Synthesis and structure-activity relationship of 4-alkoxy-thieno[2,3-b]pyridine derivatives as potent alkaline phosphatase enhancers for osteoporosis treatment

Article abstract of DOI:10.1016/j.bmcl.2019.05.014

The synthesis and structure-activity relationships of a novel series of 3-aminothieno[2,3-b]pyridine-2-carboxamides were explored. Our efforts were focused on modifying the C-4 substituent of the thienopyridine ring to develop orally available bone anabolic agents. 4-Alkoxy derivatives were found to be novel ALPase enhancers without inhibitory effect on P450 activity. Among these derivatives, compound 6k was orally administered to ovariectomized rats, and it was found to significantly improve areal bone mineral density at a dose of 30 mg/kg/day.

Full text of DOI:10.1016/j.bmcl.2019.05.014

Accepted Manuscript
Synthesis and structure-activity relationship of 4-alkoxy-thieno[2,3-b]pyridine
derivatives as potent alkaline phosphatase enhancers for osteoporosis treatment
Keiji Saito, Tsuyoshi Shinozuka, Akira Nakao, Toshihiro Kiho, Tomonori
Kunikata, Takeshi Shiiki, Yoko Nagai, Satoru Naito
PII:
S0960-894X(19)30300-2
https://doi.org/10.1016/j.bmcl.2019.05.014
BMCL 26434
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 March 2019
4 May 2019
7 May 2019
Please cite this article as: Saito, K., Shinozuka, T., Nakao, A., Kiho, T., Kunikata, T., Shiiki, T., Nagai, Y., Naito,
S., Synthesis and structure-activity relationship of 4-alkoxy-thieno[2,3-b]pyridine derivatives as potent alkaline
phosphatase enhancers for osteoporosis treatment, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://
doi.org/10.1016/j.bmcl.2019.05.014
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Synthesis and structure-activity relationship of 4-alkoxy-thieno[2,3-b]pyridine derivatives as potent
alkaline phosphatase enhancers for osteoporosis treatment
Keiji Saitoa, Tsuyoshi Shinozukab, Akira Nakaob, Toshihiro Kihoc, Tomonori Kunikatad, Takeshi Shiikie,
Yoko Nagaie, Satoru Naitof
a Venture Science Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b End-Organ Disease Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Modality Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
d Rare Disease Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
e Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
f R&D Administration & Support Department, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
Corresponding author: Tel.: +81 3 3492 3131; fax: +81 3 5436 8563; e-mail:
saito.keiji.e2@daiichisankyo.co.jp (K. Saito).
Abbreviations: PTH, parathyroid hormone; HTS, high throughput screening; PK, pharmacokinetic; AUC,
area under the curve
ABSTRACT
The synthesis and structure-activity relationships of a novel series of 3-aminothieno[2,3-b]pyridine-2-
carboxamides were explored. Our efforts were focused on modifying the C-4 substituent of the
thienopyridine ring to develop orally available bone anabolic agents. 4-Alkoxy derivatives were found to be
novel ALPase enhancers without inhibitory effect on P450 activity. Among these derivatives, compound 6k
was orally administered to ovariectomized rats, and it was found to significantly improve areal bone mineral
density at a dose of 30 mg/kg/day.
Graphical Abstract
Keywords: osteoporosis, bone anabolic agents, ALPase enhancer, time-dependent inhibition, thieno[2,3-
b]pyridine

Osteoporosis is a major public health concern observed most frequently in postmenopausal women and
aging men. It is estimated that over 200 million people worldwide have osteoporosis, and the incidence of
fracture is increasing as the population ages.1, 2, 3 The bones turn over every 6 to 10 years throughout life.
During this process, old bones are resorbed by osteoclasts and new bones are formed by osteoblasts. There
is a balance between the amount of bone resorbed and formed under normal physiological conditions. In
osteoporosis, bone resorption exceeds bone formation, resulting in total bone loss. There are two main
categories of osteoporosis treatments: antiresorptive agents and anabolic agents.4, 5, 6, 7, 8, 9 Current
therapeutic agents are mainly antiresorptive agents, such as bisphosphonates, calcitonins, estrogens,
selective estrogen receptor modulators, and the RANKL monoclonal antibody.10 On the other hand,
recombinant human PTH has long been used as a bone anabolic agent. PTH therapy has shown that anabolic
agents could increase bone mass to a greater degree than antiresorptive agents, resulting in a reduction in
fracture incidence. However, the administration period of PTH is limited owing to safety concerns. 11, 12, 13,
14, 15 An anti-sclerostin monoclonal antibody was recently approved as the second anabolic agent for the
treatment of osteoporosis in patients at high risk of fracture. Owing to the well-known disadvantages of
peptide- and protein-based therapies, including high cost and subcutaneous or intravenous administration,
orally active small molecules would clearly be an additional benefit to patients.
In a previous article,16 we reported the optimization of HTS-hit compound 1 by an assay evaluating alkaline
phosphatase (ALPase) activity, an indicator of osteoblast differentiation,17 and identified N-phenyl-
homopiperazine derivative 2 as a potent ALPase enhancer (Fig. 1).18, 19 Compound 2 was orally active and it
showed statistically significant improvements in areal bone mineral density (aBMD) at a dose of 10
mg/kg/day; however, a subsequent test revealed that compound 2 showed direct inhibition (DI) of P450s
and time-dependent inhibition (TDI) of CYP3A4, one of the major metabolic enzymes, which may cause
drug-drug interaction (DDI) and lead to serious side effects, especially in elderly patients with
polypharmacy.20, 21
N
N
NH2
O
N
NH2
S
NH2
O
N
S
NH2
N
1
2
EC200 = 0.17 g/mL
EC200 = 0.051 g/mL


CYPs DI
CYP3A4 TDI
Figure 1. Thieno[2,3-b]pyridine derivatives 1 and 2.16 EC200, concentration required to enhance alkaline
phosphatase activity to 200% of that of control; DI, direct inhibition; TDI, time-dependent inhibition.
Because HTS-hit compound 1 did not inhibit P450s in the TDI study, it is fair to say that 3-
aminothieno[2,3-b]pyridine-2-carboxamide,22, 23, 24, 25, 26 which was confirmed to be a necessary core
structure for enhancing ALPase activity in our previous SAR study, was not a cause of TDI. We thought

that we could avoid TDI by replacing the N-phenyl-homopiperazine part with other moieties. Thus, we
embarked on an investigation of C-4 alkoxy/sulfanyl derivatives as a new template to discover novel, orally
active anti-bone osteoporosis agents.
Because the mechanism of action and the target molecule have not been clarified, a classical SAR study
strategy was adopted. To execute such derivatization, we previously developed an efficient method to
synthesize C4-alkoxy thieno[2,3-b]pyridine,27 which made it possible to flexibly change the C-4 substituent.
The synthetic route is described in Scheme 1. Various alcohols or thiols were reacted with 3-cyano-2,4-
dichloropyridines 3a28 and b29, 30 in the presence of a base to regioselectively obtain 4-alkoxy or sulfanyl
pyridines 4. Next, 2-chloropyridines 4 were treated with 2-(acetylthio)acetamide to obtain thienopyridines
6a-c, 6e-f, and 6h-j in one-pot. An alternative method for constructing the thienopyridine ring involved a
stepwise method. 2-Chloropyridine 4 was treated with thiourea to provide 2-thiopyridone 5, and following
reaction with 2-chloroacetamide, thienopyridines 6d, 6g, 6k-n, 7, and 8 were obtained. Thienopyridines
were prepared in high yield using both methods.
R2
R2
Cl
N
X
N
X
c
a
CN
Cl
CN
Cl
CN
S
R1
R1
R1
N
H
1
3a
3b
: R = H
: R = Me
X = O or S
5
2
4
b
X
d
R2
NH2
O
R1
N
S
NH2
6 - 8
Scheme 1. Reagents and conditions: (a) R2XH (X = O or S), NaH or DBU, DMF, rt; (b) 2-
(acetylthio)acetamide, NaOH, H2O, DMF, rt; (c) thiourea, HCl, EtOH, reflux; (d) 2-chloroacetamide, aq.
NaOH, DMF, rt.
The effects of test compounds on cellular ALPase activity were evaluated using stromal cell line ST2
derived from mouse bone marrow. The results are shown in Table 1, 3 and 4, and the potency is expressed
as EC200, which is the concentration required to enhance ALPase activity to 200% of that of control.
Initially, various alkoxy and sulfanyl groups were introduced at the C4-position of thienopyridine. The
results are summarized in Table 1 and 2. Although methoxy analog 6a lost its activity, ethoxy derivative 6b
retained its potency. Introduction of an isopropoxy group (6c) decreased compound activity, whereas
cyclohexyloxy analog 6d showed an activity of EC200 = 0.14 μg/mL. Thus, a methyl group was introduced
to the cyclohexane ring of compound 6d, and C-2 or 4 substitution was confirmed to be optimal. The

relative configuration was found to be critical, because trans-configuration showed better results (6e vs. 6h).
One carbon elongation resulted in a significant decrease in compound activity (6i). Introduction of
piperidine was detrimental (6j), whereas tetrahydropyran was tolerated (6k). Several potent compounds
were selected for evaluation of solubility and metabolic stability (Table 2). Incorporation of tetrahydropyran
ring resulted in better solubility at neutral pH and preferable metabolic stability in human and rat liver
microsomes. Thus, a methyl group was incorporated on the tetrahydropyran ring to increase compound
activity. In contrast to the SAR of cyclohexane analog, the introduction of a methyl group led only to
retention of compound activity (6l). Regarding linker, O-linked analog provided better activity than the
corresponding S-linked analog (6d vs 6m). Because cyclohexyloxy analog 6d showed a potent activity, we
synthesized phenoxy analog 6n, which was 4-fold more potent than hit compound 1. Compound 6n seemed
amenable to rapid modifications; thus, we chose this compound as a new lead compound, and the
substituents on its phenyl group were examined in detail, as shown in Table 3.
Table 1. Effects of C4-substituents on ALPase activity in ST2 cellsa and physicochemical properties
R
NH2
4
3
O
2
S
NH2
N
Comp.
-R
EC200 (μg/mL)b
cLogPc
1.7
1
-NMe2
-OMe
0.17
>1.0
6a
1.3
6b
6c
-OEt
0.27
0.72
1.9
2.2
-Oi-Pr
6d
6e
0.14
3.4
3.9
O
0.037
O
6f
>1.0
3.9
O
O
6g
6h
6i
0.045
>1.0
>0.30
>1.0
0.25
3.9
3.9
4.4
1.4
1.0
O
O
N
6j
O
O
6k
O

O
6l
6m
6n
0.27
>1.0
1.5
3.7
3.1
O
S
0.046
O
a Values are the arithmetic means of at least two experiments. Standard deviation in the assay is less than ±10%.
b Concentration required to enhance ALPase activity in ST2 cells to 200% of that of control.
c cLogP values were calculated by the CLOGP software (CLOGP Daylight Version 4.83, Daylight Chemical Information Systems,
Inc.).
Table 2. Effects of C4-substituents on physicochemical properties
Metabolic stability (% remaining)b
Comp.
6d
Solubility (μg/mL)a
human
rat
28
1
2
6g
6k
6n
42
180
6.6
20
92
11
7
85
36
a Solubility in Japanese pharmacopoeia 2nd-fluid (pH 6.8) (JP2) solution.
b Stability described as % remaining after 30-min incubation of test compounds in 0.5 mg/mL human/rat liver microsomes.
We synthesized more than 100 compounds from commercially available phenols to investigate the effects of
substitution on the benzene ring. Representative data are shown in Table 3. When F or Cl was incorporated
(7a-d), 2-substituted compounds showed potent enhancement in activities, especially, the EC200 value of 2-
Cl analog 7a was 8 ng/mL, which was 6-fold higher than that of unsubstituted compound 6n. In the case of
ethyl or methoxy group (7e-h), 4-substitution yielded better activities than 2-substitution. The introduction
of isopropyl (7i) resulted in a significant loss of compound activity. Introduction of electron withdrawing
groups, such as cyano (7j), ester (7k), or amide (7l), was also detrimental. Next, 2-, 4-substituted compound
7m was synthesized. Although compound 7m indicated potent activity, its calculated lipophilicity was
highly predictive of poor solubility. Solubility is one of the key physicochemical parameters when selecting
compounds in drug discovery.31, 32 In general, poorly soluble compounds are often associated with poor
bioavailability and higher probability of attrition. Thus, we modified our design toward improving
physicochemical properties, as shown in Table 4.
Table 3. Effects of substituents of phenyl group on ALPase activity in ST2 cellsa and physicochemical
properties
X
3
2
4
O
N
NH2
O
S
NH2

Comp.
6n
7a
X-
H-
EC200 (μg/mL)b
0.046
cLogPc
3.1
3.5
3.8
3.8
3.0
4.1
4.1
2.6
3.0
4.5
2.5
3.0
1.5
4.3
3.6
2-Cl-
0.008
7b
7c
3-Cl-
0.149
4-Cl-
0.021
7d
7e
2-F-
0.022
2-Et-
0.201
7f
4-Et-
0.036
7g
2-MeO-
4-MeO-
4-i-Pr-
0.114
7h
7i
0.020
>0.30
0.227
7j
4-CN-
7k
7l
4-MeOC(O)-
4-Me2NC(O)-
2,4-di-Cl-
2-Cl-4-MeO-
0.090
>0.30
0.014
7m
7n
0.008
a Values are the arithmetic means of at least two experiments. Standard deviation in the assay is less than ±10%.
b Concentration required to enhance ALPase activity in ST2 cells to 200% of that of control.
c
cLogP values were calculated by the CLOGP software (CLOGP Daylight Version 4.83, Daylight Chemical Information Systems,
Inc.).
Because compounds 7f and 7h showed potent activities, several hydrophilic functional groups were
introduced via ethylene or methyleneoxy linker to improve the physicochemical properties of compounds,
as shown in Table 4 and 5. When hydroxyl group was introduced, compound solubility was improved along
with retention of in vitro activity (8a). Because this result suggested that polar substituents are tolerated
around this position, amino or amide group was incorporated to obtain amide analog 8c, which exhibited
potent enhancement activity. The incorporation of oxygen linker resulted in reduced compound activity
(8d), whereas elongation of the linker of amide 8c increased compound activity (8e, EC200 = 4 ng/mL).
Next, we synthesized C-6 methyl analog 8f, which presented a 3-fold increase in activity. Based on the
superior activity of 7n to that of 7h, a 2-fluoro or 2-chloro group was introduced into the benzene ring. As
expected, these modifications improved compound in vitro activity by several folds, and compounds 8g and
8h were the most potent compounds with EC200 = 0.5 and 0.4 ng/mL, respectively. These compounds were
approximately 300-fold more potent than hit compound 1. Compared to compound 6n, alcohol 8a showed
better solubility, good metabolic stability. On the other hand, amide derivatives 8g and 8h had poor
solubility at neutral pH. Although compound 8g was moderately stable in human liver microsomes, it was
unstable in rat microsomes. Rat metabolic stability, as well as that of humans, was thought to be important
because ovariectomized (OVX) rats, a model of postmenopausal osteoporosis, were used for
pharmacological evaluation.
Table 4. Effects of substituents on ALP activity in ST2 cellsa and physicochemical properties

R2
N
NH2
4
O
6
R1
R2-
S
NH2
Comp.
7f
R1-
EC200 (μg/mL)b
cLogPc
H-
0.036
4.1
O
HO
8a
8b
8c
8d
8e
8f
H-
H-
0.025
0.058
0.010
0.023
0.004
0.0011
0.0005
0.0004
2.3
3.0
2.2
2.2
2.6
3.1
3.0
3.6
O
N
O
O
N
H-
O
O
O
N
H-
O
N
N
H-
O
O
O
Me-
Me-
Me-
O
O
O
N
F
8g
8h
O
N
Cl
O
a Values are the arithmetic means of at least two experiments. Standard deviation in the assay is less than ±10%.
b Concentration required to enhance ALPase activity in ST2 cells to 200% of that of control.
c
cLogP values were calculated by the CLOGP software (CLOGP Daylight Version 4.83, Daylight Chemical Information Systems,
Inc.).
Table 5. Effects of substituents on physicochemical properties
Metabolic stability (% remainig)b
human rat
Comp.
Solubility (μg/mL)a

8a
8g
8h
39
0.3
0.2
93
60
25
75
12
11
a Solubility in Japanese pharmacopoeia 2nd-fluid (pH 6.8) (JP2) solution.
b Stability described as % remaining after 30-min incubation of test compounds in 0.5 mg/mL human/rat liver microsomes.
Next, several compounds selected based on their in vitro activity and physicochemical properties were
evaluated for inhibitory effect on P450 activity (Table 6). Tetrahydropyran (6k) did not show DI nor TDI of
P450s. Although unsubstituted phenoxy analog 6n did not show TDI of CYP3A4, it showed weak inhibition
of CYP1A2. Alcohol 8a was improved compared to compound 6n, and did not show DI nor TDI of P450s.
Amide derivatives 8g and 8h showed TDI probably owing to their somewhat high lipophilicity
Table 6. P450s inhibition data for thieno[2,3-b]pyridine derivatives
DI of P450 isozymes (% inhibition)a
CYP3A4 TDI
Compound
(% remaining) b
1A2
2C9
2C19
2D6
3A4
1
2
61
0
0
1
0
94
28
14
11
41
1
71
1
96
1
34
0
49
2
6k
6n
8a
8g
8h
89
93
88
47
25
13
7
15
8
5
9
3
0
10
6
17
30
10
11
10
15
20
40
a % inhibition of P450s activities with test compounds at 10 μM using typical substrates of each P450s in P450s expression system
b
% remaining activity of CYP3A4 in human liver microsome after pre-incubation of test compounds (100 μM) compared with the
activity after pre-incubation of solvent
Next, we evaluated PK parameters in rats treated with several compounds selected based on their in vitro
activity and physicochemical properties (Table 7). As expected, tetrahydropyran 6k, which had good
physicochemical properties, showed good PK profiles. Alcohol 8a exhibited low Cmax and AUC despite its
good metabolic stability in rat liver microsomes. This may occur because its solubility was still insufficient,
or metabolism by an enzyme other than P450s was possibly involved. Amide 8g, the most potent compound
in this series, also suffered from poor oral exposure correlated to poor physicochemical properties.
Table 7. PK parametersa
Dose
(mg/kg)
30
Cmax
(μg/mL)
10.4
AUC0-8h
(μg h/mL)
29.8
Compound
6k
8a
8g
60
1.54
3.58
60
0.096
0.591

a F344 rats. Mean (N = 2).
Finally, compound 6k, which showed slightly less potent in vitro activity but had much better PK profiles,
was selected for in vivo pharmacological study. Compound 6k was orally administered to OVX rats at a
dose of 30 mg/kg/day. After five weeks, their aBMD of the distal and proximal femurs were compared with
that of the vehicle control and sham-operated rats. As shown in Figure 2, vehicle control OVX rats showed
significant decreases in aBMD compared to sham-operated rats, and treatment with compound 6k
significantly increased aBMD of all parts, which recovered to almost equal to that of sham-operated rats. In
addition to the previous results of N-phenyl homopiperazine derivative 2,5 the structurally different alkoxy
compound 6k showed significant improvements in aBMD as well, which provide confidence in our lead
discovery and optimization strategy of novel bone anabolic agents.
Figure 2. Effects of compound 6k (30 mg/kg) on areal bone mineral density (aBMD) in ovariectomized
(OVX) rats. Data are presented as the mean ± SE (n = 8). ##p < 0.01 versus the Sham group, *p < 0.05, **p
< 0.01 versus vehicle (Vehi.) group, as determined using Student’s t-test.
In summary, we successfully discovered 4-alkoxy-thieno[2,3-b]pyridine derivatives as novel ALPase
enhancers without TDI of CYP3A4. Among these derivatives, compound 6k exhibited statistically
significant improvements in aBMD at 30 mg/kg. Furthermore, extensive SAR studies led to the discovery of
phenoxy analog 8h, which showed more than 300-fold increase in ALPase activity. However, phenoxy
derivatives did not show good PK profiles. These compounds are considered as useful chemical probes
requiring further investigation of mechanism of action, and thus, mechanistic studies are underway.
Furthermore, the drug discovery program is being continued, and further structure optimization and
biological evaluations of the new template will be reported in due course.
Acknowledgments
We would like to thank Dr. Kazumasa Aoki and Dr. Shigeo Yamanoi for their encouragement and helpful
support.
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