Design and synthesis of substituted pyridine derivatives as HIF-1α prolyl hydroxylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.08.026

Structure-guided de novo drug design led to the identification of a novel series of substituted pyridine derivatives as HIF-1α prolyl hydroxylase inhibitors. Pyridine carboxyamide derivatives bearing a substituted aryl group at the 5-position of the pyrid

Full text of DOI:10.1016/j.bmcl.2006.08.026

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5616–5620
Design and synthesis of substituted pyridine derivatives
as HIF-1a prolyl hydroxylase inhibitors
Namal C. Warshakoon,a,* Shengde Wu,a Angelique Boyer,a Richard Kawamoto,a
Justin Sheville,a Ritu Tiku Bhatt,a Sean Renock,a Kevin Xu,a Matthew Pokross,a
Songtao Zhou,a Richard Walter,a Marlene Mekel,a
Artem G. Evdokimova and Stephen Eastb
aLead Discovery, Procter and Gamble Pharmaceuticals, 8700 Mason-Montgomery Rd, Mason, OH 45040, USA
bEvotec (UK) Ltd, 111 Milton Park, Abingdon, Oxfordshire, OX14 4RZ, UK
Received 29 June 2006; revised 2 August 2006; accepted 2 August 2006
Abstract—Structure-guided de novo drug design led to the identification of a novel series of substituted pyridine derivatives as HIF-
1a prolyl hydroxylase inhibitors. Pyridine carboxyamide derivatives bearing a substituted aryl group at the 5-position of the pyridine
ring show appreciable activity, while constraining the side chain by placing a pyrazole carboxylic acid generated a potent lead series
with consistent activity against EGLN-1.
Ó 2006 Elsevier Ltd. All rights reserved.
Hypoxia inducible factor 1-alpha (HIF-1a) is a heterodi-
meric transcription factor that plays a key role in oxygen
homeostasis in mammals, including humans.1 Under
hypoxic conditions (low partial pressure of oxygen),
HIF-1 activates genes responsible for hypoxic response,
such as angiogenesis (VEGF), erythropoiesis, and gly-
colysis.2 Both heterodimer components, HIF-1a and
HIF-1b (ARNT), are constitutively expressed and regu-
lation is achieved by the selective destruction of HIF-1a.
Therefore, HIF-1a plays a major role in cellular re-
sponse to hypoxia.
tions hydroxylation is inhibited—as a result, HIF-1a
binds to HIF-1b to form a functional transcriptional
activator which turns on genes with hypoxic response
elements (e.g., VEGF, EPO, and glycolytic enzymes).6
The upregulation of HIF activity has potential thera-
peutic significance for ischemic diseases including myo-
cardial infarction, stroke, peripheral arterial disease,
heart failure, diabetes, and anemia. We are interested
in pharmacologically eliciting the hypoxic response by
means of small-molecule prolyl hydroxylase inhibitors.
Previously described HIF-1a prolyl hydroxylase inhibi-
tors (compound 2: approximate IC50 is 1 lM) have
largely been 2-oxoglutarate (2-OG) mimetics (Fig. 1).7
HIF-1a is hydroxylated on prolines 564 and 402 by a
family of non-heme, iron-containing prolyl hydroxylases
(EGLN-1, EGLN-2, and EGLN-3) under normoxic
conditions.3–5 Upon hydroxylation, HIF-1a binds to
the complex of proteins comprised of VHL (Von Hippel
Lindau tumor suppressor protein), elongin C/B, CuI2,
Rbx1, and ubiquitin ligase. This binding event targets
HIF-1a for cytoplasmic proteasomal degradation.
EGLN-mediated proline hydroxylation is strictly depen-
dent on oxygen and consequently, under hypoxic condi-
Interestingly, the isoquinoline 3, a recently disclosed
HIF-1a prolyl hydroxylase inhibitor, consists of a non-
peptidic-like structure and may inhibit EGLN enzymes
by acting as a 2-OG mimetic (Fig. 2).8
In a significant breakthrough, we were recently able to
solve the previously unknown crystal structure of
EGLN-1 in complex with isoquinoline 3.9,10
The key interactions between compound 3 and the
EGLN-1 active site are: the salt bridge between the car-
boxylic acid and Arg383, the coordination of iron via
the isoquinoline nitrogen and the amide carbonyl, and
Keywords: Prolyl hydroxylase inhibitors; HIF-1a; Hypoxia; Ischemia;
Peripheral arterial disease (PAD); Anemia.
*
Corresponding author. Tel.: +1 513 622 2284; fax: +1 513 622
0523; e-mail: Warshakoon.nc@pg.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.026

N. C. Warshakoon et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5616–5620
5617
Figure 1. Small-molecule HIF-1a prolyl hydroxylase inhibitors.
the hydrogen bond between the phenolic hydroxyls of
the compound and of Tyr303. While the in vitro activity
of isoquinoline 3 on EGLN-1 assay was 1.4 lM, when
either of the coordinating elements are absent (quinoline
nitrogen or the amide carbonyl), those compounds
exhibited no activity toward EGLN-1 enzyme in our
hands. Thus, we decided to explore the metal coordinat-
ing ability of pyridine carboxyamides (see Fig. 3).
Figure 3. Binding of compound 3 to the active site of EGLN-1.
In our initial modeling studies, the substituted pyridine
derivative 4 exhibited a reasonable binding orientation
when docked into the EGLN-1 active site (Fig. 4).
In this binding orientation, the inhibitor carboxylate
interacts with Arg383, while the pyridine nitrogen and
the amide carbonyl coordinate iron. In addition,
Met299 and the chloro-substituted benzene ring interact
hydrophobically. It should be noted that the hydrogen
bonding interaction that was observed with isoquinoline
3 is absent in this case, however, we observe an addition-
al p-stacking interaction between the pyridine ring and
Tyr310.
The substituted pyridine derivatives were synthesized
according to the schemes below.
Figure 4. The possible binding mode of compound 4 to the active site
of EGLN-1.
5-Bromopicolinic acid 5 was coupled with glycine meth-
yl ester and a subsequent Suzuki coupling and a hydro-
lysis of the methyl ester generated the desired 5-aryl
pyridine carboxyamides (Scheme 1). The 4-substituted
pyridine carboxyamides were generated according to
Scheme 2.
were synthesized via a microwave-assisted condensation
reaction between 5-bromo-2-chloropyridine 11 and ethyl
1H pyrazolecarboxylate 12. Suzuki coupling generated
the desired analogs with concomitant hydrolysis of the
ethyl ester under basic conditions (Scheme 3).
Next, we explored constraining the side chain with
appropriate glycine isosteres to elicit more rigid interac-
tions with the enzyme active site. The side-chain con-
strained pyridine analogs containing a pyrazole group
The substituted pyridine derivatives were screened against
EGLN-1 enzyme assay (Table 1). The unsubstituted
Figure 2. Possible binding modes of 2-OG and compound 3.

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N. C. Warshakoon et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5616–5620
Scheme 1. 5-Substituted pyridine derivatives. Reagents and conditions: (a) SOCl2/Gly-OMe/iPr2EtNH (90%); (b) 1—ArB(OH)2/Pd(dppf)Cl2/K3P
O4/dioxane/EtOH; 2—LiOH/THF/Water (65%).
Scheme 2. 4-Substituted pyridine derivatives. Reagents and conditions: (a) EDAC/HOBt/Gly-OMe/iPr2EtNH (52%); (b) 1—ArB(OH)2/Pd(dppf)Cl2/
K3P O4/dioxane/EtOH; 2—LiOH/THF/water (65%).
Scheme 3. Reagents and conditions: (a) 12/Cs2CO3/DMF/145 °C/MW/20 min (75%); (b) ArB(OH)2/Pd(PPh3)4/K2CO3/CH3CN/water/135 °C/MW/
15 min (70%).
pyridine carboxyamide (Table 1, entry 1) was completely
inactive. Similarly, the 4-substituted pyridine carboxya-
mide (Table 1, entry 2) did not show any activity.
The 5-substituted pyridine carboxyamides showed
discernable activity (Table 1, entries 3, 4, and 5). The
para-chloro phenyl-substituted 5-pyridine carboxyamide
(Table 1, entry 5) showed the best activity (15 lM).
Interestingly, the meta-chloro derivative was completely
inactive.
ed laser desorption ionization, time-of-flight MS, MAL-
DI-TOF MS). The HIF-1a peptide corresponding to
residues 556–574 (DLDLEALAPYIPADDDFQL) was
used as substrate. The reaction was conducted in a total
volume of 50 lL containing Tris–Cl (5 mM, pH 7.5),
ascorbate (120 lM), 2-oxoglutarate (3.2 lM), HIF-1a
(8.6 lM), and bovine serum albumin (0.01%). EGLN-
1, quantity predetermined to hydroxylate 20% of sub-
strate in 20 min, was added to start the reaction. Where
inhibitors were used, compounds were prepared in
dimethylsulfoxide at 10-fold final assay concentration.
After 20 min at room temperature, the reaction was
stopped by transferring 10 lL of reaction mixture to
50lL of a mass spectrometry matrix solution (a-cya-
no-4-hydroxycinnamic acid, 5 mg/ml in 50% acetoni-
trile/0.1% TFA, 5 mM NH4PO4). Two microliters of
the mixture was spotted onto a MALDI-TOF MS target
plate for analysis with an Applied Biosystems (Foster
City, CA) 4700 Proteomics Analyzer MALDI-TOF
MS equipped with a Nd:YAG laser (355 nm, 3 ns pulse
width, 200 Hz repetition rate). Hydroxylated peptide
product were identified from substrate by the gain of
16 Da. Data defined as percent conversion of substrate
to product was analyzed in GraphPad Prism 4 to calcu-
late IC50 values.
The side-chain-constrained, pyrazole substituted pyri-
dine analogs exhibited consistently good activity. In this
instance, chloro substitutions at the ortho and meta po-
sition at the outer benzene ring (Table 1, entries 8 and 9)
were about three times more active than the para-chloro
substituted compound (Table 1, entry 7). It was interest-
ing to note that other heterocyclic glycine isosteres (e.g.,
oxazoles, imidazoles, and oxadiazole) exhibited weaker
activity compared to the pyrazole isosteres.
In the case of electron-donation methoxy substitution
the para methoxy substitution (Table 1, entry 13) was
slightly more active than ortho and meta substitutions
(Table 1, entries 15 and 16). Lipophilic substituents,
for example, isopropyl (Table 1, entry 14) showed
good activity while heteroaromatics, for example,
thiophene (Table 1, entry 11), furan (Table 1, entry
10) and pyrimidine (Table 1, entry 12), showed a
decrease in activity.
In conclusion, structure-based design approach and
efficient chemistry strategy led us to discover a novel
series of substituted pyridine derivatives as new HIF-
1a prolyl hydroxylase inhibitors. This series served
as initial lead compounds for a more potent com-
pound series that were discovered by introducing
EGLN-1 activity assay. The EGLN-1 enzyme activity
was determined using mass spectrometry (matrix-assist-

N. C. Warshakoon et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5616–5620
Table 1. Activity of substituted pyridine derivatives against EGLN-1 (95% CI)
5619
Entry
Structure
EGLN-1 IC50 (lM)
1
>100
2
3
>100
46
39
4
5
15
>100
20
6
7
8
4.9
9
6.5
10
15
(continued on next page)
Table 1 (continued)

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N. C. Warshakoon et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5616–5620
Table 1 (continued)
Entry
Structure
EGLN-1 IC50 (lM)
11
12
18
15
13
7.3
14
6.8
15
19
16
10
5. Kondo, K.; Kaelin, W. G., Jr. Exp. Cell. Res. 2001, 264,
117.
appropriate substitutions on the pyridine ring (details
would follow). The EGLN-1 active compounds are
currently under investigation for HIF-1 stabilization
and cell-based VEGF induction (downstream bio-
marker for HIF-1 stabilization).
6. Semenza, G. Biochem. Pharmacol. 2002, 64, 993.
7. (a) McDonough, M. A.; McNeill, L. A.; Tilliet, M.;
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