[(4-Hydroxyl-benzo[4,5]thieno[3,2-c]pyridine-3-carbonyl)-amino]-acetic acid derivatives; HIF prolyl 4-hydroxylase inhibitors as oral erythropoietin secretagogues

Article abstract of DOI:10.1016/j.bmcl.2013.08.067

A new series of PHD (HIF prolyl 4-hydroxylase) inhibitors was designed based on the X-ray co-crystal structure of FG-2216. Using a lead generation process, a series of [(4-Hydroxyl-benzo[4,5]thieno[3,2-c]pyridine-3-carbonyl)- amino]-acetic acid derivatives was developed as potent PHD2 inhibitors. This class of compounds also showed the ability to stabilize HIF-α, to stimulate EPO secretion in in vitro studies, and to increase hematocrit, red blood cell count, and hemoglobin levels in an animal efficacy study.

Full text of DOI:10.1016/j.bmcl.2013.08.067

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5953–5957
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
[(4-Hydroxyl-benzo[4,5]thieno[3,2-c]pyridine-3-carbonyl)-amino]-
acetic acid derivatives; HIF prolyl 4-hydroxylase inhibitors as oral
erythropoietin secretagogues
a
b
Yong Rae Hong a,b, Hyun Tae Kim a, Seung-Chul Lee a, Seonggu Ro a, , Joong Myung Cho , In Su Kim ,
⇑
Young Hoon Jung b,
⇑
a CrystalGenomics, Inc., 5F, Tower A, Korea Bio Park 694-1, Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Republic of Korea
b School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 June 2013
Revised 31 July 2013
Accepted 14 August 2013
Available online 23 August 2013
A new series of PHD (HIF prolyl 4-hydroxylase) inhibitors was designed based on the X-ray co-crystal
structure of FG-2216. Using a lead generation process, a series of [(4-Hydroxyl-benzo[4,5]thieno[3,2-
c]pyridine-3-carbonyl)-amino]-acetic acid derivatives was developed as potent PHD2 inhibitors. This
class of compounds also showed the ability to stabilize HIF-a, to stimulate EPO secretion in in vitro stud-
ies, and to increase hematocrit, red blood cell count, and hemoglobin levels in an animal efficacy study.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
HIF-PH (PHD)
Stabilizer
Inhibitor
Erythropoiesis
Anemia is a common feature of advanced kidney disease and of
cancer therapy that destroys the bone marrow. Recently, treatment
of anemia has begun to focus on the application of small molecule
erythropoiesis-stimulating agents (ESAs) to increase the blood’s
capacity for oxygen transport. The advent of orally available
small-molecule ESAs such as hypoxia-inducible factor (HIF) stabi-
lizers in the development of novel anti-anemia therapies has been
revolutionary in terms of convenience and cost reduction of ane-
mia treatments.1–3
Hypoxia-inducible factor (HIF) is a transcription factor and a
key regulator of the body’s local response to oxygen deprivation
(hypoxia). Wang and Semenza revealed in 1992 that HIF binds to
the hypoxia-responsive element (HRE) in the enhancer region of
the erythropoietin gene. The following transcriptional activation
was shown to regulate various physiological processes such as
angiogenesis, erythropoiesis, energy utilization, vascular tone,
apoptosis, and cellular proliferation.4,5
Under normoxic conditions, these PHDs specifically hydroxylate
converse proline residues (Pro402 and Pro564 on human HIF- ).
These hydroxylated prolines of HIF- create recognition sites for
a
a
the von Hippel–Lindau protein (VHL) for proteasomal degradation.
Thus, under adequate oxygen levels, the body continually ex-
presses and degrades HIF-a 7
.
Stabilization of HIF-a by inhibition of PHDs could be a potential
therapy for anemia, neuro-protection, ischemic disease including
stroke, and complications of diabetes.8–11 For these reasons, many
small molecule drug candidates have been proposed as PHD2
inhibitors with a wide range of potencies measured from the dis-
covery stages to clinical trial stages, as shown in Figure 1.12,13
One of the pioneers in the field of commercial research on pro-
pyl hydroxylase inhibition is Fibrogen, which runs clinical trials for
the therapeutic use of HIF stabilizers in the most advanced stages.
FG-2216, the lead drug candidate, has completed Phase II trials,
while the second generation FG-4592 is currently enrolling for
Phase IIa and IIb trials in the USA.14–16
GlaxoSmithKline has been testing its investigational HIF prolyl
hydroxylase inhibitor GSK1278863 for the treatment of anemia
since 2008. It has completed several Phase I studies in the USA
and is currently running Phase II studies with pre-dialysis and
hemodialysis-dependent patients in Australia, New Zealand, India,
and the Russian Federation. Recently, the drug candidate GSK360A
entered the stage of a pre-clinical development program. GSK360A
was reported to mediate cardioprotective effects and to raise EPO
and Hb levels in rats.
HIF itself is primarily regulated by prolyl hydroxylases (PHD1, 2,
3) as well as asparaginyl hydroxylase (FIH).
The HIF transcription factor consists of an HIF-a/b heterodimer
that binds to hypoxia-response elements within the HIF target
gene to control transcription.6
⇑
Corresponding authors. Tel.: +82 31 628 2801; fax: +82 31 628 2701 (S.R.);
tel.: +82 31 290 7711; fax: +82 31 292 8800 (Y.H.J.).
E-mail addresses: sgro@cgxinc.com (S. Ro), yhjung@skku.edu (Y.H. Jung).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.067

5954
Y. R. Hong et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5953–5957
OH
N
O
O
NH2
O
O
O
H
N
HO
N
O
HO
OH
HO
N
H
O
O
NOG
FG-0041
N
O
NH
O
NH
O
O
Cl
O
O
HO
H
N
DFO
O
N
O
O
N
H
OH
FG-041
OH
N
MOG
OH
N
O
O
OH
O
F
N
H
OH
AKB-6548
FG-4592
N
H
O
N
O
Cl
FG-2216
(Structure undisclosed)
GSK360A
(Structure undisclosed)
Figure 1. Examples of the known PHD2 inhibitors.
140
120
100
80
EPO Immunoassay
50uM
100uM
60
40
20
0
DMSO FG-2216
1
2
3
4
5
Compounds
Figure 2. The known binding mode of FG-2216 with the catalytically active domain
of human PHD2 (PDB: 2HBU) and its additional optimization points to increased
hydrophobic interactions; the active site is small and narrow, so the testing
compounds of the new class were designed to fill the empty spaces with
hydrophobic functional groups.
Figure 3. EPO secretion by PHD2 inhibition after 24hr from each compound
treatment in Hep3B cell.
Table 2
Inhibitory activities of the [(benzo[4,5]thieno[3,2-c]pyridine-3-carbonyl)-amino]-
acetic acid analogs against the PHD2 enzyme
Table 1
Compounds
R2
R3
R4
R5
PHD2 IC50
(
lM)
SD
Inhibitory activities of compounds designed to increase additional interactions
against the PHD2 enzyme
FG-2216
4
3.9
1.5
1.8
4.1
2.8
1.7
0.5
1.3
0.1
0.1
0.1
0.2
0.2
0.1
0.0
0.1
H
H
F
H
H
H
H
H
H
H
F
H
H
OH
H
N(Et)2
H
H
H
H
H
H
H
F
Compounds
X
R1
PHD2 IC50
(lM)
Stdev.
14
15
16
17
18
19
FG-2216
–
O
S
S
S
S
–
H
H
Me
Cl
CN
3.9
45.5
8.6
1.9
1.5
0.7
0.1
6.4
0.8
0.1
0.1
0.1
1
2
3
4
5
OH
H
H
H
Akebia Therapeutics, Inc., founded in 2007, has in-licensed pre-
clinical programs on the HIF prolyl hydroxylase inhibitor (AKB-
6548) at Proctor & Gamble. Akebia is currently recruiting CKD
(Chronic Kidney Disease) patients for Phase II trials to test the
change in Hb levels following once-daily administration.
In 2006, two independent working groups succeeded in co-crys-
tallizing a catalytically active domain of human PHD2 with
FG-2216 or its derivative and analyzed the purified complexes
by X-ray crystallography.17 The solved structures provided a new
foundation for the understanding of the molecular mechanism of
PHD catalysis and inhibition and offered new perspectives for the
development of novel, more specific PHD inhibitors (Fig. 2).
In the binding observed, we can find two optimization points
that are well suited to fill the predominantly hydrophobic pockets.
Additional interactions of these pockets have not been reported in
known co-crystal structures. The optimization study predicted
new analogs of compounds 1–5 and these were synthesized to test
these predictions. The synthesized analogs were tested for their
activities of PHD2 inhibition using a fluorescence polarization
method.18 These structure activity relationships for ‘X’ and ‘R1’
are highlighted in Table 1. When ‘X’ is a sulfur atom rather than
an oxygen atom, the inhibitory activity of PHD2 is substantially in-
creased, and compounds 3–4 show higher activities than FG-2216
due to additional interactions, as expected.

Y. R. Hong et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5953–5957
5955
O
O
F
R
R
S
S
R
O
OMe
OMe
c
a
b
H
N
O
O
O
HS
BMD
OMe
d, e
6
OMe
Br
7
O
OH
OH
O
R
S
R
S
R
OMe
O
S
f
OMe
OMe
N
N
N
R
Cl
BMD
10
8
9
OMe
OH
O
OH
O
R
S
S
OMe
g
h, i
O
OH
N
H
N
N
O
OMe
H2N
Cl
Cl
12
11
S
OH
O
R
OH
j
N
H
N
O
Cl
13
Scheme 1. Reagents and conditions: (a) NaH/THF, 0 °C, then reflux, 45–57%; (b) NBS, (PhCO2)2/dichloromethane, reflux, 74–85%; (c) K2CO3/dichloromethane, reflux, 70–86%;
(d) t-BuOK/THF, ꢀ20 °C; (e) SOCl2/dichloromethane, 0 °C then 40 °C, 25–46% for 2 steps; (f) NCS, (PhCO2)2/dichloromethane, reflux, 65–72%; (g) LiOH/H2O, MeOH, THF, 40 °C,
85–90%; (h) oxalyl chloride/dichloromethane, rt; (i) Et3N/dichloromethane, rt, 61–75% for 2 steps; (j) LiOH/H2O, MeOH, THF, 40 °C, 72–80%.
FG-2216
Compound 4
Compound 16
EPO Immunoassay
140
120
100
80
50uM
100uM
HIF-1α
HIF-2α
60
40
Beta-
Tubulin
20
Figure 5. HIF-a stabilization in Hep3B cells by PHD2 inhibition due to compound 4
and 16 after 24 h incubation.
0
DMSO FG-2216
4
14
15
16
17
18
19
Compounds
The initial SAR (from Table 1 and Fig. 3) of the synthesized ana-
Figure 4. EPO secretion by PHD2 inhibition after 24 hr from each compound 4
analogs treatment in Hep3B cell.
logs revealed that compound 3 at 100
EPO secretion; however, compound 4 is a more desirable candidate
since it showed greater EPO secretion at 50 M. We enabled SAR
lM resulted in the greatest
l
The expression of EPO due to PHD2 inhibition was determined
using an EPO ELISA method19 in Hep3B cells. The EPO expression
results for each compound are presented in Figure 3.
exploration of novel classes of PHD2 inhibitors like compound 4
by successfully developing an optimal synthetic route and prepar-
ing analogs of various ‘R’ according to Scheme 1.
Table 3
Metabolic stability study and Mouse PK study for three compounds
Item
Sub-item/Compound
Compound 4
Compound 14
Compound 16
Metabolic stability, remaining % at 60 min
Method/Species
Microsomal
S9 fraction
Parameters/Route
Dose
Mouse
68.60%
125.70%
IV
4
–
Human
118.60%
100.90%
PO
20
0.5
15.1
53.7
–
Mouse
81.70%
85.00%
IV
4
–
–
6.3
3.4
Human
78.50%
77.30%
PO
20
0.5
3.9
13.7
–
–
–
Mouse
84.40%
106.70%
IV
4
–
Human
75.90%
126.50%
PO
Mouse PK
20
Tmax (h)
0.17
39.6
90.1
–
–
–
Cmax
(lg/mL)
–
–
AUC0–t
(lg h/mL)
17.0
2.79
235.3
503.4
63.2
31.1
3.23
128.2
401.6
57.7
T1/2 (h)
CL (mL/h kg)
Vss (mL/kg)
F (%)
–
–
–
635.8
831.6
43.6
–
–

5956
Y. R. Hong et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5953–5957
D5, QDx4
Secondary SAR (Table 2 and Fig. 4) of the new synthesized
analogs revealed that treatment with compounds 14 and 16 re-
sulted in good EPO secretion at 50 M and 100 M. A significant
Hematocrit
D8, QDx4
D13, QDx12
D16, QDx12
l
l
80
70
60
50
40
30
20
10
0
**
**
increase in EPO secretion was seen with the introduction of dieth-
ylamine to R4 (compound 14); this analog showed a nine-fold
*
**
**
*
improvement in EPO secretion compared to FG-2216 at 50 lM.
However, compound 14 did not show a continuous increase in
EPO secretion until it was added at a high concentration, while
EPO secretion was decreased at 100 lM. Further optimization of
compound 14 was achieved as shown by compound 16, which
demonstrated a greater than 3.4-fold and 1.6-fold increase in po-
tency compared to FG-2216 at 50 lM and 100 lM, respectively.
Compound 16 also showed no diminution of EPO secretion as
the concentration of the inhibitor increased, unlike the case for
compound 14. Therefore, compound 16 was considered to be a
more ideal candidate than compound 14. Additionally, we
checked the HIF stabilization effects of compound 4 and com-
Vehicle
FG2216
(50 mpk)
Cpd. 16
(3 mpk)
Cpd. 16
(10 mpk)
Cpd. 16
(30 mpk)
*; P<0.05; **; P<0.01 vs. vehicle of each blood sampling time; these
D5, QDx4
RBC
D8, QDx4
D13, QDx12
D16, QDx12
pound 16 for HIF-1
a and HIF-2a in Hep3B cells, and confirmed
superior effects of new synthesized analogs compared with
FG-2216 (Fig. 5).
Three compounds (compounds 4, 14, and 16) were also com-
pared for metabolic stability and mouse PK following exposure in
order to select a reasonable candidate. The results are presented
in Table 3.
12
10
8
*
**
*
6
All three compounds are quite metabolically stable in mouse
and human species. The bioavailabilities and half-lives of the three
compounds were very similar. However, among the three com-
pounds, compound 16 showed the best activity with respect to
Cmax and AUC in the mouse PK studies.
4
2
0
Vehicle
FG2216
(50 mpk)
Cpd. 16
(3 mpk)
Cpd. 16
(10 mpk)
Cpd. 16
(30 mpk)
Ultimately, compound 16 was selected as the final candidate for
an animal study. The efficacy study was designed to perform daily
administration for 4 days (QDx4) and 12 days (QDx12) in normal
ICR mice (male, 6 weeks old) like Table 4. The mice were divided
into 4 groups by blood sampling times (D5, D8, D13, and D16) with
respect to treatment with vehicle, FG-2216, or compound 16. Com-
pound 16 was also used at three doses (3 mpk, 10 mpk, and
30 mpk) in this efficacy study. The results for each group are pre-
sented in Figure 6.
The PD studies of FG-2216 and compound 16 revealed that
compound 16 had superior efficacy at a lower dose of 30 mpk
than did FG-2216 at 50 mpk. The hematocrit, red blood cell
counts, and hemoglobin levels, which all participate in erythro-
poiesis, were all increased by 30 mpk treatment with compound
16. A long-term treatment of 12 days was the most efficacious
method for treating anemia with compound 16, compared to
shorter-term treatments.
*; P<0.05; **; P<0.01 vs. vehicle of each blood sampling time
D5, QDx4
Hb
D8, QDx4
D13, QDx12
D16, QDx12
20
16
12
8
**
*
**
**
**
4
0
Vehicle
FG2216
(50 mpk)
Cpd. 16
(3 mpk)
Cpd. 16
(10 mpk)
Cpd. 16
(30 mpk)
*; P<0.05; **; P<0.01 vs. vehicle at each blood sampling time
In summary, we designed a new series of PHD2 inhibitors based
on the X-ray co-crystal structure of FG-2216. A lead generation
process was used to develop a series of [(4-Hydroxyl-benzo[4,5]
thieno[3,2-c]pyridine-3-carbonyl)-amino]-acetic acid derivatives
as potent PHD2 inhibitors. In addition, this class of compounds
Figure 6. Efficacy study by PHD2 inhibitions of FG-2216 and compound 16 in a
mouse model.
Using this synthetic route, the structure activity relationship for
phenyl group substitutions is highlighted in Table 2.
The expression of EPO following PHD2 inhibition by the new
analogs was determined using an EPO ELISA method and Hep3B
cells. The EPO expression results for each compound are presented
in Figure 4.
showed the ability to stabilize HIF-a, to stimulate EPO secretion
in in vitro studies, and to increase hematocrit, red blood cell
counts, and hemoglobin levels in an animal efficacy study.
Currently, pre-clinical studies are in progress to determine the
therapeutic potential of the selected analogs.
Table 4
Administration schedule of efficacy study
Groups/day
D1
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
D13
D14
D15
D16
p
D5, QDx4
D8, QDx4
D13, QDx12
D16, QDx12
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Y. R. Hong et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5953–5957
5. Patel, S. A.; Simon, M. C. Cell Death Differ. 2008, 15, 628.
5957
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Supplementary data
Supplementary data associated with this article can be found, in
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