Discovery and optimization of thienopyridine derivatives as novel urea transporter inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.03.060

Urea transporters (UTs) play an important role in the urine concentrating mechanism and are recognized as novel targets for developing small molecule inhibitors with salt-sparing diuretic activity. Thienoquinoline derivatives, a class of novel UT-B inhibitors identified by our group, play a significant diuresis in animal model. However, the poor solubility and low bioavailability limited its further development. To overcome these shortcomings, the structure modification of thienoquinoline was carried out in this study, which led to the discovery of novel thienopyridine derivatives as specific urea transporter inhibitors. Further optimization obtained the promising preclinical candidate 8n with not only excellent inhibition effect on urea transporters and diuretic activity on rat model, but also suitable water solubility and Log P value.

Full text of DOI:10.1016/j.ejmech.2019.03.060

Accepted Manuscript
Discovery and optimization of thienopyridine derivatives as novel urea transporter
inhibitors
Yan Zhao, Min Li, Bowen Li, Shun Zhang, Aoze Su, Yongning Xing, Zemei Ge,
Runtao Li, Baoxue Yang
PII:
S0223-5234(19)30294-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.03.060
EJMECH 11230
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 January 2019
Revised Date: 26 March 2019
Accepted Date: 28 March 2019
Please cite this article as: Y. Zhao, M. Li, B. Li, S. Zhang, A. Su, Y. Xing, Z. Ge, R. Li, B. Yang,
Discovery and optimization of thienopyridine derivatives as novel urea transporter inhibitors, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.03.060.
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ACCEPTED MANUSCRIPT
Graphical abstract

Zhao, et al. Thienopyridine urea transporter inhibitors
ACCEPTED MANUSCRIPT
Submitted to European Journal of Medicinal Chemistry
Discovery and optimization of thienopyridine derivatives as novel urea transporter inhibitors
Yan Zhao1, 3, Min Li2, Bowen Li1, Shun Zhang2, Aoze Su1, Yongning Xing1, Zemei Ge1, Runtao Li1*,
Baoxue Yang2*
1State Key Laboratory of Natural and Biomimetic Drugs, School of pharmaceutical Sciences, Peking
University, 100191, P.R. China.
2Department of Pharmacology, School of Basic Medical Sciences, Peking University Health Science
Center, 100191, P.R. China.
3College of Pharmacy, Inner Mongolia Medical University, 010110, P.R. China.
Running title: Thienopyridine urea transporter inhibitors
Corresponding author and reprint requests:
Runtao Li, Ph.D.
E-mail：lirt@bjmu.edu.cn
Baoxue Yang, M.D., Ph.D.
E-mail：baoxue@bjmu.edu.cn
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Zhao, et al. Thienopyridine urea transporter inhibitors
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Abstract：
Urea transporters (UTs) play an important role in the urine concentrating mechanism and are
recognized as novel targets for developing small molecule inhibitors with salt-sparing diuretic
activity. Thienoquinoline derivatives, a class of novel UT-B inhibitors identified by our group, play a
significant diuresis in animal model. However, the poor solubility and low bioavailability limited its
further development. To overcome these shortcomings, the structure modification of thienoquinoline
was carried out in this study, which led to the discovery of novel thienopyridine derivatives as
specific urea transporter inhibitors. Further optimization obtained the promising preclinical candidate
8n with not only excellent inhibition effect on urea transporters and diuretic activity on rat model,
but also suitable water solubility and Log P value.
Key words:
Urea transporter inhibitor; Thienoquinoline; Thienopyridine; Diuretics; Structure-activity
relationship
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Zhao, et al. Thienopyridine urea transporter inhibitors
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1. Introduction
Diuretics are mainly used to treat diseases such as edema, hypertension and heart failure and are
often used for long-term therapy[1]. At present, the commonly used diuretics in clinical include loop
diuretics (e.g. furosemide), thiazides (e.g. hydrochlorothiazide), aldosterone antagonists (e.g.
spironolactone) and osmotic dehydrating agents (e.g. mannitol)[2]. All of these drugs induce a
diuresis by mechanism in which water excretion following salt excretion, which may result in several
adverse effects, involving hypokalemia, hyponatremia, hyperuricemia, hyperlipidemia and a decrease
in glucose tolerance, while long-term using[3-7]. Moreover, electrolyte imbalance may increase risks
of arrhythmia and sudden death[8,9]. Therefore, it would be desirable to find novel diuretics that do
not cause electrolyte disturbances.
Except for electrolytes, urea is also a major solute in hyperosmolar renal medulla and plays a
critical role in urinary concentrating mechanism and in maintenance of water balance[10]. The
intrarenal urea recycling is facilitated by transmembrane urea transporters (UTs) expressed in renal
tubule epithelial cells (UT-A isoforms) and renal vasa recta microvessels (UT-B isoform)[11-13].
Study on knock-out mice lacking of UT-B[14] or various UT-A isoforms[15-17] showed that UT
functional deletion led to urea-selective urinary concentrating defects associated with urinary
hypoosmolality. Meanwhile, the glomerular filtration rate and clearance rate of the principal solutes
(Na+, K+, Cl-), except for urine, were not changed[18,19]. These evidences suggested that UT
inhibitors would be developed as novel diuretics with a mechanism different from currently used
salt-excreting diuretics, therefore would not disturb the balance of electrolyte metabolism in vivo as
they did and would be more suitable for long-term therapy [20].
Several classes of UT inhibitors, including phenyl-sulfoxyoxozoles(a, Fig. 1)[21],
benzenesulfon-anilides (b, Fig. 1)[21], phthalazinamines (c, Fig. 1)[21,22], aminobenzimidazoles (d,
Fig. 1)[21], thiazolothienopyrimidines (e, Fig. 1)[23-26], hydroxyquinolines (f, Fig. 1)[25],
aminothiazolones (g, Fig. 1)[25], thioureas (h, Fig. 1)[27,28], fluorenones (i, Fig. 1)[29] and so on,
were identified by Verkman’s group over the past decade via high-throughput screening of
small-molecule libraries or preliminary structural modification of the hits. Although some of the
molecules mentioned above exhibited potent inhibitory effect to UTs with IC50 values at micromolar
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Zhao, et al. Thienopyridine urea transporter inhibitors
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or submicromolar level in vitro, they were shown to have very low or no diuretic effect in
experimental animals, which obstructed their further development.
Fig. 1. Chemical structures of several known UT inhibitors.
In our previous work, we identified a thienoquinoline UT inhibitor named PU-14 (j, Fig. 1) [30]
by screening a commercially available collection of drug-like compounds with erythrocyte lysis
model. PU-14, containing a new thienoquinoline scaffold different from those reported by Verkman’s
group, exhibited an excellent inhibitory effect on both rat and human UT-B in vitro with IC50 value
of 3.51 and 1.72 µmol/L, respectively. Based on these results, we conducted an extensive screen of
thienoquinolin analogs and got a more superior small-molecular UT inhibitor PU-48 (k, Fig. 1) [31],
which was over 10 times more potent than PU-14. PU-48 significantly increased urine output and
decreased urine osmolality in an in vivo rat model without affecting electrolyte metabolism and
notable toxicity. Moreover, the poor solubility and low bioavailability limited further development of
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Zhao, et al. Thienopyridine urea transporter inhibitors
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PU-48. To overcome these shortcomings, the systematical structure modification of PU-48 was
firstly carried out in the present study, which led to the discovery of novel thienopyridine derivatives
as UT-B inhibitor. Further optimization obtained the promising preclinical candidate 8n with not
only excellent inhibition effect on UT-B and diuretic activity on rat model, but also suitable water
solubility and Log P value (Fig. 2).
Fig.2. Overview of present study.
2. Results and discussions
Optimization of PU48
In our previous study, the modification of amino group in PU14 had already been investigated.
All the changes including removing amino group, replacing it by hydroxyl group, alkylating or
acetylating it led to a dramatically decrease or loss of the urea transporter UT-B inhibition activity.
So in present study, the further optimization of PU-48 was mainly focused on two aspects, ester
group at 2-position of the thiophene ring (R1) and substituents at benzene ring (R2) (Fig. 3).
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Zhao, et al. Thienopyridine urea transporter inhibitors
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Fig. 3. The structural modification strategy of lead PU-48.
For the modification of R1, ethyl ester group (6a) was firstly selected to replace methyl ester of
PU-48 to investigate the effect of chain length on the UT-B inhibition activity. Secondly, as ester
group usually easily hydrolyze in vivo, it was modified with its bioisosteres such as amide groups
(6b-6e), sulfonamides (6f) and acetyl groups (6g, 6h) to improve the stability in vivo and in vitro.
Finally, since carboxyl acid was thought to be the metabolite of ester in vivo, the possible metabolites
6i was designed to exam whether this metabolite could inhibit UT-B or not.
For the optimization of R2, methyloxy group of PU-48 was removed (7a), replaced by
electro-donating (7b) or electro-withdrawing (7c, 7d) groups to test the influence of different
substituents on the UT-B inhibition activity. Furthermore, multi-substituted derivative (7e) was also
designed to examine the steric effect on activity.
As outlined in Scheme 1, the preparation of targeted compounds 6 and 7 referred to the synthesis
of PU-48 in our previous work [32]. A variety of substituted acetanilide (1a-1f) was treated with
DMF and phosphorus oxychloride at 75 ℃ to obtain 2a-2f, which was condensed with
hydroxylamine to afford oxime and subsquently treated with thionyl chloride to yield the
corresponding 3-cyano-quinolone derivatives 3a-3f. Without further purification, sulfhydrylation of
3a-3f with thiourea afforded the 2-mercapto-3-cyanoquinoline derivatives 4a-4f. Then,
2-mercapto-3-cyano-6-methoxyquinoline (4f) was reacted with corresponding materials containing
chloroacetyl or chloromethanesulfonyl (5a-5i) under basic conditions in methanol to generate the
target compounds 6a-6i. Similarly, 4a-4e was reacted with methyl chloroacetate (5j) under the same
conditions to afford 7a-7e.
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Compd.
R1
R2
Compd.
R1
R2
6-OCH3
6-OCH3
6a
6h
6-OCH3
6-OCH3
H
6b
6i
6-OCH3
6-OCH3
6c
7a
6d
6-CH3
7b
6-OCH3
6-OCH3
6-Br
6e
6f
7c
7d
7e
6-F
6-CH3,8-CH3
6-OCH3
6g
Scheme 1. Synthesis of compounds 6a-6j and 7a-7e. Reagents and conditions: (a) DMF, POCl3,
75 ℃; (b) NH2OH·HCl, H2O, THF, r.t.; SOCl2, CHCl3, reflux; (c) NH2CSNH2, EtOH, reflux; (d)
Et3N or MeONa, MeOH, reflux.
The UT-B inhibition activity of target compounds 6a-6i and 7a-7e was evaluated with an
erythrocyte lysis assay established with rat erythrocyte as previously reported [31]. The lead
compound PU-48 was used as the positive control. The results are shown in Table 1.
Unfortunately, almost all the modification of 2-ester group in PU48 resulted in the loss of
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Zhao, et al. Thienopyridine urea transporter inhibitors
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activity, even a small change in methyl ester to ethyl ester (6a). Replacement of R1 with acetyl group
(6g) caused a 5-fold decrease in efficiency compared to PU-48. These results implicated that
methoxycarbonyl at this position was necessary for the inhibition effect on UT-B.
Table 1. Inhibition potency of compounds 6a-6i and 7a-7e to rat UT-B.
Compd.
PU48
R1
R2
IC50 (µM) Compd. R1
R2
IC50 (µM)
COOCH3
6-OCH3 0.22
6-OCH3
>10
6h
COOC2H5
CONH2
6-OCH3 >10
6-OCH3 >10
6-OCH3 >10
COOH
6-OCH3
H
>10
0.22
0.23
6a
6b
6c
6i
COOCH3
COOCH3
7a
7b
CONHCH3
6-CH3
6-OCH3 >10
6-OCH3 >10
COOCH3
6-Br
6-F
0.27
0.16
6d
6e
7c
COOCH3
COOCH3
7d
7e
SO2N(CH3)2
COCH3
6-OCH3 >10
6-OCH3 1.63
6-CH3,8-CH3 0.61
6f
6g
Results of optimization on R2 indicated that no-substituted (7a) and methyl-substituted (7b) at
the 6-position maintained the activity, bromine-substituted (7c) caused a slight decrease in activity,
and fluorine-substituted (7d) resulted in a slight improvement in activity, comparing with PU-48.
These results suggested that the electronic properties of substituents attached to the phenyl ring
exerted little influence on the inhibition activity. However, the 6-,8-disubstitued compound 7e was
lower the activity by almost 3-fold compared with the 6-monosubstitued 7b, manifesting that the
large steric hindrance at 8-position of quinoline ring is unfavorable for the activity.
Discovery of thienopyridines UT inhibitors.
Based on the above SAR results that altering substituents on the benzene ring led to little
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Zhao, et al. Thienopyridine urea transporter inhibitors
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changes in potency, we surmised that removing benzene ring could maintain the UT-B inhibition
activity. To prove this conjecture, thienopyridine analogue 8a was designed by replacing the benzene
ring of PU48 with methyl group. It is really delightful that 8a showed a favorable IC50 value of 1.30
µM, remaining at micromolar level (Table 2). This encouraging finding illustrated our hypothesis and
inspired us to turn our focus to thienopyridines. For one point of view, it was a new and simplified
structural skeleton used as UT inhibitors. On the other hand, the strong intermolecular π-π action due
to the tricyclic aromatic conjugation system led to poor solubility of PU48. Removing one aromatic
benzene ring could reduce such π-π action thus improve solubility to some extent.
SAR study of thienopyridines UT inhibitors.
Based on the results of SAR study on PU48, the structural modification of compound 8a was
mainly focused on the substituent at pyridine ring. The methyl group was modified with various
groups at different position of pyridine ring while the thiophene ring remained intact. The detailed
synthetic routes for thienopyridine analogs are delineated in Scheme 2. Various of substituted
2-oxopyridine-3-carbonitrile 9, which was prepared by literature procedures [33], was treated with
phosphorus oxychloride in hot 1,4-dioxane to afford the corresponding 10[34]. The synthesis of
8a-8c, 8i-8m, 8s and 8u were performed by treating substituted 2-chloronicotinonitriles (10) with
methyl thioglycolate under basic conditions [35] while 8d and 8e were prepared from substituted
2-thioxopyridine-3-carbonitrile (11)[32] reacted with methyl chloroacetate. However, for some
compounds such as 8o-8r, the substituents at pyridine ring derived from amino group. In such cases,
functional group transformation should be implemented before thiophene ring closing in order to
avoid additional side reactions. Therefore in regard to preparation of 8o-8r, 11d, obtained by
reduction of 11c, was converted to immediate 12b first via reacting with methyl chloroacetate under
mild conditions. 12b then underwent conversion of functional groups to afford 12c-12f followed by
ring-closing reaction using sodium methoxide as base. For other compounds such as 8f-8h and 8n,
the functional group transformation was conducted after the ring closing of thiophene. Under the
catalysis of palladium-tetrakis, 8s coupled with triethylsilane or phenylboronic acid to afford 8f and
8g, respectively. 8h was acquired by treating 8ha with sodium methoxide with copper powder as
catalyst in DMF and methanol at 90℃. Finally, reduction of the nitro group of 8t using Fe/NH4Cl to
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Zhao, et al. Thienopyridine urea transporter inhibitors
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obtain the corresponding amino analogue 8n.
Compd. R3
Compd. R3
6-CH3
5-CONHCH3, 6-CH3
8a
8b
8c
8d
8e
8f
8l
6-Ph
5-(morpholine-N-formyl), 6-CH3
5-NH2, 6-CH3
8m
8n
8o
8p
8q
8r
8s
6-(morpholine-N-formyl)
4-CH3, 6-CH3
4-CF3, 6-CH3
5-N(CH3)2, 6-CH3
5-NHCOCH3, 6-CH3
5-NHSO2CH3, 6-CH3
5-F, 6-CH3
5-CH2CH3, 6-CH3
5-Ph, 6-CH3
8g
8h
8i
5-OCH3,6-CH3
5-COCH3, 6-CH3
5-CO2C2H5, 6-CH3
5-CONHC2H5, 6-CH3
5-Br, 6-CH3
5-NO2, 6-CH3
8t
5-COOC2H5
8j
8u
8k
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NH2
R4
R3
CN
O
R4
R3
R4
CN
Cl
O
O
a
d
S
N
N
R3
N
H
10a
8a R3 = CH3, R4 = H
R3 = Me, R4 = H
9a
R3 = Me, R4 = H
8b R3 = Ph, R4 = H
10b R3 = Ph, R4 = H
9b R3 = Ph, R4 = H
b
c
O
O
9c
9d
9e
9f
R3 = Me, R4 = COMe
R3 = Me, R4 = COOEt
R3 = H, R4 = COOEt
R3 = Me, R4 = Br
10c R3 =
, R4 = H
8c
R3 =
, R4 = H
O
N
O
N
8f
8g
8ha
10d
10e
R3 = Me, R4 = Et
R3 = Me, R4 = Ph
R3 = Me, R4 = I
R3 = Me, R4 = COMe
R3 = Me, R4 = COOEt
10f R3 = Me, R4 = CONHEt
g
9h R3 = Me, R4 = I
9g
10g
10h
R3 = Me, R4 = CONHMe
8h R3 = Me, R4 = OMe
R3 = Me, R4 = NO2
O
8i
8j
8k
R3 = Me, R4 = COMe
R3 = Me, R4 = COOEt
R3 = Me, R4 = CONHEt
O
R3 = Me, R4 =
N
e
f
10i
10j
10k
10l
R3 = H, R4 = COOEt
R3 = Me, R4 = Br
R3 = Me, R4 = I
8l R3 = Me, R4 = CONHMe
O
8m
R = Me, R =
O
N
3
4
R3 = Me, R4 = NO2
8s R3 = Me, R4 = Br
8u
R3 = H, R4 = COOEt
R5
R5
NH2
CN
O
S
O
O
h
i
+
NC
NH2
R
S
O
N
N
H
11a
S
8d
R5 = Me
R5 = Me
11b R5 = CF3
8e R5 = CF3
NH2
R4
CN
R4
CN
R4
O2N
CN
O
p
l
j
O
N
S
N
H
S
S
O
N
N
Cl
O
10l
8n
R4 = NH2
12a
12b
12c
12d
R4 = NO2
R4 = NH2
R4 = -NMe2
11c
R4 = NO2
8o
8p
R4 = -NMe2
k
11d R4 = NH2
m
R4 = -NHCOMe
k
n
8q R4 = -NHSO2Me
8r R4 = F
o
R4 = -NHCOMe
12e R4 = -NHSO2
12f R4 = F
8t
R4 = NO2
Scheme 2. Synthetic routes for thienopyridines 8a-8u. Reagents and conditions: (a) POCl3,
1,4-dioxane, 80 ℃, 3 h; (b) CrO3, H2SO4, r.t., 24 h; SOCl2, reflux, 8 h; morpholine, Et3N, CH2Cl2, r.t.,
1 h; (c) LiOH, THF, r.t., 30 min; SOCl2, CH2Cl2, DMF, reflux, 8 h; amine, Et3N, 0 ℃ ~ r.t., 30 min;
(d) HSCH2COOCH3, Et3N, MeOH, reflux, 6 h; (e) triethylsilane, (PPh3)4Pd, BINAP, K3PO4,
toluene-H2O, Ar protect, 100 ℃, 16 h; (f) phenylboronic acid, (PPh3)4Pd, K2CO3, 1,4-dioxane-H2O,
100 ℃, 8 h; (g) Cu, MeONa, DMF, MeOH, 90 ℃, 24 h; (h) DABCO, EtOH, reflux, 6 h; 1N HCl, r.t.,
2 h; (i) ClCH2COOMe, MeONa, MeOH, reflux, 2 h; (j) thiourea, EtOH, reflux, 2 h; (k) Fe, NH4Cl,
MeOH-H2O, 60 ℃, 2 h; (l) ClCH2COOMe, Et3N, MeOH, reflux, 6 h; (m) HCHO, Na(CN)BH3,
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CH3COOH, MeCN, 0 ℃ ~ r.t., 12 h; (n) MeCOCl or MeSO2Cl, Et3N, CH2Cl2, 0 ℃~ r.t., 30 min; (o)
NaNO2, HBF4, H2O, 0 ~ 5 ℃, 3 h; toluene, 90 ℃, 1 h; (p) MeONa, MeOH, r.t., 1 h.
The inhibition effects of all these thienopyridines to rat UT-B were presented in Table 2. Our
optimization of 8a was started with investigation of monosubstitution at the 6-position of pyridine
ring. Replacement of methyl group at 6-position in 8a with phenyl (8b) resulted in a
two-fold-decrease in activity while 8c with morpholine-4-carbonyl at this position dramatically
reduced the inhibitory potency (IC50 > 10 µM). This finding indicated that alkyl with low steric
hindrance substituted at the 6-position was more beneficial. With this result in hand, we next
incorporate substituents at 4- or 5-position of pyridine ring in 8a while keeping methyl at 6-position
intact. As illustrated in Table 2, introduction of electro-donating alkyl (8d) or electro-withdrawing
trifluoromethyl (8e) at the 4-position caused 2-fold and 5-fold losses of potency respectively.
However, 8f (IC50 = 0.71 µM), obtained by adding alkyl at the 5-position of pyridine ring, turned out
to be more potent than 8a. In light of this result, we focused our attention to the moiety at 5-position
of 8a.Various types of substituents including halide, electron-withdrawing, electron-donating and
sterically crowded ones were tested. As shown in Table 2, substitution at 5-position with
electron-donating groups such as alkyl (8f, 8g), methoxyl (8h), amino (8n), dimethylamino (8o) and
acetamino (8p) resulted in only slightly reduced or even improved (IC50 < 1 µM for 8f and 8n)
inhibitory activity to UT-B compared to 8a. However, most of the derivatives with
electro-withdrawing groups, such as sulfonamido (8q), carbamoyl (8k-8m), halide (8r, 8s) and nitro
group (8t), at 5-position of pyridine ring were much less potent (IC50 > 10 µM for 8k-8m, 8r and 8t)
than 8a. Interestingly, incorporation of acetyl (8i) or ethoxycabonyl (8j), which are also
electro-withdrawing groups, led to 2-3 fold increase in potency. These findings implied that
inhibition activities to UT-B of thienopyridines were influenced remarkably by electric effects of
group at 5-position of pyridine ring. Finally, the necessity of methyl at 6-position of pyridine ring
was detected. Deletion of the methyl group at 6-position of the pyridine ring, exemplified by the
5-monosubstituted analogue (8u), resulted in a substantial loss (>10 fold) of potency, suggesting that
substitution of methyl at 6-position of pyridine ring is required for activity.
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Table 2. Inhibitory potency of compounds with thienopyridine core to rat UT-B.
R3
Compd.
IC50 (µM)
4-
H
H
5-
H
H
6-
CH3
Ph
1.30
3.36
8a
8b
H
H
>10
8c
CH3
CF3
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
2.54
6.12
0.71
3.29
5.46
0.91
0.47
>10
>10
8d
8e
8f
H
C2H5
H
Ph
8g
8h
8i
H
OCH3
COCH3
COOC2H5
CONHC2H5
CONHCH3
H
H
8j
H
8k
8l
H
H
CH3
>10
8m
H
H
H
H
H
H
H
H
NH2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
0.54
3.75
3.47
6.42
>10
8.47
>10
>10
0.22
8n
N(CH3)2
NHCOCH3
NHSO2CH3
F
8o
8p
8q
8r
Br
8s
NO2
8t
COOC2H5
8u
PU-48
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Solubility and Log P prediction of represented compounds.
Water solubility and Log P value, served as important parameters influencing PK profile of a
molecule, were predicted using the Qikprop module from Schrödinger for the compounds with IC50
value under 1 µM. As illustrated in Table 3, the predictive log S value of most thienopyridines (8f, 8i,
8n) is lower than that of thienoquinolines (7a-7e), indicating that modification of thienoquinolines to
thienopyridines do improve the water solubility. Among them, 8n was particularly noteworthy since
it was predictable to be more than 10 times soluble in water compared to PU48. In the meanwhile,
8n had a predictive Log P value of 0.646 and favorable molecular weight (237 Da) which coincided
with the drug-like properties described by Lipinski and Veber.
Table 3. Predictive water-solubility and Log P of selected compounds.
Comd.
PU48
7a
QPlogSa
-3.001
-2.772
-3.334
-3.627
-3.137
-3.838
-2.838
-2.252
-3.114
-1.753
QPlogPa
2.232
2.125
2.442
2.695
2.361
2.816
2.107
1.067
1.631
0.646
7b
7c
7d
7e
8f
8i
8j
8n
a predicted by Qikprop module from Schrödinger.
Diuretic effect on rat model of thienoquinolines.
To evaluate in vivo diuretic activity of 8n, rats were kept in metabolic cages with being fed ad
libitum. Urine output was checked before experiments to make sure for urine output to be similar in
each rat. 8n at 100 mg/kg or 40% 2-hydroxypropyl-bcyclodextrin as vehicle control was
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subcutaneously administrated. Urine samples were collected for 10 h after drug administration. The
experimental results showed 8n significantly increased urine output (Fig. 4), which suggest that 8n
has significant diuretic effect on rats.
Fig 4. Diuretic effect of 8n on rat. Urine of SD rats was collected in metabolic cages for 10 h
after subcutaneous injection of 100 mg/kg of 8n or 40% 2-hydroxypropyl-bcyclodextrin as vehicle
control. Mean + SD (n=5) *P < 0.05 compared with control.
3. Conclusion
A novel type of UT inhibitors with thienopyridine scaffold was identified in present study based
on the structural modification of PU-48. Further structure-activity analysis resulted in a promising
preclinical candidate 8n, which possesses not only excellent inhibition effect on UT-B and diuretic
activity on rat model but also favorable water solubility and suitable Log P value. These results
provide a new lead compound and support further development of thienopyridine analogs as
potential salt-sparing diuretics by targeting kidney urea transporters.
4. Experimental section
4.1 General
All reagents and solvents were purchased from commercial sources and were used without
1
further purification. Melting points were determined on X4 microscope and are uncorrected. H
NMR and 13C NMR spectra were recorded on Bruker AVANCEIII 400 MHz and 100 MHz
spectrometer respectively. High resolution mass spectrum (HRMS) was recorded on a Thermo
Scientific Orbitrap Elite MS.
4.2 General procedure for the synthesis of compounds 2a-2f
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POCl3 (9.0 mL, 96 mmol) was added dropwise to DMF (2.8 mL, 36 mmol) precooled at 0 ℃.
Followed by adding acetanilide (10 mmol), the mixture was heated to 75 ℃ and stirred at that
temperature for 8 h. After been cooled to room temperature, the mixture was poured to 100 mL of
ice-water. The precipitate was obtained by suction filtration, washed with cold water and dried to
afford the product.1H NMR and 13C NMR data of selected products are shown as follows.
4.2.1 2-Chloro-6-methylquinoline-3-carbaldehyde (2b)
Yield 83%. Mp: 125-126 ℃. 1H NMR (400 MHz, CDCl3) δ 10.57 (s, 1H), 8.69 (s, 1H), 7.98 (d, J
= 8.5 Hz, 1H), 7.73 (dd, J = 10.9, 2.2 Hz, 2H), 2.59 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 189.70,
149.63, 148.60, 139.92, 138.76, 136.31, 128.75, 128.59, 126.95, 126.67, 21.91.
4.2.2 2-Chloro-6-methoxyquinoline-3-carbaldehyde (2f)
Yield 75%. Mp: 143-145 ℃. 1H NMR (400 MHz, CDCl3) δ 10.48 (s, 1H), 8.57 (s, 1H), 7.89 (d, J
13
= 9.2Hz, 1H), 7.45 (dd, J = 9.2, 2.8 Hz, 1H), 7.12 (d, J = 2.8 Hz, 1H), 3.89 (s, 3H). C NMR (100
MHz, CDCl3) δ 189.75, 159.15, 148.01, 146.18, 138.98, 130.27, 128.12, 126.91, 126.77, 106.79,
56.12.
4.3 General procedure for the synthesis of compounds 3a-3f
To a solution of hydroxylamine hydrochloride (520 mg, 7.5 mmol) in 1 mL water, compound
2a-2f (1.65 g, 7.5 mmol) in 15 mL THF was added. The reaction mixture was stirred at room
temperature for 1 h. THF was removed under vacuum. The residue was stirred in 10 mL water for 10
min. Solid precipitated was filtered and dried. The dried solid was dissolved in 15 mL of chloroform
and SOCl2 (1.4 mL, 15 mmol) was add slowly to the solution. After refluxing for 1 h, the mixture
was concentrated and the residue was recrystallized with ethanol to give pure product. 1H NMR and
13C NMR data of selected products are shown as follows.
4.3.1 2-Chloro-6-methylquinoline-3-carbonitrile (3b)
Yield 98%. Mp: 155-156 ℃. 1H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 7.94 (d, J = 8.7 Hz, 1H),
13
7.73 (dd, J = 8.7, 1.8 Hz, 1H), 7.63 (s, 1H), 2.57 (s, 3H). C NMR (100 MHz, CDCl3) δ 147.67,
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147.20, 144.40, 139.52, 136.52, 128.83, 127.13, 125.51, 115.55, 108.03, 21.92.
4.3.2 2-Chloro-6-methoxyquinoline-3-carbonitrile (3f)
Yield 98%. Mp: 158-160 ℃. 1H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 7.94 (d, J = 8.5 Hz, 1H),
13
7.53 (d, J = 7.7 Hz, 1H), 7.10 (s, 1H), 3.96(s, 3H). C NMR (100 MHz, CDCl3) δ 159.58, 146.04,
144.78, 143.46, 130.57, 127.02, 126.80, 115.60, 108.32, 105.51, 56.23.
4.4 General procedure for the synthesis of compounds 4a-4f
A mixture of 3a-3f (1.59 g, 7.3 mmol) and thiourea (1.11 g, 14.6 mmol) in 20 mL of ethanol
was refluxed for 1 h. After cooled to room temperature, water (50 mL) was added and the mixture
was stirred for another 30 min. The product was yielded by filtration. 1H NMR and 13C NMR data of
selected products are shown as follows.
4.4.1 2-Mercapto-6-methylquinoline-3-carbonitrile (4b)
1
Yield 98%. Mp: 229-231 ℃. H NMR (400 MHz, DMSO-d6) δ 14.21 (s, 1H), 8.70 (s, 1H),
7.73-7.63 (m, 2H), 7.57 (d, J = 8.3 Hz, 1H), 2.42 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 178.26,
144.92, 139.39, 137.24, 135.59, 129.04, 121.88, 117.43, 117.22, 116.28, 21.40.
4.4.2 2-Mercapto-6-methoxyquinoline-3-carbonitrile (4f)
Yield 98%. Mp: 209-211 ℃. 1H NMR (400 MHz, DMSO-d6) δ 14.22 (s, 1H), 8.67 (s, 1H), 7.63
(d, J = 9.1 Hz, 1H), 7.50 (dd, J = 9.1, 2.8 Hz, 1H), 7.39 (d, J = 2.8 Hz, 1H), 3.87 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 176.84, 157.02, 144.48, 136.42, 126.10, 122.87, 118.93, 117.43, 116.61,
109.65, 56.63.
4.5 General procedure for the synthesis of compounds 6a-6i and 7a-7e
To a solution of 4a-4f (1.58 g, 7.3 mmol) in 15 mL of methanol, MeONa (7.3 mmol) and
corresponding material with chloroacetyl (11.0 mmol) were added. The reaction mixture was
refluxed for 3 h. After cooled to room temperature, water (15 mL) was added and the mixture was
stirred for 5 min. The precipitate was filtered and recrystallized by ethanol to afford pure product.
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4.5.1 Ethyl 3-amino-6-methoxythieno[2,3-b]quinoline-2-carboxylate (6a)
Yield 96%. Mp: 257 ℃. 1H NMR (400 MHz, DMSO-d6) δ 9.03 (s, 1H), 7.96 (d, J = 9.3 Hz, 1H),
7.52 (dd, J = 9.3, 2.3 Hz, 1H), 7.43 (s, 2H), 7.34 (s, 1H), 4.31 (q, J = 7.0 Hz, 2H), 3.95 (s, 3H), 1.33
(t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 167.41, 157.14, 157.02, 145.44, 144.98,
129.71, 129.66, 126.75, 126.13, 124.23, 106.24, 97.55, 56.04. HRMS m/z: calcd for C15H15N2O3S
[M+H]+: 303.0798; found: 303.0791.
4.5.2 3-Amino-6-methoxythieno[2,3-b]quinoline-2-carboxamide (6b)
1
Yield 96%. Mp: > 300 ℃. H NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H), 7.97 (d, J = 9.2 Hz,
1H), 7.50 (dd, J = 9.2, 2.8 Hz, 1H), 7.36 (d, J = 2.8 Hz, 1H), 7.34 (s, 2H), 7.21 (s, 2H), 3.94 (s, 3H).
13C NMR (100 MHz, DMSO-d6) δ 167.41, 157.14, 157.02, 145.44, 144.98, 129.71, 129.66, 126.75,
126.13, 124.23, 106.24, 97.55, 56.04. HRMSm/z: calcd for C13H12N3O2S [M+H]+: 274.0645; found:
274.0641.
4.5.3 3-Amino-6-methoxy-N-methylthieno[2,3-b]quinoline-2-carboxamide (6c)
Yield 95%. Mp: 225-228 ℃. 1H NMR (400 MHz, DMSO-d6) δ 8.09 (s, 1H), 7.97 (d, J = 9.2 Hz,
1H), 7.72 (d, J = 4.4 Hz, 1H), 7.50 (dd, J = 9.2, 2.8 Hz, 1H), 7.36 (d, J = 2.8 Hz, 1H), 7.35 (s, 2H),
13
3.95 (s, 3H), 2.77 (d, J = 4.4 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 165.86, 157.03, 157.01,
144.87, 144.80, 129.72, 129.69, 126.83, 126.17, 124.17, 106.21, 97.77, 56.04, 26.57. HRMS m/z:
calcd for C14H14N3O2S [M+H]+: 288.0801; found: 288.0799.
4.5.4 Methyl (3-amino-6-methoxythieno[2,3-b]quinoline-2-carbonyl)glycinate (6d)
Yield 60%. Mp: 209-210 ℃. 1H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.16 (t, J = 5.6 Hz,
1H), 7.98 (d, J = 9.6 Hz, 2H), 7.51 (dd, J = 9.2, 2.8 Hz, 1H), 7.38 (s, 2H), 7.37 (s, 1H), 3.97 (s, 2H),
3.95 (s, 3H), 3.67(s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.00, 165.76, 157.11, 156.98, 145.84,
145.07, 129.87, 129.71, 126.54, 126.19, 124.41, 106.27, 96.60, 56.06, 52.19, 41.56. HRMS m/z:
calcd for C16H16N3O4S [M+H]+: 346.0856; found: 346.0848.
4.5.5 (3-Amino-6-methoxythieno[2,3-b]quinolin-2-yl)(morpholino)methanone (6e)
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Yield 95%. Mp: 244-245 ℃. 1H NMR (400 MHz, DMSO-d6) δ 8.97 (s, 1H), 7.95 (d, J = 9.2 Hz,
1H), 7.48 (dd, J = 9.2, 2.4 Hz, 1H), 7.33 (d, J = 2.4 Hz, 1H), 6.65 (s, 2H), 3.94 (s, 3H), 3.66 (s, 4H),
3.64 (s, 4H). 13C NMR (100 MHz, DMSO-d6) δ 165.61, 157.58, 157.00, 144.42, 141.57, 129.69,
129.50, 126.76, 126.16, 124.07, 106.15, 99.45, 66.69, 56.03, 45.86. HRMS m/z: calcd for
C17H18N3O3S [M+H]+: 344.1063; found: 344.1066.
4.5.6 3-Amino-6-methoxy-N,N-dimethylthieno[2,3-b]quinolin-2-sulfonamide (6f)
1
Yield 78%. Mp: > 300 ℃. H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.00 (d, J = 9.2 Hz,
13
1H), 7.56 (d, J = 9.2 Hz, 1H), 7.36 (s, 1H), 6.89 (s, 2H), 3.96 (s, 3H), 2.80 (s, 6H). C NMR (100
MHz, DMSO-d6) δ 157.26, 156.78, 145.04, 143.91, 131.02, 129.72, 126.23, 125.97, 125.07, 106.17,
95.27, 56.09, 38.28. HRMS m/z: calcd for C14H16N3O3S2 [M+H]+: 338.0628; found: 338.0619.
4.5.7 1-(3-amino-6-methoxythieno[2,3-b]quinoline-2-yl)ethan-1-one (6g)
Yield 93%. Mp: 294-295 ℃. 1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.19 (s, 2H), 7.96 (d,
J = 9.2 Hz, 1H), 7.52 (dd, J = 9.2, 1.8 Hz, 1H), 7.32 (d, J = 1.8 Hz, 1H), 3.94 (s, 3H), 2.38 (s, 1H).
13C NMR (100 MHz, DMSO-d6) δ 192.05, 157.49, 157.13, 147.89, 145.84, 131.59, 129.73, 126.10,
125.92, 125.12, 106.28, 104.08, 56.09, 29.69. HRMS m/z:calcd for C14H13N2O2S [M+H]+: 273.0692;
found: 273.0688.
4.5.8 Methyl(3-amino-6-methoxythieno[2,3-b]quinoline-2-yl)-3-oxopropanoate (6h)
Yield 75%. Mp: > 300 ℃.1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.38 (s, 2H), 7.96 (d, J
= 9.2 Hz, 1H), 7.54 (d, J = 9.2 Hz, 1H), 3.95 (s, 3H), 3.82 (s, 2H), 3.36 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ186.50, 168.20, 157.57, 157.19, 149.54, 146.05, 131.83, 129.70, 126.09, 125.50, 125.38,
106.33, 103.15, 56.09, 52.44, 48.11. HRMS m/z: calcd for C16H15N2O4S [M+H]+: 331.0747; found:
331.0742.
4.5.9 Methyl 3-aminothieno[2,3-b]quinoline-2-carboxylate (7a)
1
Yield 71%. Mp: 238-241 ℃. H NMR (400 MHz, DMSO-d6) δ 9.20 (s, 1H), 8.23-7.95 (m, 2H),
13
7.86 (t, J = 7.3 Hz, 1H), 7.63 (t, J = 7.3 Hz, 1H), 7.51 (s, 2H), 3.82 (s, 3H). C NMR (100 MHz,
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DMSO-d6) δ 165.66, 160.74, 149.27, 148.49, 132.87, 132.12, 130.17, 128.65, 126.80, 126.25,
125.36, 93.52, 52.41. HRMS m/z: calcd for C13H11N2O2S [M+H]+: 259.0536; found: 259.0531.
4.5.10 Methyl 3-amino-6-methylthieno[2,3-b]quinoline-2-carboxylate (7b)
Yield 89%. Mp: 278-291 ℃. 1H NMR (400 MHz, DMSO-d6) δ 9.08 (s, 1H), 7.94 (d, J = 8.7 Hz,
13
1H), 7.81 (s, 1H), 7.70 (dd, J = 8.7, 1.8 Hz, 1H), 7.49 (s, 2H), 3.82 (s, 3H), 2.54 (s, 3H). C NMR
(100 MHz, DMSO-d6) δ 165.71, 158.15, 157.56, 148.34, 145.84, 131.17, 130.13, 126.45, 125.30,
110.44, 106.77, 93.88, 56.51, 52.36. HRMS m/z: calcd for C14H13N2O2S [M+H]+: 273.0692; found:
273.0688.
4.5.11 Methyl 3-amino-6-bromothieno[2,3-b]quinoline-2-carboxylate (7c)
Yield 39%. Mp: > 300 ℃. 1H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 1H), 8.32 (s, 1H), 8.06-7.84
(m, 2H), 7.55 (s, 2H), 3.86 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 164.59, 160.26, 147.24,
146.74, 134.05, 131.10, 130.79, 129.87, 125.90, 125.57, 118.53, 93.23, 51.55. HRMS m/z: calcd for
C13H10BrN2O2S [M+H]+: 336.9641; found: 336.9637.
4.5.12 Methyl 3-amino-6-fluorothieno[2,3-b]quinoline-2-carboxylate (7d)
1
Yield 23%. Mp: > 300 ℃. H NMR (400 MHz, DMSO-d6) δ 9.17 (s, 1H), 8.14 (dd, J = 9.2, 5.4
13
Hz, 1H), 8.04-7.69 (m, 2H), 7.53 (s, 2H), 3.86 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 164.65,
160.23, 159.25, 157.79, 147.09, 145.56, 131.38, 130.58, 125.83, 124.87, 121.69, 111.56, 51.52.
HRMS m/z: calcd for C13H10FN2O2S [M+H]+: 277.0441; found: 277.0436.
4.5.13 Methyl 3-amino-6,8-dimethythieno[2,3-b]quinoline-2-carboxylate (7e)
1
Yield 78%. Mp: 259-260 ℃. H NMR (400 MHz, CDCl3) δ 8.23 (s, 1H), 7.47 (d, J = 14.9 Hz,
13
2H), 6.05 (s, 2H), 3.92 (s, 3H), 2.80 (s, 3H), 2.50 (s, 3H). C NMR (100 MHz, CDCl3) δ 201.28,
166.30, 159.44, 147.71, 146.36, 136.32, 135.60, 133.67, 128.79, 125.44, 125.12, 124.91, 52.07,
21.89, 18.51. HRMS m/z: calcd for C15H15N2O2S [M+H]+: 287.0849; found: 287.0843.
4.6 The procedure for the synthesis of 3-amino-6-methoxythieno[2,3-b]quinoline-2-carboxylic acid
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(6i)
A mixture of PU-48 (288 mg, 1 mmol) and NaOH (160 mg, 4 mmol) in water (5 mL) and ethanol
(20 mL) was heated under reflux for 3 h. 25 mL of cold water was added slowly and the mixture was
stirred in an ice bath for 5 min. Add hydrochloric acid to adjust the pH to 4-5, filter with suction to
1
provide 6i as an orange solid (200 mg, 73%). Mp: 224-225 ℃. H NMR (400 MHz, DMSO-d6) δ
9.01 (s, 1H), 7.97 (d, J = 9.2 Hz, 1H), 7.52 (dd, J = 9.2, 2.4 Hz, 1H), 7.35 (d, J = 2.4 Hz, 1H), 3.95 (s,
3H). 13C NMR (100 MHz, DMSO-d6) δ 166.68, 157.76, 157.02, 147.17, 145.20, 130.38, 129.68,
126.25, 125.97, 124.65, 95.38, 56.04. HRMS m/z: calcd for C13H11N2O3S [M+H]+: 275.0485; found:
275.0483.
4.7 General procedure for the synthesis of 10a, 10b, 10d, 10e and 10i-10l.
To a solution of 9a-9h (10 mmol) in 10 mL of 1,4-dioxane, POCl3 (2.3 mL, 25 mmol) was added
dropwise. The reaction mixture was stirred at 80 ℃ for 1 h. After cooled to room temperature, the
mixture was poured to 50 mL of ice water. 20 N NaOH aqueous solution was added to adjust pH = 7.
Solid was precipitated, filtered and purified by column chromatography (eluent: petroleum
1
13
ether/ethyl acetate = 10:1) to provide the title compound. H NMR and C NMR data of selected
products are shown as follows.
4.7.1 2-Chloro-6-methylnicotinonitrile (10a)
Yield 77%. 1H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 7.9 Hz, 1H), 7.24 (d, J = 7.9 Hz, 1H), 2.63
(s, 3H). 13C NMR (100 MHz, CDCl3) δ 163.99, 151.93, 142.37, 121.96, 114.96, 107.61, 24.77.
4.7.2 5-Acetyl-2-chloro-6-methylnicotinonitrile (10d)
Yield 93%. 1H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H), 2.83(s, 3H), 2.64 (s, 3H). 13C NMR (100
MHz, CDCl3) δ 196.87, 163.84, 153.29, 142.65, 131.29, 114.15, 107.96, 29.37, 25.13.
4.7.3 Ethyl 6-chloro-5-cyano-2-methylnicotinate (10e)
1
Yield 95%. H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 4.43 (q, J = 6.6 Hz, 2H), 2.91 (s, 3H),
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13
1.43 (t, J = 7.1 Hz, 3H). C NMR (100 MHz, CDCl3) δ 165.30, 163.72, 153.77, 144.65, 124.64,
114.15, 108.12, 62.34, 25.21, 14.19.
4.7.4 Ethyl 6-chloro-5-cyanononitrile (10i)
Yield 81%. 1H NMR (400 MHz, CDCl3) δ 9.17 (d, J = 2.3 Hz, 1H), 8.59 (d, J = 2.3 Hz, 1H), 4.48
(d, J = 7.1 Hz, 2H), 1.45 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 162.58, 156.30, 153.60,
143.33, 125.48, 113.87, 110.94, 62.59, 14.19.
4.7.5 2-Chloro-6-methyl-5-nitronicotinonitrile (10l)
1
Yield 87%. H NMR (400 MHz, CDCl3) δ 8.64 (s, 1H), 2.97 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 159.03, 154.72, 143.66, 138.81, 112.85, 109.29, 24.49.
4.8 Synthesis of 2-Chloro-6-(morpholine-4-carbonyl)nicotinonitrile (10c).
To a solution of 10a (765 mg, 5 mmol) in 10 mL of concentrated H2SO4, which was pre-cooled to
0 ℃, CrO3 (750 mg, 7.5 mmol) was added batchwise. The mixture was stirred at 0 ℃ for 1 h and
then warmed to room temperature for another 12 h. The reaction mixture was added dropwise to 20
mL of ice water while stirring. After filtration, the filter cake dissolved in 10 mL of POCl3 and stirred
at 90 ℃ for 40 min. The resulted mixture was cooled and poured to ice water. The precipitate was
filtered, dried and then dissolved in DCM (15 mL). SOCl2 (357 mg, 3 mmol) was added to the
solultion followed by 5 drops of DMF. The mixture was heated at 40 ℃for 8 h. After cooled to 0 ℃,
a solution of morpholine (348 mg, 4 mmol) and Et3N (404 mg, 4 mmol) in dichloromethane (10 mL)
was added. The resulted mixture was stirred for further 30 min and then poured to 20 mL of water.
The mixture was extracted with dichloromethane (20 mL ×3), dried over anhydrous Na2SO4 and
concentrated. The residue was purified by column chromatography (eluent: petroleum ether/ethyl
acetate = 5:1) to provide the title product. Yield 88%. Mp: 118-119 ℃. 1H NMR (400 MHz, CDCl3)
δ 8.14 (d, J = 7.9 Hz, 1H), 7.81-7.73 (m, 1H), 3.80 (s, 4H), 3.71 (s, 2H), 3.63 (d, J = 2.5 Hz, 2H). 13C
NMR (100 MHz, CDCl3) δ 163.99, 156.36, 151.32, 143.70, 122.90, 114.16, 111.56, 66.76, 66.64,
47.71, 43.00. HRMS m/z: calcd for C11H11ClN3O2 [M+H]+: 252.0534; found: 252.0530.
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4.9 General procedure for the synthesis of 10f-10h.
15 mL of 3 N LiOH aqueous solution was added drop wise to the solution of 10e (2.5 mmol) in
THF (35 mL). The mixture was stirred at room temperature for 10 min and then concentrated in
vacuum to remove THF. 1 N hydrochloric acid was added to adjust pH = 3. The solid precipitated
was filtered, dried and then dissolved in DCM (15 mL). SOCl2 (357 mg, 3 mmol) was added to the
solultion followed by 5 drops of DMF. The mixture was heated at 40 ℃for 8 h. After cooled to 0 ℃,
a solution of amine (4 mmol) and Et3N (404 mg, 4 mmol) in dichloromethane (10 mL) was added.
The resulted mixture was stirred for further 30 min and then poured to 20 mL of water. The mixture
was extracted with dichloromethane (20 mL ×3), dried over anhydrous Na2SO4 and concentrated to
provide the title products without further purification.
4.10 General procedure for the synthesis of 8a-8c, 8i-8m, 8s and 8u.
A mixture of 10a-10k (1 mmol), methyl thioglycolate (159 mg, 1.5 mmol) and Et3N (152 mg, 1.5
mmol) in 10 mL of methanol was stirred at 80 ℃ for 6~8 h. Methanol was removed under vacuum.
The residue was purified by column chromatography to provide the title compound.
4.10.1 Methyl 3-amino-6-methylthieno[2,3-b]pyridine-2-carboxylate (8a)
Yield 75%. Mp: 137 ℃. 1H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 8.3 Hz, 1H), 7.41-7.11 (m,
13
3H), 3.79 (s, 3H), 2.58 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 165.30, 160.63, 159.88, 152.94,
148.49, 136.10, 131.96, 123.67, 119.93, 113.89, 92.72, 51.75, 24.72. HRMS m/z: calcd for
C10H10N2O2S [M+H]+: 223.0536; found: 223.0530.
4.10.2 Methyl 3-amino-6-phenylthieno[2,3-b]pyridine-2-carboxylate (8b)
Yield 70%. Mp: 220-222 ℃. 1H NMR (400 MHz, CDCl3) δ 8.12 (d, J = 7.0 Hz, 2H), 7.99 (d, J =
8.5 Hz, 1H), 7.77 (d, J = 8.5 Hz, 1H), 7.50 (dq, J = 14.2, 7.0 Hz, 3H), 5.95 (s, 2H), 3.93 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 165.21, 160.45, 157.73, 148.28, 138.19, 132.90, 130.32, 129.38, 127.55,
124.88, 116.83, 51.86. HRMS m/z: calcd for C15H13N2O2S [M+H]+: 285.0692; found: 285.0688.
4.10.3 Methyl 3-amino-6-(morpholine-4-carbonyl)thieno[2,3-b]pyridine-2-carboxylate (8c).
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Yield 70%. Mp: 188-189 ℃. 1H NMR (400 MHz, CDCl3) δ 8.07 (d, J = 8.3 Hz, 1H), 7.65 (d, J =
8.3 Hz, 1H), 6.06 (s, 2H), 3.92 (s, 3H), 3.85 (d, J = 1.8 Hz, 4H), 3.71 (s, 4H). 13C NMR (100 MHz,
CDCl3) δ 166.74, 165.50, 159.27, 153.61, 145.96, 130.53, 125.86, 119.18, 99.62, 66.97, 66.76, 51.85,
47.86, 42.93. HRMS m/z: calcd for C14H15N3O4S [M+H]+: 322.0856; found: 322.0854.
4.10.4 Methyl 5-acetyl-3-amino-6-methylthieno[2,3-b]pyridine-2-carboxylate (8i).
Yield 79%. Mp: 192 ℃. 1H NMR (400 MHz, DMSO-d6) δ 9.06 (d, J = 4.1 Hz,1H), 7.39 (s, 2H),
13
3.80 (s, 3H), 2.71 (d, J = 2.0 Hz, 3H), 2.64 (d, J = 1.1 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ
199.89, 165.00, 161.41, 159.57, 148.42, 133.96, 128.66, 123.69, 93.43, 51.85, 29.60, 25.61. HRMS
m/z: calcd for C12H12N2O3S [M+H]+: 265.0641; found: 265.0639.
4.10.5 5-Ethyl 2-methyl 3-amino-6-methylthieno[2,3-b]pyridine-2,5-dicarboxylate (8j).
Yield 73%. Mp: 193-194 ℃. 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 6.05 (s, 2H), 4.43 (q, J
13
= 7.1 Hz, 2H), 3.91 (s, 3H), 2.95 (s, 3H), 1.44 (t, J = 7.1 Hz, 3H). C NMR (100 MHz, CDCl3) δ
166.18, 165.60, 162.93, 160.95, 146.42, 132.11, 123.11, 121.45, 98.17, 61.56, 51.75, 25.63, 14.33.
HRMS m/z: calcd for C13H14N2O4S [M+H]+: 295.0747; found: 295.0743.
4.10.6 Methyl 3-amino-5-(ethylcarbamoyl)-6-methylthieno[2,3-b]pyridine-2-carboxylate (8k).
Yield 89%. Mp: 274-276 ℃. 1H NMR (400 MHz, DMSO-d6) δ 8.53 (s, 2H), 7.32 (s, 2H), 3.80 (s,
13
3H), 3.29 (dd, J = 13.0, 7.0 Hz, 2H), 2.61 (s, 3H), 1.16 (t, J = 7.2 Hz, 3H). C NMR (100 MHz,
DMSO-d6) δ 167.91, 165.17, 159.72, 157.65, 148.34, 130.74, 129.48, 123.40, 93.40, 51.85, 34.46,
23.31, 15.07. HRMS m/z: calcd for C13H15N3O3S [M+H]+: 294.0907; found: 294.0901.
4.10.7 Methyl 3-amino-5-(methylcarbamoyl)-6-methylthieno[2,3-b]pyridine-2-carboxylate (8l).
Yield 87%. Mp: 287 ℃(decomposed). 1H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H), 8.46 (d, J
= 4.4 Hz, 1H), 7.30 (s, 2H), 3.79 (s, 3H), 2.81 (d, J = 4.6 Hz, 3H), 2.61 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 168.62, 165.16, 159.79, 157.76, 148.31, 130.85, 129.26, 123.40, 93.47, 51.86, 26.54,
23.39. HRMS m/z: calcd for C12H13N3O3S [M+H]+: 280.0750; found: 280.0749.
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4.10.8 Methyl 3-amino-6-methyl-5-(morpholine-4-carbonyl)thieno[2,3-b]pyridine-2-carboxylate
(8m).
1
Yield 87%. Mp: 215 ℃. H NMR (400 MHz, CDCl3) δ 7.79 (s, 1H), 6.11 (s, 2H), 3.90-3.74 (m,
7H), 3.67-3.53 (m, 2H), 3.27 (d, J = 4.1 Hz, 2H), 2.63 (s, 3H),.13C NMR (100 MHz, CDCl3) δ
168.31, 165.63, 160.88, 157.77, 146.21, 127.48, 126.72, 122.90, 98.52, 66.76, 51.73, 47.45, 42.30,
22.72. HRMS m/z: calcd for C15H17N3O4S [M+H]+: 336.1013; found: 336.1017.
4.10.9 Methyl 3-amino-5-bromo-6-methylthieno[2,3-b]pyridine-2-carboxylate (8s).
Yield 83%. Mp: 187 ℃. 1H NMR (400 MHz, DMSO-d6) δ 8.80 (d, J =6.6 Hz, 1H), 7.25 (s, 2H),
3.79 (s, 3H), 2.66 (d, J = 3.9 Hz, 3H).13C NMR (100 MHz, DMSO-d6) δ 164.97, 158.15, 147.16,
135.07, 125.83, 116.99, 94.37, 51.90, 25.55. HRMS m/z: calcd for C10H9BrN2O2S [M+H]+: 300.9641;
found: 300.9639.
4.10.10 5-Ethyl 2-methyl 3-aminothieno[2,3-b]pyridine-2,5-dicarboxylate (8u).
Yield 72%. Mp: 174-176 ℃. 1H NMR (400 MHz, CDCl3) δ 9.25 (d, J = 1.8 Hz, 1H), 8.64 (d, J =
13
1.8 Hz,1H), 6.15 (s, 2H), 4.46 (q, J = 7.1 Hz, 2H), 3.92 (s, 3H), 1.45 (t, J = 7.1 Hz, 3H). C NMR
(100 MHz, CDCl3) δ 165.44, 165.21, 164.47, 150.93, 146.35, 130.82, 124.84, 121.76, 99.16, 66.76,
61.73, 51.83, 14.30. HRMS m/z: calcd for C12H12N2O4S [M+H]+: 281.0591; found: 281.0594.
4.11 General procedure for the synthesis of 8d and8e.
To a solution of acetyl- or trifuluroacetyl acetone (1 mmol) and DABCO (220 mg, 1 mmol) in 30
mL of ethanol, 2-cyanoethanethioamide (120 mg, 1.2 mmol) was added. The mixture was stirred
under reflux for 6 h. The solvent was removed in vacuum. Add 1 N HCl (50 mL) to the residue and
stirred the resulted suspension for 2 h at room temperature. The solid was filtered and washed with
water to provide 11a or 11b without further purification. Dissolve 11a or 11b in 15 mL of methanol.
Methyl chloroacetate (162 mg, 1.5 mmol) and MeONa (81 mg, 1.5 mmol) was added to the solution
and was stirred for 3 h at room temperature. Water was added and the precipitate was filtered and
recrystallized with ethyl acetate/petroleum ether to afford the pure product.
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4.11.1 Methyl 3-amino-4,6-dimethylthieno[2,3-b]pyridine-2-carboxylate (8d).
1
Yield 82%. Mp: 184-185 ℃. H NMR (400 MHz, CDCl3) δ 6.86 (s, 1H), 6.17 (s, 2H), 3.89 (s,
3H), 2.74 (s, 3H), 2.60 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 166.13, 161.41, 159.75, 149.12,
143.63, 122.33, 121.77, 119.49, 51.51, 24.40, 20.14. HRMS m/z: calcd for C11H13N2O2S [M+H]+:
237.0692; found: 237.0688.
4.11.2 Methyl 3-amino-6-methyl-4-(trifluoromethyl)thieno[2,3-b]pyridine-2-carboxylate (8e).
1
Yield 79%. Mp: 190-192 ℃. H NMR (400 MHz, CDCl3) δ 7.45 (s, 1H), 6.39 (s, 2H), 3.93 (s,
3H), 2.76 (s, 3H).13C NMR (100 MHz, CDCl3) δ 165.75, 162.79, 159.91, 145.81, 130.90, 128.84,
124.29, 116.30, 116.24, 51.84, 24.72. HRMS m/z: calcd for C11H10F3N2O2S [M+H]+: 291.0410;
found: 291.0407.
4.12
The
procedure
for
the
synthesis
of
methyl
3-amino-5-ethyl-6-methylthieno[2,3-b]pyridine-2-carboxylate (8f).
A mixture of 8s (301 mg, 1 mmol), B(C2H5)3 (66 mg, 0.67 mmol), (PPh3)4Pd (58 mg, 0.05 mmol),
BINAP (31 mg, 0.05 mmol), K3PO4 (425 mg, 2 mmol), toluene (10 mL) and water (1 mL) was
stirred at 100 ℃under argon for 16 h followed by cooling to room temperature. Water (10 mL) was
added and the mixture was extracted with ethyl acetate (10 mL × 3). The combined organic phase
was washed with brine, dried over anhydrous Na2SO4 and concentrated. The residue was purified by
column chromatography (eluent: petroleum ether/ethyl acetate = 6:1) to provide 8f as a yellow solid
1
(156 mg, 62%). Mp: 191 ℃. H NMR (400 MHz, DMSO-d6) δ 8.29 (s, 1H), 7.23 (s, 2H), 3.78 (s,
3H), 2.70 (d, J = 7.0 Hz, 2H), 2.56 (s, 3H), 1.25 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 165.37, 159.54, 157.01, 148.50, 133.64, 130.12, 124.47, 92.93, 51.68, 25.24, 22.84, 13.95. HRMS
m/z: calcd for C12H14N2O2S [M+H]+: 251.0849; found: 251.0843.
4.13
The
procedure
for
the
synthesis
of
methyl
3-amino-6-methyl-5-phenylthieno[2,3-b]pyridine-2-carboxylate (8g).
A mixture of 8s (301 mg, 1 mmol), PhB(OH)2 (244 mg, 2 mmol), (PPh3)4Pd (58 mg, 0.05 mmol),
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K2CO3 (277 mg, 2 mmol), 1,4-dioxane (5 mL) and water (1 mL) was stirred at 100 ℃ for 8 h
followed by cooling to room temperature. Water (10 mL) was added and the mixture was extracted
with ethyl acetate (10 mL × 3). The combined organic phase was washed with brine, dried over
anhydrous Na2SO4 and concentrated. The residue was purified by column chromatography (eluent:
petroleum ether/ethyl acetate = 6:1) to provide 8g as a yellow solid (272 mg, 91%). Mp: 201 ℃. 1H
NMR (400 MHz, CDCl3) δ7.74 (s, 1H), 7.53-7.39 (m, 3H), 7.36 (dd, J = 5.2, 3.0 Hz, 2H), 3.91 (s,
13
3H), 2.61 (s, 3H). C NMR (100 MHz, CDCl3) δ 165.90, 159.35, 158.37, 146.46, 139.44, 133.41,
129.89, 129.21, 128.57, 127.79, 124.48, 97.97, 51.67, 24.08. HRMS m/z: calcd for C16H14N2O2S
[M+H]+: 299.0849; found: 299.0848.
4.14
The
procedure
for
the
synthesis
of
methyl
3-amino-5-methoxy-6-methylthieno[2,3-b]pyridine-2-carboxylate (8h).
A mixture of compound 8ha (348 mg, 1 mmol, prepared from 10k by the method similar to 4.10),
MeONa (162 mg, 3 mmol), copper powder (107 mg, 2 mmol), methanol (15 mL) and DMF (15 mL)
was stirred at 90 ℃ for 24 h followed by cooling to room temperature. Water (10 mL) was added and
the mixture was extracted with ethyl acetate (10 mL × 3). The mixture was filtered over diatomite
and concentrated. Water (30 mL) was then added to the residue and the mixture was extracted with
ethyl acetate (20 mL × 3). The combined organic layer was washed with brine, dried over anhydrous
Na2SO4 and concentrated. The residue was purified by column chromatography (eluent: petroleum
ether/ethyl acetate = 3:1) to provide 8h as a yellow solid (212 mg, 84%). Mp: 179 ℃. 1H NMR (400
MHz, DMSO-d6) δ 8.09 (s, 1H), 7.20 (s, 2H), 3.87 (s, 3H), 3.78 (s, 3H), 2.46 (s, 3H). 13C NMR (100
MHz, DMSO-d6) δ 165.31, 152.06, 151.90, 150.56, 148.37, 124.83, 111.46, 93.91, 56.22, 51.69,
20.33. HRMS m/z: calcd for C11H12N2O3S [M+H]+: 253.0641; found: 253.0639.
4.15 The procedure for the synthesis of 6-methyl-5-nitro-2-thioxo-1,2-dihydropyridine-3-carbonitrile
(11c).
A mixture of 10l (3.916 g, 20 mmol), thiourea (3.04 g, 40 mmol) and ethanol (50 mL) was
refluxed for 3 h. Water (50 mL) was added. The mixture was cooled to room temperature and filtered.
The filter cake was dried to provide 11c as a bright yellow solid (3.705 g, 75%), which was not
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purified and directly used in the next reaction.
4.16
The
procedure
for
the
synthesis
of
5-amino-6-methyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile (11d).
A mixture of compound11c (1.95 g, 10 mmol), ferric powder (1.84 g, 33 mmol), NH4Cl (2.67 mg,
50 mmol), methanol (20 mL) and water (10 mL) was drastically stirred at 60 ℃for 3 h. After cooled
to room temperature, the mixture was filtered over diatomite. Add 30 mL of water to the filtrate and
extract with ethyl acetate (30 mL × 3). The combined organic phase was washed with brine, dried
over anhydrous Na2SO4 and concentrated to provide 11d as a yellow solid (1.58 g, 96%), which was
not purified and directly used in the next reaction.
4.17 The procedure for the synthesis of 12a and 12b.
A mixture of 11c or 11d (5 mmol), methyl chloroacetate (810 mg, 7.7 mmol), Et3N (760 mg, 7.6
mmol) and methanol (20 mL) was stirred at 50 ℃for 2 h. Methanol was removed under vacuum and
the residue was purified by column chromatography (eluent: petroleum ether/ethyl acetate = 5:1) to
provide 12a or 12b.
1
Methyl 2-((5-amino-3-cyano-6-methylpyridin-2-yl)thio)acetate (12b). Yield 84%. H NMR (400
MHz, CDCl3) δ 7.04 (s, 1H), 3.95 (s, 2H), 3.77 (s, 5H), 2.41 (s, 3H).
4.18
The
procedure
for
the
synthesis
of
methyl
2-((3-cyano-5-(dimethylamino)-6-methylpyridin-2-yl)thio)acetate (12c).
To a solution of compound 12b (356 mg, 1.5 mmol) in 15 mL of acetonitrile was added aqueous
solution of formaldehyde (15 mL, w = 37%), NaCNBH3 (284 mg, 4.5 mmol) and 10 drops of acetic
acid. The mixture was stirred at room temperature for 12 h. Water (20 mL) was added and the
suspension was extracted with dichloromethane (20 mL × 3). The combined organic phase was
washed with brine, dried over anhydrous Na2SO4 and concentrated to afford 12c without further
purification.
4.19
The
procedure
for
the
synthesis
of
methyl
28