Thieno[2,3-b]pyridines - A new class of multidrug resistance (MDR) modulators

Article abstract of DOI:10.1016/j.bmc.2014.09.023

To identify new potent multidrug resistance modulators, we have synthesized a series of novel thieno[2,3-b]pyridines and furo[2,3-b]pyridines, and examined their stucture-activity relationships. All synthesized compounds were tested to determine BCRP1, P-gp, and MRP1 inhibitor activity, and most potent MDR modulators were also screened for their toxicity, cytotoxicity and Ca²⁺ channel antagonist activity. Among these compounds, thieno[2,3-b]pyridine (6r) was found to exhibit a potent P-gp inhibitory action with EC₅₀ = 0.3 ± 0.2 μM, MRP1 inhibitory action with EC₅₀ = 1.1 ± 0.1 μM and BCRP1 inhibitory action with EC₅₀ = 0.2 ± 0.05 μM and may represent suitable candidate for further pharmacological studies.

Full text of DOI:10.1016/j.bmc.2014.09.023

Accepted Manuscript
Thieno[2,3-b]pyridines – a new class of multidrug resistance (MDR) modulators
Aivars Krauze, Signe Grinberga, Laura Krasnova, Ilze Adlere, Elina Sokolova,
Ilona Domracheva, Irina Shestakova, Zigmars Andzans, Gunars Duburs
PII:
S0968-0896(14)00669-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.09.023
BMC 11810
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 June 2014
11 August 2014
11 September 2014
Please cite this article as: Krauze, A., Grinberga, S., Krasnova, L., Adlere, I., Sokolova, E., Domracheva, I.,
Shestakova, I., Andzans, Z., Duburs, G., Thieno[2,3-b]pyridines – a new class of multidrug resistance (MDR)
modulators, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.09.023
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Bioorganic & Medicinal Chemistry
jo u rn al h ome p ag e : w ww .e lse vie r .c om
Thieno[2,3-b]pyridines – a new class of multidrug resistance (MDR) modulators
Aivars Krauze∗, Signe Grinberga, Laura Krasnova, Ilze Adlere, Elina Sokolova, Ilona Domracheva, Irina
Shestakova, Zigmars Andzans and Gunars Duburs
Latvian Institute of Organic Synthesis, 21 Aizkraukles St., Riga LV-1006, Latvia
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
To identify new potent multidrug resistance modulators , we have synthesized a series of novel
thieno[2,3-b]pyridines and furo[2,3-b]pyridines, and examined their stucture-activity
relationships. All synthesized compounds were tested to determine BCRP1, P-gp, and MRP1
inhibitor activity, and most potent MDR modulators were also screened for their toxicity,
cytotoxicity and Ca2+ channel antagonist activity. Among these compounds, thieno[2,3-
b]pyridine (6r) was found to exhibit a potent P-gp inhibitory action with EC50 = 0.3 0.2 µM,
MRP1 inhibitory action with EC50 = 1.1 0.1 µM and BCRP1 inhibitory action with EC50 = 0.2
0.05 µM and may represent suitable candidate for further pharmacological studies.
Available online
Keywords:
Thieno[2,3-b]pyridine
Multidrug resistance modulators
P-glycoprotein
2014 Elsevier Ltd. All rights reserved.
Multidrug resistance-associated protein
Breast cancer resistance protein
1. Introduction
gastrointestinal tract at pharmacological barrier sites, in stem
cells and cells of immune system.2,3,4
Chemotherapy is one of the most frequently used forms of
cancer therapy and has found clinical application in the treatment
of almost every type of cancer. One of the major problems in
cancer chemotherapy is the development of resistance to
cytotoxic drugs. Patients who did not respond to a first course of
chemotherapy are in even more serious condition because tumor
cells develop resistance against chemotherapeutic agents or
resistance to cytotoxic agents used in a previous treatment. A
tumor may also manifest resistance to a cytotoxic agent to which
it has not been previously exposed.
MRP1 is a member of ABCC subfamily and confers resistance
to several hydrophobic compounds that are also P-gp substrates.
But MRP1 can export glutathione, glucuronate or sulfate
conjugates of organic anions. MRP1 is expressed in wide range
of tissues, tumors and cancer cell lines.4
BCRP1 is a member of ABCG subfamily. The substrate
specificity of BCRP1 overlaps considerably with that of P-gp.
BCRP1 is involved in the mechanism of resistance to a
topoisomerase I inhibitor (topotectan) or topoisomerase II
inhibitor (mitoxantrone).5,6 BCRP1 does not act on Paclitaxel or
Vincristine transport, which are excreted by P-gp. BCRP1 is
involved in excretion of a camptothecin derivative, which is
barely transported by P-gp.2,7 BCRP1 is expressed in many
normal tissues, including liver, placenta, brain, hematopoietic
stem cells and other types of stem cells.
Multidrug resistance (MDR) in tumor cells has a significant
impact on the efficacy of cancer chemotherapy and appears as a
major obstacle in the modern cancer treatment. MDR is mainly
related to the expression of the adenosine triphosphate (ATP)-
binding cassette (ABC) transporters. P-glycoprotein (P-gp) (the
best studied target for reverting MDR), multidrug resistance-
associated protein (MRP1) and the breast cancer resistance
protein (BCRP1) as major MDR proteins actively transport a
wide variety of structurally different substrates out of the tumor
cells, thereby decreasing their intracellular concentrations. Many
actual chemotherapeutic agents are considered as potential P-gp,
MRP1 and BCRP1 substrates.1,2
From all numerous efforts to overcome MDR like
transcription control of P-gp expression the most promising
approach has been the development of MDR modulators, which
are able to increase the intracellular drug levels in co-application
with MDR substrates by the effect of efflux pump inhibition.
Substances of different groups have been used as P-gp inhibitors.
Ca2+ channel blocker Verapamil is the most investigated and
often used as reference compound, but unfortunately in
combination with actual anticancer drugs cardio toxicity is
observed.8
P-gp, a member of the ABCB subfamily, confers the strongest
resistance to the wide variety of compounds. P-gp transports
vinca alkaloids, anthracyclines, epipodophyllotoxins and taxanes.
P—-g—p is—normally expressed in epithelium of the liver, kidney and
∗ Corresponding author. Tel.: +(371)67014870; fax: +(371)67550338; e-mail: krauze@osi.lv

Extensive computational models, based on pharmacophore
modeling and molecular docking techniques, have been
developed to predict P-gp inhibitors or substrates. Unfortunately
only limited in silico models can give satisfactory predictions.
The improvement of prediction accuracy still remains a
significant challenge.9
Rational approach of drug design – structural analogy with
known medicines – was used to develop more efficacious MDR
modulators on the basis of thieno[2,3-b]pyridines. As part of our
research interest towards bioactive N,S-containing heterocyclic
compounds, we postulated that thieno[2,3-b]pyridine scaffold
might be suitable for the linked pharmacophore approach. Model
was created assuming one part of Verapamil as linker and
methoxyphenyl groups as essential for pharmacophore (Fig. 1).
„Linked pharmacophore” model was used in development of
such P-gp ligands as anthranilamide derivatives (tariquidar),23-25
2,3-dehydrosilybin derivatives,26 naphthalenyl derivatives,27
tetrahydroisoquinoline derivatives28 and others.
In recent years intensive efforts have been taken to develop
small-molecule inhibitors with a favorable resorption in contrast
to complex natural compounds with high and critical molecular
weights which are unfavorable also because the synthetic access
to such structures is difficult and expensive.10,11
There are no literature data when multidrug resistance
modulating properties are searched in a series of thieno[2,3-
b]pyridines which in last decade are characterized by a broad
spectrum of biological activities. Some of them possess cytotoxic
activity,12-16 antiinflammatory,16,17 antiviral18 or antibacterial19,20
activity. Others are useful as hypolipoproteinemic and
antiatherosclerotic agents21 and inhibitors of Mitogen-activated
protein kinase enzymes, and are accordingly of benefit as
pharmaceutical agents, especially in the treatment of adverse
inflammatory, autoimmune, cardiovascular, proliferative and
nociceptive conditions.22
As Ca2+ channel blocker Verapamil in combination with
anticancer drugs reveals cardio toxicity,8 influence of obtained
thieno[2,3-b]pyridines on cardiovascular system as well as their
cytotoxicity were tested. Compounds possessing insignificant
calcium antagonist properties as side effect and LD50 >2000
mg/kg were tested as potential MDR modulators. To reveal
structure-activity relationship in these series, three compounds
with LD50 <2000 mg/kg were tested
OMe
OMe
OMe
OMe
CN
NH2
R
Linker
Linker =
OMe
S
O
N
N
OMe
OMe
OMe
Verapamil
Figure 1. Pharmacophore approach with modified linker
32
2-Cyanothioacetamide 1 was reacted with α,β-unsaturated
ketones 1 in presence of catalytic amount of triethylamine
(method A) to give thioxopyridines 5 in 42-80 % yield. As
alternative method for synthesis of intermediates 5 was used
condensation of ketones 3 with 3-aryl-2-cyanothioacrylamides 4
catalyzed by piperidine (method B), and intermediates were
obtained in 41-60 % yield.
2. Results and discussions
2.1. Chemistry
The synthesis of thieno[2,3-b]pyridines is very straightforward
and demonstrated in Scheme 1. The key intermediates, 1,6-
dihydro-6-thioxopyridines 5a-r, were synthesized according to
slightly modified synthesis protocols described in the literature.29-
3
R
2
3
3
R
R
2
2
CN
R
CN
S
NC
R
i
R
ii
+
+
1
1
R
1
S
NH2
R
N
H2N
S
O
R
O
H
3
1
4
2
5
iii
v, vi,
vii
O
3
3
H
R
R
N
NH2
4
R
2
2
R
iv
R
O
1
R
4
1
R
S
S
R
O
N
N
6
7
Scheme 1. Reagents and conditions: (i) 0.07 equiv Et3N , EtOH, reflux, 14 h; (ii) 0.1 equiv piperidine, EtOH 15 h; (iii) 1.0 equiv bromoacetophenone, 2.4 equiv
NaOH/H2O, EtOH, short reflux, rt, 30 min; (iv) ClCOMe, 2.0 equiv NaOH, reflux, 1 h; (v) 1.1 equiv piperidine, EtOH, rt, 12 h; (vi) 1.0 equiv
bromoacetophenone, EtOH, rt, 3h (vii) 1.0 equiv 3M NaOH/H2O, EtOH, rt, 30 min

Table 1. Structure and yields of thioxodihydropyridines 5a-r
Yield, %
Compound
R1
R2
R3
A
B
5a
5b
5c
5d
5e
5f
5g
5h
5i
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
COMe
COOMe
COOMe
COOMe
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
3,4,5-(OMe)3C6H2
4-OMeC6H4
3,4-(OMe)2C6H3
3,4,5-(OMe)3C6H2
Ph
42
53
42
45
4629
4-OMeC6H4
6030
3,4-(OMe)2C6H3
3,4,5-(OMe)3C6H2
4-OEtC6H4
4-n-OBuC6H4
3,4,5-(OMe)3C6H2
4-OMeC6H4
4331
4532
54
59
46
53
5j
5k
5l
COOn-Bu
COOC2H4OMe
5m
5n
5o
5p
5q
5r
Me
Me
COOC2H4OMe 3,4,5-(OMe)3C6H2
COOC2H4On-Pr 3,4,5-(OMe)3C6H2
42
57
3,4,5-(OMe)3C6H2
COOEt
CN
H
3,4,5-(OMe)3C6H2
3,4,5-(OMe)3C6H2
Me
44
41
NH2
Me
Ph
6733
8034
H
3-pyridyl
The synthesis of the corresponding thieno[2,3-b]pyridines was
performed, using slightly modified synthesis protocol described
in the literature.35 First alkylation takes place by treatment of 1,6-
N-Acetylated derivative 7 was prepared, by treatment of
thieno[2,3-b]pyridine 6r with acetylchloride in presence of
sodium hydroxide. 5-Unsubstituted thieno[2,3-b]pyridine 6y was
prepared by treatment of chalcone 1 (R1 = Me, R2 = H, R3 =
3,4,5-(OMe)3C6H2)36 with 2-cyanothioacetamide 2 in presence of
piperidine, in situ alkylation with bromoacetophenone and
sequential base catalyzed cyclization.
dihydro-6-thioxopyridines
5a-r
with
corresponding
bromoacetophenones in presence of excess of sodium hydroxide.
The formed intermediates 2-alkylsulfanyl-3-cyanopyridines easy
undergo Thorpe-Ziegler cyclization giving thieno[2,3-b]pyridines
6a-g, i-x, z in high yields.
OCHF2
OCHF2
OCHF2
O
O
O
N
N
OMe
OMe
i, ii, iii
iv
+
EtO
EtO
EtO
O
O
N
H
9
S
N
S
O
O
10
3a
8
OCHF2
v
O
NH2
O
EtO
S
N
6h
OMe
Scheme 2. Reagents and conditions: (i) 0.8 equiv piperidine, EtOH, rt, 30 min; (ii) 1.0 equiv 2-cyanothioacetamide, 0.32 equiv piperidine, EtOH, short reflux,
then rt 3 h; (iii) 1.0 equiv 2-bromo-4'-methoxyacetophenone, EtOH, short reflux; (iv) 2.0 equiv Mn(OAc)3 dihydrate, glacial AcOH, reflux, 2 h; (v) 1.0 equiv 3M
NaOH/H2O, EtOH, short reflux, then rt 30 min
In regard to synthesis of thieno[2,3-b]pyridine 6h containing
lipophilic C6H4OCHF2 moiety in position 4, first 1,4-
dihydropyridine 9 was built by four component condensation.
Then 1,4-DHP 9 was oxidized with manganese(III) acetate
dihydrate to pyridine 10 and transformed to the target product 6h
with sodium hydroxide (Scheme 2).

O
R3
R3
O
R3
N
O
NH2
CN
O
CN
O
O
iii
i, ii
EtO
EtO
EtO
+
R4
O
N
H
NH2 H2N
11
12a,b
14a,b
13a,b
a) R3 = 3,4,5-(OMe)3C6H2, R4 = 4-OMeC6H4; b) R3 = 4-OMeC6H4, R4 = 3,4,5-(OMe)3C6H2
Scheme 3. Reagents and conditions: (i) glacial acid/ethanol, reflux, 4h; (ii) HNO3, ethanol, reflux, 48h; (iii) 4.0 equiv K2CO3, 1.0 equiv bromoacetophenone,
DMF, 80 oC, 4h
In order to evaluate core structure, furo[2,3-b]pyridines as
bioisosters of thieno[2,3-b]pyridines were investigated. The
synthesis of furo[2,3-b]pyridine derivatives has been
accomplished as described in Scheme 3. Aminocrotonic acid
ethyl ester 11 was reacted with 3-cyano-4-(3,4,5-
trimethoxyphenyl)- or 4-(4-methoxyphenil)acrylamide 12a,b in
mixture of absolute ethanol and glacial acetic acid. The formed
intermediates 1,4,5,6-tetrahydro-6-oxopyridines undergo cycle
oxidation in ethanol-nitric acid media to give the 1,6-dihydro-6-
oxopyridines 13a and 13b37. In presence of potassium carbonate
and bromoacetophenones, compounds 13a,b consequently
undergo O-alkylation and Thorpe-Ziegler cyclization reaction to
give the corresponding furo[2,3-b]pyridines 14a,b.
Table 2. Structure and yields of synthesized thieno- and furo[2,3-b]pyridines 6, 7 and 14 as MDR modulators.
Compound
6a
R1
Me
R2
COMe
R3
R4
Yield, %
78
3,4,5-(OMe)3C6H2
4-OMeC6H4
6b
Me
COOMe
4-OMeC6H4
4-OMeC6H4
78
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
COOMe
COOMe
COOMe
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
3,4-(OMe)2C6H3
3,4-(OMe)2C6H3
3,4,5-(OMe)3C6H2 3,4-(OMe)2C6H3NH
C6H5
4-OMeC6H4
4-OCHF2C6H4
3,4-(OMe)2C6H3
3,4,5-(OMe)3C6H2
3,4,5-(OMe)3C6H2
4-OMeC6H4
2,4-(OMe)2C6H3
84
93
97
94
86
60
70
4035
89
96
96
89
93
90
90
82
80
81
86
95
41
70
89
9134
71
19
38
4-OMeC6H4
4-OMeC6H4
4-OMeC6H4
Me
C6H5
4-OMeC6H4
3,4,5-(OMe)3C6H2 2,4-(OMe)2C6H3
COOEt
COOEt
4-OEtC6H4
4-OBu(n)C6H4
3,4,5-(OMe)3C6H2
4-OMeC6H4
4-OMeC6H4
4-OMeC6H4
4-OMeC6H4
4-OMeC6H4
Ph
COOBu
COOC2H4OMe
COOC2H4OMe 3,4,5-(OMe)3C6H2
COOC2H4OMe 3,4,5-(OMe)3C6H2
COOC2H4OMe 3,4,5-(OMe)3C6H2 2,4-(OMe)2C6H3
COOC2H4OMe 3,4,5-(OMe)3C6H2 3,4,5-(OMe)3C6H2
COOC2H4OPr 3,4,5-(OMe)3C6H2
4-OMeC6H4
6s
6t
Me
Me
Me
6u
6v
6w
6x
6y
6z
7
4-OMeC6H4
4-OMeC6H4
4-OMeC6H4
2,4-(OMe)2C6H3
4-OMeC6H4
Ph
4-OMeC6H4
4-OMeC6H4
3,4,5-(OMe)3C6H2
3,4,5-(OMe)3C6H2
COOEt
CN
H
3,4,5-(OMe)3C6H2
3,4,5-(OMe)3C6H2
Me
NH2
Me
Me
Ph
Me
Me
Me
H
3,4,5-(OMe)3C6H2
3-pyridyl
H
COOC2H4OMe 3,4,5-(OMe)3C6H2
14a
14b
COOEt
COOEt
3,4,5-(OMe)3C6H2
4-OMeC6H4
2.2. Biological evaluation, structure-activity relationships, P-gp,
MRP1 and BCRP1 modulating activity
several fluorescent compounds are transported by P-gp, MRP1
and BCRP1 such as rhodamine 123 (P-gp), calcein (MRP1) and
Hoechst 33342 (P-gp, BCRP1). These fluorescent compounds are
used during studying ABC transporters in cell lines.
The tested compounds showed multidrug resistance
modulating activities which are listed in Table 3. MK-571,
Reversan, and Verapamil were used as reference inhibitors of P-
gp, MRP1 and BCRP1. Besides the clinically important drugs,

Table 3. MDR modulating activity and calculated logP values of tested compounds
MDR, EC50, µM
Ca2+, A7R5, LD50,
Compound logP
IC50, µM
mg/kg
P-gp
MRP1
BCRP1
Verapamil
MK-571
Reversan
962
752
885
7.1 2.0 27.8 0.8 37.3
7
0.3 0.1
n.e.
> 100
n.e.
-
-
12.4 2.2
9.8 0.5
18.4
6a
6b
4.10
4.80
>2000
>2000
3.8 0.1 6.6 1.0 2.6 0.6
n.e.
5.6 0.2 11.9 1.3 3.6 0.6
14.0 0.9
15.4 2.0
4.5 0.2
0.7 0.1
6c
4.70
2808
6.0 0.8
6d
6e
6f
6g
6h
6i
4.58
4.05
5.29
5.17
5.88
3.27
5.04
4.92
4.79
5.51
6.41
5.82
4.67
4.55
4.42
4.30
4.17
5.25
5.93
4.00
3.82
4.76
5.35
4.13
3.55
3.55
41.4
n.e.
n.e.
n.e.
-
1045
-
-
>2000
-
10.3 1.5
n.e.
4.1 0.9
n.e.
n.e.
5.6 1.4
-
-
n.e.
6.5 0.9
n.e.
n.e.
0.4 0.1 100.0 11.0
-
-
-
-
n.e.
-
6j
>2000
>2000
>2000
>2000
-
>2000
>2000
>2000
2097
2983
-
9.0 0.5 5.2 0.8 1.5 0.0
0.3 0.1 5.2 0.6 0.7 0.3
6.4 0.6 12.4 0.4 2.6 0.3
14.0 1.1
19.0 3.0
6k
6l
46.0 1.4
>100.0
-
6m
6n
6o
6p
6q
6r
n.e.
n.e.
n.e.
n.e.
4.2 0.7
n.e.
1.3 0.2
n.e.
1.5 0.1
1.0 0.1
0.4 0.08
0.8 0.1
5.0 0.7
21.0 4.0
2.2 0.3
3.1 0.4
1.4 0.1 3.9 0.6 1.3 0.2
0.3 0.2 1.1 0.1 0.2 0.05
6s
6t
2.0 0.0
n.e.
0.6 0.1
22.0 0.5
n.e.
7.0 1.0 2.5 0.5
9.0
1
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
-
n.e.
n.e.
n.e.
n.e.
0.6 0.1
3.0 0.3
n.e.
n.e.
n.e.
-
6u
6v
6w
6x
6y
6z
7
>100.0
n.e.
>2000
-
-
-
-
-
-
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
959
-
-
-
n.e.
3.4 0.7
n.e.
-
-
14a
14b
1770
>2000
1.0 0.1
-
n.e. - No effect
A7R5 - rat aorta smooth muscle
As shown in Table 3, some 3-amino-thieno[2,3-b]pyridines
displayed similar or more potent P-gp and MRP1 modulating
activities in comparison with control inhibitors (Verapamil, MK-
571, Reversan), and our target compounds significantly exceeded
BRCP1 inhibitor activity of reference compound Verapamil.
Only thieno[2,3-b]pyridines possessing at least one 4-
methoxyphenyl group in position 4 (substituent R3) and free
amino group at position 3 exhibit a potent P-gp, MRP1 and
BCRP1 inhibitory action. In case of 3,4-dimethoxyphenyl group
potency decreases, but modulators containing 3,4,5-
trimethoxyphenyl group have shown the most promising results.
So, para-OMe substitution at one of the two aryl rings present in
pharmacophore model is required for optimum MDR inhibition
potency and selectivity. Introduction of two 3,4,5-
trimethoxyphenyl rings is undesirable, and inhibitory action of
compounds 6t, 6v has partially or completely disappeared.
Compounds 6f (R3 = Ph), 6x (R3 = Me) and 6z (R3 = 3-pyridyl)
are completely inactive.
Aroyl group (substituent R4) in position 2 of thieno[2,3-
b]pyridine ring is essential for MDR modulating activity, its
substitution with acetyl and aromatic amide function (compounds
6i and 6e) results in loss of activity. Results show that
benzoylderivative 6j is relatively potent, introduction of one
electron-donating methoxy group in para-position of the
aromatic ring increases activity, while introducing additional
methoxy groups to the ortho-position or two methoxygroups in
meta-position leads to decrease in potency. Acylation of amino
group in position 3 of thieno[2,3-b]pyridine ring (compound 7)
resulted in loss of activity and these results suggest that the free
amino group as hydrogen bond donor contributes largely to the
biological activity.
As seen from Table 3, 4-(4-methoxyphenyl) group containing
compounds 6b, 6g and 6p selectively inhibit only P-gp and
BCRP1. 4-(4-Ethoxyphenyl) group containing compound 6m
shows similar activity to corresponding 6g, while butyloxyphenyl
ether 6n (calculated logP 6.41) has lost MDR modulating activity
which indicate that optimum lipophylicity is required.

Introduction of lipophilic C6H4OCHF2 moiety in position 4
(compound 6h, calculated logP 5.88) confirms this requirement
because activity is lost in this case, too.
of 3-amino-thieno[2,3-b]pyridine scaffold with hydrophobic aryl
groups in positions 2 and 4, ester groups in position 5 (reaching
optimum lipophilicity - logP range 4.4 - 5.8), amino group in
position 3 (hydrogen bond donor) and methoxyphenyl groups
(bearing appropriate amount of hydrogen bond acceptors) has led
to potent P-glycoprotein, multidrug resistance-associated protein
and the breast cancer resistance protein inhibitors which
significantly exceeded activity of Verapamil, MK-571, Reversan.
Significantly cheaper furo[2,3-b]pyridines as bioisosteres of
thieno[2,3-b]pyridines have shown no activity on P-gp and
MRP1 inhibition, but have found to be selective on BCRP1
inhibition.
Replacement of hydrogen at the position 5 of thieno[2,3-
b]pyridine ring (substituent R2) with acetyl or ester group results
in significant increase of activity, reaching maximum in cases of
ethyl or 2-methoxyethylester ester groups. As thienopyridine 6u
with lipophylic 2-propoxyethylester ester group (logP 5.25) is
approximately 2 times less active in comparison with 6r (logP
4.42), optimum lipophylicity has to be reached by modification
of substituent R2, too. Exchange of methyl group in position 6
with aryl or amino groups (substituent R1) leads to diminution of
activity.
SAR data in thieno[2,3-b]pyridine series indicate necessity of
both hydrogen bond donor (acylation of amino group in position
3 resulted in loss of activity) and hydrogen bond acceptors (four
to five OMe groups in different positions) to reach optimum
activity.
Among furo[2,3-b]pyridines 14 only compound 14a as
bioisostere of thieno[2,3-b]pyridine 6k has shown selective
activity against BCRP1 and was completely inactive on P-gp and
MRP1 lines. Nevertheless oxygen bioisostere 14a, being ten
times more active than verapamil, is five times less active than
compound 6k and, in addition, undesired Ca2+ channel blocking
activity has revealed.
4. Experimental
4.1. Chemistry
Unless noted otherwise, all reagents and solvents were used as
purchased without further purification. Melting points were
determined on OptiMelt MPA100 apparatus and are uncorrected.
Analytical TLC was performed using silica gel 60 F254 plates
Lipophilicity is an important indicator of how target
molecules can cross cell membranes. Predicted lipophilicity
(logP) of the compounds was calculated with the computer
program logP using ChemBioDraw® Ultra 12.0 suite. Calculated
logP values are in range of 3.27 to 6.41 (Table 3). Most of potent
compounds of thieno[2,3-b]pyridine series have logP
approximately 4.4-5.8. Among tested compounds, those with
lipophilicity lower than 4.0 and higher than 5.9 have shown
significantly weaker or no effect of MDR modulating activity.
1
(Merck). H NMR spectra were recorded on a Varian Mercury
BB 400 MHz spectrometer, and 13C NMR spectra were recorded
on Varian Mercury BB (100 MHz) spectrometer using CDCl3 or
DMSO-d6 as the solvents. The chemical shifts of the atoms are
reported in parts per million (ppm) relatively to the residual
signals of the solvent: CDCl3 (d:7.26) or DMSO-d6 (2.50) for 1H
NMR spectra and CDCl3 (d:77.16) or DMSO-d6 (39.52) for 13C
NMR spectra. Multiplicities are abbreviated as: br = broad, s =
singlet, d = doublet, t = triplet, q = quartet, qui = quintet, m =
multiplet. The coupling constants are expressed in Hz. The IR
spectra have been recorded on Shimadzu IR Prestige-21
spectrometer in nujol or KBr tablet and peak positions νmax are
expressed in cm-1. Compounds 5e,29 5r,34 6j,34 6z34 have been
described in our previous work. Compounds 5f,30 5g,31 5h,32 5r33
and 13b37 are known.
It is known that H-bond donor as well as acceptor functions
play an important role in the binding possibilities of P-gp
substrates. Association of the P-gp substrate (inhibitor) complex
is determined by the number and strength of the hydrogen bonds
formed between the substrate and the transporter.38 The
thieno[2,3-b]pyridine series bearing R1 = Me, R2 = COOAlk, R3 =
3,4,5-(OMe)3C6H2 and R4 = 4-OMeC6H4CO are the most potent
MDR modulators. Amino group in position 3 serves as hydrogen
bond donor, but OMe groups in different positions as hydrogen
bond acceptors.
4.1.1. General procedure for the preparation of 6-
thioxo-1, 6-dihydropyridines from chalcones
(method A)
Thieno[2,3-b]pyridines 6a, 6g, 6n, 6o, 6p and 6u selectively
inhibited P-gp and BCRP1, but compounds 6a, 6c, 6k, 6l, 6q, 6r
and 6s were potent and efficacious as they inhibited three MDR
transport proteins: P-gp, MRP1 and BCRP1. In particular,
compound 6r was found to exhibit a potent P-gp inhibitory action
A
mixture of corresponding chalcone 1 (5 mmol), 2-
cyanothioacetamide 2 (5 mmol) and triethylamine (0.35 mmol) in
ethanol was refluxed for 15 h. The precipitated crystals were
separated by filtration and purified by washing with ethanol and
water during the filtration.
with EC50 = 0.3 0.2 µM, MRP1 inhibitory action with EC50
=
1.1 0.1 µM and BCRP1 inhibitory action with EC50 = 0.20
0.05 µM and may be promising leads for development of MDR-
reversal drugs.
4.1.1.1. 5-Acetyl-6-methyl-2-thioxo-4-(3,4,5-
trimethoxyphenyl)-1,2-dihydropyridine-3-
carbonitrile (5a)
Thieno[2,3-b]pyridines not only are significantly potent
MDR-blockers in comparison with reference compounds, but in
addition reveal more than ten times lower Ca2+ antagonist effect
than Verapamil (less cardiovascular side effects expected) and
are non-toxic (LD50 > 2000 mg/kg). They are potential agents to
overcome MDR problem in clinic and increase the effectiveness
of chemotherapy in cancer treatment.
1
Yellow crystals; yield 42 %; mp 244 - 246 oC. H NMR (CDCl3)
δ 1.83 (3H, s); 2.50 (3H, s); 3.87 (3H, s) 3.92 (6H, s); 6.62 (2H,
s); 12.16 (1H, br.s). IR (KBr) ν 1685, 2224, 3233. Anal Calcd.
for C18H18N2O4S: C, 60.32; H, 5.06; N, 7.82. Found: C, 60.03; H,
5.08; N, 7.76.
4.1.1.2. 5-Cyano-4-(3, 4-dimethoxyphenyl)-2-methyl-
6-thioxo-1,6-dihydropyridine-3-carboxylic acid
methyl ester (5c)
3. Conclusion
A new class of multidrug resistance (MDR) modulators,
possessing a 3-amino-thieno[2,3-b]pyridine scaffold has been
discovered. Pharmacophore model was created assuming
thieno[2,3-b]pyridine scaffold as linker and methoxyphenyl
groups as essential for potential MDR-reversal drug. Decoration
1
Yellow crystals; yield 42 %; mp 230 - 232 oC. H NMR (CDCl3)
δ 2.58 (3H, s); 3.54 (3H, s); 3.90 (3H, s); 3.93 (3H, s); 6.90-6.98
(3H, m); 12.50 (1H, br.s). IR (KBr) ν 1721, 2226, 3241. Anal

1
Calcd. for C17H16N2O4S: C, 59.29; H, 4.68; N, 8.13. Found: C,
59.20; H, 4.52; N, 7.98.
Yellow crystals; yield 59 %; mp 175 - 176 oC. H NMR (CDCl3)
δ 0.92 (3H, t, J = 7.0 Hz), 0.99 (2H, se, J = 7.0 Hz), 1.50 (2H,
qui, J = 6.3 Hz); 1.79 (2H, t, J = 6.3 Hz); 2.58 (3H, s); 3.88 (2H,
q, J = 7.0 Hz); 4.00 (2H, t, J = 7.0 Hz); 6.96 (2H, d, J = 9.0 Hz);
7.31 (2H, d, J = 9.0 Hz); 12.41 (1H, br.s). IR (KBr) ν 1710, 2231,
3159. Anal Calcd. for C20H22N2O3S: C, 64.84; H, 5.99; N, 7.56.
Found: C, 64.48; H, 6.14; N, 7.50.
4.1.1. 3. 5-Cyano-2-methyl-6-thioxo-4-(3,4,5-
trimethoxyphenyl)-1, 6-dihydropyridine-3-carboxylic
acid methyl ester (5d)
o
1
Yellow crystals; yield 45 %; mp 233 - 234 C. H NMR (CDCl3)
δ 2.58 (3H, s); 3.54 (3H, s); 3.87 (6H, s); 3.90 (3H, s); 6.60 (2H,
s); 12.48 (1H, br.s). IR (KBr) ν 1733, 2227, 3070, 3177. Anal
Calcd. for C18H18N2O5S: C, 57.74; H, 4.85; N, 7.48. Found: C,
57.10; H, 4.61; N, 7.76.
4.1.2.4. 5-Cyano-2-methyl-6-thioxo-4-(3,4,5-
trimethoxyphenyl-1, 6-dihydropyridine-3-carboxylic
acid n-butyl ester (5k)
1
Yellow crystals; yield 46 %; mp 197 - 198 oC. H NMR (CDCl3)
δ 0.79 (3H, t, J = 7.0 Hz); 1.05 (2H, se, J = 7.0 Hz); 1.25 (2H,
qui, J = 6.3 Hz); 2.59 (3H, s); 3.87 (3H, s); 3.90 (6H, s); 3.92
(2H, t, J = 6.3 Hz); 6.61 (2H, s); 12.40 (1H, br.s). IR (KBr) ν
1705, 2228, 3303. Anal Calcd. for C21H24N2O5S: C, 60.56; H,
5.81; N, 6.73. Found: C, 60.02; H, 5.78; N, 6.55.
4.1.1. 4. 5-Cyano-2-methyl-6-thioxo-4-(3,4,5-
trimethoxyphenyl)-1, 6-dihydropyridine-3-carboxylic
acid 2-methoxyethyl ester (5m)
o
1
Yellow crystals; yield 42 %; mp 192 - 194 C. H NMR (CDCl3)
δ 2.58 (3H, s); 3.21-3.23(5H, m); 3.86 (6H, s); 3.89 (3H, s); 4.07
(2H, t, J = 4.7 Hz); 6.59 (2H, s); 12.57 (1H, s). IR (KBr) ν 1738,
2232, 3178. Anal Calcd. for C20H22N2O6S: C, 57.40; H, 5.30; N,
6.69. Found: C, 57.08; H, 5.33; N, 6.75.
4.1.2.5. 5-Cyano-2-methyl-6-thioxo-4-(4-
methoxyphenyl)-1,6-dihydropyridine-3-carboxylic
acid 2-methoxyethyl ester (5l)
1
Yellow crystals; yield 53 %; mp 210 - 211 oC. H NMR (CDCl3)
4.1.1. 5. 5-Cyano-2-methyl-6-thioxo-4-(3,4,5-
trimethoxyphenyl)-1, 6-dihydropyridine-3-carboxylic
acid 2-propoxyethyl ester (5n)
δ 2.57 (3H, s); 3.24-3.28 (5H, m); 3.86 (3H, s); 4.09 (2H, t, J =
4.7 Hz); 6.99 (2H, d, J = 8.6 Hz); 7.34 (2H, d, J = 8.6 Hz). IR
(KBr) ν 1726, 2226, 3167, 3479, 3579. Anal Calcd. for
C18H18N2O4S: C, 60.32; H, 5.06; N, 7.82. Found: C, 59.78; H,
5.13; N, 7.78.
Yellow crystals; yield 57 %; mp 140 - 141 oC. 1H NMR (CDCl3)
δ 0.87 (3H, t, J = 7.0 Hz); 1.51 (2H, se, J = 7.0 Hz), 2.60 (3H, s);
3.25-3.30 (4H, m) 3.87 (6H, s); 3.90 (3H, s); 4.10 (2H, t, J = 4.7
Hz); 6.61 (2H, s); 12.71 (1H, br.s). IR (KBr) ν 1730, 2228, 3178.
Anal Calcd. for C22H26N2O6S: C, 59.18; H, 5.87; N, 6.27. Found:
C, 59.10; H, 5.78; N, 6.21.
4.1.2.6. 5-Cyano-6-thioxo-2,4-bis-(3,4,5-
trimethoxyphenyl)-1,6-dihydropyridine-3-carboxylic
acid ethyl ester (5o)
1
Yellow crystals; yield 44 %; mp 178 - 179 oC. H NMR (CDCl3)
4.1.2. General procedure for the preparation of 6-
thioxo-1,6-dihydropyridines from thioacrylamides
(method B)
δ 0.87 (3H, t, J = 7.0 Hz); 3.86 (9H, s); 3.90 (9H, s); 4.00 (2H, q,
J = 7.0 Hz); 6.57 (2H, s); 6.88 (2H, s); 8.67 (1H, s). IR (KBr) ν
1727, 2223, 3148. Anal Calcd. for C27H28N2O8S: C 59.99; H
5.22; N 5.18. Found: C 60.12; H 5.11; N 5.28.
A mixture of corresponding thioacrylamide 4 (5 mmol),
acetoacetate 3 (5 mmol) and piperidine (0.5 mmol) in ethanol
was refluxed for 15 h. The precipitated crystals were separated by
filtration and purified by washing with ethanol and water during
the filtration.
4.1.2.7. 6-Amino-2-thioxo-4-(3, 4,5-
trimethoxyphenyl)-1,2-dihydropyridine-3,5-
dicarbonitrile (5p)
4.1.2. 1. 5-Cyano-4-(4-methoxyphenyl)-2-methyl-6-
thioxo-1,6-dihydropyridine-3-carboxylic acid
methyl ester (5b)
Yellow crystals; yield 41 %; mp 197 - 198 oC. 1H NMR (DMSO-
d6) δ 3.70 (6H, s) and 3.77 (3H, s); 6.84 (2H, s); 8.11 (2H, s);
13.25 (1H, s). IR (KBr) ν 2212, 3189, 3341. Anal Calcd. for
C16H14N4O3S: C 56.13; H 4.12; N 16.36. Found: C 56.00; H 4.22;
N 16.28.
o
1
Yellow crystals; yield 53 %; mp 183 - 185 C. H NMR (CDCl3)
δ 2.58 (3H, s); 3.53 (3H, s); 3.86 (3H, s); 6.99 (2H, d, J = 8.6
Hz); 7.32 (2H, d, J = 8.6 Hz); 12.65 (1H, br.s). IR (KBr) ν 1722,
2214, 2949, 3000. Anal Calcd. for C16H14N2O3S: C, 61.13; H,
4.49; N, 8.91. Found: C, 60.99; H, 4.22; N, 7.49.
4.1.3. General procedure for the preparation of
thieno[2,3-b]pyridines
To a solution of corresponding 6-thioxo-1,6-dihydropyridine 5 (1
mmol) in ethanol 3M sodium hydroxide water solution (2.4
mmol) was added. The reaction mixture was shortly refluxed to
form thiolate. Then corresponding 2-bromoacetophenone (1
mmol) was added, the reaction mixture was refluxed for 5 min
and stirred at room temperature for 30 min. The precipitated
crystals were separated by filtration and purified by washing with
ethanol and water during the filtration.
4.1.2. 2. 5-Cyano-4-(4-ethoxyphenyl)-2-methyl-6-
thioxo-1,6-dihydropyridine-3-carboxylic acid ethyl
ester (5i)
o
1
Yellow crystals; yield 54 %; mp 184 - 185 C. H NMR (CDCl3)
δ 0.91 (3H, t, J = 7.0 Hz); 1.44 (3H, t, J = 7.0 Hz); 2.57 (3H, s);
3.98 (2H, q, J = 7.0 Hz); 4.07 (2H, q, J = 7.0 Hz); 6.97 (2H, d, J
= 8.6 Hz); 7.31 (2H, d, J = 8.6 Hz) ); 12.06 (1H, br.s). IR (KBr) ν
1709, 2230, 3034, 3160. Anal Calcd. for C18H18N2O3S: C, 63.14;
H, 5.30; N, 8.18. Found: C, 63.05; H, 5.25; N, 8.18.
4.1.3.1. 5-Acetyl-3-amino-2-(4-methoxybenzoyl)-6-
methyl-4-(3,4,5-trimethoxyphenyl)-thieno[2,3-
b]pyridine (6a)
4.1.2. 3. 4-(4-Butoxyphenyl)-5-cyano-2-methyl-6-
thioxo-1,6-dihydropyridine-3-carboxylic acid ethyl
ester (5j)
Yellow crystals; yield 78 %; mp 162 - 164 oC. 1H NMR (CDCl3)
δ 2.06 (3H, s); 2.60 (3H, s); 3.86 (6H, s); 3.88 (3H, s); 3.95 (3H,
s); 6.59 (2H, s); 6.79 (2H, br.s); 6.97 (2H, d, J = 8.6 Hz); 7.87

1
(2H, d, J = 8.6 Hz). 13C NMR (CDCl3) δ 23.3, 32.3, 55.4, 56.4,
61.2, 105.2, 106.4, 113.6, 119.0, 128.5, 130.1, 133.4, 134.1,
139.0, 142.9, 150.1, 153.7, 155.3, 162.1, 189.6, 204.9. IR (nujol)
ν 1699, 3302, 3468. Anal Calcd. for C27H26N2O6S: C, 64.02; H,
5.17; N, 5.53. Found: C, 64.07; H, 5.18; N, 5.46.
Yellow crystals; yield 94 %; mp 150 - 152 oC. H NMR (CDCl3)
δ 0.94 (3H, t, J = 7.0 Hz); 2.70 (3H, s); 3.88 (3H, s); 4.01 (2H, q,
J = 7.0 Hz); 6.57 (2H, br.s); 6.97 (2H, d, J = 8.6 Hz), 7.41-7.54
(5H, m); 7.86 (2H, d, J = 8.6 Hz). 13C NMR (CDCl3) δ 23.4,
55.4, 61.5, 105.2, 107.8, 109.8, 113.6, 119.3, 128.5, 128.8, 129.5,
130.0, 133.4, 145.0, 147.9, 150.2, 162.1, 189.5. IR (nujol) ν
1725, 3301, 3477. Anal Calcd. for C25H22N2O4S: C, 67.25; H,
4.97; N, 6.27. Found: C, 67.02; H, 4.82; N, 6.15.
4.1.3. 2. 3-Amino-2-(4-methoxybenzoyl)-4-(4-
methoxyphenyl)-6-methyl-thieno[2, 3-b]pyridine-5-
carboxylic acid methyl ester (6b)
4.1.3.7. 3-Amino-2-(4-methoxybenzoyl)-4-(4-
methoxyphenyl)-6-methyl-thieno[2,3-b]pyridine-5-
carboxylic acid ethyl ester (6g)
o
1
Yellow crystals; yield 78 %; mp 179 - 180 C. H NMR (CDCl3)
δ 2.67 (3H, s); 3.58 (3H, s); 3.88 (3H, s); 3.90 (3H, s); 6.68 (2H,
br.s); 6.97 (2H, d, J = 8.6 Hz); 7.04 (2H, d, J = 8.6 Hz); 7.32 (2H,
d, J = 8.6 Hz); 7.86 (2H, d, J = 8.6 Hz). 13C NMR (CDCl3) δ
23.3, 52.3, 55.4, 105.0, 113.6, 114.3, 119.7, 125.7, 126.9, 129.7,
130.0, 133.4, 145.0, 150.3, 156.6, 160.4, 162.1, 162.6, 168.3,
189.5. IR (nujol) ν 1606, 1730, 3302, 3474. Anal Calcd. for
C25H22N2O5S: C, 64.92; H, 4.79; N, 6.06. Found: C, 64.55; H,
4.78; N, 5.94.
1
Yellow crystals; yield 86 %; mp 150 - 151 oC. H NMR (CDCl3)
δ 1.02 (3H, t, J = 7.0 Hz); 2.68 (3H, s); 3.88 (3H, s); 3.89 (3H, s);
4.06 (2H, q, J = 7.0 Hz); 6.67 (2H, br.s); 6.97 (2H, d, J = 8.6 Hz);
7.04 (2H, d, J = 8.6 Hz), 7.33 (2H, d, J = 8.6 Hz); 7.86 (2H, d, J
= 8.6 Hz). 13C NMR (CDCl3) δ 13.8, 23.3, 55.4, 61.5, 105.6,
113.6, 114.2, 119.7, 125.8, 127.1, 129.8, 130.0, 133.5, 144.9,
150.4, 156.6, 160.4, 162.1, 162.5, 167.8, 189.5. IR (nujol) ν
1606, 1723, 3299, 3474. Anal Calcd. for C26H24N2O5S: C, 65.53;
H, 5.08; N, 5.88. Found: C, 65.62; H, 4.93; N, 5.57.
4.1.3. 3. 3-Amino-4-(3,4-dimethoxyphenyl)-2-(4-
methoxybenzoyl)-6-methyl-thieno[2,3-b]pyridine-5-
carboxylic acid methyl ester (6c)
4.1.3.8. 2-Acetyl-3-amino-4-(3,4-dimethoxyphenyl)-
6-methyl-thieno[2,3-b]pyridine-5-carboxylic acid
ethyl ester (6i)
o
1
Yellow crystals; yield 84 %; mp 192 - 194 C. H NMR (CDCl3)
δ 2.61 (3H, s); 3.54 (3H, s); 3.82 (3H,s); 3.83 (3H, s); 3.91 (3H,
s); 6.67 (2H, br.s); 6.83-6.95 (3H, m); 6.91 (2H, d, J = 8.6 Hz);
7.80 (2H, d, J = 8.6Hz). 13C NMR (CDCl3) 23.3, 52.4, 55.4, 55.9,
56.1, 105.0, 111.1, 111.4, 113.6, 119.5, 120.9, 125.9, 126.8,
130.0, 133.4, 144.9, 149.0, 149.8, 150.2, 156.7, 162.1, 162.6,
168.4, 189.5. IR (nujol) ν 1699, 3463. Anal Calcd. for
C26H24N2O6S: C, 63.40; H, 4.91; N, 5.69. Found: C, 62.91; H,
4.79; N, 5.62.
Yellow crystals; yield 75 %; mp 191 - 193 oC. 1H NMR (CDCl3)
δ 0.96 (3H, t, J = 7.0 Hz); 2.37 (3H, s); 2.62 (3H, s); 3.81 (3H,
s); 3.89 (3H, s); 4.01 (2H, q, J = 7.0 Hz); 6.60 (2H, br.s); 6.80-
6.93 (3H, m). 13C NMR (CDCl3) δ 13.9, 23.1, 29.2, 56.0, 56.1,
61.6, 106.2, 111.0, 111.5, 119.9, 120.9, 125.8, 127.1, 145.0,
148.2, 149.0, 149.9, 156.5, 161.0, 167.7, 193.0. IR (nujol) ν .
Anal Calcd. for C21H22N2O5S: C, 60.85; H, 5.35; N, 6.76. Found:
C, 60.71; H, 5.11; N, 6.69.
4.1.3. 4. 3-Amino-2-(2,4-dimethoxybenzoyl)-4-(3,4-
dimethoxyphenyl)-6-methyl-thieno[2,3-b]pyridine-
5-carboxylic acid methyl ester (6d)
4.1.3.9. 3-Amino-4-(3, 4,5-trimethoxyphenyl)-6-
methyl-2-(4-methoxybenzoyl)-thieno[2,3-b]pyridine-
5-carboxylic acid ethyl ester (6k)
o
1
Yellow crystals; yield 93 %; mp 168 - 169 C. H NMR (CDCl3)
δ 2.58 (3H, s); 3.53 (3H, s); 3.75 (3H, s); 3.80 (3H, s); 3.83 (3H,
s); 3.91 (3H, s); 6.45-6.48 (2H, m); 6.64 (2H, br.s); 6.82-6.94
(3H, m); 7.20 (1H, d, J = 8.6 Hz). 13C NMR (CDCl3) δ 23.3,
52.3, 55.4, 55.6, 98.9, 104.3, 111.0, 111.4, 119.6, 120.9, 123.6,
126.0, 126.6, 129.9, 144.9, 149.0, 149.4, 149.8, 156.4, 157.9,
162.5, 162.9, 168.4, 189.9. IR (nujol) ν 1725, 3459. Anal Calcd.
for C27H26N2O7S: C, 62.06; H, 5.01; N, 5.36. Found: C, 61.50; H,
4.97; N, 5.22.
1
Yellow crystals; yield 89 %; mp 204 - 206 oC. H NMR (CDCl3)
δ 0.96 (3H, t, J = 7.0 Hz); 2.63 (3H, s); 3.80 (6H, s); 3.83 (3H, s);
3.87 (3H, s,); 4.02 (2H, q, J = 7.0 Hz); 6.55 (2H, s); 6.70 (2H, s);
6.91 (2H, d, J = 8.6 Hz); 7.80 (2H, d, J = 8.6 Hz). 13C NMR
(CDCl3) δ 13.8, 22.5, 22.7, 55.4, 56.0, 56.1, 56.4, 61.1, 61.7,
105.3, 105.6, 111.1, 111.4, 113.7, 119.8, 120.9, 125.6, 126.8,
127.3, 128.8, 130.0, 133.2, 138.8, 145.6, 149.1, 149.8, 150.1,
153.5, 156.2, 162.2, 167.2, 167.3, 189.5. IR (nujol) ν 1724, 3301,
3469. Anal Calcd. for C28H28N2O7S: C, 62.67; H, 5.26; N, 5.22.
Found: C, 62.38; H, 5.06; N, 5.16.
4.1.3. 5. 3-Amino-2-(3,4-
dimethoxyphenylcarbamoyl)-6-methyl-4-(3,4,5-
trimethoxyphenyl)-thieno[2,3-b]pyridine-5-
carboxylic acid methyl ester (6e)
4.1.3.10. 3-Amino-2-(2,4-dimethoxybenzoyl)-6-
methyl-4-(3,4,5-trimethoxyphenyl)-thieno[2,3-
b]pyridine-5-carboxylic acid ethyl ester (6l)
Yellow crystals; yield 97 %; mp 203 - 204 oC. 1H NMR (DMSO-
d6) δ 2.58 (3H, s); 3.58 (3H, s); 3.72 (3H, s); 3.73 (3H, s); 3.76
(3H, s); 3.77 (6H, s); 6.02 (2H, br. s); 6.70 (2H, s); 6.89 (1H, d, J
= 8.9 Hz); 7.23 (1H, dd, J = 2.1 Hz, J = 8.5 Hz); 7.30 (1H, d, J =
2.1 Hz); 9.40 (1H, s). 13C NMR (CDCl3) δ 22.6, 52.3, 55.3, 55.6,
56.1, 60.2, 97.5, 106.1, 106.4, 111.6, 113.2, 119.7, 126.4, 128.6,
132.1, 137.9, 143.8, 145.1, 146.5, 148.2, 152.7, 154.3, 159.2,
163.4, 167.6. IR (nujol) ν 1658, 1718, 3101, 3304. Anal Calcd.
for C28H29N3O8S: C, 59.25; H, 5.15; N, 7.40. Found: C, 59.01; H,
4.90; N, 7.40.
Yellow crystals; yield 96 %; mp 174 - 176 oC. 1H NMR (CDCl3)
δ 0.95 (3H, t, J = 7 Hz); 2.60 (3H, s); 3.75 (3H, s); 3.80 (9H, s);
3.86 (3H, s); 4.01 (2H, q, J = 7.0 Hz); 6.45-6.54 (2H, m); 7.29-
7.33 (3H, m). 13C NMR (CDCl3) δ 13.8, 23.3, 55.5, 55.6, 56.3,
61.1, 61.5, 98.9, 104.3, 105.7, 108.1, 119.3, 123.6, 126.4, 129.2,
129.9, 138.6, 144.8, 149.2, 153.4, 156.6, 157.9, 162.5, 162.8,
167.8, 189.9. IR (nujol) ν 1717, 3306, 3445. Anal Calcd. for
C29H30N2O8S: C, 61.47; H, 5.34; N, 4.94. Found: C, 60.99; H,
5.36; N, 4.62.
4.1.3. 6. 3-Amino-2-(4-methoxybenzoyl)-6-methyl-4-
phenyl-thieno[2,3-b]pyridine-5-carboxylic acid
ethyl ester (6f)
4.1.3.11. 3-Amino-4-(4-ethoxyphenyl)-2-(4-
methoxybenzoyl)-6-methyl-thieno[2,3-b]pyridine-5-
carboxylic acid ethyl ester (6m)

o
1
Yellow crystals; yield 96 %; mp 150 - 151 C. H NMR (CDCl3)
δ 1.02 (3H, t, J = 7.4 Hz); 1.48 (3H, t, J = 7.0 Hz); 2.68 (3H, s);
3.88 (3H, s); 4.06 (2H, q, J = 7.4 Hz); 4.11 (2H, q, J = 7.0 Hz);
6.66 (2H, br.s); 6.97 (2H, d, J = 8.6 Hz); 7.02 (2H, d, J = 8.6 Hz);
7.31 (2H, d, J = 8.6 Hz); 7.86 (d, J = 8.6 Hz). 13C NMR (CDCl3)
δ 13.8, 14.7, 23.3, 55.4, 61.5, 63.7, 105.0, 113.6, 114.7, 119.7,
125.6, 127.1, 129.8, 130.0, 133.5, 145.0, 150.4, 156.6, 159.8,
162.1, 167.8, 189.5. IR (nujol) ν 1608, 1725, 3302, 3474. Anal
Calcd. for C27H26N2O5S: C, 66.11; H, 5.34; N, 5.71. Found: C,
66.03; H, 5.34; N, 5.64.
Anal Calcd. for C28H28N2O7S: C, 62.67; H, 5.26; N, 5.22. Found:
C, 62.08; H, 5.66; N, 5.00.
4.1.3.16. 3-Amino-2-(4-methoxybenzoyl)-6-methyl-
4-(3,4,5-trimethoxyphenyl)-thieno[2,3-b]pyridine-5-
carboxylic acid 2-methoxyethyl ester (6r)
1
Yellow crystals; yield 82 %; mp 154 - 155 oC. H NMR (CDCl3)
δ 2.70 (3H, s), 3.28 (3H, s); 3.35 (2H, t, J = 4.7 Hz); 3.87 (6H, s);
3.89 (3H, s); 3.95 (3H, s); 4.17 (2H, t, J = 4.7 Hz); 6.62 (2H, s);
6.78 (2H, br.s); 6.97 (2H, d, J = 8.6 Hz); 7.87 (2H, d, J = 8.6 Hz).
13C NMR (CDCl3) δ 23.3, 55.4, 56.3, 58.7, 61.1, 64.4, 70.0,
105.1, 113.6, 119.2, 126.3, 129.0, 130.0, 133.4, 138.6, 144.8,
150.1, 153.4, 156.8, 162.1, 162.6, 167.8, 189.5. IR (nujol) ν
1727, 3304, 3467. Anal Calcd. for C29H30N2O8S: C, 61.47; H,
5.34; N, 4.94. Found: C, 61.06; H, 5.12; N, 5.09.
4.1.3. 12. 3-Amino-4-(4-butoxyphenyl)-2-(4-
methoxybenzoyl)-6-methyl-thieno[2,3-b]pyridine-5-
carboxylic acid ethyl ester (6n)
o
1
Yellow crystals; yield 89 %; mp 105 - 106 C. H NMR (CDCl3)
δ 0.99-1.04 (6H, m); 1.49-1.59 (2H, m), 1.79-1.86 (2H, m); 2.68
(3H, s); 3.88 (3H, s); 4.02-4.09 (4H, m); 6.67 (2H, br.s); 6.97
(2H, d, J = 8.6 Hz); 7.02 (2H, d, J = 8.6 Hz); 7.31 (2H, d, J = 8.6
Hz); 7.86 (2H, d, J = 8.6 Hz). 13C NMR (CDCl3) δ 13.8, 19.2,
23.3, 31.2, 55.4, 61.5, 67.9, 105.0, 113.6, 114.7, 119.7, 125.6,
127.1, 129.7, 130.0, 133.5, 145.0, 150.4, 156.6, 160.0, 162.1,
162.5, 167.8, 189.5. IR (nujol) ν 1608, 1726, 3296, 3474. Anal
Calcd. for C29H30N2O5S: C, 67.16; H, 5.83; N, 5.40. Found: C,
67.00; H, 5.80; N, 5.36.
4.1.3.17. 3-Amino-2-(2,4-dimethoxybenzoyl)-6-
methyl-4-(3,4,5-trimethoxyphenyl)-thieno[2,3-
b]pyridine-5-carboxylic acid 2-methoxyethyl ester
(6s)
Yellow crystals; yield 80 %; mp 129 - 131 oC. 1H NMR (CDCl3)
δ 2.67 (3H, s); 3.28 (3H, s); 3.27 (2H, t, J = 4.7 Hz); 3.82 (3H,
s); 3.87 (9H, s); 3.94 (3H, s); 4.16 (2H, t, J = 4.7 Hz); 6.52-6.55
(2H, m); 6.60 (2H, s); 6.72 (2H, br.s); 7.38 (1H, d, J = 8.2 Hz).
13C NMR (CDCl3) δ 23.3, 55.5, 55.6, 56.3, 58.7, 61.1, 64.3, 70.0,
99.0, 104.3, 105.6, 108.2, 119.3, 123.6, 126.1, 129.1, 130.0,
138.5, 144.9, 149.2, 153.4, 156.7, 162.5, 163.0, 167.8, 189.9. IR
(nujol) ν 1728, 3307, 3469. Anal Calcd. for C30H32N2O9S: C,
60.39; H, 5.41; N, 4.70. Found: C, 60.11; H, 5.32; N, 4.60.
4.1.3. 13. 3-Amino-4-(3,4,5-trimethoxyphenyl)-6-
methyl-2-(4-methoxybenzoyl)-thieno[2,3-b]pyridine-
5-carboxylic acid butyl ester (6o)
o
1
Yellow crystals; yield 93 %; mp 149 - 150 C. H NMR (CDCl3)
δ 0.85 (3H, t, J = 7.0 Hz), 1.20 (2H, se, J = 7.0 Hz), 1.38 (2H,
qui, J = 6.7 Hz); 2.69 (3H, s); 3.86 (6H, s); 3.89 (3H, s); 3.94
(3H, s); 4.02 (2H, t, J = 6.7 Hz); 6.61 (2H, s); 6.77 (2H, br.s);
6.97 (2H, d, J = 8.6 Hz); 7.87 (2H, d, J = 8.6 Hz). 13C NMR
(CDCl3) δ 13.5, 19.0, 23.3, 30.3, 55.4, 56.3, 61.1, 65.5, 105.1,
105.7, 113.6, 119.2, 126.7, 129.1, 130.0, 133.4, 138.6, 144.6,
150.1, 153.4, 156.7, 162.1, 162.5, 168.0, 189.5. IR (nujol) ν
1582, 1723, 3305, 3468. Anal Calcd. for C30H32N2O7S: C, 63.81;
H, 5.71; N, 4.96. Found: C, 63.62; H, 5.60; N, 5.02.
4.1.3.18. 3-Amino-6-methyl-2-(3,4,5-
trimethoxybenzoyl)-4-(3,4, 5-trimethoxyphenyl)-
thieno[2,3-b]pyridine-5-carboxylic acid 2-
methoxyethyl ester (6t)
Yellow crystals; yield 81 %; mp 172 - 173 oC. 1H NMR (CDCl3)
δ 2.71 (3H, s); 3.29 (3H, s); 3.55 (2H, t, J = 4.7 Hz); 3.87 (6H,
s); 3.92 (9H, s); 3.95 (3H, s); 4.18 (2H, t, J = 4.7 Hz); 6.62 (2H,
s); 6.82 (2H, br.s); 7.15 (2H, s). 13C NMR (CDCl3) δ 23.4, 56.2,
56.3, 58.7, 61.0, 61.1, 64.4, 70.0, 104.7, 105.5, 105.6, 106.0
119.1, 126.5, 128.9, 135.8, 138.7, 140.7, 145.0, 150.7, 153.0,
153.5, 157.1, 162.7, 167.7, 189.3. IR (nujol) ν 1582, 1728, 3464.
Anal Calcd. for C31H24N2O10S: C, 59.41; H, 5.47; N, 4.47.
Found: C, 59.37; H, 5.42; N, 4.39.
4.1.3. 14. 3-Amino-2-(4-methoxybenzoyl)-4-(4-
methoxyphenyl)-6-methyl-thieno[2, 3-b]pyridine-5-
carboxylic acid 2-methoxyethyl ester (6p)
Yellow crystals; yield 90 %; mp 125 - 126 oC. 1H NMR (CDCl3)
δ 2.69 (3H, s); 3.30 (3H, s); 3.36 (2H, t, J = 4.7 Hz); 3.88 (3H,
s); 3.90 (3H, s); 4.15 (2H, t, J = 4.7 Hz); 6.64 (2H, br.s); 6.97
(2H, d, J = 8.6 Hz); 7.04 (2H, d, J = 8.6 Hz); 7.34 (2H, d, J = 8.6
Hz); 7.86 (2H, d, J = 8.6 Hz). 13C NMR (CDCl3) δ 23.3, 55.4,
58.8, 64.2, 70.0, 105.0, 113.6, 114.2, 119.6, 125.8, 126.8, 129.8,
130.0, 133.4, 145.0, 150.3, 156.7, 160.4, 162.1, 162.6, 167.8,
189.5. IR (nujol) ν 1607, 1729, 3304, 3474. Anal Calcd. for
C27H26N2O6S: C, 64.02; H, 5.17; N, 5.53. Found: C, 63.50; H,
5.15; N, 5.35.
4.1.3.19. 3-Amino-2-(4-methoxybenzoyl)-6-methyl-
4-(3,4,5-trimethoxyphenyl)-thieno[2,3-b]pyridine-5-
carboxylic acid 2-propoxyethyl ester (6u)
o
1
Yellow crystals; yield 86 %; mp 79 - 80 C. H NMR (CDCl3) δ
0.89 (3H, t, J = 7.0 Hz); 1.51-1.59 (2H, m); 2.70 (3H, s); 3.33
(2H, t, J = 6.7 Hz); 3.41 (2H, t, J = 4.7 Hz); 3.86 (6H, s); 3.89
(3H, s); 3.95 (3H, s); 4.17 (2H, t, J = 4.7 Hz); 6.62 (2H, s,); 6.78
(2H, br.s); 6.98 (2H, d, J = 9 Hz); 7.87 (2H, d, J = 9.0 Hz). 13C
NMR (CDCl3) δ 10.4, 22.7, 23.3, 55.4, 56.3, 61.1, 64.6, 68.1,
73.0, 105.1, 105.6, 113.6, 119.2, 126.4, 129.0, 130.0, 133.4,
138.6, 144.8, 150.1, 153.4, 156.8, 162.1, 162.6, 167.8, 189.5. IR
(nujol) ν 1582, 1729, 3303, 3467. Anal Calcd. for C31H34N2O8S:
C, 62.61; H, 5.76; N, 4.71. Found: C, 61.99; H, 5.77; N, 4.60.
4.1.3. 15. 3-Amino-2-benzoyl-6-methyl-4-(3,4,5-
trimethoxyphenyl)-thieno[2,3-b]pyridine-5-
carboxylic acid 2-methoxyethyl ester (6q)
o
1
Yellow crystals; yield 90 %; mp 100 - 101 C. H NMR (CDCl3)
δ 2.69 (3H, s); 3.28 (3H, s); 3.35 (2H, t, J = 4.7 Hz); 3.87 (6H, s);
3.95 (3H, s); 4.17 (2H, t, J = 4.7 Hz); 6.62 (2H, s); 6.82 (2H,
br.s); 7.46-7.55(3H, m); 7.83 (2H, d, J = 8.2 Hz). 13C NMR
(CDCl3) δ 23.3, 56.3, 58.7, 61.1, 64.4, 70.0, 105.2, 105.6, 119.0,
126.4, 127.8, 128.4, 128.9, 130.0, 131.2, 138.6, 140.8, 145.0,
150.4, 153.5, 157.0, 162.8, 167.7, 190.6. IR (nujol) ν 1745, 3466.
4.1.3.20. 3-Amino-2-(4-methoxybenzoyl)-4,6-bis-
(3,4,5-trimethoxyphenyl)-thieno[2,3-b]pyridine-5-
carboxylic acid ethyl ester (6v)
Yellow crystals; yield 95%; mp 196 - 197 °C. 1H NMR (CDCl3)
δ 0.87 (3H, t, J = 7.4 Hz); 3.86 (9H, s); 3.97 (9H, s); 3.89 (3H,

s); 3.94 (2H, q, J = 7.4 Hz); 6.65 (2H, s); 6.96 (2H, s); 6.98 (2H,
d, J = 9.0 Hz); 7.87 (2H, d, J = 9.0 Hz). 13C NMR (CDCl3) δ
13.7, 55.4, 56.1, 56.3, 60.9, 61.1, 61.6, 105.8, 105.9, 113.6,
119.9, 126.3, 128.7, 130.1, 133.3, 134.2, 138.7, 139.2, 145.6,
149.8, 153.2, 153.4, 156.7, 162.2, 162.5, 167.7, 189.5. IR (nujol)
ν 1583, 1728, 3300, 3466. Anal Calcd. for C36H36N2O10S: C,
62.78; H, 5.27; N, 4.07. Found: C, 62.69; H, 5.21; N, 4.05.
7.19 (1H, s); 7.94 (2H, d, J = 8.6 Hz). 13C NMR (CDCl3) δ 22.9,
23.1, 55.5, 56.3, 58.7, 60.9, 64.4, 70.0, 106.3, 113.7, 114.6,
123.1, 127.4, 129.1, 129.2, 129.9, 130.2, 132.2, 138.1, 143.5,
153.0, 154.7, 159.8, 163.8, 167.7, 167.8, 188.0. IR (nujol) ν
1732, 3272, 3467. Anal Calcd. for C31H32N2O9S: C, 61.17; H
5.30; N 4.60. Found: C, 61.25, H 5.22, N 4.66.
4.1.6. Preparation of 5-cyano-4-(4-
difluoromethoxyphenyl)-6-[2-(4-methoxyphenyl)-2-
oxo-ethylsulfanyl]-2-methyl-1,4-dihydropyridine-3-
carboxylic acid ethyl ester (9)
4.1.3. 21. 3,6-Diamino-2-(4-methoxybenzoyl)-4-
(3,4,5-trimethoxyphenyl)-thieno[2,3-b]pyridine-5-
carbonitrile (6w)
4-Difluoromethoxybenzaldehyde (2.5 mmol) and ethyl 3-
oxobutanoate (2.5 mmol) were dissolved in 10 ml of ethanol and
piperidine was added (2.0 mmol). Mixture was stirred at room
temperature for 30 min. Then 2-cyanothioacetamide (2.5 mmol)
and piperidine (0.8 mmol) were added and reaction mixture was
shortly refluxed for 30 seconds and stirred at room temperature
for 3 hours. 2-Bromo-1-(4-methoxyphenyl)ethanone (2.5 mmol)
was added and reaction mixture was shortly refluxed and stirred
at room temperature for 30 min. The formed precipitate was
filtered and purified by washing with 3 ml of cold ethanol and
water during the filtration. White crystals; yield 85 %; mp 148 -
149 oC. 1H NMR (CDCl3) δ 1.13 (3H, t, J = 7.0 Hz); 2.41 (3H, s);
3.89 (3H, s); 4.03 (2H, q, J = 7.0 Hz); 4.66 (1H, s); 6.44 (1H, t, J
= 73.2 Hz); 6.96 (2H, d, J = 9.0 Hz); 7.22 (2H, d, J = 9.0 Hz);
7.22 (2H, d, J = 8.6 Hz); 7.93 (2H, d, J = 8.6 Hz); 8.61 (1H, s).
13C NMR (CDCl3) δ 14.1, 19.5, 37.9, 42.0, 55.7, 60.1, 90.5,
101.6, 114.4, 118.7, 119.5, 127.5, 128.8, 131.5, 142.0, 144.9,
150.3, 165.1, 166.7, 195.8. IR (nujol) ν 1668, 2199, 3282. Anal
Calcd. for C26H24N2O5S: C, 60.69, H, 4.70, N, 5.44. Found: C,
60.59, H, 4.65, N, 5.77.
Yellow crystals; yield 41 %; mp 241 - 242 oC. 1H NMR (DMSO-
d6) δ 3.72 (3H, s); 3.78 (6H, s); 3.84 (3H, s); 6.89 (2H, s); 7.06
(2H, d, J = 9.0 Hz); 7.70 (2H, br.s); 7.89 (2H, br.s); 8.02 (2H, d,
J = 9.0 Hz). 13C NMR (CDCl3) δ 55.3, 56.2, 60.1, 90.8, 99.2,
105.4, 112.5, 113.6, 115.5, 128.3, 129.1, 133.3, 138.3, 151.0,
153.3, 153.6, 159.1, 161.2, 165.8, 186.6. IR (nujol) ν 1709, 2237,
3290, 3453. Anal Calcd. for C25H22N4O5S: C, 61.21; H, 4.52; N,
11.42. Found: C, 61.05; H, 4.55; N, 11.21.
4.1.3. 22. 3-Amino-4,6-dimethyl-2-(2,4-
dimetoxybenzoyl)-thieno[2,3-b]pyridine (6x)
o
1
Yellow crystals; yield 70 %; mp 184 - 186 C. H NMR (CDCl3)
δ 2.51 (3H, s); 2.70 (3H, s); 3.75 (3H, s); 3.80 (3H, s); 6.46-6.48
(2H, m); 6.89 (1H, s); 7.14 (2H, br.s); 7.33 (1H, d, J = 9.0 Hz).
13C NMR (CDCl3) δ 20.5, 55.5, 55.6, 98.9, 104.4, 108.5, 121.9,
123.5, 129.9, 150.1, 157.9, 162.5, 190.1. IR (nujol) ν 1597, 3297,
3467. Anal Calcd. for C18H18N2O3S: C, 63.14; H, 5.30; N, 8.18.
Found: C, 62.87; H, 5.17; N, 8.09.
4.1.4. Preparation of 3-amino-6-methyl-4-(3,4,5-
trimethoxyphenyl)-2-(4-methoxybenzoyl)-
thieno[2,3-b]pyridine (6y)
4.1.7. Preparation of 5-cyano-4-(4-
difluoromethoxyphenyl)-6-[2-(4-methoxyphenyl)-2-
oxo-ethylsulfanyl]-2-methyl-nicotinic acid ethyl
ester (10)
A
mixture of 4-(3,4,5-trimethoxyphenyl)-3-buten-2-one (1
mmol), 2-cyanothioacetamide 2 (1 mmol) and piperidine (1.1
mmol) in 10 ml of ethanol was stirred at room temperature for 12
h. Then 2-bromo-1-(4-methoxyphenyl)ethanone (1 mmol) was
added and reaction mixture was shortly heated until reflux and
stirred for 3 h at room temperature. Then 3M sodium hydroxide
water solution (1 mmol) was added. Reaction mixture was
refluxed for 1 min and stirred at room temperature for 30 min.
The precipitates was filtered and purified by washing with 1 ml
of cold ethanol and water during the filtration. Yellow crystals;
yield 89 %; mp 234 - 235 C. H NMR (CDCl3) δ 2.67 (3H, s);
3.87 (3H, s); 3.88 (6H, s); 3.93 (3H, s); 6.63 (2H, s); 6.90 (2H,
br.s); 6.96 (2H, d, J = 9.0 Hz); 6.98 (1H, s); 7.87 (2H, d, J = 9.0
Hz). 13C NMR (CDCl3) δ 24.7, 55.4, 56.3, 61.1, 104.6, 105.5,
113.5, 119.3, 121.2, 130.1, 132.0, 133.6, 138.5, 147.8, 150.1,
153.5, 160.1, 162.0, 189.5. IR (nujol) ν 3303, 3467. Anal Calcd.
for C25H24N2O5S: C, 64.64, H, 5.21, N, 6.03. Found: C, 64.52, H,
5.14, N, 5.93.
5-Cyano-4-(4-difluoromethoxyphenyl)-6-[2-(4-methoxyphenyl)-
2-oxo-ethylsulfanyl]-2-methyl-1,4-dihydropyridine-3-carboxylic
acid ethyl ester (1 mmol) was dissolved in 8 ml glacial acetic
acid and manganese (III) acetate dihydrate (2 mmol) was added.
Reaction mixture was refluxed for 2 hours, then poured into ice
water, neutralized, extracted with DCM, and dried over MgSO4.
Resulting oil was was recrystallized from ethanol. White crystals;
1
yield 30 %; mp 126 - 127 oC. H NMR (CDCl3) δ 0.90 (3H, t, J
o
1
= 7.0 Hz); 2.36 (3H, s); 3.89 (3H, s); 3.98 (2H, q, J = 7.0 Hz);
4.66 (2H, s); 6.55 (1H, t, J = 73.2 Hz); 6.97 (2H, d, J = 9.0 Hz);
7.18 (2H, J = 8.6 Hz); 7.32 (2H, d, J = 9.0 Hz); 8.05 (2H, d, J =
9.0 Hz). 13C NMR (CDCl3) δ 13.5, 23.2, 37.2, 55.5, 61.8, 104.5,
112.9, 113.9, 114.3, 115.5, 118.1, 119.5, 125.2, 129.2, 129.9,
130.7, 131.6, 151.2, 152.1, 158.9, 162.3, 163.9, 166.5, 191.3. IR
(nujol) ν 1684, 1724, 2224. Anal Calcd. for C26H22F2N2O5S: C,
60.93; H 4.33; N 5.47. Found: C, 60.25, H 4.22, N 5.66.
4.1.5. Preparation of 3-acetylamino-2-(4-
methoxybenzoyl)-6-methyl-4-(3,4,5-
trimethoxyphenyl)-thieno[2,3-b]pyridine-5-
carboxylic acid 2-methoxyethylester (7)
4.1.8. Preparation of 3-amino-4-(4-
difluoromethoxyphenyl)-2-(4-methoxybenzoyl)-6-
methyl-thieno[2, 3-b]pyridine-5-carboxylic acid
ethyl ester (6h)
3-Amino-2-(4-methoxybenzoyl)-6-methyl-4-(3,4,5-
5-Cyano-4-(4-difluoromethoxyphenyl)-6-[2-(4-methoxyphenyl)-
2-oxo-ethylsulfanyl]-2-methyl-nicotinic acid ethyl ester (0.2
mmol) was dissolved in 3 ml of ethanol and 3M sodium
hydroxide water solution (0.2 mmol) was added. Reaction
mixture was shortly refluxed and stirred at room temperature for
1 hour. The precipitate was filtered and purified by washing with
1 ml of cold methanol and water during the filtration. Yellow
trimethoxyphenyl)-thieno[2,3-b]pyridine-5-carboxylic acid 2-
methoxyethyl ester (0.3 mmol) and sodium hydroxide (0.6 mmol)
in 2 ml acethylchloride was refluxed for 2 h under argon.
Reaction mixture was poured into ice water, neutralized,
extracted with DCM, and dried over MgSO4. Resulting oil was
recrystallized from ethanol. Yellow crystals; yield 71%; mp 183 -
1
184 oC. H NMR (CDCl3) δ 1.51 (3H, s); 2.73 (3H, s); 3.28 (3H,
o
1
crystals; yield 60 %; mp 140 - 141 C. H NMR (CDCl3) δ 0.96
s); 3.36 (2H, t, J = 4.7 Hz); 3.87 (6H, s); 3.88 (3H, s); 3.90 (3H,
s); 4.20 (2H, t, J = 4.7 Hz); 6.60 (2H, s); 6.94 (2H, d, J = 8.6 Hz);
(3H, t, J = 7.0 Hz); 4.00 (2H, q, J = 7.0 Hz); 2.68 (3H, s); 3.87

(3H, s); 6.61 (1H, t, J = 73.2 Hz); 6.94 (2H, d, J = 9.0 Hz); 6.82
(2H, br.s); 7.27 (2H, d, J = 8.6 Hz); 7.41 (2H, d, J = 8.6 Hz); 7.85
(2H, d, J = 9.0 Hz). 13C NMR (CDCl3) δ 13.7, 23.4, 55.4, 61.6,
113.6, 115.3, 119.2, 119.8, 130.1, 131.1, 133.3, 143.7, 149.8,
156.8, 162.2, 162.5, 167.5, 189.6. IR (nujol) ν 1610, 1724, 3296,
3465. Anal Calcd. for C26H22F2N2O5S: C, 60.93; H 4.33; N 5.47.
Found: C, 60.86, H 4.22, N 5.43.
4.2.1. Cell cultures
NIH3T3 (normal mouse fibroblasts), MES-SA (human uterine
sarcoma), MES-SA/Dx5 (doxorubicin resistant, 300 ng/ml),
MES-SA/MX2 (mitoxantrone resistant), H69 (human lung
carcinoma), H69/AR (doxorubicin resistant) and A7R5 (rat aorta
smooth muscle) cells were obtained from the ATCC cell
collection.
The potency of P-glycoprotein-mediated MDR modulator in vitro
was evaluated in the drug sensitive human sarcoma MES-SA
cells and doxorubicin (DOX) resistant cells by accumulation of
fluorescent substrate - rhodamine 123. Effects on MRP1-
mediated drug efflux were observed in the DOX resistant human
lung carcinoma H69AR cells using calcein AM assay. BCRP1-
mediated drug efflux was also evaluated using fluorescent
substrate Hoechst 33342 in the mitoxantrone resistant human
sarcoma MES-SA/MX2 cells. The half maximal effective
concentrations (EC50) were calculated from dose response curve
as the most effective method to compare MDR-modulating
activities of compounds.39,40
4.1.9. Preparation of 5-cyano-2-methyl-6-oxo-4-
(3,4,5-trimethoxyphenyl)-1,6-dihydropyridine-3-
carboxylic acid ethyl ester (13a)
A
mixture of 3-cyano-4-(3,4,5-trimethoxyphenyl)acrylamide
(2.62 g, 10 mmol), aminocrotonic acid ethyl ester (1.29g, 10
mmol), 10 ml of glacial acid and 10 ml of absolute ethanol was
refluxed for 4h. Then reaction mixture was evaporated under
reduced pressure and resulting slurry was washed with water.
Then reaction mixture was diluted with 10 ml of ethanol, and 1
ml of concentrated nitric acid was added. The resulting mixture
was refluxed for 8 h, then cooled to the room temperature, diluted
with water and neutralized with 2M NaHCO3 solution in water.
The resulting crude product was recrystallized from ethanol.
White crystals; yield 32 %; mp 223 - 224 oC. 1H NMR (CDCl3) δ
0.85 (3H, t, J = 7.0 Hz); 2.54 (3H, s); 3.81 (6H, s); 3.84 (3H, s);
3.93 (2H, q, J = 7.0 Hz); 6.54 (2H, s); 13.53 (1H, s). IR (nujol) ν
1659, 1717, 2226, 3012, 3147. Anal Calcd. for C19H20N2O6: C,
61.28; H 5.41; N 7.52. Found: C, 60.73, H 5.27, N 7.21.
4.2.2. Measurement of P-glycoprotein activity
About 50000 cells of the human uterine sarcoma cell line MES-
SA and MES-SA/Dx5 were seeded into the 96-well plates and
incubated for 24 h. The MDR modulators were incubated with
the cells for 15 min. Then rhodamine-123 (2.6 µM) was added
o
and the cells were incubated for 1h, 37 C, 5% CO2. The cells
were washed twice with phosphate buffered saline (PBS) and
fluorescence values were measured with excitation at 487 nm and
emission at 557 nm by using the fluorescence reader Tecan
infinite M1000. Verapamil, the known P-glycoprotein inhibitor
was used as a positive control.41 The EC50 values was calculated
using the program Graph Pad Prism® 4.0. Verapamil was
dissolved in water, while MK-571 and tested compounds in
DMSO (0.5% in final concentration).
4.1.10. General procedure for the preparation of
furo[2,3-b]pyridines
To a solution of corresponding 6-oxo-1,6-dihydropyridine (1
equiv) in N,N-dimethylformamide, potassium carbonate (4 equiv)
was added and reaction mixture was stirred for 5 min at room
temperature. Then corresponding bromoacetofenone (1 equiv)
was added and reaction mixture was stirred at 80°C for 4h. Then
reaction mixture was cooled to room temperature, diluted with
water and neutralized with 2M HCl water solution. The
precipitate was filtered and recrystallized from ethanol.
4.2.3. Measurement of MRP1 activity
About 60000 cells of the human lung carcinoma cell line H69
and H69AR were seeded into the 96-well plates and incubated
for 24 h. The compounds were incubated with the cells for 15
min. Then Calcein AM (0.25 µM) was added and the cells were
incubated for 30 min in a humidified atmosphere 5% CO2 at 37
oC. The cells were washed twice with phosphate buffered saline
(PBS) and fluorescence values were measured with excitation at
494 nm and emission at 517 nm by using the fluorescence reader
Tecan infinite M1000. MK-571 was used as the known inhibitor
of MRP1.42 The EC50 values was calculated using the program
Graph Pad Prism® 4.0.
4.1.10.1. 3-Amino-2-(4-methoxybenzoyl)-6-methyl-
4-(3, 4,5-trimethoxyphenyl)-furo[2,3-b]pyridine-5-
carboxylic acid ethyl ester (14a)
White crystals; yield 19 %; mp 174 - 176 oC. 1H NMR (CDCl3) δ
0.94 (3H, t, J = 7.0 Hz); 2.40 (3H, s); 3.86 (6H, s); 3.89 (3H, s);
3.93 (3H, s); 4.00 (2H, q, J = 7.0 Hz); 5.72 (2H, s); 6.61 (2H, s);
6.98 (2H, d, J = 9.0 Hz); 7.92 (2H, d, J = 9.0 Hz). 13C NMR
(CDCl3) δ 13.8, 23.4, 55.4, 56.3, 61.1, 61.7, 105.4, 108.2, 113.6,
125.7, 129.3, 129.9, 131.6, 133.4, 138.7, 140.6, 145.1, 153.5,
156.9, 159.4, 162.9, 168.0, 181.9. IR (nujol) ν 1723, 3337, 3478.
Anal Calcd. for C28H28N2O8: C, 64.61; H 5.42; N 5.38. Found: C,
64.23, H 5.17, N 5.18.
4.2.4. Measurement of BCRP1 activity
About 50000 cells of the MES-SA and MES-SA/MX2 were
seeded into the 96-well plates and incubated for 24 h. The MDR
modulators were incubated with the cells for 15 min. Hoechst
33342 (5 µg/ml) was added and the cells were incubated for 90
min, 37 oC, 5% CO2. The cells were washed twice with
phosphate buffered saline (PBS) and fluorescence values were
measured with excitation at 360 nm and emission at 450 nm.
Verapamil was used as a positive control.43 The EC50 values was
calculated using the program Graph Pad Prism® 4.0.
4.1.10.2. 3-Amino-4-(4-methoxyphenyl)-6-methyl-2-
(3,4,5-trimethoxybenzoyl)-furo[2,3-b]pyridine-5-
carboxylic acid ethyl ester (14b)
White crystals; yield 38 %; mp 188 - 190 oC. 1H NMR (CDCl3) δ
0.90 (3H, t, J = 7.0 Hz); 2.37 (3H, s); 3.78 (3H, s); 3.88 (9H, s);
3.95 (2H, q, J = 7.0 Hz); (2H, b.s); 6.91 (2H, d, J = 8.6 Hz); 7.18
(2H, s); 7.27 (2H, d, J = 8.6 Hz). 13C NMR (CDCl3) δ 13.6, 23.2,
55.3, 56.4, 61.0, 61.6, 68.6, 94.1, 105.4, 114.1, 114.4, 124.2,
127.0, 129.5, 129.6, 143.2, 153.3, 155.0, 158.5, 160.8, 162.5,
167.1, 191.6. IR (nujol) ν 1617, 1719, 3481. Anal Calcd. for
C28H28N2O8: C, 64.61; H 5.42; N 5.38. Found: C, 64.33, H 5.27,
N 5.24.
4.2.5. Intracellular Ca2 + measurements
Changes in intracellular [Ca2+]i concentration were studied using
Fluo-4 NW Calcium Assay Kit (“Invitrogen”, Sweden)
accordingly to manufacturer’s instructions. The A7R5 (rat aorta
smooth muscle) cells were grown at 37 oC in a humidified
atmosphere with 5% CO2/95% air in DMEM medium containing
2 mM glutamine and supplemented with 10% FBS. The cells
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This work was financially supported by European Regional
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Graphical Abstract
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Thieno[2,3-b]pyridines – a new class of
multidrug resistance (MDR) modulators
Leave this area blank for abstract info.
Aivars Krauze, Signe Grinberga, Laura Krasnova, Ilze Adlere, Elina Sokolova, Ilona Domracheva, Irina
Shestakova, Zigmars Andzans, Gunars Duburs
Latvian Institute of Organic Synthesis, 21 Aizkraukles St., Riga, LV-1006, Latvia
OMe
MeO
OMe
NH2
O
O
P-gp inhibitor EC50 = 0.3 0.2 µM
MRP1 inhibitor EC50 = 1.1 0.1 µM
BCRP1 inhibitor EC50 = 0.2 0.05 µM
O
O
S
N
OMe