Discovery of Clinical Candidate (5-(3-(4-Chlorophenoxy)prop-1-yn-1-yl)-3-hydroxypicolinoyl)glycine, an Orally Bioavailable Prolyl Hydroxylase Inhibitor for the Treatment of Anemia

Article abstract of DOI:10.1021/acs.jmedchem.0c01161

The design and discovery of a new series of (5-alkynyl-3-hydroxypicolinoyl)glycine inhibitors of prolyl hydroxylase (PHD) are described. These compounds showed potent in vitro inhibitory activity toward PHD2 in a fluorescence polarization-based assay. Remarkably, oral administration of 17, with an IC50 of 64.2 nM toward PHD2, was found to stabilize HIF-α, elevate erythropoietin (EPO), and alleviate anemia in a cisplatin-induced anemia mouse model with an oral dose of 25 mg/kg. Rat and dog studies showed that 17 has good pharmacokinetic properties, with oral bioavailabilities of 55.7 and 54.0%, respectively, and shows excellent safety profiles even at a high dose of 200 mg/kg in these animals. Based on these results, 17 is currently being evaluated in a phase I clinical trial for anemia.

Full text of DOI:10.1021/acs.jmedchem.0c01161

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Article
Discovery of Clinical Candidate (5-(3-(4-Chlorophenoxy)prop-1-
yn-1-yl)-3-hydroxypicolinoyl)glycine, an Orally Bioavailable
Prolyl Hydroxylase Inhibitor for the Treatment of Anemia
Xiaojin Zhang, Yonghua Lei, Tianhan Hu, Yue Wu, Zhihong Li,
Zhensheng Jiang, Changyong Yang, Lianshan Zhang, and Qi-Dong You
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c01161 • Publication Date (Web): 28 Jul 2020
Downloaded from pubs.acs.org on July 28, 2020
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Discovery of Clinical Candidate (5-(3-(4-
Chlorophenoxy)prop-1-yn-1-yl)-3-
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hydroxypicolinoyl)glycine, an Orally Bioavailable
Prolyl Hydroxylase Inhibitor for the Treatment of
Anemia
Xiaojin Zhang, a,c,1,* Yonghua Lei,a,b,1 Tianhan Hu, Yue Wu, Zhihong Li, Zhensheng Jiang,
a,b
a,b
c
a,b
d
d
a,b,*
Changyong Yang, Lianshan Zhang, Qidong You
a
State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and
Optimization, China Pharmaceutical University, Nanjing 210009, China
b
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 211198, China
Department of Chemistry, China Pharmaceutical University, Nanjing 211198, China
Shanghai Hengrui Pharmaceutical Co., Ltd., Shanghai 200245, China
c
d
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ABSTRACT
The design and discovery of a new series of (5-alkynyl-3-hydroxypicolinoyl)glycine inhibitors of
prolyl hydroxylase (PHD) is described. These compounds showed potent in vitro inhibitory
activity toward PHD2 in a fluorescence polarization-based assay. Remarkably, oral administration
of 17, with an IC50 of 64.2 nM toward PHD2, was found to stabilize HIF-α, elevate erythropoietin
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(
EPO), and alleviate anemia in a cisplatin-induced anemia mouse model with an oral dose of 25
mg/kg. Rat and dog studies showed that 17 has good pharmacokinetic properties, with an oral
bioavailability of 55.7% and 54.0%, respectively, and shows excellent safety profiles even at a
high dose of 200 mg/kg in these animals. Based on these results, 17 is currently being evaluated
in a phase I clinical trial for anemia.
KEYWORDS
HIF, PHD inhibitor, anemia, EPO
1. INTRODUCTION
Anemia is a condition characterized by abnormally reduced levels of circulating red blood cells
(
RBCs), or by the presence of RBCs that do not contain enough hemoglobin (Hb); as a result, an
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insufficient amount of oxygen reaches tissues and organs. Anemia is highly prevalent in patients
with chronic kidney disease (CKD) and cancer patients receiving chemotherapy. Over recent
decades, intravenous (iv) or subcutaneous injection of recombinant human erythropoietin (rhEPO)
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or its analogs has been considered as the standard of care for renal anemia. However, exogenous
rhEPO injection may cause serious cardiovascular side effects and intravenous iron as an adjuvant
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therapy is generally required. Recently, orally-active small molecule inhibitors of prolyl
hydroxylases (PHDs) have been clinically validated as novel and effective therapies for anemia.
Their mechanism of action is through the upregulation of endogenous EPO, thus overcoming the
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side effects of rhEPO therapy.4, 5
PHDs (PHD1-3 enzymes) are a family of iron-, 2-oxoglutarate (2-OG)-, and oxygen-
dependent dioxygenases. PHDs act as direct cell oxygen sensors to control the degradation of
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hypoxia-inducible factor (HIF), a crucial heterodimeric gene transcription factor involved in
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endogenous EPO formation and erythropoiesis. Under normoxic conditions, HIF-α, the regulatory
subunit of the HIF dimer, can be post-transcriptionally hydroxylated on certain proline residues,
catalyzed by PHD enzymes; the hydroxyl form of HIF-α is precisely recognized by the von
Hippel−Lindau tumor suppressor protein (pVHL), causing ubiquitin-mediated proteasomal
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degradation. Among the three PHD subtypes (PHD1, PHD2, and PHD3), PHD2 is the most
widely distributed isoform, playing a dominant role in regulating steady-state HIF-α and its
downstream gene expression, such as EPO.9, 10 Inhibiting PHD2 is considered to be an effective
strategy to upregulate the endogenous EPO level for the treatment of anemia.
Over the past two decades, an increasing number of PHD2 inhibitors have been
developed.5,10,11 To date, over ten PHD2 inhibitors have been subjected to clinical trials and three
of them have been approved.12-16 FG-2216 (1a) (Figure 1A) was the first PHD2 inhibitor to enter
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clinical trials from FibroGen. FG-4592 (1b, roxadustat) (Figure 1A), structurally related to FG-
2216 and also originally from FibroGen, was approved by the National Medical Products
Administration (NMPA) in China in December 2018 as a first-in-class PHD inhibitor for the
treatment of CKD-related anemia.12-14 Recently, on June 29, 2020, AKB-6545 (1c, vadadustat)15
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and GSK1278863 (1d, daprodustat) (Figure 1A), originally from Akebia and GlaxoSmithKline,
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respectively, were approved in Japan for the treatment of anemia due to CKD in dialysis-dependent
and non-dialysis-dependent adult patients.
Structurally, all of the PHD2 inhibitors in clinical trials or approved act as mimetics of the
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endogenous cofactor 2-OG. The co-crystal structure (PDB ID: 2G19) of a reported FG-2216-
like inhibitor with PHD2 revealed that the 3-hydroxylpicolinoylglycine moiety (marked in blue,
Figure 1A) in 1a-1c share three key interactions with the PHD2 catalytic pocket, including
bidentate coordination with ferrous ion, an ionic bonding interaction between carboxylic and
Arg383, and a hydrogen bonding interaction between phenol and Tyr303 (Figure 1B and 1E).17, 18
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Figure 1. (A) Chemical structures of FG-2216 (1a), the approved drugs FG-4592 (1b), AKB-
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548 (1c), and GSK1278863 (1d), and the alkynyl-containing compound (2) designed in this work.
(B) Binding pose of a reported FG-2216-like inhibitor (yellow) with PHD2 (brown) from the X-
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ray co-crystal structure (PDB ID: 2G19) . (C) Predicted binding pose of FG-4592 (yellow) with
PHD2. (D) Predicted binding pose of AKB-6548 (yellow) with PHD2. (E) Binding mode of the
FG-2216-like inhibitor (yellow) with the key residues (blue) in PHD2 catalytic sites (PDB ID:
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G19) and a superimposition with HIF peptide (green) that has been extracted from a PHD2-
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HIF-α peptide complex (PDB ID: 3OUJ) . The binding mode indicates that the 3-
hydroxylpicolinoylglycine moiety is the key pharmacophoric group binding to the PHD2 catalytic
site while the fused benzene ring and the phenyloxy substituent in FG-4592 and the substituted
benzene ring in AKB-6548 are probably responsible for preventing HIF from approaching the
PHD2 catalytic sites. (F) Predicted binding pose of 2 (yellow) with PHD2. (G) Predicted binding
mode of 2 (yellow) with the key residues (blue) in PHD2 catalytic sites (PDB ID: 2G19) and a
superimposition with HIF peptide (green) that has been extracted from a PHD2-HIF-α peptide
complex (PDB ID: 3OUJ).
Currently, structurally diverse PHD2 inhibitors have been developed by replacing the
isoquinone/pyridine cores in FG-4592/AKB-6548 with other heterocycles, such as quinolinone,19
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naphthyridinone, imidazopyridine, aza-benzimidazole, and benzoimidazole, or by changing
the formylglycine side chain into thiazole- or pyrazole-substituted carboxylic acids.22-24 Non-
carboxylic acid-type inhibitors for PHD2 have also been discovered by high-throughput
screening.25-27 With regard to the fact that most of these diverse PHD2 inhibitors are still under
preclinical study, the 3-hydroxylpicolinoylglycine moiety in FG-4592/AKB-6548 appears to be a
privilege scaffold for PHD2 inhibition. Here, we present a new series of alkynyl-containing PHD2
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inhibitors (2) designed through the scaffold hopping approach by replacing the rigid and linear
isoquinoline core/phenyl pyridine core in FG-4592/AKB-6548 with a rigid and linear 3-akynyl
pyridine core (Figure 1A). A fluorescence polarization (FP)-based assay, developed previously
using FITC-labeled HIF-α peptide as the probe, was utilized for structure-activity relationship
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(
SAR) optimization in our research. Based on the PHD2 inhibitory activity identified in vitro,
compounds of interest were selected for pharmacodynamic (PD) evaluation to assess their effects
on serum EPO in vivo. Oral administration of candidate compound 17 was found to stabilize HIF-
α, upregulate the endogenous formation of EPO, and effectively mitigate anemia in a cisplatin-
induced anemia mouse model. Further, preclinical evidence indicated that 17 has good
pharmacokinetic (PK) properties in rats and dogs and excellent early safety profiles in these
animals. Thus, 17 appears to be an orally-active clinical candidate for the treatment of anemia.
2
2
. RESULTS AND DISCUSSION
.1. Design and SAR optimization by in vitro PHD2 inhibitory activity.
When we conducted this research, the structures of FG-2216 and FG-4592 were reported and the
scaffold of AKB-6548 was disclosed. Based on the X-ray structure of a reported FG-2216-like
17
inhibitor (yellow) with PHD2 (brown) (PDB ID: 2G19) (Figure 1B), we performed molecular
docking studies using GOLD 5.1 software to predict the binding mode of FG-4592 (Figure 1C)
and AKB-6548 (Figure 1D); as seen, their picolinoylglycine moiety fit deeply into the catalytic
pocket of PHD2 (Figure 1B-1D). Further superimposition analysis (Figure 1E) demonstrated the
fused benzene ring and phenyloxy substituent in FG-4592 and the substituted benzene ring in
AKB-6548 oriented to the HIF binding region, suggesting that these rigid fused or substituted
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rings on the 3-hydroxypicolinoylglycine scaffold are probably responsible for their inhibitory
activity preventing HIF from approaching PHD2 catalytic sites.
In light of these findings, we initially designed compound 2 (Figure 1A), which maintains
the key pharmacophoric 3-hydroxylpicolinoylglycine moiety for PHD2 binding and bears a rigid
alkynyl substitution at the C5 site on the picolinoylglycine moiety to allow the extension of suitable
substituents and prevent HIF from approaching PHD2 catalytic sites (Figures 1F and 1G). It is
worth noting that the methyl group in FG-4592 was removed because it did not form any important
interaction with PHD2 (Figure 1C) and the rigid alkynyl substitution was set at the C5 site because
steric crashes with surrounding key residues will arise with C4 or C6 substitutions (Figure S1B-
D).
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Our efforts started with compound 3 with the simplest R group of H. In vitro FP-based
assays revealed that 3 was a moderately potent inhibitor toward PHD2 with an IC50 value of 2.9
μM (Table 1). Nevertheless, this starting compound represented a new scaffold for PHD2
inhibition deserving further optimization. Considering that the ethynyl group in 3 oriented to the
solvent region of PHD2 and played the role of preventing the HIF substrate from approaching the
PHD2 catalytic site, steric substituents such as tert-butyl, cyclopropyl, and phenyl groups were
introduced to the end of the ethynyl moiety, as seen in 4-6, respectively. Interestingly, their
inhibitory activities toward PHD2 were significantly improved when compared with 3; the phenyl
substituted 6 showed an IC50 value of 820 nM. We then introduced an additional phenyl group to
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, leading to great loss in activity, as seen in 7 (IC50 = 8103 nM). Because the phenyloxy moiety
3
in 1 is relatively flexible, we further introduced an sp -hybrid methylene group together with an
oxygen atom as a rotatable linkage. Encouragingly, the resulting compound, 8, possessed an IC50
value of 169 nM on PHD2 inhibition, turning out to be significantly more active than 6 and even
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more potent in vitro than the approved drug 1 (IC50 = 591 nM). Replacing the O atom in the linkage
of 8 to the NH group was detrimental for activity; compound 9 showed an IC50 value toward PHD2
of 326 nM, about two-fold less active than 8. Introducing methyl or phenyl to the NH moiety, as
in 10 and 11, resulted in further decreases in activity, with IC50 values of 454 nM and 651 nM,
respectively. Thus, these preliminary SAR data demonstrated that the phenyloxy methyl moiety
installed at the end of the ethynyl picolinoylglycine scaffold is preferable for PHD2 inhibitory
potency as compared to rigid and bulky substituents.
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Table 1. Preliminary SAR on the R group
compound
R
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PHD2 IC50 (nM)
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835 ± 3
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169 ± 1
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FG-4592 (1)
Subsequent SAR optimization set out to focus on the phenyl group of 8 (Table 2).
Substituents with different electronic properties, including fluoro, chloro, methyl, trifluoromethyl,
and methoxy, as in 12-26, were introduced to orthro, meta, and para sites of the phenyl. It was
found that, in all the cases except for methyl, para substitution was preferable compared to ortho
and meta substitution for PHD2 inhibitory activity. In particular, 17, with a para-chloro substituent,
showed a considerable increase in potency, with an IC50 of 64.2 nM toward PHD2; this made it
the most active compound among them almost nine-times more active than clinical drug 1. The
introduction of a weak electron-donating methyl group (18-20) seemed to be beneficial for activity;
the IC50 values of these compounds ranged from 70.4 nM to 125 nM, showing improved potency
as compared to 8. However, introducing a strong electron-withdrawing trimethyl group (21-23)
resulted in great loss in potency, with IC50 values ranging from 514 nM to 1073 nM. Methoxy-
substituted compounds (24-26) were found to have slightly decreased inhibitory activity as
compared to 8. In addition, para-substitutions with ethyl (27), cyano (28), and phenyl (29) were
all detrimental for PHD2 inhibition. Notably, 29, with a bulky phenyl substitution, was much less
active than 8. Moreover, replacing phenyl in 8 with a naphthyl group as in 30 caused about a two-
fold decrease in potency. The above results suggest that a strong electron-withdrawing or steric
moiety around the phenyl group of 8 is unfavorable and para-chloro substitution, as in 17,
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contributes greatly to the compound’s inhibitory activity. However, further introduction of another
chloro group, as in 31 and 32, resulted in decreased potency as compared to 17. Furthermore,
bringing a methylene link between the phenyl and oxygen atom (33) retained its activity, while
further extending to a two-atom linker (34) led to a significant loss in potency as compared to 17.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. SAR on the phenyl group of 8
compound
n
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
R
2-F
3-F
4-F
PHD2 IC50
444 ± 24
256 ± 4
186 ± 8
175 ± 3
587 ± 22
64.2 ± 1.1
70.4 ± 0.6
125 ± 5
110 ± 2
1073 ± 8
877 ± 5
1
1
1
1
1
2
3
4
5
6
2-Cl
3-Cl
4-Cl
2-Me
3-Me
4-Me
2-CF
3-CF
4-CF
2-MeO
3-MeO
4-MeO
4-Et
4-CN
4-Ph
17
1
1
2
2
2
8
9
0
1
2
3
3
3
23
24
514 ± 6
218 ± 21
428 ± 3
2
2
2
2
2
5
6
7
8
9
186 ± 4
378 ± 33
263 ± 15
825 ± 3
346 ± 1
450 ± 20
527 ± 6
30
31
-
2,4-di-Cl
3,4-di-Cl
4-Cl
3
3
2
3
84.3±12.7
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3
8
4
2
0
4-Cl
H
408 ± 7
169 ± 1
591 ± 13
FG-4592 (1)
0
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2.2. Molecular modeling.
To obtain deeper insight into the critical structural features of the binding mode between these
alkynyl compounds and the PHD2 enzyme, molecular docking studies were performed utilizing
the protein ligand docking software GOLD 5.1 for representative compounds 6 and 17. The
optimizing settings for docking procedures were established using the X-ray crystallographic
structure of a complex of wild-type PHD2 and an FG-2216-like inhibitor (PDB ID: 2G19; Figure
1
7
S1). Docking poses were analyzed using the visualization program PyMOL 2.3.0.
The binding mode of 6 with PHD2 was firstly analyzed by docking (Figure 2A). The results
showed that the pyridine nitrogen atom and the amide oxygen atom formed a 5-membered ring
with Fe(II) that was coordinated by the residues of His313, Asp315, and His375, together with
water, generating an octahedral structure. The terminal carboxy of the glycine moiety could form
an interaction network with the Tyr329 and Arg383 residues through hydrogen-bonding and
electrostatic interactions, respectively. Additionally, the phenolic hydroxyl of 6 showed hydrogen-
bonding with Tyr303 and the phenyl extending to the solvent area exhibited an orthogonal π-π
stacking with Trp258.
To understand the reason why a minor change in structure led to such a great increase in
potency, we compared the docked poses of 17 and the less active 6 into the PHD2 binding sties
(
2G19). As shown in Figure 2, the docking poses of 6 and 17 possessed a critical chelating
interaction with Fe (II), an interaction network with Arg383 and Tyr329, and a hydrogen-bonding
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interaction with Tyr303. In contrast to 6, the phenyl of 17 was capable of forming an additional
potential interaction with a pocket formed by the residues of Arg322, Trp389, Trp258, and Phe391.
T-shaped π-π stacking interactions between Trp258, Trp389, and the phenyl of 17 were present.
In addition, a cation-π interaction between the phenyl of 17 and Arg322 was observed. Further,
the 4-substituted chlorine atom on the phenyl retained a hydrogen-bonding interaction with the
positive center of Arg322; this may lead to a strong synergistic effect in enhancing the interaction
between phenyl and Arg322. This effect can explain, to some extent, why 4-substituted chlorine
plays a significant role in the remarkable increase in inhibitory activity toward PHD2.
1
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5
5
5
5
5
5
6
Figure 2. Proposed binging modes: structure of 6 and 17 docked into PHD2 (PDB ID 2G19). A,
B (6, yellow carbons), C, D (17, yellow carbons) in the PHD2 active site. The key interactions
between inhibitors and PHD2 are depicted as yellow dashes.
0
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2
.3. Synthesis of target compounds 3-34.
The synthetic route of target compounds 3-34 is depicted in Scheme 1. The key intermediate, 38,
29
was obtained from starting material 35, according to the method reported previously. The nitro
group of 35 was substituted with benzyl alcohol under the strong base condition to give 36.
Subsequent hydrolysis of the cyano group of 36 in the presence of 30% aqueous NaOH yielded
the carboxylic acid, 37. The condensation reaction between 37 and GlyOMe·HCl was applied in
the presence of EDC·HCl and HOBT to obtain the key intermediate, 38. Following that reaction,
3
8 was treated with BF
3
·Et
2
O to give 39, which was coupled with trimethylsilylacetylene (40)
using Pd(PPh Cl and CuI as the catalysts under microwave irradiation condition to yield 41.
3
)
2
2
Target compound 3 was obtained by deprotection of trimethylsilyl using tetrabutylammonium
fluoride (TBAF) followed by hydrolysis of the methyl ester. Further, 39 was treated with different
ethynyl fragments (42a-42ae) under a Sonogashira reaction followed by hydrolysis of the methyl
group, yielding the desired alkynyl-containing PHD2 inhibitors (4-34).
Scheme 1 a
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a
o
Reagents and conditions: (i) BnOH, NaH, THF, rt to 35 C, 1.5 h, 86.7%; (ii) NaOH, CH
3
OH,
2
H O, reflux, 2 h, 86.8%; (iii) GlyOMe·HCl, TEA, HOBT, EDCI, DCM, rt, 6 h, 46.5%; (vi)
o
BF
3
·Et
2
O, DCM, 45 C, 6 h, 73.5%; (v) Pd(PPh
3
)
2
Cl
2
, CuI, Et
3
N, CH
3
CN, microwave irradiation,
o
o
1
8
20 C, 300W, 7 min, 90.0%; (vi) TBAF, MeOH, reflux, 1h; then, LiOH, THF, H
2
O, 35 C, 1 h,
o
4.1% over two steps; (vii) Pd(PPh
3
2
) Cl
2
, CuI, DIPEA, DMF, microwave 120 C, 300 W, 15 min;
o
then, LiOH, THF, H
2
O, 35 C, 1h, 31.0−82.3% over two steps.
2
.4. Selectivity of alkynyl compounds represented by 17 to other PHD subtypes, 2-OG
oxygenases, and hERG.
To investigate whether the alkynyl-containing PHD2 inhibitor (17) selectively inhibited PHD2, its
potency against other PHD subtypes (PHD1, PHD3) and representative 2-OG oxygenases, such as
JmjC-domain-containing lysine histone demethylases (KDMs), were determined by O-
3
0
phenylenediamine (OPD)-based fluorescent assay (Table S1) and AlphaScreen assay (Table
1
9
S2), respectively. The results indicated that 17 also showed significant PHD3 inhibitory activity
(SR(PHD3) = 2.6) and exhibited considerable selectivity to the PHD1 subtype (SR(PHD1) = 29) (Table
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3
). The subtype selectivity of 17 is similar to that of the approved drug FG-4592, thus, alkynyl-
containing PHD2 inhibitors can modulate the HIF pathway, mainly by inhibiting PHD2 and PHD3,
thereby efficiently stabilizing HIF-α. Moreover, the AlphaScreen assay demonstrated that 17
retains 156-fold more selectivity for PHD2 over typical JmjC-KDMs such as KDM3A, KDM4A,
and KDM6B (Table 3). To verify whether 17 has the potential for cardiac toxicity, a hERG assay31
was performed. No hERG inhibition was observed for 17, which showed a 467-fold selectivity to
PHD2 over hERG (Tables 3 and S3). These enzyme-based results indicate that 17 is a promising
PHD inhibitor with good selectivity windows for HIF stabilization.
0
1
2
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0
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Table 3. Selectivity Ratio of the Representative Compound to PHD2 Over PHD1, PHD3, JmjC-
KDMs, and hERG
compound SR(PHD1) a SR(PHD3) a SR(KDM3A) b SR(KDM4A) b SR(KDM6B) b SR(hERG) c
1
7
29
2.6
1.1
>1556
>169
1556
169
>156
>169
>467
ND d
FG-4592 (1)
5.0
a
SR = IC50 (PHDs)/IC50 (PHD2). The SR was generated using the IC50 values towards PHDs
b
determined by OPD assay. SR = IC50 (JmjC-KDMs) / IC50 (PHD2). The SR was generated using
the IC50 values towards PHD2 determined by FP assay and towards typical JmjC-KDMs
c
determined by AlphaScreen assay. SR = IC50 (hERG)/IC50 (PHD2). The SR was generated using
the IC50 values towards PHD2 determined by FP assay and those towards hERG determined by
d
hERG patchliner assay. Not determined.
2.5. Representative compound 17 stabilized HIF-α and upregulated the expression and
secretion of EPO.
To validate the HIF-α stabilization effect of 17, the immunostaining method was performed to
visualize changes in 17-treated Hep3B cells. The results demonstrated that 17 and FG-4592 up-
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regulated the levels of HIF-α subunits, both HIF-1α and HIF-2α, in a dose-dependent manner
(Figure 5A). Considering that HIF can consequently bind to the hypoxia-responsive element (HRE)
motif on DNA, thereby promoting the expression of EPO, we used an HRE-luciferase dual-
1
1
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32
reporter assay to further validate the stabilizing effect of 17 to HIF-α. Before that, a 3-(4, 5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to investigate
whether 17 kills typical normal cells and experimental cells; no lethal effect of 17 was observed
towards these typical cells, indicating it has a good cell-based safety profile (Figure S2). As shown
in Figure 3B, 17 exhibited stabilization activity for HIF-α comparable to FG-4592; the HIF-α level
was upregulated four-fold with the treatment of 25 μM 17 and seven-fold with 50 μM 17.
To further confirm the effects of 17 on the EPO gene, RT-PCR was used to evaluate the
mRNA level of the EPO gene. Treatment of Hep3B cells with 17 dose-dependently upregulated
the EPO mRNA level in Hep3B cells, showing an approximate seven-fold upregulation with a
concentration of 50 μM (Figure 3C). Subsequently, we examined the level of EPO production to
further reveal the potency of 17 in regulating the HIF pathway. EPO ELISA assay revealed that
1
7 was capable of significantly improving EPO expression in a dose-dependent manner in Hep3B
cells (Figure 3D). Based on these results, the PHD inhibitor 17 exhibits remarkable efficacy in
stabilizing HIF-α, leading to the promotion of EPO expression.
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Figure 3. Evaluation of the potency of 17 in HIF-α stabilization and EPO upregulation. (A)
Representative western blot visualization of HIF-α levels in 17-treated Hep3B cells; Lamin B was
used as the control for HIF-α loading. (B) Expression of HIF target genes after treatment MCF-7
cells with 17. (C) Expression of EPO genes in Hep3B cells by RT-PCR after incubation with 17
for 10 h. (D) EPO release in 17-treated Hep3B cells after 24 h. P values were analyzed by two-
way ANOVA with the control group as the comparison (*, P < 0.05; **, P < 0.01; ***, P < 0.001;
*
***, P < 0.0001).
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.6. Evaluation of the effects of representative compounds on the plasma EPO levels in vivo.
Inspired by the PHD2 inhibition activity in vitro, in vivo pharmacodynamic (PD) evaluation in
mice was carried out. In light of their potency of PHD2 inhibition, nine representative compounds
(8, 14, 15, 17, 18, 19, 20, 26, and 33) with IC50 values less than 200 nM toward PHD2, together
with the approved drug FG-4592, were selected. C57BL/6 mice were treated with each compound
at a dose of 15 mg/kg via iv administration. Then, the levels of plasma EPO were investigated
using an EPO-based ELISA Kit.33, 34 As shown in Figure 4A, compounds 15, 17, 19, and 26 exerted
obvious EPO upregulation at more than 1000 pg/mL. Further, the pharmacodynamics of the four
effective compounds were evaluated with po administration in C57BL/6 mice at 15 mg/kg. The
results indicated that 17 produced the best upregulation of the plasma EPO levels; its effect was
even better than the positive control FG-4592 (Figure 4B).
1
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9
0
Figure 4. In vivo upregulation of EPO. (A) Plasma EPO levels in mice (C57BL/6) treated with
1
5mg/kg of each compound; levels were tested 4 h after iv administration. (B) The typical
compounds (15mg/kg) produced plasma EPO responses in mice (C57BL/6) 4 h after po
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administration. Mean ± SEM. P values were analyzed by two-way ANOVA with the control group
as the comparison (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
2.7. Compound 17 dose-dependently increases the plasma EPO and reticulocytes in vivo.
0
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Dose-dependent PD assays for EPO induction in mice were further conducted for 17 and FG-
23
4
592. Increases in plasma EPO were observed at 5, 10, 25, and 50 mg/kg, with doses given four
times for three consecutive days by po administration (Figure 5). Notably, stabilization of HIF by
po administration of 17 resulted in dose-dependent elevation of plasma EPO levels in C57BL/6
mice, which was similar to the FG-4592-treated group. Interestingly, in the case of the 25 mg/kg
dosage, 17 was more efficacious in the upregulation of plasma EPO as compared to FG-4592. The
effects on reticulocyte generation were also evaluated. After 72 h (po administration of the
compound three times) of treatment with 17 or FG-4592, RBC% (the reticulocyte count/the red
blood cell count) increased under both treatments in a dose-dependent manner (Figure 6). These
results suggest that the orally-active PHD inhibitor 17 upregulates EPO formation and stimulates
erythropoiesis.
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Figure 5. 17- and FG-4592-mediated plasma EPO response in mice (C57BL/6) after three days
of po administration of each compound (5, 10, 25, and 50 mg/kg). Mean ± SEM. P values were
analyzed by two-way ANOVA with the control group as the comparison (*, P < 0.05; **, P < 0.01;
*
**, P < 0.001; ****, P < 0.0001).
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2
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4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 6. Reticulocytes (RBC %) were measured 72 h (after po administration of each compound
three times) after treatment with 17 or FG-4592. Mean ± SEM. P values were analyzed by two-
way ANOVA with the control group as the comparison (*, P < 0.05; **, P < 0.01; ***, P < 0.001;
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
*
***, P < 0.0001).
2
.8. Pharmacokinetic and metabolic profiles of 17.
Pharmacokinetic studies of 17 in healthy rats and beagle dogs were performed to evaluate
absorption, distribution, metabolism, and excretion (ADME) properties. As shown in Table 4, 17
exhibited acceptable T1/2 in rats (6.44 ± 3.08 h) and beagle dogs (4.19 ± 1.98 h) with ig administration.
Suitable bioavailability was observed both in rats (55.7%) and beagle dogs (55.4%), verifying the
potential application of this compound for further research in vivo. In addition, the in vitro
metabolic stability of 17 was assessed in liver microsomes from different species (Table 5). The
results showed that 17 possessed suitable stability in these five liver microsomes, especially in
human microsomes. The metabolic stability of 17 was further assessed in rat/mice hepatocytes
and human L02 cells; the metabolism of 17 in human L02 cells seemed to be much slower than in
mice and rat hepatocytes (Table 6), suggesting that 17 may possess suitable pharmacokinetic
behavior with superior stability in humans. 17 was assayed with cytochrome p450 (CYP450)
enzymes; no significant inhibition in vitro was observed toward CYP450 enzyme isomers,
including CYP1A2, 2C1, 2C9, 2D6, and 3A4, with all IC50 values above 30 μM (Table 7); this
suggests the low potential for 17 to produce clinical drug-drug interactions.
Table 4. Pharmacokinetic Profiles of 17 in Rats and Beagle Dogs.a
rat (iv, 1 mg/kg)
dog (ig, 2
dog (iv, 0.5
rat (ig, 2 mg/kg)
mg/kg)
mg/kg)
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−
−
−
−
T
max (h)
0.63 ± 0.25
1027 ± 374
4795 ± 2150
0.67 ± 0.29
735 ± 493
1357 ± 329
C
max (ng/mL)
4
4
709±346
864±337
611±67
AUC0-t (ng/ml*h)
AUC0-∞
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
636±58.3
5
418 ± 2517
1374 ± 343
(
ng/ml*h)
8
3
2
.43±1.70
.44±0.23
515±540
2.91±1.11
13.2±1.3
T
1/2 (h)
6.44 ± 3.08
7.30 ± 3.34
3409 ± 434
4.19 ± 1.98
25.5 ± 7.3
CL (mL/min/kg)
(mL/kg)
3340±1305
2.27±0.69
V
z
8417 ± 2535
3.44±0.55
MRT0-∞ (h)
F
9.43 ± 2.62
55.7%
4.69 ± 1.07
54.0%
a
For rats, N = 4; equal number of males and females; for dogs, N = 3, all males.
Table 5. In Vitro Metabolic Stability of 17 in Liver Microsomes.
liver microsome
CLint
mouse
<10.00
rat
dog
monkey
<13.00
human
<10.00
<20.00
<10.00
（
μL/min/mg）
CL
<
27.00
<26.00
>67.00
<9.60
<13.00
>99.00
<6.30
（
mL/min/kg）
T
1/2
>
139.00
>139.00
>139.00
（
min）
Table 6. In Vitro Metabolic Stability of 17 in Rat/Mice Hepatocytes and Human L02 Cells.
hepatocyte
CL
mouse
2.16
rat
human
1.54
8
35.62
（mL/min/kg）
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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4
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T
1/2
8
.68
32.3
1062.7
（
min）
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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8
9
0
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9
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9
0
Table 7. Inhibition of Cytochrome P450 (CYP450) Enzymes by 17.
isozyme a
IC50（μM）
CYP1A2 (phenacetin)
CYP2C1 (9-(S)-mephenyt)
>30
>30
>30
>30
>30
>30
CYP2C9 (diclofenac)
CYP2D6 (dextromet horphan)
CYP3A4 (testosterone)
CYP3A4 (midazolam)
a
CYP450 isozymes and the corresponding substrate drugs.
2.9. Oral administration of 17 improves cisplatin-induced anemia.
Encouraged by its promising in vivo pharmacodynamic (PD) and pharmacokinetic (PK) properties,
7 was further assessed in a cisplatin-induced anemia C57BL/6 mouse model to evaluate its anti-
1
3
5
anemia efficacy. The hemoglobin (Hb) level in the cisplatin-administered mice was decreased to
130 g/L as compared with the normal control. In the male mice model, 17 and FG-4592 treatment
at dosages of 10 and 25 mg/kg every other day for 30 days produced a dose-dependent increase in
Hb levels. Similar to FG-4592, treatment with 17 prevented the development of anemia induced
by cisplatin; plasma Hb levels under 17 treatment recovered to levels observed without cisplatin
stimulation (Figure 7A). Interestingly, 17 was found to restore Hb to normal levels while FG-4592
caused a slight shift to above the normal level (Figure 7B), indicating that 17 may possess a
preferable safety profile for anemia treatment, particularly at high doses for long periods of time.
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Anti-anemia potential in vivo. (A) Changes in Hb after oral administration of 17 or FG-
4
592 in a C57Bl/6 mouse model of anemia induced by cisplatin. (B) Anti-anemia activities of 17
or FG-4592 in a C57Bl/6 mouse model of anemia induced by cisplatin. Mean ± SEM. P values
were analyzed by two-way ANOVA with the control group as the comparison (*, P < 0.05; **, P
<
0.01; ***, P < 0.001; ****, P < 0.0001).
2
.10. Subacute toxicity and genetic safety assessment of 17.
To further evaluate its in vivo safety profile, the acute toxicity of 17 was determined in SD
rats at doses of 15, 50, and 200 mg/kg. The treatment period involved iv administration of 17 for
two weeks. During this time, none of the animals showed any abnormal behavior as compared to
the no-treatment group. During the14 days of treatment, food intake, body weight, and organ/body
weight ratios were recorded (Figure 8). It was observed that body weights and food intake were
relatively stable and the growth trend was consistent with the blank control. Furthermore, no
obvious changes were observed after 17 treatment with respect to the organ/body weight ratio of
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the adrenal gland, brain, heart, kidney, liver, thymus, thymus, ovary (female), uterus with cervix
(female), testes (male), and epididymis (male); however, a slight increase in the weight of the
spleen (male) occurred which may be ascribed to the pharmacological effect of the compound
0
1
2
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9
0
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0
36
along with the elevation of EPO levels. Additionally, blood biochemistry studies revealed that
1
7 had no obvious nephrotoxicity and hepatotoxicity in SD rats compared to the control group
(Figure 9), as determined by kidney function indicators (urea; CRE, creatinine) and liver function
indicators (AST, aspartate aminotransferase; ALT, alanine amiotransferase; A/G, albumin/
globulin; GLO, globulin; ALB, albumin; TP, total protein). Only a slight increase in AST was
observed at a dosage of 200 mg/kg in male rats (Figure 9F). The above results indicate that 17 has
a preferable safety profile with no acute toxicity to normal tissues and a wide therapeutic window.
Moreover, the genotoxicity of 17 was also evaluated by AMES assay and no significant
genotoxicity of 17 was observed (Table S4). Taken together, these findings indicate that 17 has an
acceptable safety profile in vivo and is suitable for further clinical development.
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Figure 8. The effects of compound 17 on body weight (A/B), food intake (C/D), and organ/body
weight ratio (E/F) in rats (iv; 15, 50, and 200 mg/kg). (A) Changes in bodyweight (male rats); (B)
changes in bodyweight (female rats); (C) changes in food intake (male rats); (D) changes in food
intake (female rats); (E) changes in organ/body weight ratio (male rats); (F) changes in organ/body
weight ratio (female rats).
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Figure 9. Blood biochemistry analysis of TP, ALB, GLO, A/G, ALT, AST, CRE, and urea
indicators in rats. Mean ± SEM. P values were analyzed by two-way ANOVA with the control
group as the comparison (*, P < 0.05).
3
. CONCLUSION
In this work, we developed a novel series of alkynyl-containing PHD inhibitors by replacing the
rigid isoquinoline core in FG-4592 with a rigid and linear 5-akynyl pyridine core. Of these, 17
was identified as the most active compound both in vitro and in vivo. On the basis of its overall
pharmacological, pharmacokinetic, and preliminary safety profile, 17 has been advanced to human
clinical studies as an oral treatment for anemia.
4
. EXPERIMENTAL SECTION
4.1. General Methods and Materials. All materials were purchased from commercial sources
and used as received. Organic solvents were concentrated via a rotary evaporator (EYELA OBS-
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100) under reduced pressure below 50 °C. The microwave reactions were performed on a
microwave reactor from Biotage, Inc. Reactions were monitored using TLC silica gel plates
(GF254, 0.25 mm) and visualized under UV light. Hanon MP420 automatic melting point
1
1
1
1
1
1
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1
1
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2
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0
1
apparatus was used to determine the melting points. The Proton nuclear magnetic resonance ( H
13
NMR) and carbon nuclear magnetic resonance ( C NMR) spectra were determined using Bruker
AV-300 instruments. EI-mass spectra and high-resolution mass spectra (HRMS) were recorded on
a Shimadzu GCMS-2010 mass spectrometer and a Water Q-Tof micro mass spectrometer,
respectively. The purity (≥95%) of the target compounds used for biological testing were evaluated
by high-performance liquid chromatography (HPLC) analysis, performed on an Amethyst C18-P
(4.6 × 150 mm, 5 μM, Merck) column eluting with methanol/water (80:20 v:v) containing 0.1%
trifluoroacetic acid at a flow rate of 0.5 mL/min; the peaks were detected at 254 nm under UV.
The experimental cells were purchased from Cell Bank of Shanghai, Institute of Biochemistry and
Cell Biology, Chinese Academy of Sciences. FITC-HIF-1α (556-574) was purchased from
Shanghai Apeptide Co., Ltd., the typical proteins from Nanjing Zoombio biotechnology, and the
experimental mice were obtained from Beijing Vital River Laboratory Animal Technology Co.,
Ltd. All animal-related experiments were conducted under a China Pharmaceutical University
IACUC approved protocol (No. 20160622-1) in compliance with the guide for the care and use of
laboratory animals.
4
.2. Synthesis. 4.2.1. Preparation of 3-(Benzyloxy)-5-bromopicolinonitrile (36). A solution of
o
benzyl alcohol (59 mL, 570 mmol) in THF (1.8 L) was cooled to 0 C. NaH (14.4 g, 600 mmol)
o
was added slowly to the stirring mixture at 0 C. Then, the mixture was stirred at room temperature
o
for 1 h and at 35 C for another 0.5 h. The resulting solution was added to a solution of 5-bromo-
3
-nitropicolinonitrile (35, 100.0 g, 438 mmol) in THF (1.3 L) and then the mixture was stirred at
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