Discovery of novel 2-[2-(3-hydroxy-pyridin-2-yl)-thiazol-4-yl]-acetamide derivatives as HIF prolyl 4-hydroxylase inhibitors; SAR, synthesis and modeling evaluation

Article abstract of DOI:10.1016/j.bmcl.2014.05.003

The design, synthesis, and capacity to inhibit HIF prolyl 4-hydroxylases (PHDs) are described for 2-[2-(3-hydroxy-pyridin-2-yl)-thiazol-4-yl]-acetamide analogs. These analogs revealed two kinds of novel scaffolds as PHD2 inhibitors. Synthetic routes were

Full text of DOI:10.1016/j.bmcl.2014.05.003

Accepted Manuscript
Discovery of Novel 2-[2-(3-Hydroxy-pyridin-2-yl)-thiazol-4-yl]-acetamide De-
rivatives as HIF Prolyl 4-Hydroxylase Inhibitors; SAR, Synthesis and Modeling
Evaluation
Yong Rae Hong, Hyun Tae Kim, Seonggu Ro, Joong Myung Cho, Sang Hwi
Lee, In Su Kim, Young Hoon Jung
PII:
S0960-894X(14)00490-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.003
BMCL 21616
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 November 2013
22 April 2014
2 May 2014
Please cite this article as: Hong, Y.R., Kim, H.T., Ro, S., Cho, J.M., Lee, S.H., Kim, I.S., Jung, Y.H., Discovery of
Novel 2-[2-(3-Hydroxy-pyridin-2-yl)-thiazol-4-yl]-acetamide Derivatives as HIF Prolyl 4-Hydroxylase Inhibitors;
SAR, Synthesis and Modeling Evaluation, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/
10.1016/j.bmcl.2014.05.003
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Graphical Abstract
Discovery of Novel 2-[2-(3-Hydroxy-pyridin-2-yl)-thiazol-4-yl]-
acetamide Derivatives as HIF Prolyl 4-Hydroxylase Inhibitors;
SAR, Synthesis and Modeling Evaluation
Yong Rae Honga, Hyun Tae Kima, Seung-Chul Leea, Seonggu Roa*, Joong Myung Choa,
Sang Hwi Lee b, In Su Kim b and Young Hoon Jungb**
H313
H374
R322
D315
Fe2+
Y329
R383
Y303

Discovery of Novel 2-[2-(3-Hydroxy-pyridin-2-yl)-thiazol-4-yl]-
acetamide Derivatives as HIF Prolyl 4-Hydroxylase Inhibitors;
SAR, Synthesis and Modeling Evaluation
Yong Rae Honga,b
,
Hyun Tae Kima
, , ,
Seonggu Roa,* Joong Myung Choa Sang Hwi Leeb,
In Su Kimb, Young Hoon Jungb,**
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
This is where the receipt/accepted dates will go; Received Month XX, 2000; Accepted Month XX, 2000 [BMCL RECEIPT]
Abstract—The design, synthesis, and capacity to inhibit HIF prolyl 4-hydroxylases (PHDs) are described for 2-[2-(3-hydroxy-pyridin-2-
yl)-thiazol-4-yl]-acetamide analogs. These analogs revealed two kinds of novel scaffolds as PHD2 inhibitors. Synthetic routes were
developed for the preparation of their analogs containing the new scaffolds. In addition, the structure-activity relationship (SAR) of the 2-
[2-(3-hydroxy-pyridin-2-yl)-thiazol-4-yl]-acetamide derivatives and their biological activities were reported. The complex structure of
compound 18 with PHD2 was also obtained for the purpose of more efficient lead optimization.
Hypoxia–inducible factor (HIF) is a transcription factor
and key regulator for the body’s systemic and local
responses to oxygen deprivation (hypoxia). It was
identified in 1992 by Wang and Semenza and found to
bind to the hypoxia-responsive element (HRE) in the
enhancer region of the erythropoietin gene. Through
transcriptional activation, HIF regulates various
physiological processes such as angiogenesis,
erythropoiesis, energy utilization, vascular tone,
apoptosis, and cellular proliferation.1, 2
HIF itself is primarily regulated by prolyl hydroxylases
(PHD1, 2, 3) as well as asparginyl hydroxylase (FIH).
The HIF transcription factor consists of an HIF-α/β
heterodimer that binds to hypoxia-response elements
within the HIF target genes to control transcription.3
HIF-α is a constitutively produced protein whose
stability is primarily regulated by cellular pO2. Under
normoxic conditions, the PHDs specifically hydroxylate
converse proline residues (Pro402 and Pro564 on
human HIF-1α, Pro405 and Pro531 on human HIF-2α),
creating recognition sites for the von Hippel-Lindau
protein (VHL) for the purpose of proteasomal
degradation. Thus, under adequate oxygen levels, the
body continually expresses and degrades HIF-α.4
Proline hydroxylation of HIF-α is catalyzed by PHDs in
a reaction involving Fe (II), 2-Oxoglutarate (2-OG) and
O2. As pO2 falls, the prolyl group of HIF-α is poorly
hydroxylated and protein degradation is slowed;
consequently, HIF-α mediated transcription occurs.
Therefore, stabilization of HIF-α by inhibition of PHDs
could be a potential therapy for anemia, neuro-
protection, and ischemic diseases including stroke.5, 6, 7, 8
For this reason, many research programs are at various
stages in the investigation of small molecule inhibitors
of PHDs, as listed in Table 1.9, 10
Table 1. Development status of the known PHDs inhibitors
Cpds.
No.
Chemical
structure
Name
Company
Clinical stage
1
2
3
4
5
6
DFO
NOG
-
Prototype
Prototype
Prototype
Phase II
Phase II
Phase II
Disclosed
Disclosed
-
FG-0041
FG-2216
FG-4592
AKB-6548
Fibrogen
Fibrogen
Fibrogen
Akebia
Disclosed
Disclosed
Undisclosed
Undisclosed
Palkyon
(CrystalGenomics)
7
PN-3602
Pre-clinical Undisclosed
Pre-clinical Disclosed
__________________
Keywords: HIF-PHs (PHDs); stabilizer; inhibitor; EPO;
* Corresponding author. Tel.: +82 31 628 2801; fax: +82 31 628
2701; e-mail: sgro@cgxinc.com
** Corresponding author. Tel.: +82 31 290 7711; fax: +82 31 292
8800; e-mail: yhjung@skku.edu
8
9
GSK360A
GSK
GSK
Bayer
GSK1278863
Phase II
Phase I
Undisclosed
Undisclosed
10 BAY85-3934

One of the pioneers in commercial research on prolyl
hydroxylase inhibition is Fibrogen, which is running
some of the most advanced stage clinical trials for the
therapeutic use of the HIF stabilizers FG-2216 and FG-
4592. The second generation FG-4592 is currently
enrolling for Phase IIa and IIb trials in the USA.11, 12, 13
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 in several countries.14, 15, 16
GSK360A has also entered the pre-clinical development
stage.17
compounds were synthesized, we tested these
predictions using the fluorescence polarization method
to determine the capacity of the compounds to inhibit
PHD2.19 The structure activity relationship (SAR) from
hit to lead is highlighted in Table 2. The introduction of
CN to interact with Y329 and R383 substantially
increased the inhibition of PHD2 activity. Compound
16 showed similar activity to FG-2216 due to these
additional interactions, as might be expected.
Table 2. Inhibitory activities of designed compounds to increase
additional interactions against the PHD2 enzyme
Compounds
Structure
IC50 (μM)
Stdev.
0.1
Akebia is also currently recruiting CKD (Chronic
Kidney Disease) patients for Phase II trials on AKB-
6548 administered once daily to test changes in Hb
levels.18
OH
O
OH
N
H
FG-2216
3.9
N
O
Cl
In the present paper, we present novel scaffolds for
PHD inhibitors that differ from PN-3602. The initial
hits were designed based on the binding moiety of 2-OG
with PHD2 as a substrate mimic. Our studies were
initiated by overlapping the designed hits to the 2-OG
complex structure [PDB: 3OUJ], as shown in Figure 1.
OH
OH
O
HN
11
27.5
0.7
N
N
N
OH
O
OH
N
12
13
195.0
81.0
7.1
1.4
O
S
H
N
OH
N
S
O
Fe2+
N
R322
OH
O
HN
N
Y329
14
15
13.5
6.0
2.1
0.7
NC
N
OH
O
OH
OH
N
NC
O
H2O
R383
S
N
R252
D254
H
N
OH
N
S
16
5.4
0.6
Y303
NC
O
N
EPO expression due to PHD2 inhibition was determined
in Hep3B cells using the EPO ELISA method20 EPO
expression results for each compound is presented in
Figure 2.
Figure 1. Overlap of the stick form of compound 11 (yellow), compound
12 (blue), and 2-OG (cyan) showing hydrogen bonding interactions with
amino acids in the active site of PHD2. [PDB: 3OUJ] Black dotted lines
indicate interactions between 2-OG and PHD2 in complex with 2-OG.
Red dotted lines indicate expected interactions between each compound
and PHD2. Pink circle shows that these charge-charge interactions of 2-
OG are absent in compounds 11 and 12. PDB code 3OUJ
50
EPO Immunoassay
45
In overlapping studies, the designed initial hits of
compound 11 and compound 12 in Figure 1 had similar
fits to the active site as seen with 2-OG, but enzymatic
activities of these compounds for PHD2 were no better
than those of FG-2216 of Fibrogen (Table 2). The
binding showed an additional interaction point where
charge-charge interactions could occur with Y329 and
R383, similar to 2-OG. A lead generation study based
on the hits was designed to prepare new analogs of
compounds 14~16 by the introduction of a CN (nitrile)
group in the 5-position of the pyridine ring for the
interaction with Y329 and R383. After these
40
50uM
35
100uM
30
25
20
15
10
5
0
DMSO
FG-2216
11
12
13
14
15
16
Compounds
Figure 2. EPO secretion in Hep3B cells due to PHD2 inhibition 24hr
after treatment with each compound

R4
OH
The initial structure-activity relationship (from Table 2
and Figure 2) of the synthesized analogs revealed that
compounds 11 and 16 at 50μM caused the greatest EPO
secretion; however, compound 11 is an undesirable
candidate since it showed significantly decreased EPO
secretion at 100μM. An SAR exploration of the PHD2
inhibitor for novel two classes was enabled by the
successful development of optimal synthetic routes.
Compound 11 analogs were prepared for various ―R1‖
according to Scheme 1.
R6
N
S
NC
O
R5
N
IC50
(µM)
Analogs
R4
R5
R6
NHCH2Ph
Stdev.
16
17
H
H
H
H
3.9
2.2
0.6
0.0
NHCH2COOH
O
18
H
H
7.3
0.4
HN
OH
H
N
R1
R1
OH
NH
NH2
OH
R1
F
O
O
b
O
N
HN
OH
N
N
N
CN
a
19
H
H
2.8
0.0
NH2
(R1=H or CN)
P1
P2
O
O
OH
OH
EtO
OH
OEt
OH
O
N
N
O
N
O
d
EtO
c
R1
HN
OH
R1
N
OH
20
H
H
2.6
0.4
N
N
OEt
P4
P3
Scheme 1. Reagents and conditions of the synthetic route for compound
11 analogs: (a) t-BuOK/DMF, r.t., 62~71%; (b) 5% Pd-C/ MeOH, under
H2 gas, r.t., 92~98%; (c) NaOEt, NaBH4, NiCl2/ EtOH, 70ºC, 20~33%;
(d) LiOH/H2O, MeOH, THF, 70ºC, 52~63%;
HN
21
22
H
H
H
H
2.1
2.4
0.0
0.2
Compound 16 analogs for various ―R2 and R3‖ were
also produced by the synthetic route shown in Scheme 2.
OH
HN
S
OEt
OEt
O
O O
Br
O
Br
OH
NH2
Cl
Br
NO2
CN
HS
P
OH
a
N
CN
N
c
N
23
24
H
H
H
H
NHCH2CH2Ph
3.4
3.0
0.3
0.0
b
S
P5
P6
HN
HN
HN
HN
HN
HN
Br
OH
NC
OH
N
N
N
N
N
N
d
N
N
N
N
O
S
O
S
P8
O
25
26
27
28
H
H
CH3
Ph
1.2
24.0
0.7
0.0
1.4
0.1
0.1
O
P7
NC
R2
R3
OH
NC
OH
HN
e
N
N
R2
N
N
OH
S
N
f
S
R3
O
O
P10
(R2, R3= H or Alkyl)
P9
CH3
H
Scheme 2. Reagents and conditions of the synthetic route for compound
16 analogs: (a) NaOMe/MeOH, reflux, 76~82 %; (b) H2O, 90ºC,
35~48%; (c) LiI/n-BuOH, 140ºC, 55~64%; (d) Zn(CN)2, Pd(PPh3)4/DMF,
microwave (160ºC, 150W, 25min.), 63~69%; (e) NaOH/THF, EtOH, H2O,
r.t., 87~93%; (f) TBTU, Et3N/DMF, r.t., 68~76%;
CH3 CH3
0.8
29
30
31
CH3
Ph
5.7
1.4
2.0
0.2
0.1
0.1
With the preliminary SAR established, our strategy was
to maintain compound 16 as our core scaffold to expand
its derivatives and examine the substitution effects on
the amide side chain and aromatic rings. As shown in
Table 3, replacement of the hydrogen with a methyl at
the R4 position and introduction of a branched
acetamide chain at the R6 position increased the
inhibitory activity, whereas R5 substitutions had no
influence on the activity. Compound 33 showed the best
activity of these analogs, with an IC50 of 0.4μM.
CH3 CH3
NHCH2CH3
HN
H
H
H
HN
N
N
32
33
CH3
3.8
0.4
0.3
0.1
HN
CH3 CH3
EPO secretion following PHD2 inhibition by the new
analogs was determined in Hep3B cells using the EPO
ELISA method. EPO secretion results for each
Table 3. Inhibitory activities of compound 16 analogs against the PHD2
enzyme

21
22
23
24
25
26
27
28
29
30
31
32
33
>188
>188
13.6
>181
78.4
117
Medium (5.1)
Medium (5.0)
Medium (5.4)
Medium (5.8)
Medium (5.8)
High (4.3)
Low (10.0)
High (3.0)
High (3.0)
High (4.5)
compound are presented in Figure 3.
EPO Immunoassay
50
45
40
35
30
25
20
15
10
5
11.2
6.4
0.74
13.9
107.5
>185
85.9
50µM
Medium (5.7)
Low (10.0)
Low (10.0)
100µM
We attempted to confirm the binding elements
responsible for the potent inhibition of the target protein
with the analogs of the series shown in Table 3 by
0
Compounds
obtaining
a co-crystal structure of PHD2 with
Figure 3. EPO secretion by PHD2 inhibition after 24hr from each
compound treatment in Hep3B cells
compound 18 from this series. The co-crystal structure
of compound 18 with PHD2 [PDB: 4KBZ] is presented
in Figure 4 and is overlapped with FG-2216 in [PDB:
2HBT] to determine any similarities or differences in
the interactions with the PHD2 protein.
The secondary SAR (Table 3 and Figure 3) of the newly
synthesized analogs revealed that compounds of 21 and
22 generated superior EPO secretion than did FG-2216
at 50μM. However, neither compound showed a
continuous increase in EPO secretion until they were
added at a high concentration, and EPO secretion
somewhat decreased at 100μM. Although the reason for
this is still not clear, EPO secretion seen at 50μM may
be sufficient to cure a disease like anemia because the
blood concentration of the inhibitor would not reach
100μM in the real body system. Otherwise, some
derivatives (compound 23, 24, 25, 27, 28 and 29) that
R6 position is replaced with phenethyl amine or pyridine
ethylene amine showed similar EPO secretion than did
FG-2216 at 50μM and showed a continuous increased
EPO secretion until high concentration treatment. But
they did not show superior EPO secretion than did FG-
2216 at 100μM. Also, according to PHD2 inhibitory
activities from compound 24 to compound 29 in Table 3,
we know that methyl showed better the inhibitory
activity than hydrogen in R5 position and bulky group
like phenyl decreased the inhibitory activity in R4
position. In case of compound 33 which showed highest
inhibitory activity for PHD2, it did not show increased
EPO secretion until high concentration treatment. This
disagreement between enzymatic and cellular activities
can be explained by its physicochemical properties. In
Table 4, the permeability of compound 33 through
PAMPA assay is very low therefore compound 33
seems hard to transmit a cell wall. Low EPO secretions
of compound 17, 19, 20 and 32 are also explained with
the same reason.
H313
H374
R322
D315
Fe2+
Y329
R383
Y303
Figure 4. Superimposition of FG-2216 (magenta) in [PDB: 2HBT] over
PHD2 (cyan) in complex with compound 18 (grey) [PDB: 4KBZ].
Compound 18 binds to the Fe(II) by bidentate
coordination through nitrogen of its hydroxyl pyridine
ring and nitrogen of the thiazole ring. This mode of
binding enables the formation of a hydrogen bond
between the hydroxyl moiety of compound 18 and the
conserved Y303.The charge-charge interaction between
Y329 and R383 and the CN group of compound 18,
which is similar to the interaction shown by the
carboxylate of FG-2216, is important for increasing the
inhibitory action. This novel interaction could explain
the improved potency of the 2-[2-(3-hydroxy-pyridin-2-
yl)-thiazol-4-yl]-acetamide derivatives relative to the
compounds lacking a CN group at the 5-position of the
pyridine ring.
Table 4. Physicochemical properties for the compounds of Table 3
Solubility
(μg/mL at pH7.4)
Permeability in
PAMPA (-logPe)
Analogs
16
17
18
19
20
24.1
105.8
28.9
>195
>195
Medium (5.6)
Low (10.0)
Medium (5.7)
Low (10.0)
In summary, we established the structure-activity
relationship of the 2-[2-(3-hydroxy-pyridin-2-yl)-
thiazol-4-yl]-acetamide analogs, which were designed
Low (10.0)

based on the 2-OG of the PHD substrate. The use of
these analogs revealed two kinds of novel scaffolds as
PHD2 inhibitors and synthetic routes were also
developed for the preparation of analogs based on the
new scaffolds. In addition, their biological activities
were reported; some compounds showed superior
inhibition of enzymatic activities and promoted greater
EPO secretion than did the FG-2216 lead compound of
Fibrogen. The complex structure of compound 18 with
PHD2 was obtained to permit more efficient lead
optimization. Further studies on the therapeutic
potential of selected analogs are in progress.
14.GlaxoSmithKline. A study to investigate the safety,
tolerability, Pharmacokinetics and pharmaco-
dynamics of GSK1278863A in Healthy Subjects.
Available
at:
http://clinicaltrials.gov/ct2/show/
NCT00750256 [4 August 2011].
15. GlaxoSmithKline. Relative bioavailability study for
GSK1278863A. Available at: http://clinicaltrials.
gov/ct2/show/NCT01319006 [4 August, 2011].
16. GlaxoSmithKline. Repeat dose safety and efficacy
study for compound to treat anemia. Available at:
http://clinicaltrials.gov/ct2/show/NCT01047397 [4
August 2011].
17. Bao, W.; Qin, P.; Needle, S.; Erickson-Miller, C. L.;
Duffy, K. J.; Ariazi, J. L.; Zhao, S.; Olzinski, A. R.;
David, J.; Teg Pipes, G. C.; Jucker, B. M.; Hu, E.;
Lepore, J. J.; Willette, R. N. J. Cardiovasc. Pharm.
2010, 56, 147.
18. Akebia Therapeutics Inc. Akebia announces
positive results for AKB-6548 phase 2A clinical
study. Available at: http://www.akebia.com/news/
AKB6548_Phase2a_SD_Completion.pdf [28 July
2011].
Acknowledgments
The authors would like to acknowledge the assistance of
Dr. Dongkyu Shin for the help in preparation of co-
crystal figures for this work. We also wish to express
our gratitude for the initial support and efforts from
CrystalGenomics in in vitro studies.
19. Cho, H.; Park, H.; Yang E. G. Biochem. Biophys.
Res. Commun. 2005, 337, 275.
20. Chang, K. H.; Stevenson, M. M. J. Immunol.
Methods 2002. 262, 129.
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