Synthesis, biological active molecular design, and molecular docking study of novel deazaflavin-cholestane hybrid compounds

Article abstract of DOI:10.1016/j.bmc.2008.07.089

Novel deazaflavin-cholestane hybrid compounds, 3′,8′-disubstituted-5′-deazacholest-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-diones, have been synthesized by condensation reaction between 6-(monosubstituted amino)-pyrimidin-2,4(1H,3H)-diones and 2-hydroxymethylenecholest-4-en-3-one in presence of p-toluenesulfonic acid monohydrate and diphenyl ether. The antitumor activities against human tumor cell lines (CCRF-HSB-2 and KB cells) have been investigated in vitro, and many of these compounds showed promising antitumor activities. Furthermore, molecular docking study using LigandFit within the software package Discovery Studio 1.7 was done for lead optimization of these compounds as potential PTK inhibitors. In general, all of the synthesized steroid-hybrid compounds showed good binding affinities into PTK (PDB code: 1t46).

Full text of DOI:10.1016/j.bmc.2008.07.089

Bioorganic & Medicinal Chemistry 16 (2008) 8685–8696
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological active molecular design, and molecular
docking study of novel deazaflavin–cholestane hybrid compounds
Ajaya R. Shrestha a, Takashi Shindo a, Noriyuki Ashida b, Tomohisa Nagamatsu a,
*
a Division of Pharmaceutical Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University 1-1-1, Tsushima-naka, Okayama 700-8530, Japan
b Biology Laboratory, Research and Development Division, Yamasa Shoyu Co. Choshi, Chiba 288-0056, Japan
a r t i c l e i n f o
a b s t r a c t
Novel deazaflavin–cholestane hybrid compounds, 30,80-disubstituted-50-deazacholest-2,4-dieno[2,3-
g]pteridine-20,40(30H,80H)-diones, have been synthesized by condensation reaction between 6-(monosub-
stituted amino)-pyrimidin-2,4(1H,3H)-diones and 2-hydroxymethylenecholest-4-en-3-one in presence
of p-toluenesulfonic acid monohydrate and diphenyl ether. The antitumor activities against human
tumor cell lines (CCRF-HSB-2 and KB cells) have been investigated in vitro, and many of these compounds
showed promising antitumor activities. Furthermore, molecular docking study using LigandFit within the
software package Discovery Studio 1.7 was done for lead optimization of these compounds as potential
PTK inhibitors. In general, all of the synthesized steroid-hybrid compounds showed good binding affini-
ties into PTK (PDB code: 1t46).
Article history:
Received 15 July 2008
Revised 28 July 2008
Accepted 29 July 2008
Available online 22 August 2008
Keywords:
5-Deazaflavin
Cholestane
Hybrid compound
LigandFit
Ó 2008 Elsevier Ltd. All rights reserved.
Protein tyrosine kinase
1. Introduction
ins, 10-substituted-2-deoxo-2-phenyl-5–deazaflavins {2-phen-
ylpyrimido[4,5-b]quinolin-4(10H)-ones} and flavin-5-oxides have
Design and synthesis of new type of pharmacologically interest-
ing steroidal hybrid compounds have received much attention dur-
ing the past two decades. Recently, we have reported the synthesis
of new hybrid compounds such as androstanolone with 5-deazaf-
lavins and their potential anticoccidiosis activities.1 These com-
pounds were synthesized with the rationale that steroids can
penetrate the cell membrane, bind the cell nucleus and steroid
receptors may serve to localize and concentrate appended drug
species, mainly in hormones responsive cancers. Moreover,
5-deazaflavins (5-deazaisoalloxazines) have greatly attracted our
interest.2–7 Actually, the interest in 5-deazaflavin is by reason of
their first synthesis as potential flavin antagonists or flavin mod-
els,8 the discovery that they can serve as co-factors for several fla-
vin-catalyzed reactions9 and having the potent broad-spectrum
activity against coccidiosis.10
revealed that they can exhibit significant potential antitumor activ-
ities against NCI-H 460, HCT 116, A 431, CCRF-HSB-2, and KB cells.2
(Scheme 1).
Molecular docking methodologies are powerful tools in drug
design, which avail to predict the best mode by which a given
compound will fit into a binding site of a macromolecular target.14
The in silico-based docking methods are of practical importance
for lead-compound generation in drug discovery.15 Molecular
docking enables one to rapidly screen large chemical databases
and thereby identify promising candidate compounds for further
experimental processing.16 Many dockingprogramssuch asDOCK,17
FlexX,18 GOLD,19 AutoDock,20 GLIDE,21 QXP,22 and ICM23 have been
developed to achieve the purpose. In this study, we present an
approachof flexibledockingof molecules usingthedockingprogram
It has been reported that the redox active coenzyme Factor 420
(F420), which has 5-deazaflavin skeleton and absorbance maximum
at 420 nm, was isolated from methanogenic bacteria.11 Also,
5-deazaflavins have been known for their strong oxidizing ability
which can oxidize alcohols and amines to the corresponding car-
bonyl compounds, and they behave as autorecycling turnover cata-
lysts in the redox reaction.12,13 In addition, our previous study on the
selective protein kinase C (PKC) inhibitory activities of 5-deazaflav-
C8H17
17
16
CH3
12
H3C11
O
N
O
4'
13
14
5'
9 H
R1
O
1
R1
3'N
10
X
N
6'
2
8 H
15
H
3
7'
7
5
O 2' N
N
N
R2
8'
6
4
1'
R2
X= CH: 5-Deazaflavin 3',8'-Disubstituted-5'-deaza-cholest-2,4-
X= N: Flavin
dieno[2,3-g]pteridine-2',4'(3'H,8'H)-diones
* Corresponding author. Tel.: +81 86 251 7931; fax: +81 86 251 7926.
E-mail address: nagamatsu@pheasant.pharm.okayama-u.ac.jp (T. Nagamatsu).
Scheme 1. Flavin, 5-deazaflavin and 30,80-disubstituted-50-deazacholest-2,4-die-
no[2,3-g]pteridine-20,40(30H,80H)-diones.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.089

8686
A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 8685–8696
LigandFit within the software package Discovery Studio 1.7. Ligand-
Fit24 is said to provide a rapid accurate protocol for docking small
molecule ligands into protein active sites by considering shape com-
plimentarity between the ligand and the protein active site. Ligand
docking is a method in which the conformational selection of com-
pounds is of high impact for the feasibility of the proposed binding
poses. The program processes compound collections in two steps
viz. the docking step, which uses combination of a shape-based filter
with a Monte-Carlo conformational search and the scoring step,
which can be performed with one or several scoring functions.25
In the present study, we describe the novel synthesis of new
archetype of deazaflavin–steroid hybrids which are biologically
and pharmacologically different active compounds. As a new trial
for the active molecular design of the drug, we have selected cho-
lesterol as the steroid moiety. The target molecule is composed of a
six-membered ring system having deazaflavin moiety incorporated
onto the A-ring of cholesterol. Also, the compounds were docked
into the binding pocket of PTK using LigandFit within the software
package Discovery Studio 1.7.
and p-toluenesulfonic acid monohydrate (0.01 equiv) in diphenyl
ether (1 mL) under an environment of nitrogen with constant stir-
ring at 180 °C for 45 min. It has been found that temperature higher
than the 180 °C could lead to the formation of other side products,
and lower temperature causes the reduction in the overall yield of
the product. Also, diphenyl ether has been found to be appropriate
solvent for the reaction than other usual solvents like dioxane,
dimethylformamide, and acetic anhydride, acetic acid, etc. The
products (5, 7, and 9) thus obtained were separated by flash column
chromatography on silica gel (230–400 mesh, Fuji Silysia Co. Ltd,
BW-300; eluent: ethyl acetate).
UV–vis, IR, NMR spectra, and elemental analyses were used for
the determination and identification of the synthesized novel
steroidal hybrid compounds. The structures of the compounds, in
particular, were confirmed by 1H NMR and 13C NMR, where choles-
terol moiety and deazaflavin moiety both showed their character-
0
istic peaks. Particularly, the characteristic singlet signal of C5 -H at
the lower field at dH 8.2–8.6 strongly supports the presence of
deazaflavin moiety in the skeleton of the hybrid compounds. The
appearance of the peak at such downfield region indicates that
0
the methine at C5 -position is more electron-deficient than other
methine. It has been found that the aryl substituents in the 80-po-
2. Result and discussion
2.1. Chemistry
0
sition of the hybrid compound cause the deshielding of the C5 -pro-
ton, exhibiting the signal at d H8.35–8.46. This may probably be
due to the electron-withdrawing inductive effect of the aromatic
ring. Steroidal moiety of the hybrid compound had appeared at
the upper field at dH 0.7–3.3, showing the presence of characteristic
cholesterol peaks. The geminal protons at the C1-position of
cholestane-2,4-diene appeared in the region dH 2.6–3.0 with the
coupling constant value of 15.6–16.2 Hz, in agreement with
usually found value for geminal coupling constant (12–18 Hz).
The requisite precursors, 6-(monosubstituted amino)-3-meth-
ylpyrimidin-2,4(1H,3H)-diones (4a–j), 6-(monosubstituted amino)
-pyrimidin-2,4(1H,3H)-diones (6a–i), and 6-(monosubstituted
amino)-3-phenylpyrimidin-2,4(1H,3H)-diones (8a–g), were pre-
pared by direct fusion of 6-chloro-3-methylpyrimidin-2,6(1H,
3H)-dione,26 6-chloropyrimidin-2,6(1H,3H)-dione26 and 6-chloro-
3-phenylpyrimidin-2,6(1H,3H)-dione27 with appropriate alkyl or
aryl amino derivatives, respectively. The 6-chloro-3-methylpyrim-
idin-2,6(1H,3H)-dione was obtained by reaction of 1-methylurea
and malonic acid in presence of acetic acid and acetic anhydride,28
followed by chlorination with phosphoryl chloride. The 6-chloro-
pyrimidin-2,6(1H,3H)-dione was obtained by chlorination of barbi-
turic acid, first forming 2,4,6-trichloropyrimidine and then
hydrolysis by sodium hydroxide. And the 6-chloro-3-phenylpyrim-
idin-2,6(1H,3H)-dione was obtained by reaction of 1-phenylurea
and diethyl malonate in presence of sodium ethoxide, forming 1-
phenylbarbituric acid.29 After chlorination by phosphoryl chloride,
three chloro derivatives of phenyl barbituric acid were obtained,
from which 6-chloro-3-phenylpyrimidin-2,6(1H,3H)-dione was
isolated following the method as described in the literature.30
The amino derivatives of the corresponding starting compounds
were prepared from appropriate alkyl or aryl amines by direct fu-
sion reaction. The derivatives containing aryl amines could be pre-
pared by the fusion of appropriate amines with corresponding
starting compounds at higher temperature (180–200 °C) for
approximately 10–15 min, whereas the preparation of the deriva-
tives containing alkyl amines having the lower boiling point such
as methylamine and ethylamine, requires rather strenuous condi-
tions and is carried out in a stainless steel bomb. Usually reaction
of alkyl amines needs longer time and those having lower molecu-
lar weight and boiling points require more than 15 h for the com-
pletion of the reaction (Scheme 2).
The downfield doublet was assigned for axial proton (1a-H), and
the upfield doublet was assigned for equatorial proton (1b-H). It
0
is also noteworthy that substituents at the C3 -position have less
effect on the shifting of the peaks than the substituents at the
0
0
C8 -position. Moreover, aryl substituents at the 8 -position were
generally found to be deshielding the protons, causing the appear-
ance of the peaks at downfield region than that of corresponding
compounds possessing alkyl substituents. In 13C-NMR, the charac-
0
teristic peak of C5 -H appeared in the downfield region of dC
140–142, exhibiting the high electrophilicity of the carbon.
The structural identity of the synthesized hybrid compounds
was verified by UV–vis absorption spectra. Generally, all of the
synthesized compounds showed three absorption maxima at
210–235, 253–291, and 421–457 nm. In case of 30-methyl-80-(alkyl
or aryl)-50-deazacholest-2,4-dieno[2,3-g]pteridine-20,40(30H,80H)-
diones (5), the absorption peaks appeared in the longer wavelength
region than 80-(alkyl or aryl)-50-deazacholest-2,4-dieno[2,3-g]pter-
idine-20,40(30H,80H)-diones (7) and 30-phenyl-80-(alkyl or aryl)-50-
deazacholest-2,4-dieno[2,3-g]pteridine-20,40(30H,80H)-diones (9).
An aryl substituent at 80-position was found to cause the blue shift-
ing (hypsochromic shift) of the absorption peaks in the region of
210–235 nm and red shifting (bathochromic shift) in the region
of 421–457 nm. It is due to the presence of absorption maxima
at the region of 421–457 nm that all of the hybrid compounds
are colored, ranging from yellow to red.
2.2. In vitro antitumor activities of 30,80-disubstituted-50-
The key intermediate, 2-hydroxymethylenecholestenone (3),
was synthesized by treating cholest-4-en-3-one (2), with ethyl
formate in the presence of sodium hydride.31 The precursor, cho-
lest-4-en-3-one (2) was prepared by the oxidation of cholesterol
using aluminum isopropoxide as the oxidizing agent and acetone
as proton acceptor as described by Oppenauer.32 The target hybrid
compound was then prepared by heating the key intermediate,
2-hydroxymethylenecholest-4-en-3-one (3) (1 equiv) with 3,6-
disubstituted-pyrimidin-2,6(1H,3H)-diones (4, 6, and 8) (0.9 equiv)
deazacholest-2,4-dieno[2,3-g ]pteridine-20,40(30H,80H)-diones
The synthesized compounds were tested in vitro for growth
inhibitory activities against various cultured tumor cell lines.
Two human tumor cell lines viz, human T-cell acute lymphoblas-
toid leukemia cell line (CCRF-HSB-2) and human oral epidermoid
carcinoma cell line (KB) were used for the test using the antitumor
agent with cytosine arabinoside (Ara-C) as the positive control in

A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 8685–8696
8687
C8H17
C8H17
C8H17
OH
HC
H
H
H
i
ii
H
H
H
H
H
H
HO
O
O
1
2
3
O
R1
O
N
R2
C8H17
N
H
N
H
O
N
H
R1
4a—j, 6a—i, 8a—g
N
H
H
O
N
R2
iii
5a–j, 7a–i, 9a–g
4, 5 a: R1 = Me ; R2 = Me
6, 7 a: R1 = H ; R2 = Me
b: R1 = H ; R2 = Et
8, 9 a: R1 = Ph ; R2 = Me
b: R1 = Ph ; R2 = Ph
1
R = Me ; R2 = Et
b:
c: R1 = Me ; R2 = Bu
d: R1 = Me ; R2 = Ph
1
2
1
2
c:
R = H ; R = Bu
d: R1 = H ; R2 = Ph
c:
R = Ph ; R = 3-Me-C6H4
d: R1 = Ph ; R2 = 4-OMe-C6H4
e: R1 = Ph; R2 = 3,4-Me2-C6H3
R
1 = Me ; R2 = 3-Me-C6H4
e: R1 = H ; R2 = 4-OMe-C6H4
e:
f: R1 = Me ; R2 = 4-OMe-C6H4
1 = H ; R2 = 3-Me-C6H4
f:
R
1= Ph ; R 2= 4-F-C6H4
g: R1 = Ph ; R2 = 4-Cl-C6H4
f:
R
g: R1 = Me; R2 = 3,4-Me2-C6H3
g: R1 = H ; R2 = 4-F-C6H4
h: R1 = H ; R2 = 4-Cl-C6H4
1
R = Me ; R2 = 4-F-C6H4
h:
i: R1 = Me ; R2 = 4-Cl-C6H4
j: R1 = Me ; R2 = 4-Br-C6H4
1
2
i:
R = H ; R = 3,4-Me2-C6H3
Scheme 2. General procedure for the preparation of 30,80-disubstituted-50-deazacholest-2,4-dieno[2,3-g]pteridine-20,40(30H,80H)-diones (5a–j, 7a–i, 9a–g). Reagents and
conditions: (i) Al(i-PrO)3, acetone, toluene, 85 °C, 8 h, 79%; (ii) HCOOEt, NaH, toluene, 3 days, rt, 92%; (iii) 0.01 equiv p-TsOH, Ph2O, 180 °C, 45 min.
the study. The IC50 values [the concentration (
50% inhibition of cell growth] for these compounds are shown in
Table 1.
l
g/mL) required for
(IC50) against CCRF-HSB-2 cell line. Also, almost all of the com-
pounds exhibited appreciable potential growth inhibitory activities
of ca. 4.0–8.0 lg/mL (IC50) against KB cell line. The SAR revealed
As can be seen in Table 1, many deazaflavin–cholestane hybrids
derivatives have been found to show fairly good antitumor activi-
ties. Although the tested steroidal hybrid compounds exhibited
that the substituents at the 30-position of the hybrid compounds
figure out the potential inhibitory activities of the compounds to-
wards tumor cell lines. In general, any substitution at the 30-posi-
tion was found to reduce the antitumor activities of the
compounds. It has been revealed that compounds possessing
methyl substituent at the 30-position exhibited less potent antitu-
mor activities than those possessing phenyl substituent or no sub-
stituents. The compounds with aryl groups at the 80-position also
showed more efficient antitumor activities than those with alkyl
groups in case of both CCRF-HSB-2 and KB cell lines.
poor antitumor activities than that of the control, Ara-C (IC50
0.017 g/mL), appreciably many of the compounds showed the
2.1–4.3 g/mL potential growth inhibitory activities against
:
l
l
CCRF-HSB-2 cell line revealing their promising potential inhibitory
activities and less cytotoxicities. Among them, the compounds 7e
and 9c are found to be better inhibitors with less than 2.5
(IC50). Other compounds 7f–h also exhibited promising antitumor
activities of 2.7 g/mL (IC50). Similarly, in case of KB cell line, they
exhibited good growth inhibitory activities of about one-third anti-
tumor potency of Ara-C (IC50: 0.73 g/mL), that is, the IC50 value of
the compounds 7e, 7g, 7h, and 9c were 1.7, 2.1, 1.8, and 1.9 g/mL,
respectively. Moreover, almost all of the compounds exhibited rea-
lg/mL
l
2.3. Molecular docking study
l
l
The in silico automated docking studies were performed
using the molecular docking software LigandFit within the
software package Discovery Studio 1.7. The docking study of
sonable potential growth inhibitory activities of ca. 4.0–8.0 lg/mL
Table 1
Growth inhibitory activities of 30,80-disubstituted-50-deazacholest-2,4-dieno[2,3-g]pteridine-20,40(30H,80H)-diones (5, 7, and 9) against human tumor cell lines (CCRF-HSB-2 and
KB)
Compound
Inhibitory activity [IC50
(
lg/mL)]
Compound
Inhibitory activity [IC50 (lg/mL)]
R1
R2
CCRF-HSB-2
KB
40.9
>100.0
7.5
7.6
7.4
7.4
8.3
8.4
9.2
14.2
8.4
40.3
5.7
R1
R2
CCRF-HSB-2
KB
5a
5b
5c
5d
5e
5f
5g
5h
5i
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
Me
Et
Bu
Ph
36.8
>100.0
10.0
6.2
5.4
4.5
5.6
6.9
7.6
14.0
8.8
37.9
4.3
7e
7f
7g
7h
7i
9a
9b
9c
9d
9e
9f
H
H
H
H
4-OMe-C6H4
3-Me-C6H4
4-F-C6H4
4-Cl-C6H4
3,4-Me-C6H3
Me
2.1
2.7
2.7
2.7
4.7
5.5
2.4
5.4
7.5
7.7
5.5
9.0
0.017
1.7
4.4
2.1
1.8
7.8
7.4
1.9
8.0
8.7
9.1
8.4
9.3
0.73
3-Me-C6H4
4-OMe-C6H4
3,4-Me-C6H3
4-F-C6H4
4-Cl-C6H4
4-Br-C6H4
Me
H
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
3-Me-C6H4
4-OMe-C6H4
3,4-Me-C6H3
4-F-C6H4
4-Cl-C6H4
5j
7a
7b
7c
7d
H
H
Et
Bu
9g
Ara-C
H
Ph
5.4
7.9

8688
A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 8685–8696
30,80-disubstituted-50-deazacholest-2,4-dieno[2,3-g]pteridine-20,40
(30H,80H)-diones (5, 7, and 9) was carried out, which were docked
within the protein tyrosine kinase (PDB code: 1t46). Protein tyro-
sine kinases (PTKs) are enzymes, which catalyze the phosphoryla-
tion of tyrosine residues, and these enzymes are involved in
cellular signaling pathways and regulate key cell functions such
as proliferation, differentiation, antiapoptotic signaling, and neuri-
tis outgrowth. Unregulated activation of these enzymes can lead to
various forms of cancer as well as benign proliferative conditions.33
Therefore, efforts have been concentrated to develop potential
inhibitors which can inhibit the activity of tyrosine kinase and thus
could lead to the development of a new class of therapeutics of
cancers as well as other proliferative diseases. In the present study,
we have studied the in silico binding affinities of the synthesized
steroid-hybrids, 30,80-disubstituted-50-deaza-cholest-2,4-dieno[2,3-
g]pteridine-20,40(30H,80H)-diones (5, 7, and 9) into PTK.
As can be seen in Figure 1, the docked ligand (shown in ball and
stick, colored by element) has been exactly superimposed on the
native bound one (shown in a red line) with same number of
hydrogen bonds. It has been revealed that the docked ligand
formed four hydrogen bondings with the same four amino acids
viz., Cys 673, Thr 670, Glu 640, and Asp 810, involved in the native
bound ligand. The RMSD of the docked ligand was 1.15 Å and the
calculated binding free energy (DGb ) of the docked ligand was
ꢀ10.42 kcal/mol. These results exhibited the high accuracy and
success of the LigandFit simulation in comparison with the biolog-
ical methods.
2.3.2. Calculation of binding affinities of the synthesized
deazaflavin–cholestane hybrid compounds into PTK
The binding affinity was evaluated by the binding free energies
(DGb, kcal/mol), inhibition constant, hydrogen bonding and RMSD
values. The synthesized deazaflavin–cholestane hybrid com-
pounds are composed to two parts, one having more hydrophilic
deazaflavin moiety and another having highly lipophilic choles-
tane moiety. Due to the presence of bulky lipophilic cholestane
moiety, the main interaction involved in the docking simulation
was van der Waals forces. The hydrophilic interaction by hydro-
gen bonding was contributed by deazaflavin moiety, and most
of the compounds exhibited only one hydrogen bonding with
the PTK receptor. Almost all compounds exhibited similar binding
affinities comparable with the native bound STI-571 ligand. Some
compounds exhibited lower binding energies than STI-571 ligand,
such as 5j, 7h, and 9c, and 9c exhibited binding free energies as
lower as ꢀ11.72 kcal/mol in comparison with the STI-571 ligand,
that is, ꢀ10.42 kcal/mol. This shows that the deazaflavin–choles-
tane hybrid compounds have possessed better affinities towards
PTK than the native bound one. This affinity of the hybrid com-
pounds may be attributed to the high lipophilicity of the steroid
moiety of the compound, due to which better van der Waals
interaction was prospective between ligand and the receptor. In
Table 2, the compounds with higher binding affinities, that is,
lower binding energies and their hydrogen bond interactions into
the target macromolecules are listed. As shown in the Table 2,
two compounds 7c and 9f exhibited multiple hydrogen bonding;
2.3.1. Validation of the accuracy and performance of LigandFit
As a general rule, if the best-docked conformation of a ligand
resembles the bound native ligand in the experimental crystal
structure, the used scoring function is said to be successful.
According to the literature,34 the successful scoring function is
the one in which the RMSD (root mean square deviation) of the
best docked conformation is 62.0 Å from the experimental one.
Therefore, before doing actual docking simulation experiment with
synthesized compounds, the validation of the function achieved in
LigandFit/Discovery Studio 1.7 was validated by docking of the na-
tive ligand into its binding site. Then the docked result was com-
pared with the crystal structure of the bound ligand–protein
complex. It has been confirmed that the accuracy and performance
of the LigandFit/Discovery Studio 1.7 was highly satisfactory, as
shown in Figure 1. In the study, the STI-571 ligand [4-[(4-methylpi-
perazin-1-yl)methyl]-N-[4-methyl-3-[(4-pyridin-3-ylpyrimidin-2-
yl)amino]-phenyl]-benzamide, commonly known as Imatinib or
Gleevec was docked into its C-kit receptor PTK (PDB code: 1t46).
From our previous studies,2,3 we have found that the deazaflavin
derivatives were favorably fitted into the binding pocket of PTK
which was comparable with the native ligand STI-571. Therefore,
the STI-571 was selected as a reference ligand for the comparative
study of binding action of the synthesized compounds in this
study, where we used deazaflavin–cholestane hybrid molecules
as ligands.
compound 7c (
bonds with Thr 670 and Val 668 and compound 9f
D
Gb: ꢀ 9.59 kcal/mol) exhibited two hydrogen
(D
Gb:
ꢀ8.75 kcal/mol) also exhibited two hydrogen bonds with two
NH moieties of Arg 684.
The molecular docking study revealed that the majority of the
compounds docked into the c-Kit receptor PTK (PDB code: 1t46)
0
with its bound ligand STI exhibited hydrogen bonds via their C4
0
and C2 -oxo groups as illustrated for compounds 7b and 7c in Fig-
ure 2 and for compounds 5a, 7e, and 9a in Figure 3.
The study of correlation between the growth inhibitory activities
(IC50, l
g/mL) of the synthesized 30,80-disubstituted-50deazacholest-
2,4-dieno[2,3-g]pteridine-20,40(30H,80H)-diones against tumor cells
and binding affinities predicted by LigandFit showed fairly good
correlation for some compounds. In case of the growth inhibition
against CCRF-HSB-2 cells, the fairly good correlation was obtained
for compounds 5c, d, f–i, 7c, d, h, 9a, b, d, f, and 9g with correlation
coefficient (R2) of 0.5185, as shown in Figure 4. Similarly, the growth
inhibition against KB cells exhibited also similar correlation with
LigandFit binding free energies for compounds 5c–e, h, 7c–f, h, i,
and 9b, d, f, g with correlation coefficient (R2) value of 0.572 as
shown in Figure 5.
3. Conclusion
Figure 1. The validation of accuracy and performance of LigandFit. The docked STI
ligand (ball and stick, colored by elements) nearly superimposed on the native STI
ligand (red line) exhibiting same number of hydrogen bonds (green dotted lines)
In this study, various novel deazaflavin–cholestane hybrid com-
pounds, 30,80-disubstituted-50-deazacholest-2,4-dieno[2,3-g]pteri-
dine-20,40(30H,80H)-diones ( 5a–j, 7a–i, and 9a–g) have been
with same amino acids involved by the native one.
1.15 Å.
D
Gb: ꢀ10.42 kcal/mol; RMSD:

A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 8685–8696
8689
Table 2
The best docking results based on the binding free energies (
between compounds and amino acids involved in PTK
DGb) and inhibition constants (Ki) of compounds docked into PTK, and the distances and angles of hydrogen bonds
0
a
b
Compound
D
Gb (kcal/mol)
Ki
Hydrogen bonds between atoms of compounds and amino acids
RMSDc (AÅ)
0
Atom of compound
Amino acid
Distance
Angle (°)
(AÅ)
0
5a
5b
5c
5f
ꢀ9.60
ꢀ9.66
0.12
0.11
0.45
0.05
C
C
C
4 -Oxo
HN of Lys 623
HN of Lys 623
HN of Cys 673
HN of Ile 789
HN of Lys 593
HN of Lys 593
HN of Lys 623
O of Thr 670
O of Val 668
HN of Ile 789
HN of Ile 571
HN of Lys 593
—
HN of Asn 680
HN of Lys 593
HN of Arg 684
HN of Asn 680
HN of Arg 684
HN of Arg 684
HN of Cys 673
O of Thr 670
O of Glu 640
HN of Asp 810
2.28
2.01
1.75
2.18
1.76
1.77
1.75
2.13
2.08
2.20
2.30
1.75
—
2.50
1.80
2.45
2.45
2.02
2.27
2.03
1.87
2.18
1.79
122.60
126.40
178.10
133.70
147.50
150.70
130.90
151.40
157.20
132.60
157.90
148.20
8.75
8.71
13.76
6.12
9.68
8.60
0
4 -Oxo
0
ꢀ8.81
4 -Oxo
ꢀ10.15
ꢀ11.58
ꢀ10.29
ꢀ9.23
Ar-OCH3
C2-Oxo
5j
4.40Eꢀ03
0
7a
7b
7c
0.04
C
C
C
2 -Oxo
0
0.22
0.12
4 -Oxo
10.26
3.64
0
ꢀ9.59
4 -Oxo
30NH
7e
7g
7h
9a
9b
9c
9d
9e
9f
ꢀ9.77
ꢀ8.85
0.09
0.42
Ar-OCH3
Ar-F
6.06
8.56
0
ꢀ11.60
ꢀ9.81
4.30Eꢀ03
C2 -Oxo
10.20
11.78
10.49
10.49
11.01
10.74
11.15
d
0.08
—
C
C
—
0
ꢀ10.04
ꢀ11.72
ꢀ9.16
0.06
4 -Oxo
102.40
147.90
115.70
106.80
133.20
128.90
146.00
116.50
113.10
145.40
0
3.50Eꢀ03
2 -Oxo
0.25
Ar-OCH3
0
ꢀ10.42
ꢀ8.75
0.03
0.49
C4 -Oxo
Ar-F
Ar-F
N5-N
NH (H56)
NH (H66)
036
STIe
ꢀ10.42
0.03
1.15
a
Binding free energy.
Inhibition constant.
Root mean square deviation.
No hydrogen bond.
The docked ligand (STI-571) of PTK (PDB code: lt46).
b
c
d
e
Figure 2. Compound (7b) (colored by elements, ball and stick) is bound into the PTK receptor site via hydrophobic interaction with Asn 680, Lys 593, Leu 595, Asp 677, Val
603, Gly 676, Leu 799, Tyr 672, Ala 621, and Phe 811 in addition with one hydrogen bond (green line) between C4-oxo and NH of Lys 623, while compound (7c) (blue stick)
exhibited two hydrogen bonds with Thr 670 and Val 668. The binding pocket is shown in the solid surface with labeled amino acids, and the STI ligand is shown as a red line.
synthesized by condensation reaction between 6-(monosubsti-
tuted amino)-pyrimidin-2,4(1H,3H)-diones (4a–j, 6a–i, and 8a–g)
and 2-hydroxymethylenecholest-4-en-3-one (3) in presence of p-
toluenesulfonic acid monohydrate and diphenyl ether. Compounds
5a–j were synthesized in better yield than compounds 7a–i and
9a–g. Our strategy for the study was to synthesize three parallel
derivatives of 3-substituted deazaflavin–cholestane hybrid com-
pounds and the comparative study of their antitumor activity po-
tential and in silico binding affinities. From the study it has been
revealed that the 3-unsubstituted hybrid compounds (7a–i) pos-
sessed better antitumor potencies than 3-methyl and 3-phenyl
derivatives. Also, 3-methyl derivatives (5a–j) exhibited the least
potential antitumor activities in vitro against CCRF-HSB-2 and KB
cells. Compound 7e exhibited best antiproliferative potencies with
IC50 value of 2.1
KB cells. Other derivatives like 7f–h exhibited fairly similar
activities with IC50 value of 2.7 g/mL against CCRF-HSB-2
and 1.8–4.4 g/mL against KB cells. These results showed that
lg/mL against CCRF-HSB-2 and 1.7 lg/mL against
l
l

8690
A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 8685–8696
Figure 3. Stereo view for comparative binding affinities of 30,80-dimethyl-50-deazacholest-2,4-dieno[2,3-g]pteridine-20,40(30H,80H)-diones (5a, ball and stick), 80-(4-
methoxyphenyl)-50-deazacholest-2,4-dieno[2,3-g]pteridine-20,40(30H,80H)-diones (7e, blue stick), and 80-methyl-3-phenyl-50-deazacholest-2,4-dieno[2,3-g]pteridine-
20,40(30H,80H)-diones (9a, yellow stick). Compounds 5a and 7e exhibited one hydrogen bond shown as a green line, respectively, and compound 9a exhibited no hydrogen
bond. The binding pocket of PTK is shown in the wire mesh surface with labeled amino acids, the PTK protein structure is shown in solid ribbon view, and the STI ligand is
shown as a red line.
The in silico docking study of the synthesized deazaflavin–cho-
lestane derivatives (5a–j, 7a–i, and 9a–g) was carried out for lead
optimization using the software LigandFit within the software
package Discovery Studio 1.7. The synthesized hybrid compounds
were docked into the c-kit protein tyrosine kinase. Almost all of
the compounds exhibited good binding affinities within the PTK
receptor. The overall correlation between the growth inhibitory
activities of the synthesized deazaflavin–cholestane hybrid com-
pounds against tumor cells and the binding affinities predicted
by LigandFit was fairly good for some compounds. In case of the
growth inhibition against CCRF-HSB-2 cells, fairly good correlation
was obtained for compounds 5c, d, f–i, 7c, d, h, 9a, b, d, f, and 9g
Figure 4. Correlation between the binding energy (DGb) and IC50 (l
g/mL) of 30, 80-
with correlation coefficient (R2) of 0.5185. Similarly, the growth
inhibition against KB cells also exhibited similar correlation with
LigandFit binding free energies for compounds 5c–e, h, 7c–f, h, i,
9b, d, f and 9g with correlation coefficient (R2) value of 0.572.
Thus, the overall studies by computational design, chemical
synthesis, and biological investigation revealed that the deazafla-
vin–cholestane hybrid compounds are the new archetype of hybrid
compounds with promising antiproliferative activities with less
cytotoxicities. Further, the in silico binding affinity investigation
revealed that due to the higher lipophilicity of the hybrid com-
pounds, they possess good binding affinities within the protein
tyrosine kinase.
disubstituted-50-deazacholest-2,4-dieno[2,3-g]pteridine-20,40(30H,80H)-diones (5c,
d, f–i, 7c, d, h, and 9a, b, d, f, g) against human T-cell acute lymphoblastioid
leukemia cells (CCRF-HSB-2).
4. Experimental
4.1. Chemistry
Figure 5. Correlation between the binding energy (DGb) and IC50 (l
g/mL) of 30,80-
disubstituted-50-deazacholest-2,4-dieno[2,3-g]pteridine-20,40(30H,80H)-diones (5c–
e, h, 7c–f, h, i, and 9b, d, f, g) against human oral epithelial carcinoma cells (KB).
Mps were obtained on a Yanagimoto micro melting point
apparatus and were uncorrected. Microanalyses were measured
by Yanaco CHN corder MT-5 apparatus. The specific optical
rotations were measured by JASCO DIP-1000 digital polarimeter,
3-unsubstituted deazaflavin–cholestane hybrid compounds 7c–i
could be the potential candidate for antiproliferative activities with
better antitumor activities with less cytotoxicities.

A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 8685–8696
8691
and absolute ethanol was used as the solvent. IR spectra were re-
corded on a JASCO FT/IR-200 spectrophotometer as Nujol mulls.
1H and 13C NMR spectra were obtained using a Varian VXR 300
and 75 MHz spectrophotometer, respectively, and chemical shift
values are expressed in d values (ppm) relative to tetramethylsil-
ane (TMS) as internal standard. Coupling constants are given in
Hertz. UV spectra were measured in absolute EtOH using Beckman
DU-68S UV spectrophotometers and absorption values in italics re-
fers to wave lengths at which shoulders or inflexions occur in the
absorption. All reagents were of commercial quality and were used
without further purification. Organic solvents were dried in the
presence of appropriate drying agent and were stored over suitable
molecular sieves. Reaction progress was monitored by analytical
thin-layer chromatography (TLC) on pre-coated glass plates (Silica
gel 60F254-plate-Merck) and the products were visualized by UV
light.
and C7), 36.08 (C22), 36.39 (C8 and C20), 39.18 (C12), 39.79 (C24),
39.86 (C10), 40.77 (C1), 41.36 (80-NCH2CH3), 42.71 (C13), 53.99
0
(C14), 56.03 (C9), 56.41 (C17), 111.71 (C4), 112.76 ðC6 Þ, 118.13
0
0
0
0
ðC4 aÞ, 140.48 ðC5 Þ, 147.50 ðC7 Þ, 155.29 ðC2 @OÞ, 157.91 (C5),
0
0
163.10 ðC4 @OÞ, 168.99 ðC8 aÞ. Anal. Calcd for C35H51N3O2: C,
77.02; H, 9.42; N, 7.70. Found: C,77.09; H, 9.24; N, 7.69.
4.1.4. 80-Butyl-30-methyl-50-deazacholest-2,4-dieno[2,3-g]pter-
idine-20,40(30H,80H)-dione (5c)
Yield, (745 mg, 53%); mp 128–130 °C (from EtOH); ½a D28
ꢁ
(EtOH):
ꢀ24.80°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 255 (4.20),
289 (4.14), 422 (4.05); IR (m
max/cmꢀ1): 1685, 1633 (CO); 1H NMR
(CDCl3): dH 0.73 (3H, s, 18-CH3), 0.86 (6H, d, J = 6.6 Hz, 25-
CH(CH3)2), 0.94 (3H, d, J = 6.3 Hz, 21-CH3), 0.98 (3H, s, 19-CH3),
0.99 (3H, t, J = 7.5 Hz, 80-NCH2CH2CH2CH3), 1.11–2.11 (22H, m),
1.44 (2H, m,J = 7.5 Hz, 80-NCH2CH2CH2CH3) 2.55 (2H, m, 6-CH2),
2.68 (1H, d, J = 15.3 Hz, 1b-H), 2.88 (1H, d, J = 15.3 Hz, 1a-H), 3.45
4.1.1. General procedure for the preparation of 30,80-disubstituted-
50-deazacholest-2,4-dieno[2,3-g]pteridine-20,40(30H,80H)-diones(5,
7, and 9)
(3H, s, 30-NCH3), 4.45 (1H, br s, 80-NCHaHbCH2CH2CH3), 4.79 (1H,
br s, 80-NCHaHbCH2CH2CH3), 6.34 (1H, s, 4-CH), 8.27 (1H, s, 50-
CH); 13C NMR (CDCl3): dC 12.08 (C18), 13.93 (80-NCH2CH2CH2CH3),
17.69 (C21), 18.81 (C19), 20.09 (80-NCH2CH2CH2CH3), 21.78 (C11),
22.72 (C26), 22.97 (C27), 23.95 (C23), 24.42 (C15), 28.16 (30-NCH3),
28.30 (C16), 30.81 (80-NCH2CH2CH2CH3), 31.32 (C25), 33.67 (C6
and C7), 35.92 (C22), 36.25 (C8 and C20), 39.03 (C12), 39.63 (C24),
39.71 (C10), 40.64 (C1), 42.56 (C13), 45.72 (80-NCH2CH2CH2CH3),
A mixture of 2-hydroxymethylenecholest-4-en-3-one (3) (1.0 g)
and 3,6-disubstituted-pyrimidin-2,6-(1H,3H)-diones ( 4, 6, and 8)
(0.9 equiv) in diphenyl ether (1 mL) in the presence of p-toluene-
sulfonic acid monohydrate (0.01 equiv) was refluxed with constant
stirring under an environment of nitrogen at 180 °C for 45 min.
After cooling, the reaction mixture was diluted with ethanol, and
the products were separated using flash column chromatography
on silica gel (230–400 mesh, Fuji Silysia Co. Ltd, BW-300; eluent:
ethyl acetate). The yellow or red crystals thus obtained were recys-
tallized from an appropriate solvent to afford the corresponding
product as yellow or red needles in 30–55% yield.
0
53.82 (C14), 55.88 (C9), 56.26 (C17), 111.52 (C4), 112.77 ðC6 Þ,
0
0
0
0
118.37 ðC4 aÞ, 140.37 ðC5 Þ, 147.70 ðC7 Þ, 155.28 ðC2 @OÞ, 158.04
00
0
(C5), 162.92 ðC4 @OÞ, 169.03 ðC8 aÞ. Anal. Calcd for
C
37H55N3O2ꢂ0.5H2O: C, 76.24; H, 9.68; N, 7.21. Found: C, 76.32;
H, 9.38; N, 7.25.
4.1.5. 30-Methyl-80-phenyl-50-deazacholest-2,4-dieno[2,3-
4.1.2. 30,80-Dimethyl-50-deazacholest-2,4-dieno[2,3-g]pteridine-
20,40(30H,80H)-dione (5a)
g]pteridine-20,40(30H,80H)-dione (5d)
Yield, (720 mg, 50%); mp 197–199 °C (from EtOH); ½a D28
ꢁ
(EtOH):
Yield, (570 mg, 44%); mp 250–252 °C (from EtOH); ½a D28
ꢁ
(EtOH):
ꢀ18.12°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 234 (4.45),
ꢀ30.60°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 235 (4.24),
291 (4.38), 428 (4.34); IR (m
max/cmꢀ1): 1684, 1623 (CO); 1H NMR
289 (4.26), 422 (4.19); IR (
m
max/cmꢀ1): 1688, 1638 (CO); 1H NMR
(CDCl3): dH 0.71 (3H, s, 18-CH3), 0.86 (6H, d, J = 6.6 Hz, 25-
CH(CH3)2), 0.94 (3H, d, J = 6.3 Hz, 21-CH3), 1.01 (3H, s, 19-CH3),
1.06–2.32 (24H, m), 2.73 (1H, d, J = 15.6 Hz, 1b-H), 2.93 (1H, d,
(CDCl3): dH 0.73 (3H, s, 18-CH3), 0.88 (6H, d, J = 6.6 Hz, 25-
CH(CH3)2), 0.94 (3H, d, J = 6.3 Hz, 21-CH3), 0.98 (3H, s, 19-CH3),
1.11–2.11 (22H, m), 2.55 (2H, m, 6-CH2), 2.67 (1H, d, J = 15.6 Hz,
J = 15.6 Hz, 1a
-H), 3.41 (3H, s, 30-NCH3), 5.49 (1H, s, 4-CH), 7.13–
1b-H), 2.87 (1H, d, J = 15.6 Hz, 1
a
-H), 3.45 (3H, s, 30-NCH3), 4.04
7.24 (2H, m, 80-Ar-mH), 7.53–7.58 (3H, m, 80-Ar-o,pH), 8.38 (1H,
s, 50-CH); 13C NMR (CDCl3): dC 12.15 (C18), 18.11 (C21), 18.88
(C19), 21.86 (C11), 22.78 (C26), 23.04 (C27), 24.01 (C23), 24.45 (C15),
28.23 (30-NCH3), 28.35 (C16), 31.30 (C25), 33.30 (C6 and C7), 35.96
(C22), 36.31 (C8 and C20), 39.27 (C12), 39.70 (C24), 39.79 (C10),
40.45 (C1), 42.62 (C13), 54.06 (C14), 55.92 (C9), 56.31 (C17), 111.96
(3H, s, 80-CH3), 6.40 (1H, s, 4-CH), 8.26 (1H, s, 50-CH); 13C NMR
(CDCl3)35: dC 11.92 (C18), 17.56 (C21), 18.65 (C19), 21.62 (C11),
22.56 (C26), 22.82 (C27), 23.80 (C23), 24.26 (C15), 28.01 (30-NCH3),
28.15 (C16), 31.18 (C25), 33.49 (C6 and C7), 35.73 (80-NCH3), 35.76
(C22), 36.09 (C8 and C20), 38.95 (C12), 39.47 (C24), 39.54 (C10),
40.32 (C1), 42.40 (C13), 53.69 (C14), 55.71 (C9), 56.10 (C17), 111.26
(C4), 114.08 ðC60 Þ, 117.38 ðC4 aÞ, 127.93 (80-C300 ) and (C5 ), 130.07
0
0
0
0
0
0
0
00
00
0
00
0
(C4), 112.75 ðC6 Þ, 117.52 ðC4 aÞ, 140.18 ðC5 Þ, 147.90 ðC7 Þ, 155.62
ð80-C4 Þ, 130.34 (8 -C2 ) and ðC5 ), 137.06 ð8 -C1 Þ, 141.75 ðC5 Þ,
0
0
0
0
0
0
ðC2 @OÞ, 157.37 (C5), 162.71 ðC4 @OÞ, 168.99 ðC8 aÞ. Anal. Calcd for
C34H49N3O2ꢂ0.3H2O: C, 76.02; H, 9.31; N, 7.82. Found: C,76.04; H,
9.13; N, 7.83.
148.43 ðC7 Þ, 157.09 ðC2 @OÞ, 157.55 (C5), 162.72 ðC4 @OÞ, 168.11
0
ðC8 aÞ. Anal. Calcd for C39H51N3O2.0ꢂ0.38H2O: C, 77.99; H, 8.68; N,
7.00. Found: C,77.85; H, 8.35; N, 7.39.
4.1.3. 80-Ethyl-30-methyl-50-deazacholest-2, 4-dieno[2,3-g]pter-
idine-20,40(30H,80H)-dione (5b)
4.1.6. 30-Methyl-80-(3-methylphenyl)-50-deazacholest-2,4-
dieno[2,3-g]pteridine-20,40(30H,80H)-dione (5e)
Yield, (680 mg, 51%); mp 275–277 °C (from EtOH); ½a D28
ꢁ
(EtOH):
Yield, (770 mg, 52%); mp 194–196 °C (from EtOH); ½a D28
ꢁ
(EtOH):
ꢀ28.44°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 235 (4.36),
ꢀ21.72°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 213 (4.41),
289 (4.38), 421 (4.31); IR (
m
max/cmꢀ1): 1686, 1637 (CO); 1H NMR
291 (4.24), 428 (4.21); IR (m
max/cmꢀ1): 1686, 1631 (CO); 1H NMR
(CDCl3): dH 0.71 (3H, s, 18-CH3), 0.86 (6H, d, J = 6.6 Hz, 25-
CH(CH3)2), 0.94 (3H, d, J = 6.3 Hz, 21-CH3), 0.98 (3H, s, 19-CH3),
1.11–2.11 (22H, m), 1.40 (3H, t, J = 6.9 Hz, 80-NCH2CH3), 2.55 (2H,
m, 6-CH2), 2.68 (1H, d, J = 15.3 Hz, 1b-H), 2.88 (1H, d, J = 15.3 Hz,
(CDCl3): dH 0.71 (3H, s, 18-CH3), 0.86 (6H, d, J = 6.6 Hz, 25-
CH(CH3)2), 0.94 (3H, d, J = 6.3 Hz, 21-CH3), 1.01 (3H, s, 19-CH3),
1.06–2.32 (24H, m), 2.43 (3H, s, 80-Ar-CH3), 2.71 (1H, d,
J = 15.6 Hz, 1b-H), 2.92 (1H, d, J = 15.6 Hz, 1a
-H), 3.39 (3H, s, 30-
1a
-H), 3.45 (3H, s, 30-NCH3), 4.54 (1H, br s, 80-NCHaHbCH3), 4.85
NCH3), 5.51 (1H, s, 4-CH), 6.92–7.01 (2H, m, 80-Ar-200,500H), 7.31–
7.47 (2H, m, 80-Ar-400,600H), 8.37 (1H, s, 50-CH); 13C NMR (CDCl3):
dC 12.08 (C18), 17.99 (C21), 18.81 (C19), 21.64 (C11), 21.78 (80-Ar-
mCH3), 22.71 (C26), 22.96 (C27), 23.94 (C23), 24.38 (C15), 28.16 (30-
NCH3), 28.28 (C16), 31.25 (C25), 33.25 (C6, C7), 35.87 (C22), 36.24
(1H, br s, 80-NCHaHbCH3), 6.37 (1H, s, 4-CH), 8.27 (1H, s, 50-CH);
13C NMR (CDCl3): dC 12.24 (C18), 14.25 (80-NCH2CH3), 17.89 (C21),
18.96 (C19), 21.93 (C11), 22.87 (C26), 23.13 (C27), 24.11 (C23),
24.57 (C15), 28.32 (30-NCH3), 28.46 (C16), 31.48 (C25), 33.73 (C6

8692
A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 8685–8696
0
(C8 and C20), 39.20 (C12), 39.63 (C24), 39.72 (C10), 40.40 (C1), 42.55
(C9), 56.24 (C17), 111.92 (C4), 113.78 ðC60 Þ, 117.45 (C4 a), 80-C300 and
0
0
0
0
00
00
00
0
(C13), 54.02 (C14), 55.86 (C9), 56.24 (C17), 111.81 (C4), 114.07 ðC6 Þ,
ðC5 ), 129.89 (8 -C2 and C6 ), 132.68 ð8 -C1 Þ, 141.91 ðC5 Þ, 148.42
0
117.21 ðC4 aÞ, 124.77 ð80-C4 Þ, 128.14 (8 -ðC50 )), 130.13 (8 -C3 ),
ðC7 Þ, 157.53 ðC2 @OÞ, 161.45 (C5), 162.52 ðC4 @OÞ, 164.77 ð8 -C4 Þ,
0
0
0
00
00
0
0
0
00
130.88 (80-C6 ), 136.95 (80-C2 ), 140.49 ð80-C1 Þ, 141.54 ðC5 Þ,
168.67 ðC8 aÞ. Anal. Calcd for C39H50FN3O2: C, 73.32; H, 8.36; N,
00
00
00
0
0
0
0
0
148.45 ðC7 Þ, 157.03 ðC2 @OÞ,157.45 (C5), 162.71 ðC4 @OÞ, 167.88
6.58. Found: C, 73.14; H, 8.09; N, 6.75.
0
ðC8 aÞ. Anal. Calcd for C40H53N3O2ꢂH2O: C, 76.76; H, 8.86; N, 6.71.
Found: C, 76.90; H, 8.67; N, 6.71.
4.1.10. 80-(4-Chlorophenyl)-30-methyl-50-deazacholest-2,4-
dieno[2,3-g]pteridine-20,40(30H,80H)-dione (5i)
4.1.7. 80-(4-Methoxyphenyl)-30-methyl-50-deazacholest-2,4-
Yield, (710 mg, 47%); mp 240–242 °C (from EtOH); ½a D28
ꢁ
(EtOH):
dieno[2,3-g]pteridine-20,40(30H,80H)-dione (5f)
ꢀ42.80°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 219 (4.40),
Yield, (530 mg, 35%); mp 185–188 °C (from EtOH); ½a D28
ꢁ
(EtOH):
278 (4.15), 448 (4.06); IR (m
max/cmꢀ1): 1684, 1627 (CO); 1H NMR
ꢀ26.84°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 226 (4.67),
(CDCl3): dH 0.71 (3H, s, 18-CH3), 0.86 (6H, d, J = 6.6 Hz, 25-CH(CH3)2),
0.94 (3H, d, J = 6.3 Hz, 21-CH3), 1.01 (3H, s, 19-CH3), 1.06–2.32 (24H,
291 (4.49), 429 (4.40); IR (m
max/cmꢀ1): 1684, 1633 (CO); 1H NMR
(CDCl3): dH 0.71 (3H, s, 18-CH3), 0.86 (6H, d, J = 6.6 Hz, 25-
CH(CH3)2), 0.94 (3H, d, J = 6.3 Hz, 21-CH3), 1.01 (3H, s, 19-CH3),
1.06–2.32 (24H, m), 2.71 (1H, d, J = 15.6 Hz, 1b-H), 2.91 (1H, d,
m), 2.72 (1H, d, J = 15.6 Hz, 1b-H), 2.93 (1H, d, J = 15.6 Hz, 1a-H), 3.40
(3H, s, 30-NCH3), 5.52 (1H, s, 4-CH), 7.07–7.19 (2H, m, 80-Ar-mH),
7.51–7.56 (3H, m, 80-Ar-oH), 8.38 (1H, s, 50-CH); 13C NMR (CDCl3):
dC 12.14 (C18), 18.84 (C21) and (C19), 21.80 (C11), 22.74 (C26), 22.99
(C27), 23.96 (C23), 24.40 (C15), 28.19 (30-NCH3 and C16), 31.24 (C25),
33.30 (C6 and C7), 34.68 (C22), 35.90 (C8 and C20), 36.28 (C12), 39.27
(C24), 39.64 (C10 and C1), 42.55 (C13), 54.03 (C14), 55.85 (C9), 56.26
J = 15.6 Hz, 1
5.59 (1H, s, 4-CH), 7.03–7.06 (2H, m, 80-Ar-mH), 7.10–7.13 (2H,
m, 80-Ar-oH), 8.36 (1H, s, 50-CH); 13C NMR (CDCl3): d
12.06
a
-H), 3.41 (3H, s, 30-NCH3), 3.88 (3H, s, 80-Ar-OCH3),
C
(C18), 18.02 (C21), 18.80 (C19), 21.78 (C11), 22.70 (C26), 22.96 (C27),
23.93 (C23), 24.37 (C15), 28.14 (30-NCH3), 28.28 (C16), 31.24 (C25),
33.23 (C6 and C7), 35.88 (C22), 36.23 (C8 and C20), 39.21 (C12),
39.62 (C24), 39.70 (C10), 40.41 (C1), 42.53 (C13), 53.98 (C14), 55.67
(C17), 111.95 (C4), 113.78 ðC60 Þ, 117.32 ðC4 aÞ, 129.39 (80-C3 ) and
0
00
0
0
0
00
00
00
00
ðC5 ), 130.71 (8 -C2 and C6 ), 135.47 ð8 -C4 Þ, 136.17 ðC1 Þ, 141.98
0
0
0
0
ðC5 Þ, 148.09 ðC7 Þ, 157.17 ðC2 @OÞ, 157.39 (C5), 162.52 ðC4 @OÞ,
(80-Ar-OCH3), 55.84 (C9), 56.23 (C17), 111.81 (C4), 114.12 ðC6 Þ,
168.66 ðC8 aÞ. Anal. Calcd for C39H50ClN3O2ꢂ0.66H2O: C, 73.16; H,
0
0
115.41 (80-C3 ) and ðC5 ), 117.57 ðC4 aÞ, 125.01 ð80-C1 Þ, 128.89
8.08; N, 6.56. Found: C, 73.03; H, 7.84; N, 6.72.
00
0
0
00
(80-C2 and C6 ), 141.59 ðC5 Þ, 148.95 ðC7 Þ, 157.18 ðC2 @OÞ, 157.79
00
00
0
0
0
(C5), 160.43 ð80-C400 Þ, 162.65 ðC4 @OÞ, 168.20 ðC8 aÞ. Anal. Calcd for
4.1.11. 80-(4-Bromophenyl)-30-methyl-50-deazacholest-2,4-
0
0
C
40H53N3O3: C, 77.01; H, 8.56; N, 6.74. Found: C, 77.30; H, 8.23;
dieno[2,3-g]pteridine-20,40(30H,80H)-dione (5j)
N, 6.45.
Yield, (550 mg, 33%); mp 247–249 °C (from EtOH); ½a D28
ꢁ
(EtOH):
ꢀ64.44°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 220 (4.32),
4.1.8. 30-Methyl-80-(3,4-dimethylphenyl)-50-deazacholest-2, 4-
283 (4.04), 455 (4.01); IR (m
max/cmꢀ1): 1697, 1634 (CO); 1H NMR
dieno[2,3-g]pteridine-20,40(30H,80H)-dione (5g)
(CDCl3): dH 0.71 (3H, s, 18-CH3), 0.86 (6H, d, J = 6.6 Hz, 25-
CH(CH3)2), 0.95 (3H, d, J = 6.3 Hz, 21-CH3), 1.01 (3H, s, 19-CH3),
1.06–2.32 (24H, m), 2.71 (1H, d, J = 15.6 Hz, 1b-H), 2.92 (1H, d,
Yield, (650 mg, 43%); mp 208–210 °C (from EtOH); ½a D28
ꢁ
(EtOH):
ꢀ41.48°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 212 (4.53),
291 (4.34), 428 (4.30), 453 (4.20); IR (
m
max/cmꢀ1): 1684, 1625 (CO);
J = 15.6 Hz, 1a
-H), 3.40 (3H, s, 30-NCH3), 5.46 (1H, s, 4-CH), 7.01–
1H NMR (CDCl3): dH 0.71(3H, s, 18-CH3), 0.86 (6H, d, J = 6.6 Hz, 25-
CH(CH3)2), 0.94 (3H, d, J = 6.3 Hz, 21-CH3), 1.01 (3H, s, 19-CH3),
1.06–2.30 (24H, m), 2.31 (3H, s, 80-Ar-300CH3), 2.34 (3H, s, 80-Ar-
7.16 (2H, m, 80-Ar-mH), 7.66–7.73 (3H, m, 80-Ar-oH), 8.37 (1H, s,
50-CH); 13C NMR (CDCl3): dC 12.08 (C18), 18.87 (C21) and (C19),
21.80 (C11), 22.71 (C26), 22.96 (C27), 23.93 (C23), 25.67 (C15),
28.16 (30-NCH3 and C16), 33.00 (C25), 33.28 (C6 and C7), 34.65
(C22), 35.88 (C8 and C20), 36.23 (C12), 39.26 (C24), 39.62 (C10 and
400CH3), 2.71 (1H, d, J = 15.6 Hz, 1b-H), 2.91 (1H, d, J = 15.6 Hz, 1
a-
H), 3.41 (3H, s, 30-NCH3), 5.56 (1H, s, 4-CH), 6.82–6.94 (2H, m, 80-
Ar-200, 500H), 7.27–7.31 (1H, m, 80-Ar-600H), 8.36 (1H, s, 50-CH); 13C
NMR (CDCl3): dC 12.08 (C18), 18.06 (C21), 18.80 (C19), 19.82 (80-Ar-
mCH3), 20.16 (80-Ar-pCH3), 21.78 (C11), 22.71 (C26), 22.96 (C27),
23.94 (C23), 24.38 (C15), 28.16 (30-NCH3), 28.28 (C16), 31.24 (C25),
33.14 (C6 and C7), 35.87 (C22), 36.24 (C8 and C20), 39.63 (C12), 39.62
(C24), 39.72 (C10), 40.42 (C1), 42.54 (C13), 53.01 (C14), 55.86 (C9),
C1), 42.58 (C13), 54.02 (C14), 55.83 (C9), 56.24 (C17), 111.91 (C4),
0
113.72 ðC6 Þ, 117.99 ðC4 aÞ, 124.29 ð80-C4 Þ, 129.63 (8 -C3 ) and
0
0
00
00
0
0
00
00
00
0
ðC5 ), 133.71 (8 -C2 and C6 ), 136.17 ðC1 Þ, 142.26 ðC5 Þ, 148.03
0
0
0
0
ðC7 Þ, 158.21 ðC2 @OÞ, 160.68 (C5), 162.50 ðC4 @OÞ, 168.68 ðC8 aÞ.
Anal. Calcd for C39H50BrN3O2ꢂ0.60H2O: C, 68.53; H, 7.55; N, 6.15.
Found: C, 68.54; H, 7.34; N, 6.19.
56.23 (C17), 111.73 (C4), 114.25 ðC6 Þ, 117.29 ðC4 aÞ, 124.86 (80-
0
0
0
0
0
0
ðC5 )), 128.34 ð8 -C4 Þ, 131.34 (8 -C2 ), 134.52 ð8 -C1 Þ, 138.91 (8 -
4.1.12. 80-Methyl-50-deazacholest-2,4-dieno[2,3-g]pteridine-
20,40(30H,80H)-dione (7a)
0
00
00
00
00
00
0
0
0
C6 and C3 ), 141.47 ðC5 Þ, 148.67 ðC7 Þ, 157.08 ðC2 @OÞ, 157.56 (C5),
162.79 ðC4 @OÞ, 167.77 ðC8 aÞ. Anal. Calcd for C41H55N3O2: C, 79.18;
Yield, (510 mg, 40%); mp 295–297 °C (from EtOH); ½a D28
ꢁ
(EtOH):
0
0
H, 8.91; N, 6.76. Found: C, 79.23; H, 8.86; N, 6.74.
ꢀ15.20°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 220 (4.26),
258 (4.24), 304 (3.92), 421 (4.02); IR (m
max/cmꢀ1): 3170 (NH),
4.1.9. 80-(4-Fluorophenyl)-30-methyl-50-deazacholest-2,4-
1686, 1621 (CO); 1H NMR (CDCl3): dH 0.72 (3H, s, 18-CH3), 0.88
(6H, d, J = 6.6 Hz, 25-CH(CH3)2), 0.94 (3H, d, J = 6.3 Hz, 21-CH3),
0.99 (3H, s, 19-CH3), 1.11–2.12 (22H, m), 2.56 (2H, m, 6-CH2),
dieno[2,3-g]pteridine-20,40(30H,80H)-dione (5h)
Yield, (710 mg, 48%); mp 218–220 °C (from EtOH); ½a D28
ꢁ
(EtOH):
ꢀ33.38°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 235 (4.33),
2.68 (1H, d, J = 15.6 Hz, 1b-H), 2.86 (1H, d, J = 15.6 Hz, 1a-H), 4.05
291 (4.22), 431 (4.17); IR (
m
max/cmꢀ1): 1698, 1634 (CO); 1H NMR
(3H, s, 80-NCH3), 6.42 (1H, s, 4-CH), 8.26 (1H, s, 50-CH), 8.46 (1H,
s, exchangeable with D2O, 30-NH); 13C NMR (CDCl3): dC11.96
(C18), 17.58 (C21), 18.66 (C19), 21.61 (C11), 22.56 (C26), 22.79 (C27),
23.80 (C23 and C15), 27.99 (C16), 31.19 (C25), 33.93 (C6 and C7),
35.75 (80-NCH3 and C22), 36.05 (C8 and C20), 39.01 (C12), 39.49
(C24 and C10), 40.25 (C1), 42.40 (C13), 53.68 (C14), 55.68 (C9),
(CDCl3): dH 0.71 (3H, s, 18-CH3), 0.86 (6H, d, J = 6.6 Hz, 25-CH(CH3)2),
0.94 (3H, d, J = 6.3 Hz, 21-CH3), 1.01 (3H, s, 19-CH3), 1.06–2.32 (24H,
m), 2.72 (1H, d, J = 15.6 Hz, 1b-H), 2.93 (1H, d, J = 15.6 Hz, 1a-H), 3.41
(3H, s, 30-NCH3), 5.52 (1H, s, 4-CH), 7.01–7.16 (2H, m, 80-Ar-mH),
7.20–7.25 (3H, m, 80-Ar-oH), 8.38 (1H, s, 50-CH); 13C NMR (CDCl3):
dC 12.08 (C18), 18.06 (C21), 18.81 (C19), 21.79 (C11), 22.71 (C26),
22.97 (C27), 23.94 (C23), 24.38 (C15), 28.16 (30-NCH3 and C16), 31.23
(C25), 33.30 (C6 and C7), 34.48 (C22), 35.88 (C8 and C20), 36.24 (C12),
39.26 (C24), 39.63 (C10), 40.33 (C1), 42.54 (C13), 54.01 (C14), 55.83
0
0
0
56.10 (C17), 111.69 (C4), 112.69 ðC6 Þ, 117.94 ðC4 aÞ, 141.06 ðC5 Þ,
0
0
0
148.64 ðC7 Þ, 156.89 ðC2 @OÞ, 157.57 (C5), 162.59 ðC4 @OÞ, 168.88
0
ðC8 aÞ. Anal. Calcd for C33H47N3O2: C, 76.77; H, 9.15; N, 8.12. Found:
C, 76.62; H, 8.82; N, 8.12.

A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 8685–8696
8693
4.1.13. 80-Ethyl-50-deazacholest-2,4-dieno[2,3-g]pteridine-
4.1.16. 80-(4-Methoxyphenyl)-50-deazacholest-2,4-dieno[2,3-
20,40(30H,80H)-dione (7b)
g]pteridine-20,40(30H,80H)-dione (7e)
Yield, (450 mg, 35%); mp 270–272 °C (from EtOH);
½
a 2D8
ꢁ
Yield, (510 mg, 34%); mp 219–221°C (from EtOH); ½a D28
ꢁ
(EtOH):
(EtOH): ꢀ19.80°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1):
ꢀ38.80°; UV (EtOH): kmax/nm (log
e
/dm3molꢀ1 cmꢀ1): 223 (4.23),
235 (4.54), 289 (4.52), 420 (4.44); IR (
m
max/cmꢀ1): 3160 (NH),
262 (4.14), 456 (3.97); IR (m
max/cmꢀ1): 3195 (NH), 1683, 1618
1682, 1637 (CO); 1H NMR (CDCl3): dH 0.70 (3H, s, 18-CH3), 0.86
(6H, d, J = 6.6 Hz, 25-CH(CH3)2), 0.91 (3H, d, J = 6.3 Hz, 21-CH3),
0.96 (3H, s, 19-CH3), 1.11–2.10 (22H, m), 1.39 (3H, t, J = 6.9 Hz,
80-NCH2CH3), 2.54 (2H, m, 6-CH2), 2.65 (1H, d, J = 15.6 Hz, 1b-H),
(CO); 1H NMR (CDCl3): dH 0.72 (3H, s, 18-CH3), 0.86 (6H, d,
J = 6.6 Hz, 25-CH(CH3)2), 0.95 (3H, d, J = 6.3 Hz, 21-CH3), 1.07 (3H,
s, 19-CH3), 1.06–2.33 (24H, m), 2.94 (1H, d, J = 16.2 Hz, 1b-H), 3.05
(1H, d, J = 16.2 Hz, 1a
-H), 3.88 (3H, s, 80-Ar-OCH3), 6.61 (1H, s, 4-
2.88 (1H, d, J = 15.6 Hz, 1
a
-H), 4.52 (1H, br s, 80-NCHaHbCH3),
CH), 6.99–7.07 (4H, m, 80-Ar-H), 8.45 (1H, s, 50-CH), 8.56 (1H, s,
exchangeable with D2O, 30-NH); 13C NMR (CDCl3): dC 12.04 (C18),
18.84 (C21 and C19), 21.53 (C11), 22.71 (C26), 22.98 (C27), 23.95 (C23
and C15), 28.15 (C16), 32.99 (C25, C6 and C7), 35.82 (C22), 36.21 (C8
and C20), 37.93 (C12), 39.18 (C24), 39.61 (C10), 39.96 (C1), 42.57
(C13), 49.60 (C14), 55.75 (80-Ar-OCH3), 56.07 (C9), 56.52 (C17),
4.82 (1H, br s, 80-NCHaHbCH3), 6.35 (1H, s, 4-CH), 8.22 (1H, s,
50-CH), 8.58 (1H, s, exchangeable with D2O, 30-NH); 13C NMR
(CDCl3): dC 11.94 (C18), 13.86 (80-NCH2CH3), 17.59 (C21), 18.68
(C19), 21.62 (C11), 22.58 (C26), 22.83 (C27), 23.81 (C23), 24.27
(C15), 28.15 (C16), 31.18 (C25), 33.51 (C6 and C7), 35.76 (C22),
36.11 (C8,C20 and C12), 38.91 (C24), 39.56 (C10), 40.42 (C1), 41.68
(80-NCH2CH3), 42.41 (C13), 53.68 (C14), 55.72 (C9), 56.12 (C17),
115.49 (80-C3 ) and ðC50 ), 115.69 (C4 and C6 ), 120.10 ðC4 aÞ, 121.44
00
00
00
0
ð80-C1 Þ, 128.90 (8 -C2 and C6 ), 141.83 ðC5 Þ, 146.89 ðC7 Þ, 152.60
0
00
00
0
0
000
0
0
0
0
0
00
0
0
111.79 (C4), 112.41 ðC6 Þ, 118.19 ðC4 aÞ, 139.92 ðC5 Þ, 147.89 ðC7 Þ,
ðC2 @OÞ, 156.72 (C5), 159.49 ð8 -C4 Þ, 160.76 ðC4 @OÞ, 162.05 ðC8 aÞ.
Anal. Calcd for C39H51N3O3ꢂH2O: C, 74.61; H, 8.51; N, 6.69. Found:
C, 74.96; H, 8.58; N, 6.74.
0
0
0
156.92 ðC2 @OÞ, 157.09 (C5), 162.70 ðC4 @OÞ, 169.52 ðC8 aÞ. Anal.
Calcd for C34H49N3O2ꢂ0.25H2O: C, 76.15; H, 9.30; N, 7.84. Found:
C, 76.18; H, 9.10; N, 7.93.
4.1.17. 80-(3-Methylphenyl)-50-deazacholest-2,4-dieno[2,3-
4.1.14. 80-Butyl-50-deazacholest-2,4-dieno[2,3-g]pteridine-
20,40(30H,80H)-dione (7c)
g]pteridine-20,40(30H,80H)-dione (7f)
Yield, (470 mg, 32%); mp 213–215 °C (from EtOH); ½a D28
ꢁ
(EtOH):
Yield, (460 mg, 34%); mp 233–235 °C (from EtOH); ½a D28
ꢁ
(EtOH):
ꢀ31.24°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 225 (4.43),
ꢀ22.32°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 232 (4.12),
279 (4.39), 444 (4.07); IR (m
max/cmꢀ1): 3178 (NH), 1624, 1540
289 (4.08), 425 (3.98); IR (
m
max/cmꢀ1): 3145 (NH), 1651, 1596
(CO); 1H NMR (CDCl3): dH 0.71 (3H, s, 18-CH3), 0.86 (6H, d,
J = 6.6 Hz, 25-CH(CH3)2), 0.92 (3H, d, J = 6.3 Hz, 21-CH3), 1.12 (3H,
s, 19-CH3), 1.06–2.33 (24H, m), 2.40 (3H, s, 80-Ar-CH3), 2.93 (1H, d,
(CO); 1H NMR (CDCl3): dH 0.73 (3H, s, 18-CH3), 0.88 (6H, d,
J = 6.6 Hz, 25-CH(CH3)2), 0.94 (3H, d, J = 6.3 Hz, 21-CH3), 0.98 (3H,
s, 19-CH3), 0.99 (3H, t, J = 7.5 Hz, 80-NCH2CH2CH2CH3), 1.50 (2H,
m,J = 7.5 Hz, 80-NCH2CH2CH2CH3), 1.11–2.12 (22H, m), 2.57 (2H,
m, 6-CH2), 2.67 (1H, d, J = 15.6 Hz, 1b-H), 2.86 (1H, d, J = 15.6 Hz,
J = 16.5 Hz, 1b-H), 3.03 (1H, d, J = 16.5 Hz, 1a-H), 5.51 (1H, s, 4-CH),
6.92–7.03 (2H, m, 80-Ar-200, 500H), 7.32–7.45 (2H, m, 80-Ar-400, 600H),
8.35 (1H, s, 50-CH), 8.44 (1H, s, exchangeable with D2O, 30-NH); 13C
NMR (CDCl3): dC 11.90 (C18), 18.60 (C21 and C19), 21.49 (C11 and 80-
Ar-mCH3), 22.51 (C26), 22.62 (C27), 23.79 (C23 and C15), 28.06 (C16),
32.87 (C25), 33.13 (C6 and C7), 35.81 (C22,C8 and C20), 37.81 (C12),
39.11 (C24), 39.48 (C10), 39.78 (C1), 42.43 (C13), 53.81 (C14), 56.07
1a
-H), 4.40 (1H, m, 80-NCHaHbCH2CH2CH3), 4.74 (1H, m, 80-
NCHaHbCH2CH2CH3), 6.34 (1H, s, 4-CH), 8.25 (1H, s, 50-CH), 8.42
(1H, s, exchangeable with D2O, 30-NH); 13C NMR (CDCl3): dC
12.06 (C18), 13.93 (80-NCH2CH2CH2CH3), 17.76 (C21), 18.78 (C19),
20.11 (80-NCH2CH2CH2CH3), 21.76 (C11), 22.73 (C26), 22.96 (C27),
23.97 (C23), 24.42 (C15), 28.17 (C16), 30.75 (80-NCH2CH2CH2CH3),
31.33 (C25), 33.74 (C6 and C7), 35.90 (C22), 36.23 (C8, C20 and C12),
39.07 (C24), 39.64 (C10), 40.59 (C1), 42.56 (C13), 46.28 (80-
NCH2CH2CH2CH3), 53.81 (C14), 55.86 (C9), 56.25 (C17), 111.91 (C4),
(C9 and C17), 112.04 (C4), 115.41 ðC60 Þ, 117.67 ðC4 aÞ, 124.34 ð80-C4 Þ,
0
00
0
0
0
0
0
00
00
00
127.71 (8 -ðC5 )), 130.30 (8 -C3 ), 131.27 (8 -C6 ), 136.29 (8 -C2 ),
139.278 ð80-C1 Þ, 140.46 ðC5 Þ, 149.02 ðC7 Þ, 156.69 ðC2 @OÞ,157.12
00
0
0
0
0
0
(C5), 161.85 ðC4 @OÞ, 168.69 ðC8 aÞ. Anal. Calcd for C36H53N3O2: C,
78.88; H, 8.66; N, 7.08. Found: C, 78.71; H, 8.58; N, 7.11.
0
0
0
0
112.74 ðC6 Þ, 118.57 ðC4 aÞ, 140.07 ðC5 Þ, 148.27 ðC7 Þ, 157.23
ðC2 @OÞ, 157.57 (C5), 162.95 ðC4 @OÞ, 169.67 ðC8 aÞ. Anal. Calcd for
36H53N3O2: C, 77.24; H, 9.54; N, 7.51. Found: C, 77.23; H, 9.42;
4.1.18. 80-(4-Fluorophenyl)-50-deazacholest-2,4-dieno[2,3-
0
0
0
C
g]pteridine-20,40(30H,80H)-dione (7g)
N, 7.21.
Yield, (470 mg, 32%); mp 227–229 °C (from EtOH); ½a D28
ꢁ
(EtOH):
ꢀ58.84°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 216 (3.99),
4.1.15. 80-Phenyl-50-deazacholest-2,4-dieno[2,3-g]pteridine-
20,40(30H,80H)-dione (7d)
253 (3.87), 457 (3.67); IR (m
max/cmꢀ1): 3180 (NH), 1683, 1601 (CO);
1H NMR (CDCl3): dH 0.71(3H, s, 18-CH3), 0.88 (6H, d, J = 6.6 Hz, 25-
CH(CH3)2), 0.94 (3H, d, J = 6.3 Hz, 21-CH3), 1.02 (3H, s, 19-CH3), 1.06–
2.34 (24H, m),2.72 (1H, d, J = 15.6 Hz, 1b-H), 2.91 (1H, d, J = 15.6 Hz,
Yield, (470 mg, 33%); mp 227–229 °C (from EtOH); ½a D28
ꢁ
(EtOH):
ꢀ23.24°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 220 (4.11),
275 (4.00), 485 (3.70); IR (
m
H
max/cmꢀ1): 3190 (NH), 1617, 1560
0.71 (3H, s, 18-CH3), 0.86 (6H, d,
1a
-H), 6.52 (1H, s, 4-CH), 7.12–7.14 (2H, m, 80-Ar-mH), 7.22–7.28
(CO); 1H NMR (CDCl3): d
(3H, m, 80-Ar-oH), 8.37 (1H, s, 50-CH), 8.49 (1H, s, exchangeable with
D2O, 30-NH); 13C NMR (CDCl3): d C12.08 (C18), 18.12 (C21), 18.81 (C19),
21.46 (C11), 22.71 (C26), 22.96 (C27), 23.94 (C23 and C15), 28.16 (C16),
31.21 (C25), 33.38 (C6 and C7), 35.87 (C22), 36.22 (C8 and C20), 37.99
(C12), 39.32 (C24), 39.63 (C10), 40.27 (C1), 42.59 (C13), 55.83 (C14),
J = 6.6 Hz, 25-CH(CH3)2), 0.94 (3H, d, J = 6.3 Hz, 21-CH3), 1.02 (3H,
s, 19-CH3), 1.06–2.33 (24H, m), 2.72 (1H, d, J = 15.6 Hz, 1b-H),
2.93 (1H, d, J = 15.6 Hz, 1a-H), 5.51 (1H, s, 4-CH), 7.13–7.23 (2H,
m, 80-Ar-mH), 7.55–7.61 (3H, m, 80-Ar-o,pH), 8.37 (1H, s, 50-CH),
8.39 (1H, s, exchangeable with D2O, 30-NH); 13C NMR (CDCl3): dC
11.89 (C18), 18.63 (C21 and C19), 21.40 (C11), 22.61 (C26), 22.78
(C27), 23.82 (C23 and C15), 27.98 (C16), 32.85 (C25), 33.15 (C6 and
C7), 35.73 (C22), 36.06 (C8 and C20), 37.81 (C12), 39.11 (C24), 39.47
(C10), 40.17 (C1), 42.45 (C13), 53.83 (C14), 55.70 (C9), 56.11 (C17),
56.26 (C9), 56.52 (C17), 112.27 (C4), 113.90 ðC60 Þ, 117.94 (C4 a), 80-C300
0
0
0
0
00
00
00
0
and ðC5 ), 129.74 (8 -C2 and C6 ), 135.26 ð8 -C1 Þ, 141.81 ðC5 Þ,
0
0
0
149.16 ðC7 Þ, 157.41 ðC2 @OÞ, 161.47 (C5), 162.45 ðC4 @OÞ, 164.80
ð80-C4 Þ, 169.54 ðC8 aÞ. Anal. Calcd for C38H48FN3O2ꢂ0.2H2O: C, 75.89;
00
0
H, 8.11; N, 6.99. Found: C, 76.02; H, 8.38; N, 6.74.
112.10 (C4), 115.47 ðC60 Þ, 117.50 ðC4 aÞ, 127.48 (80-C3 ) and ðC5 ),
0
00
0
130.18 ð80-C4 Þ, 130.26 (8 -C2 ) and ðC5 ), 136.39 ð8 -C1 Þ, 141.17
4.1.19. 80-(4-Chlorophenyl)-50-deazacholest-2,4-dieno[2,3-
0
0
00
00
0
00
ðC5 Þ, 148.91 ðC7 Þ, 156.46 ðC2 @OÞ, 156.82 (C5), 162.36 ðC4 @@OÞ,
g]pteridine-20,40(30H,80H)-dione (7h)
0
0
0
0
Yield, (470 mg, 31%); mp 223–225 °C (from EtOH); ½a D28
ꢁ
(EtOH):
0
168.66 ðC8 aÞ. Anal. Calcd for C38H49N3O2: C, 78.72; H, 8.52; N,
7.25. Found: C, 78.69; H, 8.47; N, 7.32.
ꢀ35.92°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 221 (4.53),

8694
A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 8685–8696
252 (4.41), 289 (4.27), 452 (4.24); IR (
m
max/cmꢀ1): 3195 (NH), 1683,
J = 6.6 Hz, 25-CH(CH3)2), 0.93 (3H, d, J = 6.3 Hz, 21-CH3), 1.07 (3H,
s, 19-CH3), 1.11–2.33 (24H, m), 2.94 (1H, d, J = 16.2 Hz, 1b-H),
3.06 (1H, d, J = 16.2 Hz, 1 -H), 6.51 (1H, s, 4-CH), 7.20–7.23 (4H,
1620 (CO); 1H NMR (CDCl3): dH 0.71 (3H, s, 18-CH3), 0.86 (6H, d,
J = 6.6 Hz, 25-CH(CH3)2), 0.93 (3H, d, J = 6.3 Hz, 21-CH3), 1.05 (3H,
s, 19-CH3), 1.06–2.16 (24H, m), 2.93 (1H, d, J = 16.5 Hz, 1b-H),
a
m, 30, 80-Ar-mH), 7.34–7.39 (1H, m, 30-Ar-pH), 7.42–7.48 (2H, m,
30-Ar-oH), 7.55–7.58 (3H, m, 80-Ar-o,pH), 8.47 (1H, s, 50-CH); 13C
NMR (CDCl3): dC 12.04 (C18), 18.38 (C21), 18.82 (C19), 21.53 (C11),
22.67 (C26), 22.93 (C27), 23.94 (C23 and C15), 28.14 (C16), 33.02
(C25, C6 and C7), 35.85 (C22), 36.24 (C8 and C20), 37.96 (C12), 39.62
(C24 and C10), 39.96 (C1), 42.59 (C13), 49.65 (C14), 56.07 (C9),
3.07 (1H, d, J = 16.5 Hz, 1a-H), 6.50 (1H, s, 4-CH), 7.04–7.08 (2H,
m, 80-Ar-mH), 7.52–7.55 (3H, m, 80-Ar-oH), 8.41 (1H, s, 50-CH),
8.88 (1H, s, exchangeable with D2O, 30-NH); 13C NMR (CDCl3): dC
11.81 (C18), 18.22 (C21), 18.32 (C19), 21.35 (C11), 22.59 (C26),
22.76 (C27), 23.80 (C23), 23.98 (C15), 27.97 (C16), 32.83 (C25, C6
and C7), 35.67 (C22), 36.05 (C8 and C20), 37.82 (C12), 39.01 (C24),
39.45 (C10), 39.69 (C1), 42.43 (C13), 49.40 (C14), 55.92 (C9), 56.35
56.32 (C17), 115.73 (C4), 119.98 ðC6 Þ, 121.13 ðC4 aÞ, 127.67 (80-C3
0
0
000
0
0
000
00
00
0
00
00
and C5 ),128.41 (3 -C4 ), C3 and ðC5 ), 129.42 (3 -C2 and C6 ),
(C17), 115.45 (C4), 119.98 ðC60 Þ, 120.55 ðC4 aÞ, 128.97 (80-C3 ) and
130.45 ð80-C4 Þ, 130.62 (8 -C2 and C6 ), 136.45 ð30 ꢀ C1 Þ, 142.40
0
0
00
000
000
000
00
0
0
0
0
00
00
00
00
0
0
000
0
ðC5 ), 130.57 (8 -C2 and C6 ), 134.71 ð8 -C4 Þ, 136.49 ðC1 Þ, 141.96
ðC5 Þ, 146.08 ðC7 Þ, 152.49 (8 -C1 ), 156.35 ðC2 @OÞ, 157.91 (C5),
0
0
0
0
0
0
ðC5 Þ, 145.93 ðC7 Þ, 152.77 ðC2 @OÞ, 156.33 (C5), 159.04 ðC4 @OÞ,
162.13 ðC4 @OÞ, 168.57 ðC8 aÞ. Anal. Calcd for C44H53N3O2ꢂH2O: C,
0
161.64 ðC8 aÞ. Anal. Calcd for C38H48ClN3O2ꢂ0.4H2O: C, 73.44; H,
78.42; H, 8.23; N, 6.24. Found: C, 78.76; H, 8.09; N, 6.35.
7.91; N, 6.76. Found: C, 73.58; H, 7.96; N, 6.79.
4.1.23. 80-(3-Methylphenyl)-30-phenyl-50- deazacholest-2,4-
4.1.20. 80-(3,4-Dimethylphenyl)-50-deazacholest-2,4- dieno[2,3-
dieno[2,3-g]pteridine-20,40(30H,80H)-dione (9c)
g]pteridine-20,40(30H,80H)-dione (7i)
Yield, (580 mg, 35%); mp 214–216 °C (from EtOH); ½a D28
ꢁ
(EtOH):
Yield, (480 mg, 32%); mp 197–199 °C (from EtOH); ½a D28
ꢁ
(EtOH):
ꢀ32.00°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 214 (4.45),
ꢀ19.08°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 219 (4.46),
260 (4.23), 454 (3.92); IR (m
max/cmꢀ1): 1698, 1624 (CO); 1H NMR
286 (4.28), 428 (4.11); IR (
m
max/cmꢀ1): 3195 (NH), 1697, 1623 (CO);
(CDCl3): dH 0.72 (3H, s, 18-CH3), 0.88 (6H, d, J = 6.6 Hz, 25-
CH(CH3)2), 0.93 (3H, d, J = 6.3 Hz, 21-CH3), 1.13 (3H, s, 19-CH3),
1.17–2.33 (24H, m), 2.42 (3H, s, 80-Ar-CH3), 2.94 (1H, d,
1H NMR (CDCl3): dH 0.71 (3H, s, 18-CH3), 0.87 (6H, d, J = 6.6 Hz, 25-
CH(CH3)2), 0.93 (3H, d, J = 6.3 Hz, 21-CH3), 1.00 (3H, s, 19-CH3), 2.29
(3H, s, 80-Ar-300CH3), 1.06–2.29 (24H, m), 2.33 (3H, s, 80-Ar-400CH3),
J = 15.6 Hz, 1b-H), 3.12 (1H, d, J = 15.6 Hz, 1a-H), 5.15 (1H, s, 4-
2.71 (1H, d, J = 15.9 Hz, 1b-H), 2.90 (1H, d, J = 15.9 Hz, 1
a
-H), 5.58
CH), 6.91–7.17 (2H, m, 80-Ar-200,500H), 7.21 (2H, m, 30-Ar-mH),
7.36–7.40 (3H, m, 30-Ar-pH, 80-Ar-400,600H), 7.45 (2H, m, 30-Ar-oH)
8.49 (1H, s, 50-CH); 13C NMR (CDCl3): dC 11.82 (C18), 18.27 (C21),
18.65 (C19), 21.43 (C11 and Ar-mCH3), 22.52 (C26), 22.58 (C27),
23.77 (C23), 23.98 (C15), 27.99 (C16), 32.85 (C25, C6 and C7), 35.27
(C22), 35.66 (C8 and C20), 37.04 (C12), 39.12 (C24), 39.45 (C10),
39.79 (C1), 42.43 (C13), 49.51 (C14), 55.92 (C9), 56.37 (C17), 115.46
(1H, s, 4-CH), 6.84–6.94 (2H, m, 80-Ar-200, 500H), 7.27–7.29 (1H, m, 80-
Ar-600H), 8.35 (1H, s, 50-CH), 8.61 (1H, s, exchangeable with D2O, 30-
NH); 13C NMR (CDCl3): dC11.88 (C18), 17.92 (C21), 18.68 (C19), 19.64
(80-Ar-mCH3), 20.04 (80-Ar-pCH3), 21.62 (C11), 22.57 (C26), 22.84
(C27), 23.80 (C23), 24.22 (C15), 28.02 (C16), 31.06 (C25), 33.05 (C6, C7),
35.71 (C22), 36.08 (C8 and C20), 37.78 (C12), 39.08 (C24), 39.47 (C10),
40.20 (C1), 42.38 (C13), 53.81 (C14), 55.69 (C9), 56.09 (C17), 111.99
(C4), 120.06 ðC6 Þ, 124.52 ðC4 aÞ, 128.22 (30-C4 ), C3 and ðC5 ),
0
0
00
00
0
0
(C4), 114.14 ðC6 Þ, 117.55 ðC4 aÞ, 124.53 (8 -ðC5 )), 128.14 ð8 -C4 Þ,
129.04 ð80-C4 Þ, 129.30 (3 -C2 and C6 ), 135.21 (8 -C5 ), 136.08
0
0
0
0
0
0
00
000
00
00
000
0
0
0
0
0
00
00
00
00
0
00
000
00
0
131.10 (8 -C2 ), 134.43 ð8 -C1 Þ, 138.73 (8 -C6 and C3 ), 141.06 ðC5 Þ,
ð30 ꢀ C1 Þ, 137.30 (8 -C6 ), 139.14 (8 -C2 ), 140.56 ðC5 Þ, 142.39
149.18 ðC7 Þ, 157.07 ðC2 @OÞ, 158.79 (C5), 162.45 ðC4 @OÞ, 168.42
(80-C1 ), 146.14 ðC70 Þ, 151.25 (8 -C3 ), 156.57 ðC20 @OÞ, 157.62 (C5),
0
0
0
0
000
000
0
0
0
ðC8 aÞ. Anal. Calcd for C40H53N3O2: C, 79.03; H, 8.79; N, 6.91. Found:
161.94 ðC4 @OÞ, 164.66 ðC8 aÞ. Anal. Calcd for C45H55N3O2ꢂ0.5H2O:
C, 79.06; H, 8.66; N, 6.75.
C, 79.61; H, 8.31; N, 6.19. Found: C, 79.51; H, 8.54; N, 6.19.
4.1.21. 80-Methyl-3-phenyl-50-deazacholest-2,4-dieno[2,3-
4.1.24. 80-(4-Methoxyphenyl)-30-phenyl-50-deazacholest-2,4-
g]pteridine-20,40(30H,80H)-dione (9a)
dieno[2,3-g]pteridine-20,40(30H,80H)-dione (9d)
Yield, (450 mg, 31%); mp 218–220 °C (from EtOH); ½a D28
ꢁ
(EtOH):
Yield, (610 mg, 36%); mp 202–204 °C (from EtOH); ½a D28
ꢁ
(EtOH):
ꢀ16.44°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 220 (4.15),
ꢀ23.04°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 222 (4.38),
286 (4.09), 420 (3.90); IR (
m
max/cmꢀ1): 1698, 1635 (CO); 1H NMR
282 (4.15), 428 (3.88); IR (m
max/cmꢀ1): 1697, 1636 (CO); 1H NMR
(CDCl3): dH0.73 (3H, s, 18-CH3), 0.88 (6H, d, J = 6.6 Hz, 25-
CH(CH3)2), 0.94 (3H, d, J = 6.3 Hz, 21-CH3), 0.99 (3H, s, 19-CH3),
1.11–2.11 (22H, m), 2.56 (2H, m, 6-CH2), 2.69 (1H, d, J = 15.9 Hz,
(CDCl3): dH 0.71 (3H, s, 18-CH3), 0.86 (6H, d, J = 6.6 Hz, 25-CH(CH3)2),
0.94 (3H, d, J = 6.3 Hz, 21-CH3), 1.01 (3H, s, 19-CH3), 1.11–2.33 (24H,
m), 2.73 (1H, d, J = 15.6 Hz, 1b-H), 2.94 (1H, d, J = 15.6 Hz, 1a-H), 3.88
1b-H), 2.89 (1H, d, J = 15.9 Hz, 1
a
-H), 4.07 (3H, s, 80-NCH3), 6.43
(3H, s, 80-Ar-OCH3), 5.64 (1H, s, 4-CH), 7.07 (2H, d, J = 6.9 Hz, 80-Ar-
mH), 7.24 (2H, d, J = 6.9 Hz, 80-Ar-oH), 7.25–7.28 (2H, m, 30-Ar-oH),
7.35–7.39 (1H, m, 30-Ar-pH), 7.44–7.48 (2H, m, 30-Ar-oH), 8.40 (1H,
s, 50-CH); 13C NMR (CDCl3): dC 11.95 (C18), 18.65 (C21 and C19),
21.64 (C11), 22.56 (C26), 22.82 (C27), 23.77 (C23 and C15), 27.99
(C16), 31.11 (C25), 33.13 (C6 and C7), 35.72 (C22), 36.06 (C12, C8 and
(1H, s, 4-CH), 7.24 (2H, d, J = 7.8 Hz, 30-Ar-mH), 7.41 (1H, m, 30-
Ar-pH), 7.48 (2H, d, J = 7.8 Hz, Ar-oH), 8.26 (1H, s, 50-CH); 13C
NMR (CDCl3): dC 12.08 (C18), 17.78 (C21), 18.83 (C19), 21.80 (C11),
22.72 (C26), 22.99 (C27),23.97 (C23), 24.43 (C15), 28.17 (C16), 31.35
(C25, C6 and C7), 33.66 (C22), 33.87 (C8 and C20), 35.91 (80-NCH3),
36.25 (C12), 39.17 (C24), 39.63 (C10), 40.46 (C1), 42.56 (C13), 53.86
C
20), 39.11 (C24 and C10), 39.47 (C1), 42.39 (C13), 53.85 (C14), 55.68
(C14), 55.87 (C9), 56.26 (C17), 111.95 (C4), 112.98 ðC6 Þ, 117.98
(C9, 80-Ar-pOCH3), 56.07 (C17), 112.12 (C4), 113.96 ðC6 Þ, 115.22
0
0
ðC4 aÞ, 128.47 (30-C3 , C5 and C4 ),129.43 (3 -C2 and C6 ), 136.75
(80-C3 ) and ðC5 ), 117.68 ðC4 aÞ, 128.28 (30-C4 ), C3 and ðC5 ), 129.03
0
0
00
00
00
00
00
00
000
0
0
00
00
0
0
(30-C1 ), 140.74 ðC5 Þ, 148.48 ðC7 Þ, 156.32 ðC2 @OÞ, 157.01 (C5),
(80-C2 and C6 ), 129.24 (3 -C2 and C6 ), 135.41 (8 -C1 ), 136.45
0
00
0
0
0
000
00
00
000
0
0
00
0
0
0
162.93 ðC4 @OÞ, 169.68 ðC8 aÞ. Anal. Calcd for C39H51N3O2: C,
ð30-C1 Þ, 141.72 ðC5 Þ, 149.14 ðC7 Þ,157.01 ðC @OÞ, 157.52 (C5),
160.36 ð80-C4 Þ, 162.50 ðC4 @OÞ, 168.53 ðC8 2aÞ. Anal. Calcd for
C45H55N3O3: C, 78.79; H, 8.08; N, 6.13. Found: C, 79.10; H, 8.17; N,
6.13.
000
0
0
78.88; H, 8.66; N, 7.08. Found: C, 79.05; H, 8.74; N, 7.22.
4.1.22. 30-80-Diphenyl-50-deazacholest-2,4-dieno[2,3-
g]pteridine-20,40(30H,80H)-dione (9b)
Yield, (550 mg, 34%); mp 216–218 °C (from EtOH); ½a D28
ꢁ
(EtOH):
4.1.25. 80-(3,4-Dimethylphenyl)-30-phenyl-50-deazacholest-2,4-
ꢀ29.60°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1): 210 (4.29),
dieno[2,3-g]pteridine-20,40(30H,80H)-dione (9e)
258 (4.14), 303 (3.93), 446 (3.97); IR (
m
max/cmꢀ1): 1698, 1646
Yield, (740 mg, 44%); mp 197–199 °C (from EtOH);
½
a 2D8
ꢁ
(CO); 1H NMR (CDCl3): dH 0.71 (3H, s, 18-CH3), 0.88 (6H, d,
(EtOH): ꢀ36.68°; UV (EtOH): kmax/nm (log
e
/dm3 molꢀ1 cmꢀ1):

A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 8685–8696
8695
208 (4.30), 290 (4.02), 431 (3.85); IR (
m
max/cmꢀ1): 1697, 1645
4.2. Growth inhibitory activities of 30,80-disubstituted-50-
deazacholest-2,4-dieno[2,3-g]pteridine-20,40(30H, 80H)-diones
(5a–j, 7a–i and 9a–g) against human tumor cell lines
(CO); 1H NMR (CDCl3): dH 0.71 (3H, s, 18-CH3), 0.88 (6H, d,
J = 6.6 Hz, 25-CH(CH3)2), 0.94 (3H, d, J = 6.3 Hz, 21-CH3), 1.02
(3H, s, 19-CH3), 1.11–2.26 (24H, m), 2.31 (3H, s, 80-Ar-300CH3),
2.35 (3H, s, 80-Ar-400CH3), 2.73 (1H, d, J = 15.6 Hz, 1b-H), 2.94
The procedure was carried out using the modified MTT assay36
to determine the inhibitory effects of test compounds on cell
growth in vitro as mentioned in detail in our previous work.2
Two human tumor cell lines of human T-cell acute lymphoblastoid
leukemia (CCRF-HSB-2) and human oral epidermoid carcinoma
(KB) were used in this study.
(1H, d, J = 15.6 Hz, 1a-H), 5.61 (1H, s, 4-CH), 6.91–7.04 (2H, m,
80-Ar-200,500H), 7.22–7.25 (2H, m, 30-Ar-mH),7.25–7.33 (1H, m,
80-Ar-600H), 7.35–7.41 (1H, m, 30-Ar-pH), 7.44–7.46 (2H, m, 30-
Ar-oH), 8.40 (1H, s, 50-CH); 13C NMR (CDCl3): dC 11.87 (C18),
17.89 (C21), 18.66 (C19), 19.72 (80-Ar-mCH3), 20.12 (80-Ar-pCH3),
21.77 (C11), 22.59 (C26 and C27), 23.79 (C23 and C15), 28.00
(C16), 33.13 (C25, C6 and C7), 35.73 (C22), 36.06 (C12, C8 and
4.3. Protocol for molecular docking study
C20), 39.11 (C24 and C10), 39.49 (C1), 42.39 (C13), 53.85 (C14),
0
0
56.09 (C9 and C17), 112.11 (C4), 114.21 ðC6 Þ, 117.62 ðC4 aÞ,
The docking software LigandFit within the software package
Discovery Studio 1.7 was used to evaluate and predict the in silico
binding free energy of the inhibitors within the macromolecules.
First, water molecules and phosphate group of the receptor mole-
cule were removed, hydrogen was added under a condition pH
7.40, and the alternative conformations were corrected. A binding
pocket of the native STI-571 ligand was selected as the binding site
for the study and expanded to make the cavity. The cavity was par-
titioned into 10 partition sites, and the number of points of the cav-
ity was 5461. The forcefield PLP1 function was used to roughly
search the conformations when compounds dock into the PTK.
Then the conformations were optimized using a forcefield function,
CHARMm and ‘Adopted Basis NR’ was chosen for the algorithm to
optimize conformations. At last, Ludi Energy Estimate 3 was used
to estimate the binding free energies of the ligands. Among the
docked conformations, the pose with highest value of Ludi-3 was
124.71 ð80-C5 Þ, 128.30 (3 ꢀ C4 ), C3 and ðC5 ), 129.25 (8 -C2
0
0
000
00
00
0
000
and C6 ), 131.08 (80-C2 ), 134.30 (80-C1 ), 136.48 ð30 ꢀ C1 Þ,
000
00
000
00
138.83 (80-C3 and C6 ), 141.73 ðC5 Þ, 148.93 ðC7 Þ, 151.15 (8 -
0
00
00
0
0
C3 ), 151.49 ð80-C4 Þ, 157.03 ðC2 @OÞ, 157.63 (C5), 162.57
000
000
0
0
0
ðC4 @OÞ, 168.34 ðC8 aÞ. Anal. Calcd for C46H57N3O2ꢂ0.33H2O: C,
80.07; H, 8.42; N, 6.09. Found: C, 80.15; H, 8.68; N, 6.03.
4.1.26. 80-(4-Fluorophenyl)-30-phenyl-50-deazacholest-2,4-
dieno[2,3-g]pteridine-20,40(30H,80H)-dione (9f)
Yield, (580 mg, 35%); mp 212–214 °C (from EtOH);
(EtOH): ꢀ41.80°; UV (EtOH): kmax/nm (log
/dm3 molꢀ1 cmꢀ1):
max/cmꢀ1): 1697, 1636
½
a 2D8
ꢁ
e
220 (4.33), 282 (4.18), 433 (3.97); IR (
m
(CO); 1H NMR (CDCl3): dH 0.71 (3H, s, 18-CH3), 0.88 (6H, d,
J = 6.6 Hz, 25-CH(CH3)2), 0.93 (3H, d, J = 6.3 Hz, 21-CH3), 1.01
(3H, s, 19-CH3), 1.06–2.33 (24H, m), 2.72 (1H, d, J = 15.9 Hz, 1b-
H), 2.93 (1H, d, J = 15.9 Hz, 1
a
-H), 5.54 (1H, s, 4-CH), 7.19–7.23
selected for the calculation of binding free energy (DGb) and inhi-
bition constant (Ki).
(2H, m, 80-Ar-mH), 7.24–7.28 (3H, m, 30-Ar-mH), 7.36–7.39 (1H,
m, 30-Ar-pH), 7.42–7.47 (2H, m, 80-Ar-oH), 8.40 (1H, s, 50-CH);
13C NMR (CDCl3): dC 11.94 (C18), 17.93 (C21), 18.67 (C19), 21.65
(C11), 22.56 (C26), 22.82 (C27), 23.79 (C23), 24.23 (C15), 28.02
(C16), 31.09 (C25), 33.22 (C6 and C7), 35.72 (C22), 36.10 (C8 and
4.3.1. Preparation of ligands and target tyrosine kinase and
analysis of docked results
The compounds involved in this study as ligands including 5a–j,
7a–i, and 9a–g were studied for their binding activities into protein
tyrosine kinase. The three-dimensional structures of the aforemen-
tioned compounds were constructed using Chem 3D ultra 8.0 soft-
ware [Chemical Structure Drawing Standard; Cambridge Soft
Corporation, USA (2003)]. The crystal structures of protein tyrosine
kinase (1t46) complex with (STI) were retrieved from the RCSB
Protein Data Bank http://www.rcsb.org/pdb/home/home.do (PDB
code: 1t46). All bound waters and ligands were removed from
the protein, and the polar hydrogens were added to the proteins.
Accelrys, DS Studio 1.7 (Accelrys, San Diego CA, www.accelrys.
com) was used for evaluating the hydrogen bonding, for determin-
ing binding orientations, for molecular modeling, and for measur-
ing RMSD, which was measured as distance between the centroids
of the docked inhibitor and the native ligand. The native ligand
(STI-571) within PTK was used as a standard docked model for
the study of mode of interaction of the ligands with receptor and
for the calculation of RMSD.
C
20), 37.82 (C12), 39.16 (C24), 39.48 (C10), 40.15 (C1), 42.40 (C13),
0
53.87 (C14), 55.69 (C9), 56.10 (C17), 112.25 (C4), 113.67 ðC6 Þ,
117.26 ðC4 aÞ, 117.61 (80-C3 and C5 ), 128.27 (3 ꢀ C4 ), C3 and
0
0
000
000
00
00
0
0
0
00
00
000
000
ðC5 ), 129.25 (3 -C2 and C6 ), 129.82 (8 -C2 and C6 ), 132.57
(80-C1 ), 136.40 ð30 ꢀ C1 Þ, 142.11 ðC5 Þ, 148.64 ðC7 Þ,157.44
000
00
0
0
0
0
0
000
ðC2 @OÞ, 158.36 (C5), 162.37 ðC4 @OÞ, 164.66 ð8 -C4 Þ, 169.05
0
ðC8 aÞ; Anal. Calcd for C44H52FN3O2ꢂ0.4H2O: C, 77.59; H, 7.81; N,
6.17. Found: C, 77.74; H, 7.44; N, 6.22.
4.1.27. 80-(4-Chlorophenyl)-30-phenyl-50-deazacholest-2,4-
dieno[2,3-g]pteridine-20,40(30H,80H)-dione (9g)
Yield, (650 mg, 39%); mp 200–202 °C (from EtOH);
(EtOH): ꢀ31.52°; UV (EtOH): kmax/nm (log
/dm3 molꢀ1 cmꢀ1):
max/cmꢀ1): 1697, 1637
½
a 2D8
ꢁ
e
217 (4.42), 294 (4.16), 432 (4.14); IR (
m
(CO); 1H NMR (CDCl3): dH 0.71 (3H, s, 18-CH3), 0.87 (6H, d,
J = 6.6 Hz, 25-CH(CH3)2), 0.93 (3H, d, J = 6.3 Hz, 21-CH3), 1.02
(3H, s, 19-CH3), 1.11–2.33 (24H, m), 2.72 (1H, d, J = 15.6 Hz, 1b-
H), 2.93 (1H, d, J = 15.6 Hz, 1a-H), 5.54 (1H, s, 4-CH), 7.19–7.24
(4H, m, 30,80-Ar-mH), 7.37–7.39 (1H, m, 80-Ar-pH), 7.43–7.46
(2H, m, 30-Ar-oH), 7.54–7.57 (3H, m, 80-Ar-oH), 8.39 (1H, s, 50-
CH); 13C NMR (CDCl3): dC 12.11 (C18), 18.10 (C21), 18.82 (C19),
21.79 (C11), 22.73 (C26), 23.00 (C27), 23.96 (C23), 24.40 (C15),
28.17 (C16), 31.26 (C25), 33.33 (C6 and C7), 35.89 (C22), 36.10
(C12, C8 and C20), 39.30 (C24), 39.65 (C10), 40.28 (C1), 42.56 (C13),
Acknowledgments
The authors are indebted to the SC NMR Laboratory of Okayama
University for the NMR spectral measurements and the Okayama
University Information Technology Center for using Accelrys
Discovery Studio 1.7.
0
54.03 (C14), 55.85 (C9), 56.26 (C17), 112.41 (C4), 113.83 ðC6 Þ,
117.41 ðC4 aÞ, 128.25 (80-C3 and C500), 128.46 (30 ꢀ C4 ), C3 and
0
0
000
00
00
0
0
References and notes
0
00
000
000
000
ðC5 ), 129.37 (3 -C2 and C6 ), 130.66 (8 -C2 and C6 ), 135.39
ð80-C4 Þ, 136.14 (8 -C1 ), 136.69 ð30 ꢀ C1 Þ, 142.17 ðC5 Þ, 148.42
000
000
00
0
1. Nagamatsu, T.; Yamada, H.; Shiromoto, K. Heterocycles 2004, 63, 9.
2. Ali, H. I.; Tomita, K.; Akaho, E.; Kambara3, H.; Miura, S.; Hayakawa, H.; Ashida,
N.; Kawashima, Y.; Yamagishi, T.; Ikeya, H.; Yoneda, F.; Nagamatsu, T. Bioorg.
Med. Chem. 2007, 15, 242.
0
0
0
0
ðC7 Þ,156.60 ðC2 @OÞ, 157.59 (C5), 162.59 ðC4 @OÞ, 168.92 ðC8 aÞ;
Anal. Calcd for C44H52ClN3O2ꢂH2O: C, 74.60; H, 7.68; N, 5.93.
Found: C, 74.43; H, 7.48; N, 5.93.

8696
A. R. Shrestha et al. / Bioorg. Med. Chem. 16 (2008) 8685–8696
3. Ali, H. I.; Ashida, N.; Nagamatsu, T. Biorg. Med. Chem. 2007, 15, 6336.
4. Sakuma, Y.; Nagamatsu, T.; Yoneda, F. J. Chem. Soc. Chem. Commun. 1975, 977.
5. Sakuma, Y.; Matsumoto, S.; Nagamatsu, T.; Yoneda, F. Chem. Pharm. Bull. 1976,
24, 338.
6. Nagamatsu, T.; Yamato, H.; Ono, M.; Takarada, S.; Yoneda, F. J. Chem. Soc. Perkin
Trans. 1 1992, 2101.
20. Morris, G. M.; Goodsell, D. S.; Halliday, R.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
21. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.;
Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.;
Shenkin, P. S. J. Med. Chem. 2004, 47, 1739.
22. McMartin, C.; Bohacek, R. S. J. Comput.-Aided Mol. Des. 1997, 11, 333.
23. Abagyan, R.; Totrov, M.; Kuznetsov, R. J. Comput. Chem. 1994, 15, 488.
24. Venkatachalam, C. M.; Jiang, X.; Oldfield, T.; Waldman, M. J. Mol. Graph. Model.
2003, 21, 289.
25. Montes, M.; Miteva, M. A.; Villoutreix, B. O. Proteins 2007, 68, 712.
26. Yoneda, F.; Sakuma, Y.; Ichiba, M.; Shinomura, K. J. Am. Chem. Soc. 1976, 98(3),
830.
7. Nagamatsu, T.; Kuroda, K.; Mimura, N.; Yanada, R.; Yoneda, F. J. Chem. Soc.
Perkin Trans. 1 1994, 1125.
8. O0Brien, D. E.; Weinstock, L. T.; Cheng, C. C. J. Heterocycl. Chem. 1970, 7, 99.
9. Hemmerich, P.; Massey, V.; Fenner, H. FEBS Lett. 1972, 84, 5.
10. Graham, D. W.; Brown, J. E.; Ashton, W. T.; Brown, R. D.; Rogers, E. F. Experientia
1977, 33, 1274.
11. Eirich, L. D.; Vogels, G. D.; Wolfe, R. S. Biochemistry 1978, 17, 4583.
12. Yoneda, F.; Sakuma, Y.; Hemmerich, P. J. Chem. Soc. Chem. Commun. 1977, 825.
13. Yoneda, F.; Tanaka, K. Med. Res. Rev. 1987, 7, 477.
14. Chen, H.; Lyne, P. D.; Giordanetto, F.; Lovell, T.; Li, J. J. Chem. Inf. Model. 2006, 46,
401.
15. Booth, B.; Zemmel, R. Nat. Rev. Drug Discovery 2004, 3, 451.
16. Bajorath, J. Na. Rev. Drug Discovery 2002, 1, 882.
17. Makino, S.; Kuntz, I. D. J. Comput. Chem. 1997, 18, 1812.
18. Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. J. Mol. Biol. 1996, 261, 470.
19. Jones, G.; Willett, R.; Glen, R.; Leach, A. R.; Taylor, R. J. Mol. Biol. 1997, 267,
727.
27. Goldner, H.; Dietz, G.; Carstens, E. Liebigs Ann. Chem. 1966, 691, 142.
28. Stein, A.; Gregor, H. P.; Spoerri, P. E. J. Am. Chem. Soc. 1956, 78, 6185.
29. Bruckman, G.; Isaacs, S. D. J. Am. Chem. Soc. 1949, 71, 390.
30. Nagamatsu, T.; Yamasaki, H.; Yoneda, F. Heterocycles 1994, 37, 1147.
31. Weisenborn, F. L.; Remy, D. C.; Jacobs, T. L. J. Am. Chem. Soc. 1954, 76, 552.
32. Oppenauer, R. V. Org. Synth. 1941, 21, 18.
33. Robinson, D. R.; Wu, Y.; Lin, S. Oncogene 2000, 19, 5548.
34. Wang, R.; Lu, Y.; Wang, S. J. Med. Chem. 2003, 46, 2287.
35. Kovganko, N. V.; Kashkan, Zh. N.; Borisov, E. V. Chem. Nat. Compds. 2000, 36, 596.
36. Miura, S.; Yoshimura, Y.; Endo, M.; Machida, H.; Matsuda, A.; Tanaka, M.;
Sasaki, T. Cancer Lett. 1998, 129, 103.