Synthesis, reactions, characterization and biological evaluation of 2,3′-bipyridine derivatives (III)

Article abstract of DOI:10.1002/jhet.1521

1-Pyridin-3-yl-3-(2-thienyl of 2-furyl)prop-2-en-1-ones 1a, 1b reacted with 2-cyanoethanethioamide 2 to afford the corresponding 4-(thiophen-2-yl or furan-2-yl)-6-sulfanyl-2,3′-bipyridine-5-carbonitriles 3a, 3b. The synthetic potentiality of compounds 3a,

Full text of DOI:10.1002/jhet.1521

July 2014
Synthesis, Reactions, Characterization and Biological Evaluation of 2,3′-
927
Bipyridine Derivatives (III)
Fawzy A. Attaby, Azza M. Abdel-Fattah, Labeeb M. Shaif, and Mohamed M. Elsayed
a
a
a
b
*
aChemistry Department, Faculty of Science, Cairo University, Giza 12613, Egypt
bResearch Units, Hi-Care Pharmaceutical Co., Cairo, Egypt
*
E-mail: fattaby@gmail.com
Received June 28, 2011
DOI 10.1002/jhet.1521
Published online 10 December 2013 in Wiley Online Library (wileyonlinelibrary.com).
O
NC
N
EtOH/pip.
reflux 2h
X
+
H2N
HN
X
S
CN
N
S
1a,b
2
3a,b
R
Y
4a-g
Z
X=S, O
N
N
X
X
Cyclisation
N
N
NH2
CN
R
S
S
Z
Z
6a,b
5a,b
1-Pyridin-3-yl-3-(2-thienyl of 2-furyl)prop-2-en-1-ones 1a,b reacted with 2-cyanoethanethioamide (2) to
afford the corresponding 4-(thiophen-2-yl or furan-2-yl)-6-sulfanyl-2,3′-bipyridine-5-carbonitriles 3a,b. The
synthetic potentiality of compounds 3a,b were investigated in the present study via their reactions with
several active halogen containing compounds 4a–h. Our aim here is the synthesis of 4-(2-thienyl or 2-
furyl)-6-pyridin-3-ylthieno[2,3-b]pyridin-3-amines 6a–e,g–n via 6-(alkyl-thio)-4-(2-thienyl or 2-furyl)-
2,3′-bipyridine-5-carbonitriles 5a–e,i–m. The structures of all newly synthesized heterocyclic compounds
were elucidated by considering the data of IR, 1H-NMR, mass spectra, as well as that of elemental analyses.
Anti-cancer, anti-Alzheimer, and anti-COX-2 activities were investigated for all the newly synthesized
heterocyclic compounds.
J. Heterocyclic Chem., 51, 927 (2014).
INTRODUCTION
formed via a Michael addition of –CH2– in 2 on –CH═
CH– of 1a to give the reaction product 3a. Considering
the IR (cmÀ1) data of this reaction product; SH (3216),
CN (2210) groups and the mass spectrum which gave
m/z = 295 (100%) that corresponds to the molecular weight
of the assigned structure. Also, 1H-NMR (d ppm) spectrum
revealed the presence of a broad signal of SH proton at
14.19 (cf. Scheme 1 and Experimental section).
In a similar manner, 1-pyridin-3-yl-3-(2-furyl)prop-2-en-
1-one (1b) was reacted with 2-cyanoethanethioamide (2)
under the same aforementioned experimental conditions
to give the finally isolated 3b. The chemical structure of
3b was elucidated by considering the data of IR and mass
spectra as well as that of elemental analyses (cf. Experimen-
tal section). A further confirmation of 3a,b arose from
their synthesis through an alternative pathway via the
reaction of each of 1a,b, malononitrile in a dispersed sulfur,
In conjunction to our previous recent work [1–14] and
aiming to investigate and evaluate the biological activities
of the newly synthesized heterocyclic compounds, we are in-
terested here to use 3-(2-thienyl or 2-furyl)-1-pyridin-3-
ylprop-2-en-1-ones 1a,b as key compounds to synthesize
2,3′-bipyridine-5-carbonitriles 3a,b required for several
chemical transformations as well as our medicinal chemistry
programs.
RESULTS AND DISCUSSION
1-Pyridin-3-yl-3-(2-thienyl)prop-2-en-1-one (1a) reacted
with 2-cyanoethanethioamide (2) in absolute ethanol
containing the catalytic amount of piperidine under reflux
to afford the reaction product. Such reaction product
© 2013 HeteroCorporation

928
F. A. Attaby, A. M. Abdel-Fattah, L. M. Shaif, and M. M. Elsayed
Vol 51
Scheme 1
X
NH
2
O
N
S
EtOH \ Pip.
reflux 2h
+
HN
CN
X
NC
N
S
1a,b
2
3a,b
X = S, O
morpholine, and ethanol under reflux for 2 h [15]. It is
important to refer here that 3a,b obtained by the two path-
ways are identical in all physical and chemical properties
(cf. Scheme 1 and Experimental section).
The structure of 6c was further confirmed via its
preparation through the reaction of 3a with 3-chloropentan-
2,4-dione 4f (Y═Cl, R═Z═COCH3) in methanolic sodium
methoxide solution either under stirring at room temperature
or refluxing for 2 h. The reaction seemed to proceed via the
intermediate I, which converted to II that, in turn, released
acetic acid molecule to give 2-acetyl-3-amino-4-(2-thienyl)-
6-(3-pyridyl)thieno[2,3-b]pyridine (6c) (cf. Scheme 3).
In contrast to the behavior of 3a towards each of 4a–e, it
has been found that 3a was reacted with each of 4g,h either
under stirring in methanolic sodium methoxide solution at
room temperature or under reflux for 2 h in ethanolic
sodium ethoxide to give the corresponding thieno[2,3-b]
pyridine derivatives 6g,h, respectively, whose structures
The synthetic potentiality of each of 3a,b was investi-
gated through the electrophilic substitution reactions by
using several electrophilic C species. Thus, it has been
found that 3a was reacted with ethyl chloroacetate (4a) in
a stirred methanolic sodium methoxide solution at room
temperature for 15 min to give the reaction product 5a.
The IR (cm1) of this reaction product showed the bands of
CN (2206) and CO (1736) of the newly introduced COOEt
1
group. Its H-NMR (d ppm) spectrum revealed the signals
of –SCH2–, –COOCH2CH3, –COOCH2CH3 protons.
Depending on these spectral data, we concluded that the
good nucleophilicity of S in 3a facilitated the electrophilic
attack of 4a to afford 5a in very pure state. Furthermore,
structure 5a was elucidated through its cyclization in
ethanolic sodium ethoxide under reflux for 30 min to give
the reaction product 6a. The IR spectrum of 6a showed
no bands of CN group, and instead, the bands of the newly
formed NH2 group were detected. Thus, we concluded that
both –SCH2– and CN groups in 5a were involved in the
intramolecular Thrope-Zieglar cyclization step to give the
finally isolated 6a. A further confirmation of 6a structure
was obtained through its preparation authentically via the
reaction of 3a with 4a in ethanolic sodium ethoxide under
reflux for 2 h (cf. Scheme 2 and Experimental section).
Similarly, 3a was reacted with each of 4b–e in a stirred
methanolic sodium methoxide solution at room temperature
to give the corresponding 2-alkylthio derivatives 5b–e
whose structures were elucidated by considering the data
1
elucidated by considering the data of IR, H-NMR, mass
spectra, as well as that of elemental analyses (cf. Scheme 2
and Experimental section).
In continuation to our effort for the investigation of
the electrophilic substitution reaction along the SH group
in each of 3a,b, it has been found that compound 3b was
reacted with each of 4a,b,d,e,g in a stirred methanolic
sodium methoxide at room temperature for 15 min to
afford the corresponding 2-alkylthio derivatives 5i–m.
The structures of these reaction products were elucidated
by considering the data of their elemental analyses, IR,
1H-NMR (cf. Experimental section). A further elucida-
tion for these structures arose from their cyclization in
ethanolic sodium ethoxide solution under reflux for
30 min to give the corresponding thieno[2,3-b]-pyridine
derivatives 6i–m, respectively. An authentic sample of
each of 6i–m were obtained via refluxing a mixture of
3b and each of 4a,b,d,e,g in ethanolic sodium ethoxide
solution for 2 h. In contrast to this behavior, 3b was
reacted with either chloroacetone 4c or 3-chloropentan-
2,4-dione 4f either under stirring in methanolic sodium
methoxide solution at room temperature for 15 min or
refluxing for 2 h in ethanolic sodium ethoxide solution
to give the corresponding thieno[2,3-b]pyridine deriva-
tive 6n. Unexpectedly, 5m did not undergo cyclization
1
of IR, H-NMR, and elemental analyses (cf. Scheme 2 and
Experimental section). In a similar way, compounds 5b–e
were cyclized in ethanolic sodium ethoxide under reflux
for 30 min and gave the corresponding thieno[2,3-b]pyridine
derivatives 6b–e, respectively, which were obtained also via
refluxing of 3a with each of 5b–e in ethanolic sodium
ethoxide for 2 h.
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Synthesis, Reactions, Characterization and Biological Evaluation of 2,3′-Bipyridine
929
Derivatives (III)
Scheme 2
Y
X
X
N
N
R
4a-h
Z
HN
N
Stirring at room temp.
CN
CN
R
S
S
3a,b
Z
5a-m
Y
X
N
R
Z
Reflux
Reflux
N
NH
4a-h
2
2hrs
30 min.
S
Z
6a-n
X = S,O
Scheme 3
X
X
N
N
N
N
C
H
N
NH
-CH
3COOH
6c
S
S
Ac
CH
Ac
3
X = S, O
Ac
O
II
I
reaction under various experimental conditions to give
the corresponding thieno[2,3-b]pyridine derivative.
(cf. Fig. 1). It is worth to mention that as the activity
increases, both the pharmacokinetics and pharmacodynamics
properties greatly improved to be directed towards a good
bioavailability drug profiles (cf. Figs. 2 and 3).
BIOLOGICAL EVALUATION
For series 6 where pyridine is fused to another thiophene,
all the tested compounds showed moderate potent activities,
and the activity in descending order is 3a, 6d, 6g, 6c, 6h,
6b, 6a, 1a, 6e (cf. Figs. 4 and 5). Generally, compounds 6,
with fused thiophene and pyridine rings, is less active than
compounds 5.
Anti-Alzheimer activity.
For compounds 2 and 1a,b,
their relative potency individually is high enough, whereas
after their reactions to afford the corresponding bipyridine-
5-carbonitriles 3a,b, their relative potency decreased. For
series 5, the substituted pyridine derivatives have potent
activities where the compounds arranged according to
descending order of activity: 1a, 5c, 3a, 5b, 5d, 5e, 5a
Structural activity relationship of anti-Alzheimer
activity. Generally for compounds 5, we note that the
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F. A. Attaby, A. M. Abdel-Fattah, L. M. Shaif, and M. M. Elsayed
Vol 51
150
100
50
600
500
400
300
200
100
0
0
Plasma Durg Conc after 4 days {microM}
Brain Durg Conc after 4 days {microM}
Compounds
Compounds
120
100
80
60
40
20
0
Relative potency to Flurbiprofen
Figure 1. Anti-Alzheimer relative to flurbiprofen of 5 series.
% alteration Brain Abeta level2
% alteration in Plasma Abet42
80
60
40
20
0
Compounds
Figure 5. The in vivo pharmakinatic (top) and in vivo pharmacodynamic
(bottom) of 6 series.
Plasma Durg Conc after 4 days {microM}
Brain Durg Conc after 4 days {microM}
0.0002
0.00015
0.0001
0.00005
0
Compounds
Figure 2. The in vivo pharmakinatic of 5 series.
Compounds
Effect on COX-2 ( IC 50 microM)
120
100
80
60
40
20
0
Figure 6. Effect on COX-2 of 5 series.
0.00015
0.0001
0.00005
0
% alteration in Plasma Abet42
% alteration Brain Abeta level2
Compounds
Figure 3. The in vivo pharmodynamic of 5 series.
Compounds
Effect on COX-2 ( IC 50 microM)
Figure 7. Effect on COX-2 of 6 series.
600
500
400
300
200
100
0
compounds 6 are arranged in the following descending
order: 6h, 6c, 6b, 3a, 6g, 6d, 1a, 6a (cf. Fig. 7).
Structural activity relationship for anti-COX-2.
Generally for compounds 5, the thienyl moiety provides
the highest activity, whereas furyl moiety is inactive as
anti-COX-2 activity. Attaching of either –COOH or –
COMe function to the SCH2 provides the highest activity,
whereas attaching of either –CONH2 or COOEt functions
provides moderate activity. Similarly, for compounds 6, the
thienyl moiety provides the highest activity.
Relative potency to Flurbiprofen
Compounds
Figure 4. Anti-Alzheimer relative to flurbiprofen of 6 series.
thienyl derivatives exhibit anti-Alzheimer activity, whereas
their furyl analogs have no activity. The order of activity of
relative potency for S-substitution in all compounds 5 with
thienyl moiety is ketone > amide > acid > ester.
Acute toxicity of both compounds 5 and 6 is illustrated in
Figures 8 and 9.
EXPERIMENTAL
Anti-COX-2 activity. The potent activities of compounds
5 are arranged according to the following descending order:
5e, 3a, 5c, 5d, 1a, 5b, 5a (cf. Fig. 6).
The relative potency of compounds 6 are found to be more
than that of compounds 5, noting that the relative potency of
All melting points were uncorrected. IR (KBr discs) spectra were
recorded on a FTIR-8201PC spectrophotometer (Shimadzu Corp.,
Japan). 1H-NMR spectra were recorded on a Varian Mercury
300 MHz and Varian Gemini 200 MHz. spectrometers using TMS
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Synthesis, Reactions, Characterization and Biological Evaluation of 2,3′-Bipyridine
931
Derivatives (III)
3000
2500
2000
1500
1000
500
washed with water, and dried then crystallized from ethanol to
give 5a–m, respectively.
Ethyl {[5-cyano-4-(2-thienyl)-2,3′-bipyridin-6-yl]thio}acetate
Pale yellow crystals (88%), mp = 154ꢀC; IR (n cmÀ1):
0
(5a).
3079 (C–H, aromatic), 2206 (CN), 1736 (ester CO); Anal. for
C19H15N3O2S2 (381), Calcd./Found (%): C(59.82/59.64); H(3.96/
4.15); N(11.02/10.87); S(16.81/16.65).
Compounds
LD50 mg/kg
2-{[5-Cyano-4-(2-thienyl)-2,3′-bipyridin-6-yl]thio}acetamide
(5b). White crystals (77%), mp = 270ꢀC; IR (n cmÀ1): 3407,
3311 (NH2) 3053 (C–H aromatic), 2217 (CN), 1670 (amide
CO); Anal. for C17H12N4OS2 (352), Calcd./Found (%): C
(57.93/57.76); H(3.43/3.75); N(15.90/15.73); S(18.20/18.54).
6-[(2-Oxopropyl)thio]-4-(2-thienyl)-2,3′-bipyridine-5-carbonitrile
(5c). Pale yellow crystals (89%), mp = 170ꢀC; IR (n cmÀ1): 3083
(C–H, aromatic), 2206 (CN), 1720 (CO); 1H-NMR (DMSO-d6)
(d ppm): 2.288 (s, 3H, –COCH3), 4.380 (s, 2H, –SCH2–),
7.330–8.733 (m, 7H, pyridine H’s, thiophene H’s), 9.359 (s,
1H, pyridine C5(H)); Anal. for C18H13N3OS2 (351), Calcd./
Found (%): C(61.52/61.87); H(3.73/3.98); N(11.96/12.23); S
(18.25/18.01).
Figure 8. Acute toxicity of 5 series.
3000
2500
2000
1500
1000
500
0
Compounds
LD50 mg/kg
Figure 9. Acute toxicity of 6 series.
6-[(2-Oxo-2-phenylethyl)thio]-4-(2-thienyl)-2,3′-bipyridine-5-
carbonitrile (5d).
Yellow crystals (91%), mp = 190ꢀC; IR
(n cmÀ1): 3062 (C–H, aromatic), 2211 (CN), 1698 (CO); Anal.
for C23H15N3OS2 (413), Calcd./Found (%): C(66.80/66.64); H
(3.66/3.97); N(10.16/10.31); S(15.51/15.87).
as an internal standard and DMSO-d6 as solvent. Chemical shifts were
expressed as d (ppm) units. Mass spectra were recorded on Shimadzu
GCMS-QP1000EX using an inlet type at 70 eV. The Micro analytical
Center of Cairo University performed the microanalyses.
{[5-Cyano-4-(2-thienyl)-2,3′-bipyridin-6-yl]thio}acetic acid
(5e).
Orange crystals (80%), mp = 256ꢀC; IR (n cmÀ1):
Synthesis of 3a,b (general method). A solution of each of 2
(0.1 g; 1 mmole) and 1a,b (0.409 and 0.378g; 1 mmole) in absolute
ethanol (30 mL) containing the catalytic amount of piperidine
(0.4 mL) heated under reflux for 5h. The reaction mixture then
evaporated, cooled, and triturated with ethanol. The products so
formed were collected by filtration, washed with cold ethanol, and
then crystallized from the dioxane to give the corresponding 3a,b,
respectively.
4-(2-Thienyl)-6-thioxo-1,6-dihydro-2,3′-bipyridine-5-carbonitrile
(3a). Orange crystals (71%), mp 244ꢀC, IR (n cmÀ1): 3216 (NH),
3086 (aromatic-CH) and 2210 (CN); MS (m/z)=295 (M+, 100%
which is corresponding to the molecular weight), 294 (M+–H,
32.3%); 267 (M+–2H, CN, 15.1%); 262 (M+–SH, 10.4%);1H-NMR
(DMSO-d6) (d ppm): 7.340–8.997 (m, 7H, Ar H’s, pyridine
H’s, thiophene H’s), 9.225 [s, 1H, pyridine C5(H)]
and 14.19 (s, br., 1H, NH); Anal. for C15H9N3S2 (295),
Calcd./Found (%): C(60.99/61.23); H(3.07/3.32); N(14.23/
14.00); S(21.71/21.54).
4-(2-Furyl)-6-thioxo-1,6-dihydro-2,3′-bipyridine-5-carbonitrile
(3b). Orange crystals (65%), mp 244ꢀC, IR (n cmÀ1): 3112 (NH),
3030 (aromatic-CH) and 2214 (CN); MS (m/z) = 279 (M+, 100%
which is corresponding to the molecular weight), 278 (M+–H,
14.2%); 253 (M+–CN, 1.3%); 252 (M+–H, CN, 3.2%); 246 (M+–
SH, 3.2%); 246 (M+–H2S, CN 4.5%); Anal. for C15H9N3OS
(279), Calcd./Found (%): C(64.50/64.76); H(3.25/3.65); N(15.04/
15.31); S(11.48/11.18).
3446–2812 (acidic OH); 3024 (C–H, aromatic), 2213 (CN),
1612 (CO); Anal. for C17H11N3O2S2 (353), Calcd./Found (%):
C(57.77/57.43); H(3.14/3.53); N(11.89/11.65); S(18.15/18.65).
Ethyl {[5-cyano-4-(2-furyl)-2,3′-bipyridin-6-yl]thio}acetate
(5i).
Pale yellow crystals (80%), mp = 180ꢀC; IR (n cmÀ1):
3058 (C–H aromatic), 2210 (CN), 1737 (ester CO); Anal.
for C19H15N3O3S (365), Calcd./Found (%): C(62.45/62.87);
H(4.14/4.54); N(11.50/11.21); S(8.78/8.54).
6-[(Cyanomethyl)sulfanyl]-4-(2-furyl)-2,3′-bipyridine-5-
carbonitrile (5j). Yellow crystals (84%), mp = 260ꢀC; IR
(n cmÀ1): 3053 (C–H aromatic), 2212, 2192 (CN); Anal. for
C16H8N4OS (304), Calcd./Found (%): C(63.15/63.65); H
(2.65/2.21); N(18.41/18.87); S(10.54/10.23).
2-{[5-Cyano-4-(2-furyl)-2,3′-bipyridin-6-yl]thio}acetamide
(5k).
White crystals (84%), mp = 260ꢀC; IR (n cmÀ1): 3338,
3138 (NH2), 3085 (C–H aromatic), 2213 (CN), 1741 (CO);
1H-NMR (DMSO-d6) (d ppm): 4.065 (s, 2H, –SCH2–), 4.906
(br, s, 2H, –NH2), 7.212–8.729 (m, 7H, pyridinyl H’s, furyl
H’s), 9.458 [s, 1H, pyridinyl C5(H)]; Anal. for C17H12N4O2S
(336), Calcd./Found (%): C(60.70/60.43); H(3.60/3.32);
N(16.66/16.41); S(9.53/9.78).
4-(2-Furyl)-6-[(2-oxo-2-phenylethyl)thio]-2,3′-bipyridine-
5-carbonitrile (5l). Orange crystals (91%), mp = 188ꢀC; IR
(n cmÀ1): 3046 (C–H aromatic), 2213 (CN), 1689 (CO); Anal.
for C23H15N3O2S (397), Calcd./Found (%): C(69.50/69.78);
H(3.80/3.32); N(10.57/10.76); S(8.07/8.32).
{[5-Cyano-4-(2-furyl)-2,3′-bipyridin-6-yl]thio}acetic acid
(5m).
Synthesis of 5a–m (general procedure). A solution of each
of 3a,b (0.295 and 0.28 g; 1 mmole) and ethyl chloroacetate (4a),
2-chloroacetamide (4b), chloroacetone (4c), 2-bromo-1-
phenylethanone (4d), chloroacetic acid (4e), 3-chloropentane-2,4-
dione (4f), chloroacetonitrile (4g), 2-bromo-1-(4-chlorophenyl)
ethanone (4h) (0.122, 0.076, 0.093, 0.092, 0.134, 0.198, 0.233,
and 0.094 g; 1 mmole) in sodium methoxide (prepared from 0.10 g
of sodium and 25mL methanol) was stirred at room temperature
for 15 min. The formed precipitate was collected by filtration,
Orange crystals (80%), mp = 250ꢀC; IR (n cmÀ1):
3226–2852 (acidic OH), 3027 (C–H aromatic), 2217 (CN),
1611 (CO); Anal. for C17H11N3O3S (337), Calcd./Found (%):
C(60.52/60.86); H(3.29/3.65); N(12.46/12.86); S(9.50/9.21).
The synthesis of 6a–n.
Method A. Asolutionof eachof5a–e,i–m(0.38, 0.35, 0.35, 0.41,
0.35, 0.37, 0.30, 0.34, 0.40, and 0.34 g; 1 mmole) in sodium ethoxide
solution (prepared from 0.10 g of sodium and 25 mL of ethanol)
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heated under reflux for 30 min. The solid formed after cooling,
collected by filtration, washed with water and ethanol, then
crystallized from the proper solvent to give 6a–n, respectively.
CN, 17.7%); Anal. for C17H10N4S2 (334), Calcd./Found (%): C
(61.06/61.09); H(3.01/3.06); N(16.75/16.80); S(19.18/19.20).
[3-Amino-6-pyridin-3-yl-4-(2-thienyl)thieno[2,3-b]pyridin-2-yl]
(4-chlorophenyl)-methanone (6h). Orange crystals crystallized
from dioxane (90%); mp = 240 ꢀC; IR (n cmÀ1): 3454, 3281
(NH2), 3062 (C–H aromatic), 1675 (CO); MS (m/z):449 (M++2,
32.0%); 448 (M++1, 53.8%); 447 (M+, 100% which is
corresponding to its molecular weight), 446 (M+–H, 88.7%); 412
(M+–Cl, 0.1%); 336 (M+–C6H4Cl, 5.9%); 308 (M+–COC6H4Cl,
16.1%); Anal. for C23H14ClN3OS2 (447), Calcd./Found (%): C
Method B.
A solution of each of 3a,b (0.295 and 0.28 g;
1 mmole) and ethyl chloroacetate (4a), 2-chloroacetamide (4b),
chloroacetone (4c), 2-bromo-1-phenylethanone (4d), chloroacetic
acid (4e), 3-chloropentane-2,4-dione (4f), chloroacetonitrile (4g),
2-bromo-1-(4-chlorophenyl)-ethanone (4h) (0.122, 0.093, 0.092,
0.198, 0.094, 0.134, 0.75, and 0.233g; 1mmole) in sodium
methoxide (prepared from 0.10 g of sodium and 25 mL of
methanol) heated under reflux for 2 h. The solid products so
formed after cooling, collected by filtration, washed with water
and ethanol, and dried crystallized from the proper solvent to give
6a–n, respectively.
(61.67/61.70); H(3.15/3.19); Cl(7.91/7.95); N(9.38/9.42);
(14.32/14.36).
S
Ethyl 3-amino-4-(2-furyl)-6-pyridin-3-ylthieno[2,3-b]pyridine-2-
Yellow crystals crystallized from ethanol
carboxylate (6i).
(90%); mp = 210ꢀC; IR (n cmÀ1): 3499, 3377 (NH2), 3037 (C–H
aromatic), 1672 (CO); MS (m/z): 365 (M+, 100% which is
corresponding to its molecular weight), 320 (M+–OEt, 21.3%);
292 (M+–COOEt, 19.1%); Anal. for C19H15N3O3S (365),
Calcd./Found (%): C(62.45/62.21); H(4.14/4.32); N(11.50/11.76);
S(8.78/8.98).
Ethyl 3-amino-6-pyridin-3-yl-4-(2-thienyl)thieno[2,3-b]pyridine-
2-carboxylate (6a).
Yellow crystals crystallized from ethanol
(90%); mp = 218ꢀC; IR (n cm1): 3480, 3346 (NH2), 3038 (C–H
aromatic), 1672 (CO); MS (m/z): 381 (M+, 100% which is
corresponding to the molecular weight), 380 (M+–H, 5.2%); 336
(M+–OEt, 15.0%); 334 (M+–OEt, –2H, 74.8%); 308 (M+–COOEt,
8.6%); Anal. for C19H15N3O2S2 (381), Calcd./Found (%): C
(59.82/59.45); H(3.96/4.32); N(11.02/11.43); S(16.81/16.97).
3-Amino-6-pyridin-3-yl-4-(2-thienyl)thieno[2,3-b]pyridine-2-
carboxamide (6b). Yellow crystals crystallized from dioxane
(81%); mp = 296 ꢀC; IR (n cm-1) 3463, 3297, 3276, 3125
(2NH2), 1666 (CO); MS (m/z): 352 (M+, 100% which is
corresponding to its molecular weight), 336 (M+–NH2, 10.0%);
334 (M+–NH2, –2H, 79.6%); 308 (M+–CONH2, 10.0%); Anal.
for C17H12N4OS2 (352), Calcd./Found (%): C(57.93/57.95); H
(3.43/3.45); N(15.90/15.95); S(18.20/18.24).
3-Amino-4-(2-furyl)-6-pyridin-3-ylthieno[2,3-b]pyridine-2-
carbonitrile (6j).
Yellow crystals crystallized from dioxane
(85%); mp = 308ꢀC; IR (n cmÀ1): 3482, 3207 (NH2) 3057 (C–H
aromatic), 2192 (CN); MS (m/z): 318 (M+, 100% which is
corresponding to its molecular weight), 317 (M+–H, 43.8%);
Anal. for C17H10N4OS (318), Calcd./Found (%): C(64.14/64.17)
H(3.17/3.20); N(17.60/17.64); S(10.07/10.10).
3-Amino-4-(2-furyl)-6-pyridin-3-ylthieno[2,3-b]pyridine-2-
carboxamide (6k). Yellow crystals crystallized from ethanol
(80%); mp = 290ꢀC; IR (n cmÀ1): 3494, 3467, 3330, 3133
(2NH2) 1665 (amidic CO); MS (m/z): 336 (M+, 100% which
is corresponding to its molecular weight), 319 (M+–H, NH2,
30.6%); 318 (M+–NH2, –2H, 94.4%); 291 (M+–H, CONH2,
27.8%); 290 (M+–2H, –CONH2, 33.3%); Anal. for
C17H12N4O2S (336), Calcd./Found (%): C(60.70/60.73); H
(3.60/3.63); N(16.66/16.69); S(9.53/9.56).
1-[3-Amino-6-pyridin-3-yl-4-(2-thienyl)thieno[2,3-b]pyridin-
2-yl]ethanone (6c). Yellow crystals crystallized from dioxane
(86%); mp = 250ꢀC; IR (n cm-1) 3451, 3285 (NH2), 3055 (C-H
aromatic), 1622 (CO); MS (m/z): 351 (M+, 100% which is
corresponding to its molecular weight), 350 (M+–H, 40.9%),
336 (M+–CH3 65.7%); 308 (M+–COCH3 28.8%), Anal. for
C18H13N3OS2 (351), Calcd./Found (%): C(61.52/61.55); H
(3.73/3.77); N(11.96/11.99); S(18.25/18.29).
[3-Amino-4-(2-furyl)-6-pyridin-3-ylthieno[2,3-b]pyridin-2-
yl](phenyl)methanone (6l).
Orange crystals crystallized
[3-Amino-6-pyridin-3-yl-4-(2-thienyl)thieno[2,3-b]pyridin-2-yl]
from ethanol (83%); mp = 220ꢀC; IR (n cmÀ1): 3474, 3300
(NH2), 3043 (C–H aromatic), 1602 (CO); MS (m/z): 397 (M+,
22.5% which is corresponding to its molecular weight), 396
(M+–H, 18.5%); 320 (M+–Ph, 1.6%); 292 (M+–COPh, 2.8%);
Anal. for C23H15N3O2S (397), Calcd./Found (%): C(69.50/
69.54); H(3.80/3.84); N(10.57/10.60); S(8.07/8.11).
(phenyl)methanone (6d).
Orange crystals crystallized from
dioxane (86%); mp=200 ꢀC; IR (n cmÀ1): 3471, 3295 (NH2),
3030 (C–H aromatic), 1691 (CO); MS (m/z): 413 (M+, 75.2%
which is corresponding to its molecular weight), 412 (M+–H,
100%); 411 (M+–2H, 14%); 308 (M+–COPh, 24.1%); Anal. for
C23H15N3OS2 (413), Calcd./Found (%): C(66.80/66.83); H(3.66/
3.69); N(10.16/10.18); S(15.51/15.55).
[3-Amino-4-(2-furyl)-6-pyridin-3-ylthieno[2,3-b]pyridin-
2-yl](4-chlorophenyl)-methanone (6m).
Orange crystals
crystallized from ethanol (86%); mp = 235 ꢀC; IR (n cmÀ1): 3478,
3279 (NH2), 3048 (C–H aromatic); MS (m/z): 433 (M + 2 15.1%);
432 (M + 1 28.0%); 431 (M+, 39.2% which is corresponding to its
molecular weight), 430 (M+–H, 43.5%); 396 (M+–Cl, 4.8%); 292
(M+–COC6H4Cl, 6.5%); Anal. for C23H14ClN3O2S (431), Calcd./
Found (%): C(63.96/64.00); H(3.27/3.30); Cl(8.21/8.24); N(9.73/
9.77); S(7.42/7.45).
3-Amino-6-pyridin-3-yl-4-(2-thienyl)thieno[2,3-b]pyridine-2-
carboxylic acid (6e). Yellow crystals crystallized from dioxane
(71%); mp = 210 C; IR (n cmÀ1): 3473–3328 (NH2, OH), 1665
ꢀ
(CO); MS (m/z) = 353 (M+ 10.3% which is corresponding to its
molecular weight), 334 (M+–H, H2O 4.5%); 309 (M+–COOH,
1
100%); H-NMR (DMSO-d6) (d ppm): 6.531 (br, s, 2H, NH2),
7.274–8.689 (m, 7H, pyridine H’s, thiophene H’s), 9.369 [s, 1H,
pyridine C5(H)] and 12.763 (s, br., 1H, COOH); Anal. for
C17H11N3O2S2 (353), Calcd./Found (%): C(57.77/57.90); H
(3.14/3.38); N(11.89/11.62); S(18.15/18.30).
1-[3-Amino-4-(2-furyl)-6-pyridin-3-ylthieno[2,3-b]pyridin-2-
yl]ethanone (6n).
Yellow crystals crystallized from dioxane
(83%); mp = 222ꢀC; IR (n cmÀ1): 3478, 3289 (NH2), 3066 (C-H
aromatic), 1610 (CO); MS (m/z) = 335 (M+, 100% which is
corresponding to its molecular weight), 334 (M+–H, 27.5%), 320
(M+–CH3 71.6%); 319 (M+–NH2, 11.9%); 292 (M+–COCH3
3-Amino-6-pyridin-3-yl-4-(2-thienyl)thieno[2,3-b]pyridine-2-
carbonitrile (6g).
Yellow crystals crystallized from dioxane
(85%); mp = 280ꢀC; IR (n cmÀ1): 3447, 3226 (NH2) 3044 (C–H
aromatic), 2197 (CN), MS (m/z) :334 (M+, 100% which is
corresponding to its molecular weight), 333 (M+–H, 46.9%);
332 (M+–2H, 5.0%); 308 (M+–CN, 0.4%); 290 (M+–2H, NH2,
1
22.9%); H-NMR (DMSO-d6) (d ppm): 2.408 (s, 3H, COCH3),
6.842 (s, br., 2H, NH2), 7.459–8.703 (m, 7H, pyridine H’s, furan
H’s), 9.398 (s, 1H, pyridine C5(H)); Anal. for C18H13N3O2S
Journal of Heterocyclic Chemistry
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Synthesis, Reactions, Characterization and Biological Evaluation of 2,3′-Bipyridine
933
Derivatives (III)
(335), Calcd./Found (%): C(64.46/64.70); H(3.91/3.64); N(12.53/
12.38); S(9.56/9.80).
San Diego, CA, USA). The microsomal fractions were
prepared from the transfected cells and used to assay the
enzymatic activities. Briefly, the enzymes (2g) reconstituted
in a buffer (100 mM Tris–HCl, pH 8.0) containing 2 mM
phenol were preincubated with vehicle (DMSO) or test
compounds in DMSO (1%DMSO in the final assay) for
20 min at 22ꢀC. The reaction mixture was completed with 1M
hematin. The reaction was initiated adding arachidonic acid (4
and 2 m for COX-1 and COX-2, respectively) and the
mixture was incubated for 5 min at 22ꢀC for COX-1
assay or for 10 min at 25ꢀC for COX-2 assay. For control
measurements, arachidonic acid was omitted from the
reaction mixture. The reactions were stopped by the
sequential addition of 1M HCl and 1M Tris–HCl
(pH 8.0), followed by cooling to 4ꢀC. The amount of
PGE2 present in the reaction mixture was quantified
using an enzyme immunoassay.
MATERIALS AND METHODS
Ab42 and Ab40 assay. Ab42 and Ab40 were measured
in the culture medium of H4 cells, a human neuroglioma cell
line expressing the double Swedish mutation (K595N/
M596L) of human amyloid precursor protein (APPsw).
Cells were seeded onto 24-well plates (2 Â 105 cell wellÀ1
)
and allowed to grow to confluence for 24 h, in 5% CO2/95%
air in a humidified atmosphere. Increasing concentrations
(from 3 to 300–400 mM) of the compounds were added
to the cells overnight in a final volume of 0.5 mL. R-
flurbiprofen was used as positive control (3–1000 mM).
DMSO (1%) was used as negative control. At the end of the
incubation, 100 mL of supernatants were removed and
treated with a biotinylated mouse monoclonal antibody
(4G8, Signet Laboratories Inc., Dedham, MA, USA),
specifically recognizing the 17–24 amino acid region of Ab
and two rabbit polyclonal antibodies (C-term 42 and C-term
40, BioSource International, Camarillo, CA, USA),
specifically recognizing the C-terminus of Ab42 and Ab40,
respectively. Antigen–antibody complexes were recognized
by TAG-donkey anti-rabbit IgG (Jackson Immuno Research
Laboratories, Soham, UK). Streptavidin coated magnetic
beads captured the complexes, and the signals were read by
an electrochemiluminescence instrument (Origen M8
Analyzer, BioVeris Corporation, Gaithersburg, MD, USA).
The cytotoxicity potential of test compounds was assessed
in the same cells of the Ab assay (H4) with the 3-[4,5-
dimethylthiazol-2-yl]-2,5-di-phenyltetrazol-ium bromide (MTT)
assay. MTT is a soluble pale yellow salt that is reduced by
mitochondrial succinate dehydrogenase to form an
insoluble dark blue formazan product to which the cell
membrane is impermeable. The ability of cells to reduce
MTT provides an indication of mitochondria integrity and
activity, and it may be interpreted as a measure of viability
and/or cell number. After medium removal of Ab 42 and
Ab 40 determination, cells were incubated for 3 h with
500 mL of culture medium containing 0.5 mg mLÀ1 of
MTT, at 37ꢀC, 5% CO2 and saturated humidity. After
removal of the medium, 500 mL of 100% DMSO was
added to each well. The amount of formed formazan
was determined by reading the samples at 570 nm
(background 630 nm) by using a microplater reader
(model 450, Bio-Rad, Hercules, CA, USA).
Studies in Tg2576 transgenic mice.
Young male and
female transgenic mice (Tg2576) expressing the human
APP gene with the Swedish double mutation (K670N/
M671L) under the transcriptional control of the hamster
prion protein promoter [17] were used for the in vivo
studies. Male animals were housed singly in individual
cages, whereas female animals were placed in groups of
3–5 animals per cage. The experiments were performed in
accordance with EEC Guidelines (86/609/ECC) for the use
of laboratory animals.
Study 1. Groups of mole mice 4–5 months, each group
composed of 21 male mice of 4–5 months of age, were
given by oral gavage vehicle (Kool-Aid 7.5 mLkgÀ1) or a
suspension of each individual compound (100 or
300 mgkgÀ1 dayÀ1 in Kool-Aid) once daily for 5 days.
This vehicle was selected to replicate that reported with
flurbiprofen in similar studies [17–19]. On day 5, mice
were given a final dose of 100 or 300mgkgÀ1 or vehicle
and sacrificed 3 h later, as described in the succeeding text.
Study 2. Groups of female mice of 5–7 months of age,
each group composed 17 female mice of 5–7 months of
age, were given by oral gavage vehicle (Kool-Aid
7.5 mL kgÀ1) or a suspension of individual compound (100
or 300mgkgÀ1 dayÀ1 in Kool-Aid) once daily for 4 days.
On day 4, mice were given a final dose of 100 or
300 mgkgÀ1 or vehicle and sacrificed 3 h later, as
described in the succeeding text.
Study 3. Groups of male and female mice of 4–5 months,
each group composed of 33 male and female mice of 4–
5 months were given vehicle or tested compounds or R-
flurbiprofen-supplemented chow ad libitum for 4 weeks.
There were 11 animals in each treatment group.
R-flurbiprofen (Sigma, St. Louis, MO, USA), and the
tested compounds were formulated into standard, color-
coded, rodent diet by Charles River (Calco, Italy) at a final
drug concentration of 375 ppm. The concentration of the
drugs in the diet was the same as that used for flurbiprofen
in previous studies [20–22].
COX-1 and COX-2 assay.
The inhibition of the
cyclooxygenase activity was estimated measuring
prostaglandin E2 (PGE2) production from arachidonic acid
according to a modified version of the method [16].
Recombinant human prostaglandin H2 synthase-1 (PGHS-1)
and prostaglandin H2 synthase-2 (PGHS-2) were expressed
in transfected Spodoptera frugiperda (Sf-9) cells (Invitrogen,
Journal of Heterocyclic Chemistry
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934
F. A. Attaby, A. M. Abdel-Fattah, L. M. Shaif, and M. M. Elsayed
Vol 51
Body weight and food consumption were monitored every
3–4 days.
Plasma and brain ab measurements. Twenty-four hours
then centrifuged at 15,000 rpm for 25 min at 4ꢀC. The
supernatants were collected and neutralized with 1M Tris,
pH11 at 1:20 (w/v) dilution with 3Â protease inhibitor
mixtures (Boehringer Mannheim, Mannheim, Germany).
Levels of Ab40 and Ab42 in plasma and in brain
homogenate supernatants were measured with commercial
ELISA kits (The Genetics Company, Zurich, Switzerland).
The microtitre plates were coated with capturing purified
monoclonal antibodies specifically recognizing the
C-terminus of human Ab40 (clone G2-10, reactive to amino
acid residues 31–40, isotype IgG2b, kappa) or Ab42 (clone
G2-13, reactive to amino acid residues 33–42, isotype IgG1,
before starting treatment, one blood sample was collected
by means of retrobulbar puncture for measurement of
baseline plasma, Ab40, and Ab42 concentrations. On the
last day of treatment, mice were sacrificed by decapitation.
Blood samples were collected in EDTA-coated tubes and
centrifuged at 800 rpm for 20 min to separate the plasma.
Plasma samples were divided into two aliquots of
approximately 100 mL each and stored at À80ꢀC until Ab
and drug assay. The brains were quickly removed and
placed on an ice-cold plate. The cortex and hippocampus
were dissected and immediately frozen on dry ice and
stored at À80ꢀC for Ab assay. The remaining brain was
immediately frozen on dry ice and stored at À80ꢀC for
drug level measurements. Plasma was diluted 1:4 for
Ab42 and 1:20 for Ab40. For measurement of Ab, brain
tissue samples were homogenized in 70% formic acid at
1:10 (w/v). Homogenates were agitated at 4ꢀC for 3 h and
kappa). As detection antibody,
a monoclonal biotin
conjugated antibody recognizing the N-terminus of human
Ab (clone W0-2, reactive to amino acid residues 4–10,
isotype IgG2a, kappa) was used. The assay was linear in the
range 25–500 pg mLÀ1
25 pg mLÀ1
, and the detection limit was
.
Plasma and brain drug measurements. Drugs levels in
plasma and in brain samples were measured by liquid
Table 1
Antitumor activity and novel topoisomerase II inhibitors.
The correlation coefficients with (topoisomerase Ι inhibitor), and
cisplatin, calculated according to the NCI COMPARE analysis
procedure using the tested compound as the benchmark
Inhibition of
topoisomerase
II activity
In vivo
efficacy study
Growth-inhibitory effect
Relative
Relative
Relative
Average
GI50
potency to
etoposide
potency to
etoposide
potency to
etoposide
Compound
Etoposide
Doxorubicin
SN-38
Cisplatin
1a
1b
3a
3b
5a
5b
5c
5d
5e
5i
Inactive
377
Inactive
381
Inactive
Inactive
Inactive
Inactive
Inactive
355
380
344
351
388
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
3.49E-58
341
Inactive
124.41
Inactive
125.73
Inactive
Inactive
Inactive
Inactive
Inactive
117.15
125.4
113.52
115.83
128.04
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
135.3
Inactive
4.35435
Inactive
4.40055
Inactive
Inactive
Inactive
Inactive
Inactive
4.10025
4.389
Inactive
3.48348
Inactive
3.52044
Inactive
Inactive
Inactive
Inactive
Inactive
3.2802
Inactive
2.86143
Inactive
2.89179
Inactive
Inactive
Inactive
Inactive
Inactive
2.69445
2.8842
2.61096
2.66409
2.94492
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
3.1119
Inactive
2.2393
Inactive
2.2631
Inactive
Inactive
Inactive
Inactive
Inactive
2.1087
2.2572
2.0433
2.0849
2.3047
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
2.4354
2.0255
2.3225
2.0017
2.0730
2.1740
2.1978
Inactive
128.1423
Inactive
129.5019
Inactive
Inactive
Inactive
Inactive
Inactive
120.6645
129.162
116.9256
119.3049
131.8812
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
139.359
115.9059
132.9009
114.5463
118.6251
124.4034
125.763
Inactive
118.1895
Inactive
119.4435
Inactive
Inactive
Inactive
Inactive
Inactive
111.2925
119.13
107.844
110.0385
121.638
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
128.535
106.9035
122.5785
105.6495
109.4115
114.741
115.995
5j
5k
5l
3.5112
3.9732
4.05405
4.4814
3.17856
3.24324
3.58512
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
3.7884
3.15084
3.61284
3.11388
3.22476
3.38184
3.4188
5m
6a
6b
6c
6d
6e
6g
6h
6i
6j
6k
6l
6m
6n
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
4.7355
3.93855
4.51605
3.89235
4.03095
4.2273
112.53
129.03
111.21
115.17
120.78
122.1
2.58819
2.96769
2.55783
2.64891
2.77794
2.8083
391
337
349
366
370
4.2735
Journal of Heterocyclic Chemistry
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July 2014
Synthesis, Reactions, Characterization and Biological Evaluation of 2,3′-Bipyridine
935
Derivatives (III)
chromatography as previously described [20]. Briefly,
samples were prepared by adding 300mL of acetonitrile
and 40 mL of phosphoric acid 40% to 100 mL plasma or
brain homogenate and placing the mixture in a vortex for
5 s. Plasma and brain samples were then centrifuged at
14,000 rpm for 5 min, and the supernatants (15 and 50 mL,
respectively) were injected into the HPLC system.
Equipment systems with fluorescence (Waters 474, Waters,
Guyancourt, France) or mass spectrometry (API 2000,
Applied Biosystems, Foster City, CA, USA) detectors were
used. The chromatographic conditions were adapted to
each compound to obtain good peak separation and
detection sensitivity. A mixture of ammonium formate
(20 mM) buffer–acetonitrile–methanol was used as mobile
phase for the fluorescence detector. For drugs, the assay
was linear in the range 20–4000 ng gÀ1 in the brain and
5–1000ng mLÀ1 in plasma with limits of quantitation of
20 ng gÀ1 in the brain and 5 ng mLÀ1 in plasma. For
drugs, the assay was linear between 400 and
20,000 ng gÀ1 in the brain and 100–8500 ng mLÀ1 in
plasma with limits of quantitation of 400 ng gÀ1 in the
brain and 100 ng mLÀ1 in plasma.
Antitumor activity and topoisomerase II inhibitors. DNA
topoisomerase II has been shown to be an important
therapeutic target in cancer chemotherapy. Here, we describe
studies on the antitumor activity of novel topoisomerase II
inhibitors. The tested compounds inhibited topoisomerase II
activity at 10 times lower concentration than etoposide in
relaxation assay and cells. Murine colon cancer Colon 38,
murine leukemia P388, and human lung cancer LX-1 were
obtained from the Cancer Chemotherapy Center, Japan
Foundation for Cancer Research (Tokyo, Japan). Human
lung cancer PC-1 and human gastric cancer MKN-1, MKN-
7, MKN-28, and MKN-74 were obtained from Immuno
Biology Laboratory (Gunma, Japan). Human lung cancer
A549 and human colon cancer WiDr were obtained from
Dainippon Pharmaceutical Co. Ltd (Osaka, Japan). Human
gastric cancer HGC-27 and GT3TKB were obtained from
Riken Cell Bank (Ibaraki, Japan). The other cell lines were
purchased from American Type Culture Collection
(Rockville, MD, USA) (Table 1).
seeded in 96-well plates on day 0. On day 1, 0.1 mL
aliquots of medium containing graded concentrations of
test drugs were added to the wells. After incubation at
37ꢀC for 72 h, the cell number was determined by
MTT assay .IC50 values were calculated as the drug
concentrations that reduced the number of cells to 50%
of the control number.
In vivo efficacy study. Female C57BL/6 mice (6 weeks of
age; Charles River, Atsugi, Japan) were housed in barrier
facilities on a 12-h light/dark cycle, with food and water ad
libitum. About 30 mg of Colon 38 tumor tissues was
inoculated S. C. into the flank of animals. Mice
were randomized and separated into control and treatment
(n = 5 each) groups when the tumor volume reached
approximately 300 mm3 on day 1. The tested compound
was dissolved in 5% glucose. Etoposide was diluted in
saline, and doxorubicin was dissolved in saline. The tested
compound was administrated on day 1 (single dose) by i.v.
injection into the tail vein, and etoposide and doxorubicin
were given at the maximum tolerated dose on the reported
administration schedule. Control animals received 5%
glucose. Tumor volume and body weight were measured
on the days indicated in the figures. Tumor volume was
calculated according to the following equation: tumor
volume (mm3) = (lengthÀwidth2)/2
Relaxation assay. Topoisomerase II was purified from
P388 cells. One unit of the enzyme was defined as the
activity to relax completely 0.125 mg of supercoiled pBR-
322 DNA at 30ꢀC for 1 h. For the assay, 0.125 mg of
supercoiled pBR-322 DNA (Takara Shuzo Co. Ltd,
Tokyo, Japan) was relaxed with one unit of
topoisomerase II in 20 mL of the assay buffer [50 mM
Tris–HCl(pH 8.0), 100 mM KCl, 10 mM MgCl2, 0.5 mM
DTT, 0.5 mM EDTA, 1 mM ATP, and 30 mg mLÀ1 BSA]
in the presence or absence of an inhibitor at 30ꢀC for 1 h.
Samples were subjected to electrophoresis s in 0.7%
agarose gels with tris-borate-EDTA (TBE) buffer [89 mM
Tris, 89 mM boric acid, and 2 mM EDTA (pH 8.0)].
The DNA was stained with ethidium bromide and
photographed under UV light. (pH8.0), 100 mM KCl,
10 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA, 1 mM ATP,
and 30 mg mLÀ1 BSA] in the presence or absence of an
inhibitor at 30ꢀC for 1 h. Samples were subjected to
electrophoresis s in 0.7% agarose gels with TBE buffer
[89 mM Tris, 89 mM boric acid, and 2 mM EDTA
(pH 8.0)]. The DNA was stained with ethidium bromide
and photographed under UV light.
Growth-inhibitory effect. Exponentially growing solid
tumor cells in 0.1 mL of medium were seeded in 96-well
plates on day 0. On day 1, 0.1 mL aliquots of medium
containing graded concentrations of test drugs were
added to the cell plates. After incubation at 37ꢀC for
72 h, the cell number was estimated by sulforhodamine
B assay; as described by Skehan et al. separately, the
cell number on day 1 was also measured. GI50 values
are the drug concentrations causing a 50% reduction in
the net protein increase in control cells. The antitumor
spectrum of ER-37328 was compared with those of
other drugs by means of the NCI COMPARE analysis
procedure. Exponentially growing P388 cells were
REFERENCES AND NOTES
[1] Attaby, F. A.; Abdel-Fattah, A. M.; Shaif, L. M.; Elsayed, M.
M. Phosphorus, Sulfur, Silicon, Relat. Elem. 2010, 185, 668.
[2] Attaby, F. A.; Abdel-Fattah, A. M.; Shaif, L. M.; Elsayed, M.
M. Phosphorus, Sulfur, and Silicon, Relat. Elem. 2010, 185, 129.
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Vol 51
[3] Abdel-Fattah, A. M.; Elsayed, M. M. Current Organic Chem-
istry 2009, 13, 1751.
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Journal of Heterocyclic Chemistry
DOI 10. 1002/jhet