Evaluation of 3-carbamoylpropanoic acid analogs as inhibitors of human hypoxia-inducible factor (HIF) prolyl hydroxylase domain enzymes

Article abstract of DOI:10.1007/s00044-020-02681-7

There is current interest in developing human hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) inhibitors for the treatment of anemia and other hypoxia-related diseases. We describe the synthesis of 3-carbamoylpropanoic acid derivatives and their evaluation as human PHD-2 inhibitors. MS assays indicated that derivatives with a 3-carbamoylpropanoic acids-containing benzoxazole moiety are inhibitors of PHD-2 with IC₅₀ values of 2.24 μM and 1.32 μM, respectively. However, neither the acids nor their respective ethyl esters were observed to upregulate HIF-1α levels in cells.

Full text of DOI:10.1007/s00044-020-02681-7

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:977–986
https://doi.org/10.1007/s00044-020-02681-7
ORIGINAL RESEARCH
Evaluation of 3-carbamoylpropanoic acid analogs as inhibitors of
human hypoxia-inducible factor (HIF) prolyl hydroxylase domain
enzymes
MuiPhin Chong1 LeeRoy Toh1 Anthony Tumber2 YanYing Chan3 MunChiang Chan3 Martine I. Abboud2
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Christopher J. Schofield2 KarKheng Yeoh
1
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Received: 29 May 2020 / Accepted: 2 December 2020 / Published online: 3 February 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Abstract
There is current interest in developing human hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) inhibitors
for the treatment of anemia and other hypoxia-related diseases. We describe the synthesis of 3-carbamoylpropanoic acid
derivatives and their evaluation as human PHD-2 inhibitors. MS assays indicated that derivatives with a 3-
carbamoylpropanoic acids-containing benzoxazole moiety are inhibitors of PHD-2 with IC50 values of 2.24 μM and
1.32 μM, respectively. However, neither the acids nor their respective ethyl esters were observed to upregulate HIF-1α levels
in cells.
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Keywords Hypoxia-inducible factor HIF Prolyl hydroxylase domain PHD inhibitors 2-oxoglutarate/alpha-ketoglutarate
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dependent oxygenase 3-carbamoylpropanoic acid derivatives
Introduction
half-life of HIF-α is relatively short (<5 min) and it is
rapidly degraded via the ubiquitin-proteasome pathway [4].
Two proline residues (Pro402 and Pro564) located in the
oxygen-degradation domains (ODDs) of the human HIF-1α
and HIF-2α isoforms undergo oxygen-dependent prolyl
hydroxylation. Human HIF-α prolyl hydroxylation is cata-
lyzed by the prolyl hydroxylase domain enzymes (PHD1–3)
in the presence of ferrous ion (Fe2+) and 2-oxoglutarate (2-
OG) [5–7]. HIF-α prolyl hydroxylation promotes its binding
to the von Hippel-Lindau tumor suppressor protein, which
is part of an E3 ubiquitin complex, leading to HIF-α pro-
teasomal degradation [8]. Note that only one hydroxylated
proline is sufficient to target HIF for its proteasomal
degradation. In a second type of oxygenase catalyzed
modification, β-hydroxylation of an asparagine residue
(Asn803) in HIF-1α, as catalyzed by the factor inhibiting
HIF within the C-terminal transcriptional activation domain
(C-TAD) hinders the binding of HIF-1α to co-activator
proteins p300/CBP, reducing the extent of HIF transcrip-
tional activity [9, 10].
Physiological hypoxia, which in animals involves a
decrease in oxygen supply to tissues [1], induces major
adaptive responses. The chronic response to hypoxia is
mediated by the hypoxia-inducible transcription factors
(HIFs) [2, 3]. HIF is an α,β-heterodimeric transcription
factor comprised of a constitutively expressed oxygen-
resistant β-subunit (HIF-β) and a hypoxia-regulated oxygen-
labile α-subunit (HIF-α). Under normoxia, the average
These authors contributed equally: MuiPhin Chong, LeeRoy Toh
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02681-7) contains supplementary
material, which is available to authorized users.
* KarKheng Yeoh
kkyeoh@usm.my
1
School of Chemical Sciences, Universiti Sains Malaysia,
Penang 11800, Malaysia
When cellular oxygen consumption surpasses oxygen
supply, hypoxia occurs and PHD activity is significantly
reduced; this results in the stabilization and accumulation of
HIF-α subunits. Hypoxic conditions thus elevate the half-
life of HIF-α, leading to its translocation into the nucleus
2
Chemical Research Laboratory, University of Oxford,
Oxford OX1 3TA, UK
3
Department of Molecular Medicine, University of Malaya,
Kuala Lumpur 50603, Malaysia

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Medicinal Chemistry Research (2021) 30:977–986
and dimerization with HIF-β to form transcriptionally active
HIF heterodimers, which upregulates the expression of
multiple genes in a context dependent manner [11–14]. At
the same time, the transcriptional co-activator complex
p300/CBP is recruited and binds to the C-TAD region of
HIF-α, leading to the induction of HIF target genes [15–17].
HIF does not respond directly to the cellular oxygen levels
[18]. Instead, the PHD proteins act as ‘oxygen sensors’ in
the HIF degradation pathway [4].
PHD inhibitors have been studied as a potential treatment
for ischemic disorders, inflammatory lung disease, and
diabetes, and some are in late-stage clinical trial/have been
recently approved for the treatment of renal anemia in
chronic kidney disease in China [19–28]. It is therefore of
interest to develop optimized PHD inhibitors in order to
pharmacologically upregulate HIF-α, ideally in manner that
affects specific sets of HIF target genes, for the treatment of
anemia and other hypoxia-related diseases [19, 29].
Many of the reported PHD-2 inhibitors including the
Fibrogen drug/drug candidate, 2-[(4-hydroxy-1-methyl-7-
phenoxyisoquinoline-3-carbonyl)amino]acetic acid (also
known as IOX3 or FG-2216) and 2-[(4-hydroxy-1-methyl-
7-phenoxyisoquinoline-3-carbonyl)amino]acetic acid (also
known as Roxadustat) contain a 2-formamidoacetic acid
side chain, which has been identified to be a critical func-
tional groups for chelation of active site iron and formation
of salt bridge with Arg383 residue in PHD-2 active site
[29–34].
column chromatography. Tris(hydroxymethyl) amino-
methane was from Fisher and ferrous ammonium sulfate
(FAS; 12304), 2-oxoglutarate α-ketoglutarate (αKG;
K3752) and L-ascorbic Acid (L-AA; A5960) were from
Sigma Aldrich. The melting points of all the synthesized
compounds were determined using Stuart Scientific SMP1
in glass capillary tubes. The nuclear magnetic resonance
(NMR) spectra of synthesized compounds were recorded
using Bruker Avance spectrometers, 500 MHz for 1H NMR
and 125 MHz for 13C NMR, FT-IR spectra on Perkin–Elmer
2000 spectrometer, mass spectra ESI measurements were
recorded on TSQ QuantumTMAccess MAX Triple Quad-
rupole Mass Spectrometer and elemental analysis mea-
surements were performed on Perkin Elmer series II, 240
CHN analyzer.
General procedure for the synthesis of 3CAs
(F1a–F5d)
The 3CAs were synthesized as described in the literature
with modifications [35]. To a stirred solution of the het-
erocyclic amino starting material in the requisite solvent,
i.e., chloroform, tetrahydrofuran, dimethylformamide,
ethanol, ethyl acetate, or acetone, succinic anhydride was
added. The resultant mixture was refluxed for 4 – 24 h, then
set aside until completion of the reaction. The precipitate so
formed was filtered, washed, then dried in a fume hood to
afford F1a–F5a. The preparation of compounds F5a, F5b,
F5c, and F5d required the use of additional N,N-diisopro-
pylethylamine in the reaction mixture and the use of column
chromatography for further purification using a mixture of
solvent systems including hexane, ethyl acetate, dichlor-
omethane, methanol, and/or formic acid.
3-[(1,3-Benzothiazol-2-yl)carbamoyl]propanoic acid
(F1a) 2-Aminobenzothiazole (152 mg, 1 mmol) was stirred
in a solution of tetrahydrofuran (15 mL) followed by
addition of succinic anhydride (110 mg, 1 mmol); the
reaction was stirred for 24 h at room temperature. The
precipitate so formed was collected by filtration and dried
in a fume hood to give a white solid (186 mg, yield 74%);
mp 258–260 °C; IR (KBr) νmax 3467 (N-H), 3413 (O-H),
3073 (sp2 C-H), 1690 (C=O); 1H-NMR, (DMSO-d6,
500 MHz) δ = 2.60 (2H, t, J = 6.5 Hz, CH2COOH), 2.74
(2H, t, J = 6.5 Hz, CH2CONH), 7.30 (1H, td, J = 1.0 Hz,
8.0 Hz, ArH), 7.44 (1H, td, J = 1.0 Hz, 8.0 Hz, ArH), 7.74
(1H, d, J = 8.0 Hz, ArH), 7.97 (1H, d, J = 8.0 Hz, ArH),
12.37 (1H, s, NH); 13C-NMR (DMSO-d6, 125 MHz) δ =
28.8 (CH2, CH2COOH), 30.6 (CH2, CH2CONH), 120.9
(CH, ArCH), 122.1 (CH, ArCH), 123.9 (CH, ArCH),
126.5 (CH, ArCH), 131.9 (C, ArCS), 149.0 (C, ArCN),
158.3 (C, ArCN), 171.8 (C, CONH), 174.0 (C, COOH);
HRMS (ESI) calcd. for C11H9N2O3S [M–H]-: 249.0339;
found 249.0257.
Here we report studies in which the 3-
carbamoylpropanoic acid (3CA) side chain was used in
the design of novel PHD-2 inhibitors in combination with
different heterocyclic and sulfonyl aromatic groups; the
resulting inhibitors were tested for their inhibitory effect
against PHD-2 and for their ability to induce HIF upregu-
lation in cells.
Materials and methods
Chemistry
Reactions were monitored by commercially aluminum
supported silica gel 60 F254 thin layer chromatography
(TLC) plate (Merck 1.05554.0001) using different solvent
systems. The spots on the TLC plate were then visualized
by CAMAG® Ultra Violet (UV) Lamp 4 dual-wavelength
254/366 nm. All commercial chemicals and reagents used in
the synthesis were from Sigma-Aldrich, Acros Organics,
Fisher Scientific, or Maybridge Chemical and were used
without further purification. The starting material 2-amino
and sulfonamide compounds were used as purchased. The
solvents used were all AR grade from QRëC. Silica gel 60F,
230–400 mesh ASTM (Merck 1.09385.1000) was used for

Medicinal Chemistry Research (2021) 30:977–986
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3-[(6-Fluoro-1,3-benzothiazol-2-yl)carbamoyl]propa-
noic acid (F1b) 2-Amino-6-fluorobenzothiazole (82 mg,
0.5 mmol) was stirred in a solution of tetrahydrofuran
(10 mL); succinic anhydride (104 mg, 1 mmol) was added
and the mixture refluxed for 2 h; the mixture was then set
aside for 24 h. The precipitant formed was collected by
filtration. The product was obtained as a white solid
(103 mg yield 57%); mp 268-269 °C; IR (KBr) νmax 3469
(N-H), 3413 (O-H), 3288, 3066 (sp2 C-H), 1691 (C=O);
1H-NMR, (DMSO-d6, 500 MHz) δ = 2.59 (2H, t, J =
6.6 Hz, CH2COOH), 2.73 (2H, t, J = 6.6 Hz, CH2CONH),
7.28 (1H, td, J = 2.7 Hz, 9.0 Hz, ArH), 7.74 (1H, m, ArH),
7.89 (1H, dd, J = 2.7 Hz, 9.0 Hz, ArH), 12.42 (1H, s, NH)
ppm; 13C-NMR (DMSO-d6, 125 MHz) δ = 28.7 (CH2,
CH2COOH), 30.6 (CH2, CH2CONH), 108.6 (CH, ArCH),
114.6 (CH, ArCH), 122.0 (CH, ArCH), 133.1 (C, ArCS),
145.7 (C, ArCN), 158.3 (C, ArCF), 160.0 (C, ArCN), 171.9
(C, CONH), 174.0 (C, COOH); HRMS (ESI) calcd. for
C11H8FN2O3S [M–H]-: 267.0245; found 267.0261.
3-[(6-Chloro-1,3-benzothiazol-2-yl)carbamoyl]propa-
noic acid (F1c) To a stirred solution of 2-amino-6-
chlorobenzothiazol (188 mg, 1 mmol) in chloroform
(15 mL), succinic anhydride (99 mg, 1 mmol) was added;
the mixture was stirred for 2 h at room temperature. The
reaction mixture was then set aside for 24 h. The precipitant
formed was collected by filtration. The product was
obtained as a white solid (221 mg yield 78%); mp 257-
258 °C; IR (KBr) νmax 3446 (N-H), 3181, 3061 (sp2 C-H),
2973 (O-H), 1687 (C=O); 1H-NMR, (DMSO-d6, 500 MHz)
δ = 2.59 (2H, t, J = 6.6 Hz, CH2COOH), 2.75 (2H, t, J =
6.6 Hz, CH2CONH), 7.44 (1H, dd, J = 2.0 Hz, 8.6 Hz,
ArCH), 7.71 (1H, d, J = 8.6 Hz, ArCH), 8.11 (1H, d, J =
2.0 Hz, ArCH), 12.24 (1H, s, CONH), 12.46 (1H, s,
COOH); 13C-NMR (DMSO-d6, 125 MHz) δ = 28.7 (CH2,
CH2COOH), 30.6 (CH2, CH2CONH), 121.8 (CH, ArCH),
122.1 (CH, ArCH), 126.9 (CH, ArCH), 128.0 (C, ArCCl),
133.6 (C, ArCS), 147.9 (C, ArCN), 159.2 (C, ArCN), 172.0
(C, CONH), 174.0 (C, COOH); MS (ESI) calcd. for
C11H8ClN2O3S [M–H]-: 283.00; found 283.00. Anal. calcd.
for C11H9ClN2O3S (%): C 46.40, H 3.19, N 9.84. Found: C
46.26, H 3.18, N 9.81.
(1H, d, J = 9.0 Hz, ArH), 8.27 (1H, dd, J = 2.4 Hz, 9.0 Hz,
ArH), 9.04 (1H, d, J = 2.4 Hz, ArH), 12.30 (1H, s, NH),
12.79 (1H, s, COOH); 13C-NMR (DMSO-d6, 125 MHz) δ
= 28.2 (CH2, CH2COOH), 30.3 (CH2, CH2CONH), 119.0
(CH, ArCH), 120.5 (CH, ArCH), 121.7 (CH, ArCH), 132.1
(C, ArCS), 142.9 (C, ArCNO2), 153.5 (C, ArCN), 163.4 (C,
ArCN), 172.1 (C, CONH), 173.4 (C, COOH); HRMS (ESI)
calcd. for C11H8N3O5S [M–H]-: 294.0190; found 294.0162.
3-[(1,3-Benzoxazol-2-yl)carbamoyl]propanoic
acid
(F2a) White powder (Solution of 2-aminobenzoxazole
(135 mg, 1 mmol) and succinic anhydride (103 mg,
1 mmol) in ethyl acetate (10 mL) was stirred and refluxed
for 1 h. Resulting mixture continued to stir at room tem-
perature and allowed to stand at room temperature for 24 h.
The precipitant formed was collected by filtration and
washed with ethyl acetate. The product was obtained as a
white solid (148 mg, 63%); mp 158-160 °C; IR (KBr) νmax
3445 (N–H), 3217, 3022 (sp2 C-H), 2968 (O-H), 1728,
1695 (C=O), 1681(C=N), 1646 (N-H bend), 1589, 1458
(CC); 1H-NMR, (DMSO-d6, 500 MHz) δ = 2.56 (2H, t, J =
6.5 Hz, CH2COOH), 2.78 (2H, t, J = 6.5 Hz, CH2CONH),
7.29 (2H, td, J = 1.5 Hz, 7.5 Hz, 2 × ArH), 7.58 (2H, td,
J = 1.5 Hz, 7.5 Hz, 2 × ArH), 11.69 (1H, s, NH), 12.20 (1H,
s, COOH); 13C-NMR (DMSO-d6, 125 MHz) δ = 28.2 (CH2,
CH2COOH), 30.9 (CH2, CH2CONH), 109.9 (CH, ArCH),
118.1 (CH, ArCH), 123.4 (CH, ArCH), 124.5 (CH, ArCH),
140.7 (C, ArCO), 147.5 (C, ArCN), 155.1 (C, ArCN), 173.5
(C, CONH, COOH); MS (ESI) calcd. for C11H9N2O4
[M–H]-: 233.06; found 233.05. Anal. calcd. for C11H10N2O4
(%): C 56.41, H 4.30, N 11.96. Found: C 56.50, H 4.32,
N 11.94.
3-[(5-Chloro-1,3-benzoxazol-2-yl)carbamoyl]propa-
noic acid (F2b) Solution of 2-amino-5-chlorobenzoxazole
(168 mg, 1 mmoL) and succinic anhydride (110 mg,
1.1 mmol) in ethyl acetate (15 mL) was stirred and refluxed
for 1 h. The reaction mixture then allowed to stir for 24 h for
completion of reaction. The precipitant formed was col-
lected by filtration. The product was obtained as a white
solid (243 mg, 79%); mp 173-175 °C; IR (KBr) νmax 3109,
3026 (sp2 C-H), 2930 (O-H), 1735, 1697 (C=O), 1654
(C = N), 1578, 1453, 1374 (CC), 1419 (CO), 1309 (C-N);
1H-NMR, (DMSO-d6, 500 MHz) δ = 2.56. (2H, t, J =
6.5 Hz, CH2COOH), 2.77 (2H, t, J = 6.5 Hz, CH2CONH),
7.30 (1H, dd, J = 2.1 Hz, 8.6 Hz, ArH), 7.64 (1H, d, J =
8.6 Hz, ArH), 7.68 (1H, d, J = 2.1 Hz, ArH), 11.83 (1H, s,
NH), 12.20 (1H, s, COOH); 13C-NMR (DMSO-d6,
125 MHz) δ = 28.7 (CH2, CH2COOH), 31.5 (CH2,
CH2CONH), 111.7 (CH, ArCH), 118.4 (CH, ArCH), 123.8
(CH, ArCH), 129.2 (C, ArCCl), 142.7 (C, ArCN), 146.8 (C,
ArCO), 156.9 (C, ArCN), 170.6 (C, CONH), 174.0 (C,
COOH); MS (ESI) calcd. for C11H8ClN2O4 [M–H]-:
267.02; found 267.01. Anal. calcd. for C11H9ClN2O4 (%): C
49.18, H 3.38, N 10.43. Found: C 49.34, H 3.37, N 11.40.
3-[(6-Nitro-1,3-benzothiazol-2-yl)carbamoyl]propa-
noic acid (F1d) 2-Amino-6-nitrobenzothiazole (185 mg,
1 mmol) was stirred in dichloromethane (15 mL) for 1 h
until homogeneous solution had formed. Succinic anhy-
dride (102 mg, 1 mmol) was then added and the reaction
mixture was stirred for 12 h. The precipitant formed was
collected by filtration. The product was obtained as a yellow
solid (234 mg, 69%); mp 262–263 °C; IR (KBr) νmax 3446
(N-H), 3163, 3090, 3069 (sp2 C-H), 2962 (O-H), 1692
1
(C=O), 1584 (C=N), 1566 and 1380 (N=O); H-NMR,
(DMSO-d6, 500 MHz) δ = 2.61 (2H, t, J = 6.6 Hz,
CH2COOH), 2.77 (2H, t, J = 6.6 Hz, CH2CONH), 7.88

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Medicinal Chemistry Research (2021) 30:977–986
3-{[3-(4-Chlorophenyl)-1H-pyrazol-5-yl]carbamoyl}
t, J = 6.6 Hz, CH2COOH), 2.70 (2H, t, J = 6.6 Hz,
CH2CONH), 7.50 (1H, m, ArH), 7.70 (1H, s, ArHS), 7.75
(1H, m, ArH), 7.90 (1H, m, ArH), 12.21 (1H, s, NH), 12.30
(1H, s, COOH); 13C-NMR (DMSO-d6, 125 MHz) δ = 28.3
(CH2, CH2COOH), 29.8 (CH2, CH2CONH), 108.9 (C,
ArCS), 114.4 (CH, ArCH), 117.9 (CH, ArCH), 122.3 (CH,
ArCH), 132.0 (C, ArC), 148.7 (C, ArCN), 149.9 (C, ArCF),
158.1 (C, ArCN), 170.7 (C, CONH), 173.5 (C, COOH);
HRMS (ESI) calcd. for C13H9F2N2O3S [M–H]-: 311.0307;
found 311.0346.
propanoic acid (F3a) Solution of 3-(4-chlorophenyl)-1H-
pyrazol-5-amine (186 mg, 1 mmol) and succinic anhydride
(101 mg, 1 mmol) in tetrahydrofuran (10 mL) was stirred in
room temperature for 24 h. The reaction mixture was then
left aside for 2 h. The precipitant formed was collected by
filtration. washed with tetrahydrofuran (2 mL) and dried in a
fume hood. The product was obtained as a beige solid
(187 mg, 64%); mp 195–197 °C; IR (KBr) νmax 3438 (N-H),
3241 (O-H), 3180, 3114 (sp2 CH), 1707, 1654 (C=O), 1619
(C=N), 1511, 1493 (CC); 1H-NMR, (DMSO-d6, 500 MHz)
δ = 2.53 (2H, t, J = 7.0 Hz, CH2COOH), 2.57 (2H, t, J =
7.0 Hz, CH2CONH), 6.83 (1H, s, ArCH, pyrazole ring),
7.50 (d, J = 8.6 Hz, ArCH), 7.74 (d, J = 8.6 Hz, ArCH),
10.49 (1H, s, NH), 12.45 (2H, s, COOH, NH pyrazole ring);
13C-NMR (DMSO-d6, 125 MHz) δ = 29.2 (CH2,
CH2COOH), 30.7 (CH2, CH2CONH), 94.0 (C, ArC, pyr-
azole ring), 127.1 (CH, ArCH), 129.4 (CH, ArCH), 130.2
(C, ArC), 132.9 (C, ArCCl), 142.2 (C, ArC), 147.2 (C,
ArCNH), 169.9 (C, CONH), 174.3 (C, COOH); MS (ESI)
calcd. for C13H11ClN3O3 [M–H]-: 292.05; found 292.04.
Anal. calcd. for C13H12ClN3O3 (%): C 53.16, H 4.12, N
14.31. Found: C 53.31, H 4.13, N 14.37.
3-{[4-(4-Chlorophenyl)-1,3-thiazol-2-yl]carbamoyl}
propanoic acid (F4a) 2-amino-4-(4-chlorophenyl) thiazole
(105 mg, 0.5 mmol) was stirred in chloroform (15 mL)
followed by the addition of succinic anhydride (120 mg,
1.2 mmol) and the mixture was stirred for 12 h. The pre-
cipitant formed was collected by filtration and dried at room
temperature. The product was obtained as a white solid
(94 mg, 62%); mp 235–236 °C; IR (KBr) νmax 3438 (N-H),
3184, 3040 (sp2 CH), 2980 (O-H), 1684 (C=O), 1589,
1482, 1427, 1380 (CC); 1H-NMR, (DMSO-d6, 500 MHz) δ
= 2.57 (2H, t, J = 6.5 Hz, CH2COOH), 2.69 (2H, t, J =
6.5 Hz, CH2CONH), 7.48 (2H, dt, J = 2.2 Hz, 9.2 Hz, 2 ×
ArH), 7.65 (1H, s, ArCS), 7.90 (2H, dd, J = 2.2, 9.2 Hz,
2 × ArH), 12.30 (1H, s, COOH); 13C-NMR (DMSO-d6,
125 MHz) δ = 28.3 (CH2, CH2COOH), 29.9 (CH2,
CH2CONH), 108.6 (C, ArCS), 127.3 (2CH, ArCH), 128.7
(2CH, ArCH), 132.2 (C, ArCCl), 133.2 (C, ArCCN), 147.5
(C, ArCN), 158.1 (C, ArCNH), 170.6 (C, CONH), 173.5
(C, COOH); HRMS (ESI) calcd. for C13H10ClN2O3S
[M–H]-: 309.0106; found 309.0093.
3-{[4-(5-Bromothiophen-2-yl)-1,3-thiazol-2-yl]carba-
moyl}propanoic acid (F4c) 4-(5-bromo-2-thienyl)-1,3-
thiazol-2-amine (136 mg, 0.5 mmol) was stirred in mixture
of dichloromethane (10 mL) and succinic anhydride (67 mg,
0.7 mmol) for 24 h. The precipitant formed was collected by
filtration and dried in a fume hood. The product was
obtained as a white solid (96 mg, 53%); mp 233–234 °C; IR
(KBr) νmax 3432 (N-H), 3184 (sp2 CH), 2974 (O-H), 1684
1
(C=O), 1589 (C=N), 1425, 1379 (CC), 961 (C-S); H-
NMR, (DMSO-d6, 500 MHz) δ = 2.56 (2H, t, J = 6.5 Hz,
CH2COOH), 2.68 (2H, t, J = 6.5 Hz, CH2CONH), 7.22
(2H, d, J = 4.0 Hz, ArH), 7.36 (1H, d, J = 4.0 Hz, ArH),
7.52 (1H, s, ArHS), 12.39 (1H, s, COOH); 13C-NMR
(DMSO-d6, 125 MHz) δ = 28.3 (CH2, CH2COOH), 29.8
(CH2, CH2CONH), 106.9 (CH, ArCHS), 110.7 (C, ArCBr),
124.1 (CH, ArCH), 131.4 (CH, ArCH), 140.1 (C, ArCN),
142.5 (C, ArCS), 158.2 (C, ArCN), 170.7 (C, CONH),
173.5 (C, COOH). MS (ESI) calcd. for C11H10BrN2O3S2
[M + H]+: 360.93; found 360.92. Anal. calcd. for
C11H9BrN2O3S2 (%): C 36.58, H 2.51, N 7.76. Found: C
36.36, H 2.52, N 7.78.
3-[(Benzenesulfonyl)carbamoyl]propanoic acid (F5a)
To a mixture of benzenesulfonamide (149 mg, 1 mmol) and
succinic anhydride (136 mg, 1.4 mmol) in dichloromethane
(10 mL), N,N-diisopropylethylamine (300 μL) was added
and the mixture was stirred for 12 h. The organic solvents
were removed using a rotary evaporator and the crude
compound was purified by column chromatography
(dichloromethane:methanol, 0.5:4.5). The product was
obtained as a white solid (104 mg, 40%); mp 110–112 °C;
IR (KBr) νmax 3345 (N-H), 3230 (O-H), 1736, 1717 (C=O),
1444, 1409 (CC), 1330, 1162 (S=O), 871 (S-N); 1H-NMR,
(DMSO-d6, 500 MHz) δ = 2.36 (2H, t, J = 6.5 Hz,
CH2COOH), 2.46 (2H, t, J = 6.5 Hz, CH2CONH), 7.62
(2H, t, J = 8.0 Hz, ArH), 7.71 (1H, t, J = 8.0 Hz, ArH), 7.91
(2H, d, J = 8.0 Hz, ArH), 12.14 (1H, s, COOH); 13C-NMR
(DMSO-d6, 125 MHz) δ = 28.2 (CH2, CH2COOH), 30.9
(CH2,CH2CONH), 127.8 (CH, ArCH), 129.6 (CH, ArCH),
134.0 (CH, ArCH), 140.0 (CH, ArCH), 171.1 (C, CONH),
173.7 (C, COOH); MS (ESI) calcd. for C10H10NO5S
[M–H]-: 256.02; found 256.01. Anal. calcd. for
C10H11NO5S (%): C 46.69, H 4.31, N 5.44. Found: C 46.82,
H 4.33, N 5.45.
3-{[4-(3,4-Difluorophenyl)-1,3-thiazol-2-yl]carba-
moyl}propanoic acid (F4b) To a stirred solution of 4-(3,4-
difluorophenyl)-1,3-thiazol-2-amine (110 mg, 0.5 mmol) in
acetonitrile (15 mL), succinic anhydride (60 mg, 0.6 mmol)
was added and the mixture was stirred at room temperature
for 24 h. The precipitant formed was collected by filtration.
The product was obtained as a white solid (60 mg, 38%);
mp 198–200 °C; IR (KBr) νmax 3432 (N-H), 3188 (sp2 CH),
2983 (O-H), 1685 (C=O), 1587 (C = N), 1509, 1429,
1
1377 (CC); H-NMR, (DMSO-d6, 500 MHz) δ = 2.57 (2H,

Medicinal Chemistry Research (2021) 30:977–986
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3-[(4-Nitrobenzenesulfonyl)carbamoyl]propanoic
acid (F5b) To a mixture of 4-nitrobenzene-1-sulfonamide
(202 mg, 1 mmol) and succinic anhydride (120 mg,
1.2 mmol) in dichloromethane (15 mL), N,N-diisopropy-
lethylamine (200 μL) was added and the mixture was stirred
for 12 h. The organic solution was removed using a rotary
evaporator and the crude mixture was purified by column
chromatography (ethyl acetate + 0.5% (v/v) methanol +
1% (v/v) formic acid). The product was obtained as a white
solid, (189 mg, 63%); mp 153–154 °C; IR (KBr) νmax 3681
(N-H), 3522 (O-H), 3115 (sp2 CH), 1702 (C=O), 1544
(C=C), 1311 and 1140 (S=O); 1H-NMR (Ace-d6,
500 MHz) δ = 2.55 (2H, t, J = 6.5 Hz, CH2COOH), 2.67
(2H, t, J = 6.5 Hz, CH2CONH), 8.30 (2H, d, J = 8.0 Hz,
ArH), 8.45 (2H, d, J = 8.0 Hz, ArH); 13C-NMR (Ace-d6,
125 MHz) δ = 27.4 (CH2, CH2COOH), 30.6 (CH2,
CH2CONH), 124.1 (CH, ArCH), 129.7 (CH, ArCH), 145.1
(C, ArCS), 150.8 (C, ArCN), 170.5 (C, CONH), 172.6 (C,
COOH); HRMS (ESI) calcd. for C10H10N2O7S [M]-:
302.0209; found 302.0209.
3-[(4-Chlorobenzenesulfonyl)carbamoyl]propanoic
acid (F5c) To a mixture of 4-chlorobenzene-1-sulfonamide
(383 mg, 2 mmol) and succinic anhydride (300 mg, 3 mmol)
in dichloromethane (25 mL), N,N-diisopropylethylamine
(350 μL) was added and stirred for 12 h. The organic
solution was removed using a rotary evaporator and the
crude mixture was purified by column chromatography
(ethyl acetate:hexane, 1:1 + 1% (v/v) formic acid). The
product was obtained as a white solid (315 mg, 54%); mp
151–156 °C; IR (KBr) νmax 3273 (N-H and O-H), 3090 (sp2
CH), 1697 (C=O), 1584 (C=C), 1344 and 1128 (S=O);
1H-NMR (Ace-d6, 500 MHz) δ = 2.55 (2H, t, J = 7.0 Hz,
CH2COOH), 2.65 (2H, t, J = 7.0 Hz, CH2CONH), 7.67
(2H, d, J = 9.0 Hz, ArH), 8.02 (2H, d, J = 9.0 Hz, ArH);
13C-NMR (Ace-d6, 125 MHz) δ = 27.4 (CH2, CH2COOH),
30.5 (CH2, CH2CONH), 129.1 (C, ArCH), 129.9 (C,
ArCH), 138.6 (C, ArCS), 139.2 (C, ArCN), 170.2 (C,
CONH), 172.6 (C, COOH); HRMS (ESI) calcd. for
C10H10ClNO5S [M]-: 290.9968; found 290.9969.
(1H, dd, J = 2.0 Hz, 8.0 Hz, ArH), 8.06 (1H, d, J = 8.0 Hz,
ArH), 8.14(1H, d, J = 8.0 Hz, ArH), 8.22 (1H, d, J =
8.0 Hz, ArH), 8.58 (1H, s, ArH), 12.16 (2H, s, NH, OH);
13C-NMR (DMSO-d6, 125 MHz) δ = 28.2 (CH2,
CH2COOH), 30.9 (CH2, CH2CONH), 122.9 (CH, ArCH),
128.2 (CH, ArCH), 128.3 (CH, ArCH), 129.4 (CH, ArCH),
129.7 (CH, ArCH), 129.9 (CH, ArCH), 131.9 (C, ArC),
135.1 (C, ArC), 137.0 (C, ArCS), 171.2 (C, CONH), 173.7
(C, COOH); MS (ESI) calcd. for C14H12NO5S [M–H]-:
306.04; found 306.44. Anal. calcd. for C14H13NO5S (%): C
54.72, H 4.26, N 4.56. Found: C 53.41, H 2.32, N 3.89.
Ethyl 3-[(1,3-benzoxazol-2-yl)carbamoyl]propanoate
(E1) To a mixture of 3-[(1,3-benzoxazol-2-yl)carbamoyl]
propanoic acid (240 mg, 1.03 mmol) in ethanol (5 mL), two
drops of sulfuric acid were added; the mixture was stirred at
60 °C for 2 h. The solution was then cooled to room tem-
perature and saturated sodium bicarbonate (20 mL) was
added. The solution was then extracted with ethyl acetate
(20 mL × 3). The organic solution was removed using a
rotary evaporator and crude compound was purified by
column chromatography (hexane:ethyl acetate, 7:3). The
product was obtained as a yellow solid (60 mg, 22%); mp
173–178 °C; IR (KBr) νmax 3383 (N-H), 3239 (O-H), 3152
1
(sp2 CH), 1678 (C=O), 1554 (C=C); H-NMR (Ace-d6,
500 MHz) δ = 1.23 (3H, t, J = 7.0 Hz, CH3C), 2.70 (2H, t,
J = 7.0 Hz, CH2COO), 2.83 (2H, t, J = 7.0 Hz, CH2CON),
4.11 (2H, q, J = 7.0 Hz, CH2O), 6.83 (1H, t, J = 7.5 Hz,
ArH), 6.93 (2H, m, ArH), 8.12 (1H, d, J = 7.5 Hz, ArH);
13C-NMR (Ace-d6, 125 MHz) δ = 13.6 (CH3, CH3C), 28.9
(CH2, CH2COO), 30.9 (CH2, CH2CON), 60.1 (CH2,
CH2O), 115.0 (CH, ArCH), 119.7 (CH, ArCH), 120.2 (CH,
ArCH), 123.6 (CH, ArCH), 126.7 (C, ArCN), 146.4 (C,
ArCO), 150.7 (C, ArCNH), 171.9 (C, CONH), 173.9 (C,
COO); HRMS (ESI) calcd. for C13H14N2O4 [M]−:
262.0954; found 262.0962.
Ethyl
propanoate (E2) To a mixture of 3-[(5-chloro-1,3-ben-
zoxazol-2-yl)carbamoyl]propanoic acid (270 mg,
3-[(5-chloro-1,3-benzoxazol-2-yl)carbamoyl]
1.01 mmol) in ethanol (5 mL), two drops of sulfuric acid
were added; the mixture was stirred at 50 °C for 12 h. The
solution was then cooled to room temperature and saturated
sodium bicarbonate (20 mL) was added. The solution was
then extracted with ethyl acetate (20 mL × 3). The organic
solution was removed in rotary evaporator and the crude
compound was purified by column chromatography (hex-
ane:ethyl acetate, 6.5:3.5). The products was obtained as a
yellow solid (74 mg, 25%); mp 185–190 °C; IR (KBr) νmax
3227 (N-H and O-H), 3123 (sp2 CH), 1681 (C=O), 1548
3-[(Naphthalene-2-sulfonyl)carbamoyl]propanoic
acid (F5d) To a mixture of naphthalene-2-sulfonamide
(154 mg, 0.7 mmol) and succinic anhydride (140 mg,
1.4 mmol) in dichloromethane (10 mL), N,N-diisopropy-
lethylamine (300 μL) was added; the mixture was stirred at
room temperature for 72 h. The organic solvents were
removed using a rotary evaporator. The resultant crude
mixture was washed with distilled water (5 mL), filtered and
recrystallized using tert-butyl alcohol. The product was
obtained as a white solid (99 mg, 64%); mp 153–154 °C; IR
(KBr) νmax 3442 (N-H), 3241 (O-H), 1728, 1707 (C=O),
1337, 1119 (S=O), 869 (S-N); 1H-NMR (DMSO-d6,
500 MHz) δ = 2.34 (2H, t, J = 6.5 Hz, CH2COOH), 2.48
(2H, t, J = 6.5 Hz, CH2CONH), 7.70 (2H, m, ArH), 7.88
1
(C=C); H-NMR (Ace-d6, 500 MHz): δ = 1.23 (3H, t, J =
7.0 Hz, CH3C), 2.70 (2H, t, J = 7.0 Hz, CH2COO), 2.84
(2H, t, J = 7.0 Hz, CH2CON), 4.11 (2H, q, J = 7.0 Hz,
CH2O), 6.93 (2H, m, ArH), 8.31 (1H, s, ArH); 13C-NMR
(Ace-d6, 125 MHz): δ = 13.6 (CH3, CH3C), 28.0 (CH2,

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Medicinal Chemistry Research (2021) 30:977–986
CH2COO), 30.9 (CH2, CH2CON), 60.1 (CH2, CH2O), 115.5
(CH, ArCH), 119.4 (CH, ArCH), 122.7 (CH, ArCH), 123.9
(C, ArCCl), 128.1 (C, ArCN), 144.9 (C, ArCO), 150.6 (N,
NH), 171.9 (C, CON), 174.0 (C, COO); HRMS (ESI) calcd.
for C13H13ClN2O4 [M]−: 295.0491; found 295.0507.
1H and 13C NMR spectra of the above compounds are
shown in Supplementary information.
(v/v) formic acid in water to remove nonvolatile buffer salts
and then peptide was eluted from the SPE with 85% (v/v)
acetonitrile, 15% water containing 0.1% formic acid onto
the mass spectrometer. Peptide charges state were mon-
itored in the positive ionization mode. Ion chromatogram
data were extracted for the +2 charge state and peak area
data integrated using RapidFire Integrator software (Agi-
lent). Percentage of conversion of the CODD peptide sub-
strate to the +16 hydroxylated peptide was calculated using
the equation below:
Recombinant PHD-2 production
The catalytic domain of human PHD-2181-426 (referred to as
PHD-2 in this study) was produced and purified as reported
[36, 37]. In brief, DNA encoding for a truncated form of
PHD-2181–426 (hereafter PHD-2), was cloned into the pET-
28a(+) vector. Recombinant PHD-2 was produced in
Escherichia coli BL21(DE3) cells by induction with
0.5 mM isopropyl β-D-1-thiogalactopyranoside for 6 h at
28 °C. Cells were freeze-thawed and lysed by sonication.
PHD-2 was purified by Ni2+ affinity chromatography fol-
lowed by size-exclusion chromatography (0.1 M Tris·HCl
pH 7.5, 0.1 M NaCl). Protein purity was assessed by
SDS–PAGE; proteins were characterized by electrospray
ionization mass spectrometric analyses under non-
denaturing and/or denaturing conditions.
100 Â hydroxylated
% of conversion ¼
:
hydroxylated þ non hydroxylated peptide
2-[(4-Hydroxy-1-methyl-7-phenoxyisoquinoline-3-car-
bonyl)amino]acetic acid (Roxadustat) was employed as the
positive inhibitor control in this assay [38, 39]. The IC50
data were determined from the non-linear regression plots
using GraphPad Prism 8.
Cell lines and culture conditions
The human lung cancer cell line A549 was from Addex-
Bio Technologies (San Diego, CA, USA). All cells were
cultured in Dulbecco’s modified Eagle’s medium
(DMEM; Nacalai Tesque 08459-64) supplemented with
10% Fetal Bovine Serum (FBS; TICO Europe,
FBSEU500) and 100 units/mL Penicillin/Streptomycin
(Nacalai Tesque, 09367-34) in a humidified incubator at
37 °C and 5% CO2.
Isolated PHD-2 mass spectrometric assays
As reported the MS based assay for PHD-2 as employed
here is in an endpoint assay (typical enzyme incubation time
of 15 min) [29, 38]. All solutions were prepared freshly
each day. All inhibition assays were carried out in 384-well
polypropylene plates (Greiner Bio-One). The PHD-2 assays
were performed in 50 mM Tris HCl, 50 mM NaCl, pH 7.8.
Titrations of compounds for IC50 determinations (threefold
and 11-point IC50) were performed using an ECHO 550
acoustic dispenser (Labcyte) and dry dispensed into 384-
well polypropylene assay plates. The final assay con-
centration of aqueous DMSO was kept constant at 0.5% (v/
v). PHD-2 was prepared at a concentration of 300 nM in the
assay buffer and 25 μL were dispensed across each 3840-
well assay plate. PHD-2 protein was allowed to equilibrate
with compounds for 15 min and the reaction then initiated
by dispensing 25 μL of the substrate (20 μM ammonium
iron(II) sulfate, 200 μM L-sodium ascorbate, 10 μM HIF-1α
CODD peptide and 20 μM disodium 2-oxoglutarate) in
assay buffer. Enzyme reactions were allowed to proceed for
15 min and each reaction was terminated by the addition of
10% formic acid (5 μL). Assay plates were then transferred
to a RapidFire RF360 sampling robot (Agilent) connected
to an Agilent 6530 accurate mass quadrupole-time-of-flight
mass spectrometer. Assay samples were aspirated under
vacuum and loaded onto a C4 solid-phase extraction (SPE)
cartridge. After loading, the C4 SPE was washed with 0.1%
Compound treatment and immunoblotting
Cells at about 80% confluency were incubated for 6 h in
medium supplemented with an inhibitor (dissolved in
DMSO, stock concentration of 100 mM; final concentration
1–2% (v/v)) as previously described [30, 40]. DMSO was
used as the negative control; Roxadustat was used as the
positive control. In brief, cells were rinsed in phosphate-
buffered saline (PBS) and subsequently lysed in urea/
sodium dodecyl sulfate (SDS) buffer (6.7 M urea, 10 mM
Tris-HCl (pH 6.8), 10% (v/v) glycerol, and 1% SDS).
Whole-cell extracts were resolved by SDS-PAGE (poly-
acrylamide gel electrophoresis) gel and the protein was
electroblotted onto polyvinylidene difluoride (PVDF)
membrane (IPVH00010, Immobilon). The membranes were
then stained by Ponceau S solution (22001, Biotium) to
determine equal loading and washed three times in PBS for
10 min. Subsequently, the membranes were blocked over-
night at 4 °C in 1X PBS, 0.1% Tween-20 with 5% w/v non-
fat dry milk (Marvel) and probed with mouse monoclonal
HIF-1α antibody clone 54 (610958, BD Transduction
Laboratories, 1:1000) and β-actin/HRP (A3854, Sigma
Aldrich, 1:25,000) for 1 h at room temperature. After three

Medicinal Chemistry Research (2021) 30:977–986
983
10-min washings in PBS mixed with 0.1% Tween 20 (PBS-
T buffer), the membranes probed with HIF-1α antibody
were further incubated with horseradish peroxidase (HRP)-
conjugated goat polyclonal anti-mouse immunoglobulin G
(NB7539, Novus Biologicals) at 1:10,000 for 1 h at room
temperature. Membranes were washed three times, and
immunoreactivity was visualized with Immobilon Forte
Western ECL substrate (WBLUF0100, Immobilon) using a
chemiluminescence Molecular Imager® ChemiDoc™ XRS
+ system (Bio-Rad Laboratories, Inc.) according to the
manufacturer’s instructions.
PHD-2 inhibitory assays
An established mass spectrometry based assay was then
employed to investigate the half-maximal inhibitory con-
centrations (IC50s) of the 3CA derivatives against the cat-
alytic domain of human PHD-2 [29, 38]. The PHD-2 MS
assay directly monitors the extent of reaction of the
C-terminal oxygenase dependent domain (CODD) HIF-1α
peptide substrate DLDLEMLAPYIPMDDDFQL-NH2 (all
peptides were prepared as C-terminal amides) into the
hydroxylated product by adding for a +16 Da mass shift.
All assays were performed in triplicate. Of all the 3CAs
tested, only the benzoxazole analogs, F2a and F2b were
found to significantly inhibit PHD-2 activity with IC50
values of 2.24 μM and 1.32 μM, respectively. Both com-
pounds exhibit similar inhibitory potencies to the positive
control, Roxadustat (IC50 = 2.21 μM). Other 3CA com-
pounds had IC50 values higher than 100 μM. The IC50
values of the 3CA analogs against PHD-2 are summarized
in Table 1. Plots of log of the concentrations of the inhibitor
versus the percentage of PHD-2 inhibition for F2a and F2b
are shown in the Supplementary information (Fig. S1).
Interestingly, the benzothiazole analogs, F1a and F1c,
which have similar structures to F2a and F2b did not inhibit
PHD-2 catalysis at concentrations up to 100 μM. These
results suggest that the sulfur-containing analogs might be too
big to fit into the PHD-2 active site binding pocket. The
results also show that the pyrazole ring (F3a), thiophene ring
Results and discussion
Synthesis of 3-carbamoylpropanoic acid (3CA)
analogs
Compounds containing 3CA appended onto benzothiazole
(F1a–F1d), benzoxazole (F2a–F2b), pyrazole (F3a), thio-
phene (F4a–F4c) and sulfonyl aromatic (F5a–F5d) ring
scaffolds by synthesis as shown in Scheme 1. Commercially
available amines precursors of the corresponding target
compounds were reacted with succinic anhydride in the
presence or absence of N,N-diisopropylethylamine [35].
The ethyl esters (E1 and E2) were synthesized by reacting
the acids (F2a and F2b) with ethanol in the presence of
sulfuric acid.
Scheme 1 Synthesis of 3CA
analogs. Reagents and
conditions: a CHCl3, THF,
DCM, DMF, reflux, or room
temperature 1–24 h. b DCM,
DIPEA, 12–72 h

984
Medicinal Chemistry Research (2021) 30:977–986
(F4a–F4c) and sulfonyl aromatic ring (F5a–F5d) 3CA ana-
logs do not inhibit PHD-2, further supports the importance of
an appropriately functionalized heterocyclic ring in the design
of PHD-2 inhibitors of the type under study here [41].
The somewhat higher potency of F2b versus F2a may
reflect the presence of a chlorine atom in the bicyclic ring of
F2b, which may interact with the hydrophobic elements at
the active site entrance thus enhancing binding [38]. Dock-
ing studies suggest that both F2a and F2b bind in a similar
way to the PHD-2 active site, with the docked conformation
of F2b shown in Fig. 1. As anticipated based on crystal-
lographic analyses of related compounds in the model [38],
the carboxylate of F2b is positioned to forms a salt bridge
with Arg383 and to hydrogen bond with Tyr329 as does the
C-5 carboxylate of 2-OG [29, 42]. The benzoxazole nitrogen
atom and the amide carbonyl oxygen atom in F2b are
positioned to coordination in a bidentate manner with the
active site iron. Note that since HIF-α can bind to the PHD-2
active site in the presence of some (but not all) active site
binding inhibitors, the chlorine atom in F2b may interfere
with HIF peptide substrate binding (Fig. 1), thus increasing
the inhibitory potency of F2b relative to F2a [29].
Cell-based assays
Ester derivatives, in general, are reported to be more cell-
penetrating than their respective free acids and are commonly
used as prodrugs in cell-based experiments [43, 44], including
for PHD inhibitors such as N-oxalylglycine/dimethyl oxa-
lylglycine [4, 45]. Therefore, the ethyl ester (E1 and E2)
derivatives of F2a and F2b were synthesized for studies in
cells. Human lung cancer A549 cells were treated with E1
and E2 at two concentrations (200 μM and 1 mM) for 6 h and
the resulting cell extracts were probed for HIF-1α by immu-
noblotting. F2a and F2b acids were tested for comparison.
Although the PHD-2 MS results described above show that
F2a and F2b are relatively potent PHD-2 inhibitors, neither of
these compounds, nor their corresponding esters (E1 and E2)
causes HIF-1α accumulation in the cells (see Supplementary
information (Figs. S1 and S2). It was speculated that their
solubilities might negatively affect the results due to com-
pound precipitation in the cell media during treatment (1% (v/
v) aqueous DMSO final concentration). Therefore, the
experiments were then repeated at a higher final concentration
of DMSO (2% (v/v)) with no observable precipitation of
compounds in the cell media. However, again no HIF-1α
upregulation was observed. One possible reason for this
unexpected observation is the failure of the tested compounds
to cross the cell membrane. Future studies could be focused
on structure-activity relationship studies to optimize the
penetration of PHD inhibitors into cells, including by
increasing the lipophilicity and membrane-permeability of the
compounds described here.
Table 1 The IC50 values of 3CA analogs against PHD-2. All assays
were performed in triplicate, and the data are expressed as mean
values standard deviation (SD)
Compound
IC50 (μM)
Compound
IC50 (μM)
Roxadustat
F1a
2.21 0.74
>100
F4a
F4b
F4c
F5a
F5b
F5c
F5d
>100
>100
>100
>100
>100
>100
>100
F1b
>100
F1c
>100
F1d
>100
Conclusion
F2a
2.24 1.9
1.32 1.03
>100
F2b
In this study, a novel series of 3-carbamoylpropanoic acid
analogs were evaluated as potential PHD-2 inhibitors. The
F3a
Fig. 1 LEFT: The docked conformation of F2b with 2-OG in PHD-2.
Fe(II) complex in Chimera Version 1.13.1. RIGHT: The chlorine atom
in F2b is positioned to block the entrance of the PHD-2 active site, as
shown by a PYMOL image. (Version 2.3.2 Schrödinger, LLC).
Docking was performed using Maestro Version 11.8.012 Schrödinger,
LLC and PDB: 2hbt [46]

Medicinal Chemistry Research (2021) 30:977–986
985
PHD-2 inhibitory assay results revealed that the simple
heteroaromatic derivatives F2a and F2b are relatively
potent PHD-2 inhibitors with IC50 values of 2.24 μM and
1.32 μM, respectively. These values are similar to that of
PHD inhibitors in clinical trials. However, the cell-based
results indicated that F2a, F2b and their ethyl esters (E1
and E2) failed to induce HIF-1α in cells, likely due to their
low cell permeability.
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14. Bruick RK. Oxygen sensing in the hypoxic response pathway:
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Acknowledgements We thank the Research University Grants (RUI),
Universiti Sains Malaysia (Grant number: 1001.PKIMIA.811250) and
Bantuan Kecil Penyelidikan, University of Malaya (Grant number:
BKP004-2018) for supporting the research. CJS, MIA, and AT thank
the Welcome Trust and Cancer Research UK for funding.
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containing protein inhibitors as stabilizers of hypoxia-inducible
factor: small molecule-based therapeutics for anemia. Exp Opin
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inhibitors: a potential new treatment for anemia in patients With
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Compliance with ethical standards
Conflict of interest The authors declare they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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