-oxides {2-methylthio-4-oxo-3,4-
dihydrobenzo[g]pteridine-5-oxides} (13a–e). To a stirring
solution of 6-anilino-2-methylthio-4-oxo-3,4-dihydro-
pyrimidines (11, 0.01 mol) in acetic acid (5–15 mL) at
10–15 ꢁC was added sodium nitrite (0.02–0.04 mol) by
portions, and the mixture was stirred at room tempera-
ture with occasional warming in water bath to enhance
the cyclization for 1–2 h. The solid deposited was col-
lected by suction filtration and washed with water. The
filtrate was concentrated in vacuo and the residue was
diluted with excess water or neutralized with aqueous
ammonia (pH 7) to afford the second crop. The solid
dried was crystallized from a mixture of DMF and water
to afford the corresponding products as yellow or or-
ange needles in 66–89% yields.
k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 209 (4.45), 257 (4.48),
300 (4.21); IR (mmax/cmꢀ1): 3310, 3160 (NH), 1640
1
(C@O); H NMR (CDCl3): d 2.53 (3H, s, 2-SMe), 3.81
(3H, s, 40-OMe), 5.32 (1H, s, 5-H), 6.33 (1H, s, 6-NH),
6.88 (2H, d, JA,B = 9.0 Hz, Ar-oH), 7.15 (2H, d,
JA,B = 9.0 Hz, Ar-mH), 11.98 (1H, br s, 3-NH). Anal.
Calcd for C12H13N3O2S: C, 54.74; H, 4.98; N, 15.96.
Found: C, 54.78; H, 5.02; N, 15.79.
4.1.7.5. 6-(4-Hydroxyanilino)-2-methylthio-4-oxo-3,4-
dihydropyrimidine (11e). Yield, (2.34 g, 94%); mp 293–
295 ꢁC (decomp., from DMF–H2O); UV (EtOH):
k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 209 (4.54), 252 (4.49),
296 (4.20); IR (mmax/cmꢀ1): 3400 (OH), 3380, 3210
1
(NH), 1640 (C@O); H NMR [(CD3)2SO]: d 2.45 (3H,
s, 2-SMe), 5.06 (1H, s, 5-H), 6.72 (2H, d, JA,
B = 8.7 Hz, Ar-oH), 7.13 (2H, d, JA, B = 8.7 Hz, Ar-
mH), 8.75 (1H, s, 6-NH), 9.23 (1H, s, OH), 11.86 (1H,
br s, 3-NH). Anal. Calcd for C11H11N3O2SÆ0.1H2O: C,
52.62; H, 4.50; N,16.74. Found: C, 52.53; H, 4.68; N,
16.35.
4.1.9.1. 2-Methylthio-4-oxo-3,4-dihydrobenzo[g]pteri-
dine-5-oxide (13a). Yield, (2.32 g, 89%); mp >300 ꢁC (de-
comp., from DMF–H2O); UV (EtOH): kmax/nm (loge/
dm3 molꢀ1 cmꢀ1): 244 (4.30), 270 (4.45), 292 (4.42),
355 (3.87), 446 (3.81); IR (mmax/cmꢀ1): 3200 (NH),
1
1690 (C@O); H NMR [(CD3)2SO]: d 2.44 (3H, s, 2-
4.1.8. General procedure for the preparation of 2-deoxo-2-
methylthio-5-deazaalloxazines
SMe), 7.55 (1H, d t, J6,7 = J7,8 = 8.7 Hz, J7,9 = 1.5 Hz,
7-H), 7.76–7.87 (2H, m, 6 and 8-H), 8.32 (1H, dd,
J6,7 = 8.7, J6,8 = 0.9 Hz, 6-H), 12.69 (1H, br s, 3-NH,
exchangeable with D2O). Anal. Calcd for C11H8N4O2S:
C, 50.76; H, 3.10; N, 21.53. Found: C, 50.58; H, 3.39; N,
21.30.
{2-methylthiopyrimi-
do[4,5-b]quinolin-4(3H)-ones} (12a,b). A mixture of 6-
anilino-2-methylthio-4-oxo-3,4-dihydropyrimidines (11,
0.01 mol) and phosphoryl chloride (7.7 g, 0.05 mol) in
anhydrous DMF (10 mL) was heated under stirring
at 90 ꢁC for 1 h. Then, the reaction mixture was
poured onto ice and neutralized with aqueous ammo-
nia (pH 7). The yellow crystals separated were fil-
tered off, washed with water, dried, and
recrystallized from an appropriate solvent to afford
the corresponding products as pale yellow needles
in 63–72% yields.
4.1.9.2. 7,9-Dimethyl-2-methylthio-4-oxo-3,4-dihydro-
benzo[g]pteridine-5-oxide (13b). Yield, (2.36 g, 82%); mp
>300 ꢁC (decomp., from DMF–H2O); UV (EtOH):
k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 247 (4.42), 275 (4.79),
298 (4.71), 361 (3.92), 453 (3.99); IR (mmax/cmꢀ1): 3180
1
(NH), 1680 (C@O); H NMR [(CD3)2SO]: d 2.49 (3H,
s, 7-Me), 2.61 (3H, s, 2-SMe), 2.62 (3H, s, 9-Me), 7.61
(1H, br s, 8-H), 7.98 (1H, br s, 6-H), 12.76 (1H, br s,
3-NH, exchangeable with D2O). Anal. Calcd
C13H12N4O2S: C, 54.15; H, 4.19; N, 19.43. Found: C,
54.25; H, 4.53; N, 19.37.
4.1.8.1. 2-Methylthiopyrimido[4,5-b]quinolin-4(3H)-
one (12a). Yield, (1.75 g, 72%), mp 292–294 ꢁC (de-
comp., from DMF–H2O); UV (EtOH):
kmax/nm
(loge/dm3 molꢀ1 cmꢀ1): 226 (4.49), 239sh (4.35),
273sh (4.54), 283 (4.64), 313sh (3.78), 366 (3.86), 381
(3.75); IR (mmax/cmꢀ1): 3160 (NH), 1680 (C@O); 1H
NMR (CDCl3): d 2.83 (3H, s, 2-SMe), 7.58 (1H, br
t, J6,7 = J7,8 = 7.5 Hz, 7-H), 7.86 (1H, br t, J7,8 = J8,9
= 7.5 Hz, 8-H), 7.99 (1H, br d, J8,9 = 7.5 Hz, 9-H),
8.22 (1H, br d, J6,7 = 7.5 Hz, 6-H), 9.16 (1H, s, 5-
H), 9.43 (1H, br s, 3-NH, exchangeable with D2O).
Anal. Calcd for C12H9N3OSÆ0.1H2O: C, 58.81; H,
3.78; N, 17.15. Found: C, 58.79; H, 4.08; N, 16.76.
4.1.9.3. 7-Methoxy-2-methylthio-4-oxo-3,4-dihydro-
benzo[g]pteridine-5-oxide (13c). Yield, (2.38 g, 82%);
mp >300 ꢁC (decomp., from DMF–H2O); UV (EtOH):
k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 242 (4.19), 275sh
(4.61), 285 (4.72), 352 (3.83), 457 (3.94); IR (mmax
/
cmꢀ1): 3175 (NH), 1695 (C@O); H NMR [(CD3)2SO]:
d 2.59 (3H, s, 2-SMe), 3.97 (3H, s, 7-OMe), 7.60 (1H,
dd, J8,9 = 9.3 Hz, J6,8 = 2.7 Hz, 8-H), 7.67 (1H, d,
1

H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
937
J6,8 = 2.7 Hz, 6-H), 7.93 (1H, d, J8,9 = 9.3 Hz, 9-H),
12.78 (1H, br s, 3-NH, exchangeable with D2O). Anal.
Calcd for C12H10N4O3S: C, 49.65; H, 3.47; N, 19.30.
Found: C, 49.79; H, 3.76; N, 19.33.
(1.22 g, 82%); mp 273–275 ꢁC (decomp., from EtOH);
UV (EtOH): kmax/nm (log e/dm3 molꢀ1 cmꢀ1): 246
(4.42), 266 (4.64), 287 (4.36), 322 (3.96), 366 (3.65); IR
(mmax/cmꢀ1): 3260 (NH), 1660 (C@O); 1H NMR
(CDCl3): d 2.48 (3H, s, 7-Me), 2.74 (3H, s, 9-Me), 3.35
(3H, s, 2-NMe), 3.73 (2H, br s, 2-N–CH2–CH2–OH),
3.88 (2H, br s, 2-N–CH2–CH2–OH), 5.50 (1H, br s, 2-
N–CH2–CH2–OH, exchangeable with D2O), 7.50 (1H,
s, 8-H), 7.52 (1H, s, 6-H), 8.82 (1H, s, 5-H), 11.09
(1H, br s, 3-NH, exchangeable with D2O). Anal. Calcd
for C16H18N4O2Æ0.2EtOH: C, 64.05; H, 6.29; N, 18.22.
Found: C, 63.74; H, 6.29; N, 17.83.
4.1.9.4. 7-Methyl-2-methylthio-4-oxo-3,4-dihydroben-
zo[g]pteridine-5-oxide (13d). Yield, (1.81 g, 66%); mp
>300 ꢁC (decomp., from DMF–H2O); UV (EtOH):
k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 241 (4.10), 274 (4.56),
295 (4.50), 351 (3.85), 443 (3.85); IR (mmax/cmꢀ1): 3175
1
(NH), 1695 (C@O); H NMR [(CD3)2SO]: d 2.55 (3H,
s, 7-Me), 2.59 (3H, s, 2-SMe), 7.78 (1H, d,
J8,9 = 8.4 Hz, 9-H), 7.90 (1H, br d, J8,9 = 8.4 Hz, 8-H),
8.16 (1H, br s, 6-H), 12.79 (1H, br s, 3-NH, exchange-
able with D2O). Anal. Calcd for C12H10N4O2S: C,
52.54; H, 3.67; N, 20.43. Found: C, 52.58; H, 4.06; N,
20.19.
4.1.10.3. Preparation of 7-methoxy-2-morpholino-4-
oxo-3,4-dihydrobenzo[g]pteridine (15a). A mixture of 7-
methoxy-2-methylthio-4-oxo-3,4-dihydrobenzo[g]pteri-
dine-5-oxide (13c, 2.90 g, 0.01 mol) and morpholine
(8.71 g, 0.10 mol) in DMF (30 mL) was refluxed with
stirring for 6 h. After the resulting solution was kept
in refrigerator overnight, the yellow crystals precipitated
were collected by filtration to get the product which was
washed with ethyl acetate, dried, and recrystallized from
DMF to give the yellow pure product 15a.
4.1.9.5. 7-Hydroxy-2-methylthio-4-oxo-3,4-dihydro-
benzo[g]pteridine-5-oxide (13e). Yield, (1.81 g, 66%);
mp >300 ꢁC (decomp., from DMF–H2O); UV (EtOH):
k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 222 (4.35), 280 (4.28),
422 (3.70); IR (mmax/cmꢀ1): 3260 (OH), 3100 (NH),
1
1700 (C@O); H NMR [(CD3)2SO]: d 2.61 (3H, s, 2-
SMe), 7.39 (1H, d, J8,9 = 9.0 Hz, 9-H), 8.24 (1H, dd,
J8,9 = 9.0 Hz, J6,8 = 2.7 Hz, 8-H), 8.73 (1H, d,
J6,8 = 2.7 Hz, 6-H), 11.67 (1H, s, 7-OH), 13.19 (1H, br
s, 3-NH exchangeable with D2O). Anal. Calcd for
C11H8N4O3SÆ0.6H2O: C, 46.02; H, 3.23; N, 19.52.
Found: C, 45.61; H, 3.41; N, 19.49.
Yield, (2.10 g, 67%); mp >300 ꢁC (decomp., from
DMF); UV (EtOH): kmax/nm (loge/dm3 molꢀ1 cmꢀ1):
224 (4.14), 275 (4.47), 332 (3.51), 446 (3.85); IR (mmax
/
cmꢀ1): 3135 (NH), 1698 (C@O); H NMR (CDCl3): d
3.92 (4H, br m, 20 and 60-CH2), 3.93 (4H, br m, 30 and
50-CH2), 3.98 (3H, s, 7-OMe), 7.45–7.57 (2H, m, 6 and
8-H), 7.96 (1H, d, J8,9 = 9.0 Hz, 9-H), 10.62 (1H, br s,
3-NH, exchangeable with D2O). Anal. Calcd for
C15H15N5O3: C, 57.50; H, 4.83; N, 22.35. Found: C,
57.15; H, 5.02; N, 22.44.
1
4.1.10. General procedure for the preparation of 2-
alkylaminopyrimido[4,5-b]quinolin-4-(3H)-one (14a,b). A
mixture of 2-methylthiopyrimido[4,5-b]quinolin-4(3H)-
ones (12a, 5.0 mmol) and appropriate amine (0.035–
0.05 mol) in DMF (30 mL) for compound 14a or in n-
butanol (20 mL) for compound 14b was refluxed with
stirring for 15–24 h. After the resulting clear yellow solu-
tion was kept in refrigerator overnight, the yellow crys-
tals precipitated were collected by filtration to get the
first crop. The filtrate was concentrated in vacuo to re-
move the excess amine and the residue was treated with
water to get the second crop free from amine. The col-
lected solids were dried and recrystallized from ethanol
to give the pure products as yellow needles in 82–84%
yields.
4.1.10.4. Preparation of 2-dimethylamino-4-oxo-3,4-
dihydrobenzo[g]pteridine (15b). A mixture of 2-methyl-
thio-4-oxo-3,4-dihydrobenzo[g]pteridine-5-oxide (13a,
2.6 g, 0.01 mol) and 50% aqueous dimethylamine
(50 mL) was heated in steel sealed tube at 135 ꢁC
(10 kg/cm2 pressure) for 8 h. After the reaction was com-
plete, the resulting clear yellow solution was concen-
trated in vacuo and the yellow solid residue was
washed with water, dried, and crystallized from ethanol
to afford the pure product as yellow crystals.
Yield, (2.08 g, 86%); mp >300 ꢁC (decomp., from H2O);
4.1.10.1. 2-Morpholinopyrimido[4,5-b]quinolin-4(3H)-
one (14a). Yield, (1.19 g, 84%); mp 288–291 ꢁC (de-
comp., from EtOH); UV (EtOH): kmax/nm (loge/
dm3 molꢀ1 cmꢀ1): 225 (4.53), 263 (4.45), 280 (4.57),
321sh (3.88), 408 (3.97), 434 (3.39); IR (mmax/cmꢀ1):
3260 (NH), 1660 (C@O);1H NMR (CDCl3): d 3.82–
3.90 (4H, m, 20 and 60-CH2), 3.90–4.01 (4H, m, 30 and
50-CH2), 7.47 (1H, t, J6,7 = J7,8 = 7.2 Hz, 7-H), 7.79
UV (EtOH): k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 221
(4.38), 269 (4.31), 287 (4.29), 334 (3.62), 376sh (3.58),
1
399 (3.68), 430 (3.78); IR (mmax/cmꢀ1): 1700 (C@O); H
NMR (CDCl3): d 3.39 (6H, s, 2-N (CH3)2), 7.64 (1H,
dt, J6,7 = J7,8 = 8.4 Hz, J7,9 = 1.5 Hz, 7-H), 7.79 (1H,
dt, J7,8 = J8,9 = 8.4 Hz, J6,8 = 1.5 Hz, 8-H), 8.02 (1H, d,
J8,9 = 8.4 Hz, 9-H), 8.21 (1H, d, J6,7 = 8.4 Hz, 6-H).
Anal. Calcd for C12H11N5OÆ0.1H2O: C, 59.30; H, 4.64;
N, 28.81. Found: C, 59.23; H, 4.72; N, 28.41.
(1H, t, J7,8 = J8,9 = 7.2 Hz, 8-H), 7.92 (1H, d, J8,9
=
7.2 Hz, 9-H), 7.98–8.12 (1H, m, 6-H), 8.96 (1H, s, 5-
H), 10.65 (1H, br s, 3-NH, exchangeable with D2O).
Anal. Calcd for C15H14N4O2Æ0.2H2O: C, 63.02; H,
5.08; N, 19.60. Found: C, 62.76; H, 5.19; N, 19.36.
4.1.11. General procedure for the preparation of 3-alkyl-
2-deoxo-2-methylthioalloxazine-5-oxides {3-alkyl-2-
methylthio-4-oxo-3,4-dihydrobenzo[g]pteridine-5-oxides}
(16a–e).
dihydrobenzo[g]pteridine-5-oxides (13, 5.0 mmol), alkyl
iodide or alkyl bromide (0.015–0.025 mol), and anhy-
A
mixture of 2-methylthio-4-oxo-3,4-
4.1.10.2. 2-[N-(2-Hydroxyethyl)-N-methylamino]-7,9-
dimethylpyrimido[4,5-b]quinolin-4(3H)-one (14b). Yield,

938
H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
drous potassium carbonate (2.76 g, 0.02 mol) in DMF
(30 mL) was stirred at room temperature for 3–10 h.
After the reaction was complete, the insoluble potassium
carbonate was filtered off and washed with CH2Cl2. The
combined solution of the filtrate and washing solution
of CH2Cl2 was concentrated in vacuo. The residue was
dissolved in water and extracted with CH2Cl2 (3·
50 mL), dried over anhydrous magnesium sulfate and
filtered. Then, the solvent was removed in vacuo to af-
ford the solid, which was collected by filtration, dried,
and crystallized from ethanol to afford the correspond-
ing products as yellow crystals in 71–82% yields.
J6,8 = 1.8 Hz, 6-H). Anal. Calcd for C15H14N4O4S: C,
52.02; H, 4.07; N, 16.18. Found: C, 52.24; H, 4.38; N,
15.85.
4.1.11.5. 3,7-Dimethyl-2-methylthio-4-oxo-3,4-dihy-
drobenzo[g]pteridine-5-oxide (16e). Yield, (1.14 g, 79%);
mp 211–213 ꢁC (decomp., from EtOH); UV (EtOH):
k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 229 (3.42), 278 (4.10),
299 (4.07), 338sh (3.48), 427 (3.37), 449sh (3.21); IR
(mmax/cmꢀ1): 1700 (C@O); 1H NMR (CDCl3): d 2.60
(3H, s, 7-Me), 2.78 (3H, s, 2-SMe), 3.64 (3H, s, 3-
NMe), 7.68 (1H, dd, J8,9 = 8.7 Hz, J6,8 = 2.1 Hz, 8-H),
8.02 (1H, d, J8,9 = 8.7 Hz, 9-H), 8.34 (1H, br s, 6-H).
Anal. Calcd for C13H12N4O2S: C, 54.15; H, 4.19; N,
19.43. Found: C, 54.48; H, 4.34; N, 19.17.
4.1.11.1. 3-Methyl-2-methylthio-4-oxo-3,4-dihydro-
benzo[g]pteridine-5-oxide (16a). Yield, (1.13 g, 82%);
mp 231–233 ꢁC (decomp., from EtOH); UV (EtOH):
k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 226 (3.72), 272 (4.32),
4.2. Growth inhibitory activities of test compounds against
human tumor cell lines
299 (4.40), 337sh (3.77), 422 (3.64), 445sh (3.48); IR
(mmax/cmꢀ1): 1700 (C@O); 1H NMR (CDCl3): d 2.79
(3H, s, 2-SMe), 3.64 (3H, s, 3-NMe), 7.66 (1H, d t,
J6,7 = 9.0 Hz, J7,8 = 8.7 Hz, J7,9 = 1.5 Hz, 7-H), 7.85
(1H, d t, J7,8 = J8,9 = 8.7 Hz, J6,8 = 1.5 Hz, 8-H), 8.13
(1H, br d, J8,9 = 8.7, 9-H), 8.56 (1H, br d, J6,7 = 9.0, 6-
H). Anal. Calcd for C12H10N4O2S: C, 52.54; H, 3.67;
N, 20.43. Found: C, 52.67; H, 3.94; N, 20.74.
The procedure was carried out using the modified 3-(3,4-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) colorimetric assay25 to determine the inhibitory
effects of test compounds, namely, 10-alkyl-2-deoxo-2-
methylthioflavin-5-oxides (2a–i), 10-alkyl-2-alkylami-
no-2-deoxoflavin-5-oxides (4a–e), 10-alkylflavins (5a
and b), 7-methoxy-10-methylflavin-5-oxide (6), 2-alkyla-
mino-2-deoxo-10-methyl-5-deazaflavins (8a and b), 2-
deoxo-10-methyl-2-methylaminoflavin-5-oxide (9a),
2-deoxo-2-methylthio-5-deazaalloxazine (12a), 2-deo-
xo-2-methylthioalloxazine-5-oxides (13a–d), 2-alkylami-
no-2-deoxoalloxazines (15a and b), and 3-alkyl-2-deoxo-
2-methylthioalloxazine-5-oxides (16a–e), on cell growth
in vitro as mentioned in detail in our previous paper.6
Two human tumor cell lines CCRF-HSB-2 (human
T-cell acute lymphoblastoid leukemia) and KB (human
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.
4.1.11.2. 3,7,9-Trimethyl-2-methylthio-4-oxo-3,4-dihy-
drobenzo[g]pteridine-5-oxide (16b). Yield, (1.21 g, 80%);
mp 264–266 ꢁC (decomp., from EtOH); UV (EtOH):
k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 225 (3.79), 280 (4.52),
304 (4.55), 344sh (3.72), 431 (3.72), 458sh (3.50); IR
(mmax/cmꢀ1): 1698 (C@O); 1H NMR (CDCl3): d 2.54
(3H, s, 7-Me), 2.79 (3H, s, 2-SMe), 2.81 (3H, s, 9-Me),
3.63 (3H, s, 3-NMe), 7.53 (1H, br s, 8-H), 8.20 (1H, br
s, 6-H). Anal. Calcd for C14H14N4O2S: C, 55.61; H,
4.67; N, 18.53. Found: C, 55.20; H, 4.92; N, 18.20.
4.1.11.3. 3-Ethyl-7,9-dimethyl-2-methylthio-4-oxo-3,4-
dihydrobenzo[g]pteridine-5-oxide (16c). Yield, (1.12 g,
71%); mp 210–212 ꢁC (decomp., from EtOH); UV
(EtOH): kmax/nm (loge/dm3 molꢀ1 cmꢀ1): 227 (3.63),
280 (4.49), 304 (4.56), 343sh (3.77), 432 (3.77), 457sh
(3.56); IR (mmax/cmꢀ1): 1698 (C@O); 1H NMR (CDCl3):
d 1.41 (3H, t, J = 7.2 Hz, 3-NCH2–CH3), 2.54 (3H, s, 7-
Me), 2.79 (3H, s, 2-SMe), 2.80 (3H, s, 9-Me), 4.22 (2H,
q, J = 7.2 Hz, 3-NCH2–CH3), 7.53 (1H, br s, 8-H), 8.20
(1H, br s, 6-H). Anal. Calcd for C15H16N4O2S: C, 56.94;
H, 5.10; N, 17.71. Found: C, 56.75; H, 5.25; N,
17.38.
4.3. Molecular docking study
The automated docking studies were carried out using
AutoDock version 3.0.5.26 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. The cubic grid
˚
˚
box of 80 A size (x,y,z) with a spacing of 0.375 A
and grid maps were created representing the catalytic
active target site region where the native ligand was
embedded. Then automated docking studies were car-
ried out to evaluate the binding free energy of the
inhibitors within the macromolecules. The GA-LS
search algorithm (Genetic algorithm with local search)
was chosen to search for the best conformers. The
parameters were set using the software ADT (Auto-
Dock Tool Kit) on PC which is associated with Auto-
Dock 3.0.5. For all docking parameters, default values
were used with 10 independent docking runs for each
docking case. The AutoDock performs the task of the
docking, where the 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
4.1.11.4. 3-(Ethoxycarbonylmethyl)-2-methylthio-4-
oxo-3,4-dihydrobenzo[g]pteridin-5-oxide (16d). Yield,
(1.28 g, 74%); mp 212–214 ꢁC (decomp., from EtOH);
UV (EtOH):
k
max/nm (loge/dm3 molꢀ1 cmꢀ1): 219
(4.31), 272 (4.51), 299 (4.63), 336sh (3.95), 415 (3.79),
443sh (3.60); IR (mmax/cmꢀ1): 1740, 1705 (C@O); 1H
NMR (CDCl3):
d
1.31 (3H, t, J = 7.2 Hz, 3-
CH2COOCH2CH3), 2.81 (3H, s, 2-SMe), 4.28 (2H, q,
J = 7.2 Hz, 3-NCH2COOCH2CH3), 4.91 (2H, s, 3-
NCH2COOCH2CH3), 7.68 (1H,
8.7 Hz, J7,9 = 1.8 Hz, 7-H), 7.88 (1H, d t, J7,8 = J8,9
d
t, J6,7 = J7,8
=
=
8.7 Hz, J6,8 = 1.8 Hz, 8-H), 8.14 (1H, dd, J8,9 = 8.7 Hz,
J7,9 = 1.8 Hz, 9-H), 8.55 (1H, dd, J6,7 = 8.7 Hz,

H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
939
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 measure-
ments and the Okayama University Information Tech-
nology Center for using Accelrys Discovery Studio 1.7.
4.3.1. Preparation of ligands and target protein–tyrosine
kinase. The compounds involved in this study as ligands
include 10-alkyl-2-deoxo-2-methylthioflavin-5-oxides
(2), 2-dimethylaminoalloxazine (3), 10-alkyl-2-alkylamino-
2-deoxoflavin-5-oxides (4), 10-alkylflavins (5),
7-methoxy-10-methylflavin-5-oxide (6), 2-alkylamino-2-
deoxo-10-methyl-5-deazaflavins (8), 2-alkylamino-2-
deoxo-10-methylflavin-5-oxides (9), 2-methylthio-2-deo-
xo-5-deazaalloxazines (12), 2-deoxo-2-methylthioallox-
References and notes
1. Sakuma, Y.; Nagamatsu, T.; Yoneda, F. J. Chem. Soc.
Chem. Commun. 1975, 977.
2. Sakuma, Y.; Matsumoto, S.; Nagamatsu, T.; Yoneda, F.
Chem. Pharm. Bull. 1976, 24, 338.
3. Nagamatsu, T.; Yamato, H.; Ono, M.; Takarada, S.;
azine-5-oxides
(13),
2-alkylamino-2-deoxo-5-
deazaalloxazines (14), 2-alkylamino-2-deoxoalloxazines
(15), and 3-alkyl-2-deoxo-2-methylthioalloxazine-5-oxi-
des (16) which were studied for their binding activities
into PTK. The three-dimensional structures of the afore-
mentioned compounds were constructed using Chem 3D
ultra 8.0 software [Chemical Structure Drawing Stan-
dard; Cambridge Soft corporation, USA (2003)], then
they were energetically minimized by using MOPAC
with 100 iterations and minimum RMS gradient of
Yoneda, F. J. Chem. Soc. Perkin Trans.
2101.
1 1992,
4. Nagamatsu, T.; Kuroda, K.; Mimura, N.; Yanada, R.;
Yoneda, F. J. Chem. Soc. Perkin Trans. 1 1994, 1125.
5. Nagamatsu, T.; Yamada, H.; Shiromoto, K. Heterocycles
2004, 63, 9.
6. Ali, H. I.; Tomita, K.; Akaho, E.; Kambara, H.; Miura,
S.; Hayakawa, H.; Ashida, N.; Kawashima, Y.; Yamag-
ishi, T.; Ikeya, H.; Yoneda, F.; Nagamatsu, T. Biorg. Med.
Chem. 2007, 15, 242.
7. Platz, M. S. U.S. Patent 6,933,285, 2005.
8. Kawamoto, T.; Ikeuchi, Y.; Hiraki, J.; Eikyu, Y.; Shimizu,
K.; Tomishima, M.; Bessho, K.; Yoneda, F.; Mikata, Y.;
Nishida, M.; Ikehara, K.; Sasaki, T. Bioorg. Med. Chem.
Lett. 1995, 5, 2109.
9. Kawamoto, T.; Ikeuchi, Y.; Hiraki, J.; Eikyu, Y.; Shimizu,
K.; Tomishima, M.; Bessho, K.; Yoneda, F.; Mikata, Y.;
Nishida, M.; Ikehara, K. Bioorg. Med. Chem. Lett. 1995,
5, 2115.
0.10. The Gasteiger-Huckel charges of ligands were as-
¨
signed. The crystal structures of c-Kit receptor pro-
tein-tyrosine kinase in complex with STI-571 (Imatinib
or Gleevec) were retrieved from the RCSB Protein Data
Bank http://www.rcsb.org/pdb/Welcome.do (PDB code:
1t46)27 accessed on April 4, 2006. All bound waters, li-
gand were removed from the protein and the polar
hydrogens and the Kollman-united charges were added
to the proteins. The amino acids of the ligand-target
binding site were defined using data in pdbsum
http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/
accessed on June 10, 2006.
10. Ikeuchia, Y.; Sumiya, M.; Kawamoto, T.; Akimoto, N.;
Mikata, Y.; Kishigami, M.; Yano, S.; Sasaki, T.; Yoneda,
F. Biorg. Med. Chem. 2000, 8, 2027.
11. Kanaoka, Y.; Ikeuchi, Yo.; Kawamoto, T.; Bessho, K.;
Akimoto, N.; Mikata, Y.; Nishida, M.; Yano, S.; Sasaki,
T.; Yoneda, F. Biorg. Med. Chem. 1998, 6, 301.
12. Kimachi, T.; Yoneda, F.; Sasaki, T. J. Heterocycl. Chem.
1992, 29, 763.
13. Fall, H. H.; Petering, H. G. J. Am. Chem. Soc. 1956, 78,
377.
14. Petering, H. G.; Giessen, G. J. V. J. Org. Chem. 1961, 26,
2818.
15. Petrova, L. F.; Pushkareva, Z. V.; Vavilov, G. A. KhGs
(Chem. Heterocycl. Compd.) 1967, 3, 734.
16. Wilson, J. M.; Henderson, G.; Black, F.; Sutherland, A.;
Ludwig, R. L.; Vousden, K. H.; Robins, D. J. Biorg. Med.
Chem. 2007, 15, 77.
4.3.2. Molecular modeling and analysis of the docked
results. The predicted binding free energy that includes
the intermolecular 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. Clus-
ter analysis was performed on the docked results using
˚
a root mean square (RMS) tolerance of 0.5 A. Each of
the clusters that exhibited significant negative interac-
tion energies was examined by Accelrys, DS modeling
1.7 [Accelrys Inc., San Diego CA (2006)] to determine
their binding orientations, molecular modeling, evalua-
tion of the hydrogen bonds, and for measuring
RMSD, which was measured as distance between the
centroids of the docked inhibitor and the native li-
gand. The mode of interaction of the native ligand
(STI-571) within the crystal structure of c-Kit receptor
protein–tyrosine kinase was used as a standard docked
model as well as for RMSD calculation. Regarding
the hydrogen bond interaction, the more linear hydro-
gen bond is likely to be more stronger.29 Therefore, in
our modeling results we consider the hydrogen bond
angle within about 120ꢁ to be of a reasonable
strength.
17. Sarhan, A. A. O.; Hozien, Z. A.; El-Sherief, H. A. H.
Biorg. Med. Chem. 2001, 9, 2993.
18. Ali, H. I.; Ashida, N.; Nagamatsu, T. Bioorg. Med. Chem.
2007, 15, 6336.
19. Israel, M.; Protopapa, H. K.; Schlein, H. N.; Modest, E. J.
J. Med. Chem. 1964, 7, 5.
20. Wheeler, H. L.; Jamieson, G. S. Am. Chem. J. 1904, 32,
342.
21. Curran, W. V.; Angier, R. B. J. Org. Chem. 1963, 28,
2672.
22. Ali, H. I. Ph.D. thesis, Okayama University, 2007;
Okayama, Japan.
23. Hubsch, W.; Pfleiderer, W. Helv. Chim. Acta 1988, 71,
¨
1379.
24. Nogimori, T.; Emerson, C. H.; Braverman, L. E.; Wu, C.
F.; Gambino, J.; Wrighr, G. E. J. Med. Chem. 1985, 28,
1692.

940
H. I. Ali et al. / Bioorg. Med. Chem. 16 (2008) 922–940
25. Miura, S.; Yoshimura, Y.; Endo, M.; Machida, H.;
Matsuda, A.; Tanaka, M.; Sasaki, T. Cancer Lett. 1998,
129, 103.
26. 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.
27. Mol, C. D.; Douglas, 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.
28. Yoneda, F.; Sakuma, Y.; Ichiba, M.; Shinomura, K.
J. Am. Chem. Soc. 1976, 98, 830.
29. Taylor, R.; Kennard, O. Acc. Chem. Res. 1984, 17, 320.