1,3,8-triazaspiro[4.5]decane-2,4-diones as efficacious pan-inhibitors of hypoxia-inducible factor prolyl hydroxylase 1-3 (HIF PHD1-3) for the treatment of anemia

Article abstract of DOI:10.1021/jm201542d

The discovery of 1,3,8-triazaspiro[4.5]decane-2,4-diones (spirohydantoins) as a structural class of pan-inhibitors of the prolyl hydroxylase (PHD) family of enzymes for the treatment of anemia is described. The initial hit class, spirooxindoles, was identified through affinity selection mass spectrometry (AS-MS) and optimized for PHD2 inhibition and optimal PK/PD profile (short-acting PHDi inhibitors). 1,3,8-Triazaspiro[4.5]decane-2,4-diones (spirohydantoins) were optimized as an advanced lead class derived from the original spiroindole hit. A new set of general conditions for C-N coupling, developed using a high-throughput experimentation (HTE) technique, enabled a full SAR analysis of the spirohydantoins. This rapid and directed SAR exploration has resulted in the first reported examples of hydantoin derivatives with good PK in preclinical species. Potassium channel off-target activity (hERG) was successfully eliminated through the systematic introduction of acidic functionality to the molecular structure. Undesired upregulation of alanine aminotransferese (ALT) liver enzymes was mitigated and a robust on-/off-target margin was achieved. Spirohydantoins represent a class of highly efficacious, short-acting PHD1-3 inhibitors causing a robust erythropoietin (EPO) upregulation in vivo in multiple preclinical species. This profile deems spirohydantoins as attractive short-acting PHDi inhibitors with the potential for treatment of anemia.

Full text of DOI:10.1021/jm201542d

Featured Article
pubs.acs.org/jmc
1,3,8-Triazaspiro[4.5]decane-2,4-diones as Efficacious Pan-Inhibitors
of Hypoxia-Inducible Factor Prolyl Hydroxylase 1−3 (HIF PHD1−3)
for the Treatment of Anemia
Petr Vachal,*,† Shouwu Miao,† Joan M. Pierce,† Deodial Guiadeen,† Vincent J. Colandrea,†
Matthew J. Wyvratt,† Scott P. Salowe,‡ Lisa M. Sonatore,‡ James A. Milligan,§ Richard Hajdu,§
Anantha Gollapudi,§ Carol A. Keohane,§ Russell B. Lingham,§ Suzanne M. Mandala,§ Julie A. DeMartino,§
Xinchun Tong,∥ Michael Wolff,∥ Dietrich Steinhuebel,⊥ Gerard R. Kieczykowski,⊥ Fred J. Fleitz,⊥
Kevin Chapman,# John Athanasopoulos,# Gregory Adam,# Can D. Akyuz,▽ Dhirendra K. Jena,▽
Jeffrey W. Lusen,# Juncai Meng,▽ Benjamin D. Stein,▽ Lei Xia,▽ Edward C. Sherer,○ and Jeffrey J. Hale†
†
‡
§
∥
⊥
Departments of Medicinal Chemistry, Infectious Diseases; Immunology; Drug Metabolism/Pharmacokinetics; Process
Research; Target Validation; ▽Information Technology; ○Chemistry Modeling and Informatics. Merck Research Laboratories,
#
Rahway, New Jersey 07065, United States
S
* Supporting Information
ABSTRACT: The discovery of 1,3,8-triazaspiro[4.5]decane-
2,4-diones (spirohydantoins) as a structural class of pan-
inhibitors of the prolyl hydroxylase (PHD) family of enzymes
for the treatment of anemia is described. The initial hit class,
spirooxindoles, was identified through affinity selection mass
spectrometry (AS-MS) and optimized for PHD2 inhibition
and optimal PK/PD profile (short-acting PHDi inhibitors).
1,3,8-Triazaspiro[4.5]decane-2,4-diones (spirohydantoins) were optimized as an advanced lead class derived from the original
spiroindole hit. A new set of general conditions for C−N coupling, developed using a high-throughput experimentation (HTE)
technique, enabled a full SAR analysis of the spirohydantoins. This rapid and directed SAR exploration has resulted in the first
reported examples of hydantoin derivatives with good PK in preclinical species. Potassium channel off-target activity (hERG) was
successfully eliminated through the systematic introduction of acidic functionality to the molecular structure. Undesired
upregulation of alanine aminotransferese (ALT) liver enzymes was mitigated and a robust on-/off-target margin was achieved.
Spirohydantoins represent a class of highly efficacious, short-acting PHD1−3 inhibitors causing a robust erythropoietin (EPO)
upregulation in vivo in multiple preclinical species. This profile deems spirohydantoins as attractive short-acting PHDi inhibitors
with the potential for treatment of anemia.
achieved by endogenous EPO upregulation through upregula-
tion of hypoxia-inducible factor (HIF; Figure 1). HIF is a
heterodimeric transcription factor biosynthesized from a
constitutively expressed subunit HIFβ and an oxygen-regulated
subunit HIFα. HIFα is short-lived under oxygen-normal
conditions and undergoes an oxidative degradation regulated
by prolyl hydroxylases 1−3 (PHD1−3). It has been postulated
that if PHD1−3 are downregulated, as they would naturally be
under hypoxia conditions (e.g., at high altitudes), the oxidative
degradation of HIFα should be impeded and thus the rate of
biosynthesis of HIF amplified due to the higher concentration
of rate-limiting HIFα, ultimately leading to EPO upregulation.7
This hypothesis is consistent with the recent outcome of
Fibrogen’s FG-2216 PhII clinical trial, supporting the exciting
opportunity to pursue an orally bioavailable therapy, in
principle significantly less expensive than the current treatments
INTRODUCTION
■
Anemia, generally defined as a decrease in normal levels of red
blood cells (RBCs) or/and hemoglobin (i.e., hemoglobin levels <12
g/dL), may be caused by a range of medical conditions, some of
which are potentially very serious and carry significant risk factors.
These include chronic kidney disease (CKD),1 chemotherapy-
induced anemia (CIA),2 and anemia of chronic disease (ACD).3,4
Currently available treatments of anemia typically rely on
intravenous (iv) administration of either RBCs or recombinant
erythropoietin (rhEPO).5 While administration of the former tends
to be complicated by undesired iron and RBC level fluctuations, the
latter option represents an expensive treatment that makes it
inaccessible to many patients.6 Furthermore, iv administration may
cause additional adversity among patients because it often requires
hospitalization. Discovery of a new, orally bioavailable, and
economically viable alternative to combat anemia would most
likely have a profound impact on the lives of this patient population.
It has been proposed that the RBC biosynthesis, clinically
demonstrated to be stimulated by rhEPO iv therapy, may be
Received: November 15, 2011
Published: February 27, 2012
© 2012 American Chemical Society
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0.4−8 μM range in vitro (3a−3j, Table 1). While substituent R3
appeared sterically forgiving with respect to PHD2 inhibition
because no significant difference was observed for hydrogen vs
isopropyl (3a vs 3b), both R1 and R2 groups appeared to have a
significant impact on the in vitro inhibition. In addition to the
SAR data shown in Table 1 for 3a−3j, several hundred R1 and
R2-derivatives of 3, presumably included in the initial 500000-
member collection mixtures, did not qualitatively show binding
affinity for PHD2 in the AS-MS screen (Figure 2). This fact is
consistent with the importance of the R1 and R2 substituents
and provides additional evidence for real SAR causality in the
series. These observations warranted a systematic SAR inves-
tigation of the spiroindole hit series. A general research operation
plan was established, described therein by means of the hierarchy
of biological assays utilized for SAR optimization:13
• Primary in vitro screening assays
− PHD1, PHD2, PHD3 inhibition binding assays
(HT)
Figure 1. Rationale for PHD1−3 inhibition as potential mechanism
for increasing RBC count through upregulation of EPO: impeding
oxidative degradation of HIFα through PHD1−3 downregulation
amplifies rate of HIF biosynthesis due to the higher concentration of
rate-limiting HIFα, ultimately leading to upregulation of HIF and
EPO.
− General counter screens, including hERG binding
assay and CYP’s inhibition assay (HT)
− Project-specific counter screen: factor Inhibiting
HIF (FIH)
• Primary in vivo pharmacodynamic (PD) screening assays
(Figure 3)
and thus generally more accessible, for certain types of
anemia.8−10
− Mouse pharmacodynamic erythropoietin determi-
nation (MoPED; medium throughput)
• Single dose iv, EPO (in vivo efficacy)
• Single dose po, reticulocyte readout
− Multiple dose PD (rat, dog, rhesus monkey), RBC
count
The discovery efforts centered around one of the enzymes of
the PHD family, PHD2. The enzyme had been successfully
isolated and purified,11 which allowed for the application of the
high-throughput (HT) affinity selection mass spectrometry
(AS-MS)12 technique as the basis for the initial small molecule
hit identification. Pure PHD2 enzyme was incubated with
mixtures of hundreds of compounds per well. The resulting
mixtures were treated using size exclusion techniques to
discriminate unbound protein from potential protein−ligand
complexes. Protein−ligand complexes were subsequently
trapped and dissociated, yielding unbound compounds which
were analyzed by mass spectrometry. AS-MS enabled a rapid
screen of approximately 500000 molecules, yielding a prominent
hit class of proprietary spiroindoles depicted in Figure 2.
− ALT (acute toxicity)
• Primary in vivo PK assays (mouse, rat, dog, rhesus
monkey)
Initial SAR optimization of the AS-MS hits, confirmed
independently as singleton compounds, focused predominantly
on increasing the in vitro inhibition of a single PHD enzyme
subtype, PHD2. This strategy was largely driven by the fact that
the PHD2 binding assay was of significant throughput and that
no substantial selectivity for other PHD subtypes was generally
observed (or desired) for the discovery program. The synthetic
scheme depicted in Table 1 allowed for a facile survey of R1 and
R2. Substitution at R1 proved to be restricted to a rather small
number of closely related analogues. Out of over 100 diverse
heterocyclic substituents investigated for this position (data not
shown), only 2-pyridylmethyl (e.g., 3j) and 1-methylimidazole-
2-ylmethyl (e.g., 3h) and a handful of structurally closely
related analogues were associated with a significant in vitro
RESULTS AND DISCUSSION
■
Representative examples of spirohydantoin AS-MS hits 3a−3j
were resynthesized and tested as discrete compounds in PHD2
in vitro binding assay.13 This exercise confirmed that the
binding affinity, qualitatively observed by AS-MS, in fact
translated quantitatively into PHD2 enzyme inhibition in the
Figure 2. Identification of spiroindole hit series through AS-MS screening.
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a
Table 1. In Vitro Inhibition of PHD2 by Spiroindole Derivatives; Initial SAR Work Up of the AS-MS Hit
a
Conditions: (a) LiHMDS, NMP then (ClCH2CH2)2NBoc. (b1) (R2 aromatic): R2X, CuI, K2CO3, MeNHCH2CH2NMe, MeCN, DMF. (b2) (R2 aliphatic):
R2X, NaH, DMF. (c) 4M HCl in dioxane. (d1) (R1 aliphatic): R1−1CCHO, NaBH(OAc)3, MeCN. (d2) (R1 aromatic): R1X + conditions b1.
inhibition of PHD2. Any other investigated R1 derivatives caused
a significant decrease in the in vitro activity, typically by several
orders of magnitude. By way of exemplifying the narrow SAR of
the R1-position, it is noteworthy that seemingly close analogues of
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Figure 3. The principles for the primary in vivo PD assay: the investigated compound is administered at t = 0; in vivo PD readout based on EPO
elevation (MOPED, iv single dose), reticulocyte elevation (po, single dose), or RBC count increase (chronic dosing).
3h and 3j displayed no activity in vitro (compare 3h vs 3k−l, and
3j vs 3m−o, respectively). All compounds of the series suffered
from poor PK in the rat and mouse, deeming it impossible to
conclusively test the in vivo activity of the series in the primary PD
model. It is noteworthy that 3p and 3q, for which some key
physical properties were improved in vitro, did not provide a
significantly better PK profile in vivo. Adding a methylene spacer
to the R1 substituent also proved important for the primary assay
activity but at the same time represented a metabolic liability (the
dealkylated spiropiperidine was identified as one of the major
metabolites in vitro). Attempts to alter the methylene spacer in an
effort to improve the generally poor PK profile led to a complete
lost of in vitro efficacy as evident from examples 3r−s. An
important leap forward for the R1 substituent was accomplished
through a comparison of the in vitro activity for close derivatives 3t
vs 3u; while methylene substitution was detrimental to the in vitro
activity in 3t, 6-pyridine substitution was tolerated as evident by
the measurable activity of 3u. This observation led to the design
and synthesis of 3v, the most active compound of the series
prepared thus far. Even though 3v was still lacking robust in vivo
PK in the rat, it proved to be relatively more stable in a side-by-
side comparison with 3h in the in vitro rat liver microsome (LM)
incubation (35% vs <10% of parent remaining after 30 min,
respectively), suggesting that the 2-pyridyl was a viable R1
substituent alternative to the metabolically labile 1-methylimida-
zole-2-yl. Subsequent optimization of the 6-pyridyl position of 3v
improved neither in vitro activity nor liver microsome stability (3v
vs e.g., 3w−x), which validated 6-methyl-2-pyridylmethyl as the
most promising R1 pharmacophore.
While certainly encouraging from the in vitro inhibition
stand point, compound 3ff suffered from two major drawbacks:
suboptimal PK in both rat and mouse (Table 2) and hERG off-
target activity (binding hERG assay, IC50 = 123nM). The
former represented a very significant drawback as this
prevented an in vivo proof-of-concept (POC) experiment, a
potential ultimate validation of the series as viable specific
PHD2 inhibitors. Several structurally related 3ff R2 analogues
(Table 1, 3gg−hh) and R3 analogues (3ii−kk) were prepared
in attempt to stabilize parts of the molecule identified as
metabolic hot spots, however, none of the derivatives led to
significant improvements in physical properties and metabolic
stability (the later were generally prioritized by LM homogenate
incubation in vitro followed by mouse/rat PK in vivo).
Because introduction of polar groups on the periphery of the
3ff lead (substituents R1−3) provided marginal improvement in
the overall PK profile of the molecule, core altering changes of
the spiroindole were pursued. First, the spiroindole moiety of
3ff was replaced with aza-spiroidole; both 4- and 7-
azaspiroindoles were synthesized (Table 2, 4a and 4b,
respectively). While the 4-aza derivative 4a failed to generate
a PD response in vivo, the 7-aza analogue 4b was weakly
efficacious in the MOPED PD model, raising the EPO levels by
approximately 800 ng/L after a single 50 mpk dose (Table 2).
Albeit weak, the PD response represented the first evidence of
statistically significant in vivo efficacy for the spiroindolone
derivatives and thus provided a critical validation of the series.
However, subsequent SAR optimization of 4b by means of
various pyridine, piperidine, and biphenyl substitutions did not
result in any substantial improvement of the still suboptimal PD
efficacy. More radical changes of the spiroindole core were
envisioned with the intention of identifying the minimal
pharmacophore and concomitantly reducing the lipophylicity of
the lead. Examples of such derivatives, for which the level of the
on-target in vitro inhibition was preserved, are depicted in
Table 2 (4c−g). Derivatives 4c−e proved nonefficacious in the
primary PD assay. On the other hand, both examples of
spirohydantoin analogues 4f−g exhibited an in vivo efficacy at
the level approximately equivalent to 4b. Better physical
properties and reduced molecular weight of 4f were credited for
similar efficacy despite the seemingly far inferior PK profile of
4f compared vs 4b. Furthermore, the presence of a free
carboxylic acid in 4g substantially improved the off-target
profile of the series, informing us that the off-target activity may
be derisked for a long-term developmental plan (e.g., compare
hERG IC50 of 20 μM vs 0.12 μM for 4g vs 4b, respectively).
Improving the suboptimal in vivo efficacy and poor PK of the
spirohydantoin 4f derivatives became the central focus of the
optimization effort. The analysis of hydantoins reported in the
Because of the synergistic nature of the SAR of R1 and R2
substituents, optimization of the R2 substituent was conducted
in parallel to the R1 substituent optimization described
previously. As such, one of the initial hits, 3h (Table 1), had
been selected as the starting point of the exercise.
Approximately 50 aromatic, heteroaromatic, and aliphatic
derivatives of 3h were surveyed using the synthesis outlined
in the scheme in Table 1, exemplified here by 3a−3h and 3y−
3bb. In general, introduction of aromatic substituents provided
stronger inhibition compared to their aliphatic (e.g., 3a and 3y
vs 3c) and heteroaromatic (e.g., 3a vs 3z, and 3h vs 3y)
counterparts. The in vitro activity trend observed for a series of
analogues 3z > 3aa > 3bb suggested that the 4-substitution
pattern of aromatic/heteroaromatic R2 should provide an
additional inhibition enhancement. Indeed, compounds 3cc−ee
provided significant improvement of in vitro inhibition
compared to unsubstituted 3a, yielding a strong PHD2
inhibition (3ee). Finally, combining optimized substituents R1
(in 3v) and R2 (in 3ee) produced analogue 3ff, which showed 4
nM PHD2 inhibition in vitro.
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Table 2. Attempting to Improve the PD Profile of 3ff through Significant Changes of Its Spiroindole Core; R1 = (3-
methylpyridine-1-yl)methyl, R2 = (4-phenylbenzene)-1-yl
a
b
c
Not statistically significant vs vehicle arm. Statistically significant vs vehicle arm. Racemate of a single diastereomer.
literature was consistent with our observation for 4f that
hydantoins are generally plagued with poor PK. However,
virtually all of the literature examples of hydantoins happened
to be either mono N-substituted or N,N′-disubstituted with at
least one of the substituents being aliphatic. We hypothesized
that N,N′-disubstituted hydantoins in which both N-substitu-
ents are either aromatic or heteroaromatic should be
metabolically more stable. The hypothesis was tested on 6a,
an example of an N′-pyrimidine derivative of 4f. The choice of
this specific pyrimidine substituent was inspired by another
program lead series in which it proved critical for PK properties
enhancement (5, Figure 4).14 Indeed, the PK profile of N,N′-
disubstituted 6a was substantially improved compared to its
unsubstituted parent 4f. An improved PK profile contributed to
the dramatic boost of in vivo efficacy observed for 6a (Figure 4,
MOPED: 12000 ng/L at 100 mpk).15
The groundbreaking improvement of in vivo properties
demonstrated by both the efficacy as well as favorable PK in
multiple preclinical species (Figure 4, 6a) warranted further
SAR investigation of the N′-aryl substituent (Scheme 1, Ar
group). However, reaction conditions suitable for the synthesis
of 6a via C−N coupling (9 → 10)16 failed to provide the
desired analogues 6 for virtually all attempted aryl substituents.
Because attempts to optimize step c of Scheme 1 in a
traditional linear manner (one reaction at a time) failed to yield
the desired intermediates 10, a high-throughput experimentation
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Figure 4. Improving the in vivo properties by merging the key structural features of two distinct program leads yielded a robust PD efficacy in
MOPED model.
Scheme 1. High-Throughput Optimization of Reaction Conditions for N′-Aryl Coupling (Conditions c) Critical for the Detail
a
SAR Optimization of Derivatives 6.
a
Conditions: (a) KCN, (NH4)2CO3 (95%); (b) CuI, K2CO3, MeNHCH2CH2NHMe, MeCN, DMF (78%); (c) reaction conditions optimized
through HTE (Table 3): ArX (X = Br, I), CuI, TMHD, Cs CO3, DMF, MeCN; (d) 4 M HCl in dioxane; (e) 3-methylpyridine-2-carboxaldehyde,
2
NaBH(OAc)3, MeCN.
a
Table 3. HTE Optimization of Reaction Conditions for C−N Coupling on Spirohydantoin Core (Scheme 1; Step c: 9 → 10)
b
c
d
DMEDA
DACH
proline
N,N-diMe-glycine
TMHD
1,10-phenantroline
ArX/Base B
e
f
e
f
e
f
e
f
e
f
e
f
ligand
B1
B2
B1
B2
B1
B2
B1
B2
B1
B2
98
B1
B2
PhI
32
33
3
24
95
5
1
9
1
0
0
0
0
0
5
65
0
0
1
1
0
0
0
0
0
5
13
6
1
1
1
0
0
0
0
0
46
78
1
57
45
19
1
2
3
100
2
2-iodopyridine
100
71
94
47
36
90
0
64
2
3-iodopyridine
2-I-pyridine-4-CO2H
2-bromopyrimidine
2-iodothiophene
3-iodothiophene
control (no ArX)
1
24
0
1
0
1
0
73
41
3
0
0
1
0
2
4
0
3
0
2
17
56
0
2
0
2
33
0
0
6
20
0
2
11
0
0
0
0
0
a
General screen conditions: 0.5 equiv of CuI vs 9, ligand/CuI ratio = 3.5/1, DMF/MeCN = 1/1, 110 °C, conversion after 20 h.
b
c
d
e
f
Dimethylethylenediamine. 1,2-Diaminocyclohexane (trans, racemic). 1,1,6,6-Tetramethyl-3,5-heptadione. B1 = K3PO4. B2 = Cs2CO3.
(HTE) method17 was employed for reaction optimization. HTE
enabled a rapid screen of all combinations of six ligands and two
bases against seven model substrates in a single 96-well plate
(Table 3). The object of the reaction optimization was to
overcome the inherent lack of reactivity, presumably due to the
high steric hindrance of the spirohydantoin N-coupling partner.
Screening against a diverse subset of aryl halides was critical
because our intention was to broadly investigate the SAR of 6a
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Table 4. SAR Optimization of N′-Aryl Substituent of Spiroindolones 6
derivatives. Finding conditions with a high coverage index (i.e.,
single set of conditions generally applicable to the synthesis of a
variety of substrates) was more important than finding highly
effective conditions only applicable to a limited number of
substrates. Table 3 shows reaction conversion at 20 h as assayed
by LCMS analysis normalized against an internal standard. It is
notable that while some of the reaction conditions effectively
converted 9 to the corresponding analogues 10 for some of the
investigated aryl halide substrates, only a single set of conditions
proved general for all of the aryl halides: the 1,1,6,6-tetramethyl-
3,5-heptadione (TMHD) ligand with the cesium carbonate (B2)
base (highlighted in Table 3). This result demonstrates the power
of HTE, which yielded the general conditions for the conversion
of 9 to 10 in a single parallel screen in less than 48 h total
optimization time.
Armed with the general reaction conditions for the C−N
coupling of aryl halides with spiropiperidine 9, approximately
30 aryl and heteroaryl (both 5- and 6-member heterocyclyl)
analogues of 6 were synthesized according to Scheme 1. While
most analogues exhibited promising in vitro enzyme inhibition,
only a handful proved efficacious in vivo in the MOPED PD
model, with the efficacy being strictly limited to pyridine
derivatives (efficacious spiropiperidines 6 are listed in Table 4).
Several spirohydantoins 6 (6c, 6e−g) exhibited a robust PD
response after a single dose of 15 mpk, a remarkable efficacy
considering the inherently short half-life (t1/2 = 1−2 h) and a
relatively high clearance for these molecules. It is noteworthy
that prior to our work, there were no examples of orally
bioavailable hydantoin derivatives reported in the literature. We
concluded that the lack of favorable PK properties of previously
reported hydantoin examples was consistent with the originally
proposed hypothesis that at least one of the hydantoin nitrogens
was not substituted with an aromatic or heteroaromatic
substituent, ultimately leading to metabolic liability of the
molecule.18 The new C−N coupling reaction developed though
HTE enabled us to access a completely new class of N,N′-
bisarylsubstituted hydantoin derivatives 6 with good in vivo
stability, a critical prerequisite for further development of the
series, as well as establishing a proprietary intellectual property.
Once the spirohynatoin series was established as a viable lead
class, rigorous off-target screening protocol was utilized to
differentiate among a number of leading efficacious derivatives
(6c, 6e−g, Table 5). The series proved to be generally selective
against standard off-targets (typically, PXR >50 μM; CYP 3A4,
2C9, 2D6 >10 μM; Cav1.2 >10 μM) with the exception of the
potassium channel (represented therein by the hERG binding
assay). Carboxylic acid functionality was introduced into several
suitable positions of the molecule, resulting in derivatives with
variable PHD1−3 activity but generally clean hERG profiles.
Carboxylic acid substitution of any available position of the
pyrimidine ring proved detrimental to the primary in vitro
activity (data not shown). On the other hand, both R3 and R4
carboxylic acid substitutions of 6 were tolerated, yielding
derivatives 6h/6i and 6j/6k, respectively, with good in vitro
profiles of which 6h and 6k also proved exceptionally
efficacious in vivo even at a lower dose (MED = 5 mpk, iv).
The 4-substitution pattern of 6k was critical for the in vivo PD
response as evidenced by complete loss of activity for positional
isomers 6l and 6m. Two leading subclasses with developmental
potential, represented by 6h and 6k, were differentiated based
a,b
on their PK profile (Table 5 ): 6h had a profile consistently
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Table 5. SAR Optimization of Spiroindolones 6 for Optimal off-Target Profile (hERG Binding)
PHDi IC50 (nM)
6
R1
R2
H
R3
R4
1
2
3
hERG binding (μM)
CYPs (μM)
MOPED ng/L (mpk) ↑ALT (mpk)
6c
H
H
H
1.3
2.5
11
0.22
3A4 = 69
2C9 = 65
2D6 >100
26000 (50 iv)
15000 (15 iv)
910 (5 iv)
300 (1.5 iv)
9000 (15 iv)
1500 (5 iv)
8000 (50 iv)
400 (15 iv)
200 (5 iv)
a
6h
6i
H
H
H
H
H
CO2H
CO2H
Me
1.1
0.4
3.5
>30
6j
H
H
H
CO2H
CO2H
H
H
1.1
0.3
1.1
0.4
7.2
2.1
>30
6.4
3A4, 2C9,
2D6 >10
3A4, 2C9,
2D6 >50
510 (50 iv)
210 (15 iv)
14000 (15 iv)
1700 (5 iv)
b
6k
Me
ALT 6.1x (15 iv)
ALT OK (5 iv)
260 (15 iv)
6l
3-CO2H
2-CO2H
2-Cl
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
0.2
8.2
0.9
1.2
0.4
0.2
0.4
5.0
0.8
1.0
0.4
0.2
1.7
7.0
2.4
>60
5.7
54
3A4, 2C9,
2D6 >50
3A4, 2C9,
2D6 >50
3A4, 2C9,
2D6 >50
3A4, 2C9,
2D6 >50
3A4, 2C9,
2D6 >50
3A4, 2C9,
2D6 >50
6m
6n
6o
6p
H
95
27
8.4
260 (50 iv)
CO2H
CO2H
CO2H
CO2H
11000 (100 iv)
120 (5 iv)
3-Cl
170 (15 iv)
120 (5 iv)
2-F
3.6
1.9
23000 (100 iv)
230 (5 iv)
c
6q
2-Me
13
9700 (15 iv)
470 (5 iv)
ALT 3.8x (100 iv)
ALT OK (50 iv)
5400 (15 iv)
6r
3-Me
Me
CO2H
H
0.3
0.5
5.8
23
3A4, 2C9,
2D6 >50
570 (5 iv)
ALT 5.1x (15 iv)
a
6h rat PK: Clp = 1.2 mL/min/kg, Vdss = 0.34 L/kg, cmax = 63 nM, t1/2 = 3.6 h, %F = 4.3. Mouse PK (5 po): Clp = 0.9 mL/min/kg, Vdss = 0.4 L/kg,
b
cmax = 300 nM, t1/2 = 2.1 h, %F = 7.1. 6k rat PK: Clp = 8.8 mL/min/kg, Vdss = 1.7 L/kg, cmax = 1900 nM, t1/2 = 2.6 h, %F = 92. Mouse PK (5 po):
Clp = 1.2 mL/min/kg, Vdss = 0.4 L/kg, cmax = 6100 nM, t1/2 = 3.7 h, %F = 36; 6k PPB (100%) m = 98.4, r = 98.2, d = 94.6, h = 99.4. 6q rat PK:
c
Clp = 11.9 mL/min/kg, Vdss = 4.3 L/kg, cmax = 570 nM, t1/2 = 4.1, %F = 64; 6q PPB (100%) m = 98.3, r = 98.4, d = 97.4, h = 99.7.
inferior to that of 6k across multiple species (rat and mouse)
deeming 6k more suitable for further optimization.
was shifted into the 50−100 mpk range, leaving only two
examples, 6q−r, efficacious at levels comparable to 6k (5−15
mpk). While 6r was also plagued by a lack of a measurable
significant ALT NOEL/MOPED MED window, a statistically
significant margin of at least 10-fold between the ALT elevation
and MOPED efficacy could be established in case of 6q (ALT
NOEL = 50 mpk and MOPED MED = 5mpk, Table 5). This
result warranted SAR optimization focused on increasing the
window between the NOEL ALT and MOPED MED as well as
improving the suboptimal PK profile of 6q (Table 5, footnote c).
Because even relatively minor structural changes of the biphenyl
portion of the 6k/6q structure proved to have pronounced effects
Additional in vivo profiling of the leading derivative 6k
revealed an unexpected elevation in liver enzyme levels, ALT, at
the higher iv dose experiments. Subsequent titrations
established a 4−6-fold increase in ALT levels at 15, 50, and
100 mpk (no increase at 5 mpk), effectively defining a
nonexistent window between the ALT No Observable Effect
Level (NOEL) and the desired HIF-PHD MED for in vivo PD
efficacy. Several closely related analogues of 6k (6l−r) were
prepared in an effort to probe the ALT elevation liability of the
series. For most of these analogues, the MED in the PD assay
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on both the primary MOPED efficacy as well the undesired ALT
elevation (Table 5), a detailed SAR analysis of the biphenyl
pharmacophore was conducted including a broader range of
structural variations in an attempt to expand the scope across a
larger chemical space (Table 6 exemplifies the effort). Aliphatic
replacements of the terminal aromatic ring proved detrimental to
the PD efficacy as exemplified by comparing saturated 6s (Table 6)
to unsaturated 6c (Table 4). Four- (6t), five- (6u−aa), and six-
membered (6bb−cc) heterocyclic replacements of the terminal
benzene ring, both N- (6t, 6v) and C-linked (6u, 6w−cc),
did not provide any overall advantage when compared to 6q.
Because most of those derivatives lacked carboxylic acid
functionality, methyl derivatives (R2 = Me) generally also suffered
from hERG off-target activity, while the more acidic (and more
hERG selective) proteo derivatives (R = H) provided suboptimal
PD efficacy compared to 6q. Of all investigated substitutions, only
6x proved weakly efficacious with MED at 50 mpk. Even
heterocyclic analogues closely related to 6k in which the carboxylic
functionality was placed in the pseudo-para position lost essentially
all in vivo efficacy (6y−aa). In vivo efficacy was maintained for
some biaryl (6ee−gg) and fused (6ii−kk) R1 substitutions, but
the activity was limited to high dose experiments. Given the lack of
a PD response at lower doses, it was impossible to reliably
establish the margin of the desired PD efficacy over the undesired
ALT up-regulation even for cases where no ALT increase was
observed up to 100mpk (6jj−kk).
elevation at the highest tested dose of 100 mpk, effectively
providing the largest possible ALT NOEL/MOPED MED
margin within the measured range (≥20×). There were no
significant off-target findings for 11i and 11l, which was
consistent with the series being generally selective against
Cav1.2 (>20 μM), PXR (>30 μM), CYPs induction, and hERG
binding (Table 7).
While 11i and 11l were virtually indistinguishable based on
their physiochemical properties, PPB free fraction, or PK, 11l
was superior to 11i based on better selectivity against hERG
and as such was selected for additional profiling in preclinical
species. We reason that the relatively short half-life of 11l
represents the advantage of the spirohydantoin series for the
HIF-PHD mechanism because only a very short-time inhibition
of the PHDi is sufficient for HIF stabilization and further
downstream EPO upregulation. Several in vitro experiments,
including incubation with liver microsomes (Figure 5),
correlate this favorable in vivo profile (observed in rat PK
and mouse PD experiments) to human. This notion suggests
that 11l has the potential to be efficacious in humans, placing it
in a unique position of a short-acting highly efficacious PHDi
inhibitor. The combined characteristic profile of 11l may
significantly reduce the chance of finding unpredictable adverse
effects (AEs) in the clinic, which are more likely to be caused
by the longer half-life of other known PHDi inhibitors (i.e.,
shorter duration of action might result in comparable in vivo
efficacy with a reduced risk of AEs).
Because the structure diversity-oriented probe of the
biphenyl subunit resulted in no improvement of the leading
candidate 6q, subsequent SAR investigation focused on
relatively minor changes of the 4-position of the terminal
benzene ring (6ll−oo) as well as substituting benzene with a
pyridine ring (6pp−rr). Primary, secondary, and tertiary amides
6mm−oo all proved efficacious in vivo, 6mm even as low as 15
mpk, but at the same time 6mm caused the undesired up-
regulation of the ALT levels at the 50 mpk dose and thus
provided a worse on-/off-target margin compared to 6q.
Introduction of nitrogen into one of the biphenyl benzene rings
provided the desired improvement in the overall physical
properties of the molecule as evidenced by the increased free
fraction in plasma protein binding (PPB) across preclinical
species (compare two pairs of direct N vs CH analogues, 6pp vs
6k and 6qq vs 6q, respectively, in footnotes to Tables 6 and 5).
However, the free-fraction increase did not translate into an
efficacy improvement in vivo (for most efficacious derivative 6pp,
MED = 50 mpk).
Methylpyridyl had been previously shown to be critical for
both in vitro as well as in vivo efficacy for the spiroindolone
series (Table 1), a trend subsequently verified for selected
examples of the spirohydantoin series 6 (data not shown). At
the same time, a detailed SAR optimization of the biphenyl
subunit of the 6k/6q lead series provided no significant
improvement of in vivo efficacy in MOPED nor did it improve
the undesired ALT off-target activity profile (Table 6). These
two data sets essentially left the N-heteroaryl substituent as the
only potential group to be modified with the aim to improve
the ALT off-target activity. The aim was to maintain or improve
the generally good on-target profile observed for a number of
spirohydantoins 6, such as 6k and 6q, and at the same time
improve the ALT NOEL/MOPED MED margin (Table 7).
Variation of the N-heteroaryl substituent provided a range of
efficacy in the primary MOPED in vivo PD assay with four
derivatives (11a, 11i, 11k, 11l), exhibiting MED as low as 5
mpk. Of these four, two (11i and 11l) did not cause ALT
CONCLUSION
■
In conclusion, we developed a novel class of PHDi inhibitors
exemplified by the highly efficacious, short-acting preclinical
candidate 11l (Figure 6). The initial spiroindolone program
lead (hit class represented by 3a in Figure 6) was discovered
using the AS-MS screening methodology and systematically
optimized for efficacy, off-target activity, and physical chemical
properties. Judicious effort to identify the minimal pharmaco-
phore of the spiroindolone led to the spirohydantoin class of
compounds (exemplified by 4f in Figure 6). For a thorough
SAR optimization of the spirohydantoin series targeted at
improving PK properties the need to prepare an array of
unprecedented N,N′-diarylhydantoins arose. The new chemical
space was enabled by the discovery of an efficient, general C−N
bond forming transformation through the use of HTE
methodology. SAR optimization of the series for ALT elevation
NOEL vs MOPED MED margin ultimately yielded 11l. Given
the complexity of the HIF-PHD mechanism of action for the
treatment of anemia and possible AEs associated with the
mechanism, we believe that 11l represents a very promising
clinical candidate due to its short acting PHDi inhibition
resulting into robust downstream in vivo efficacy.
EXPERIMENTAL SECTION
■
General, Chemistry. Unless specified otherwise, all materials were
purchased form commercial sources and used as received. H and 13
C
1
NMR spectra were recorded using a Bruker 400 MHz instruments.
Preparative reverse phase liquid chromatography (RPHPLC) was
performed using Waters MS directed purification system consisting of
2525 binary gradient pump, 2767 injector/collector, and 2996 PDA
UV detector. Mobile phase: gradient of water and acetonitrile (each
containing 0.1% TFA). Column: Waters Xterra (50 mm × 3 mm, 3.5
μm packing material). The purity of 1−11 has been determined by
HPLC analysis (electrospray positive ionization, Micromass ZQ single
quadrupole)19 as ≥95%.
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Table 6. SAR Optimization of the Biphenyl Portion of Spirohydantoins 6 for Optimal Desired Primary MOPED PD and
Undesired Off-Target Elevation of Liver Enzymes (ALT)
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Table 6. continued
a
b
c
6ff PPB (100%): m = 94.8, r = 99.2, d = 98.3, h = 97.3. 6pp PPB (100%): m = 93.8, r = 96.1, d = 90.6, h = 97.8. 6qq PPB (100%): m = 88.5, r =
92.9, d = 83.0, h = 98.7.
Preparation of 3ff (oxindole → 1a → 2ff → 3ff; Scheme in Table 1)
represents a typical procedure used for the synthesis of spiroindoles
3a−3kk. Preparation of 6c (8 → 9 → 10c → 6c; Scheme 1)
represents a typical procedure used for the synthesis of spiroindoles 4f,
4g, 6a−rr, 11a−11m.
room temperature. The crude mixture was diluted with ethyl acetate and
washed with 0.1 M HCl solution, dried over sodium sulfate, filtered, and
concentrated. Further purification of the desired product was
accomplished by column chromatography on silica gel, eluting with a
gradient of ethyl acetate in hexanes 0−40%/1.3 L, yielding 3.1 g (74%) of
2ff as a white solid. LCMS: 4.10 min, m/z (M-BocH)+ = 355.1.
tert-Butyl 2-Oxo-1,2-dihydro-1′H-spiro[indole-3,4′-piperidine]-1′-
carboxylate (1a, R3 = H). Oxindole (44 g, 330 mmol) was dissolved
in diemthoxyethane (DME, 880 mL), and the solution was cooled
to −78 °C. A solution of LiHMDS/DME (550 mL, prepared by
dissolving 246 g of 97% of LiHMDS in 750 mL of DME) was added
over 40 min, maintaining an internal temperature of < −55 °C. The
suspension was warmed to −30 °C over 30 min, and a solution of
N-Boc-bis(2-chloroethyl)amine20 (92.1 g, 380 mmol) in DME (120 mL)
was added. The reaction mixture was allowed to warm to room
temperature and stirred 18 h. An additional 280 mL of the LiHMDS
solution was added in portions over 2 days. The reaction mixture was
poured into 1.7 L of 2N HCl and ice and aged for 1 h. The mixture
was diluted with 2 L of ether/hexanes (1/1) and the layers separated.
The organic layer was washed with water (1 L), saturated sodium
bicarbonate solution (1.5 L), and brine (0.5 L). The organic layer was
dried over MgSO4 and treated with Darco G-60. The mixture was filtered
through MgSO4, and the filtrate was concentrated to slurry. The slurry
was filtered and the cake washed with hexanes affording 80.5 g (81%) of
1a as a white solid. LCMS: 2.4 min, m/z (M-BocH)+ = 203.2.
tert-Butyl 2-Oxo-1-(biphenyl-4-yl)-1,2-dihydro-1′H-spiro[indole-
3,4′-piperidine]-1′-carboxylate (2ff, R3 = H, R2 = 4-biphenyl). In
an oven-dried flask, 1a (2.8 g, 9.26 mmol) and 4-iodobiphenyl (3.37 g,
12.04 mmol) were dissolved in acetonitrile (100 mL), and the mixture
was degassed with stream of nitrogen through the solution at 40 °C for
20 min. Anhydrous potassium carbonate (3.84 g, 27.8 mmol), copper(I)
iodide (0.441 g, 2.315 mmol), and N,N′-dimethylethylenediamine (0.204
g, 2.315 mmol) were added sequentially, and the resulting reaction
mixture was heated to 80 °C for 15 h under nitrogen and then cooled to
1-(Biphenyl-4-yl)-1′-[(3-methylpyridin-2-yl)methyl]spiro[indole-
3,4′-piperidin]-2-(1H)-one (3ff). Hydrogen chloride (4 M) solution in
dioxane (60 mL, 240 mmol) was added to 2ff (3 g, 6.60 mmol) via
syringe in one portion, and the resulting mixture was stirred at room
temperature for 1 h, concentrated to an approximate volume of 30 mL,
and cooled to 10 °C. Solids were collected by filtration, rinsed with 10
mL of dioxane, and dried in a desiccator to yield deprotected amine
intermediate as a white solid (6.0 mmol). LCMS 2.92 min, m/z
(MH)+ = 355.1. To a solution of the amine intermediate (6.0 mmol)
in methylene chloride (120 mL), 3-methylpyridine-2-carboxaldehyde
(6.6 mmol), and sodium triacetoxyborohydride (18 mmol) were
added sequentially, and the resulting mixture was stirred at ambient
temperature for 2 h. Methanol (60 mL) was added, and the resulting
mixture stirred at room temperature for 5 min and concentrated. The
final purification was accomplished by preparative reverse phase HPLC
1
to yield 2.9 g (77%) of 3ff a white trifluoroacetic acid salt. H NMR
(500 MHz, CD3OD): 2.32 (m, 2H), 2.41 (s, 3H), 2.57 (m, 2H), 3.75
(m, 2H), 4.09 (m, 2H), 4.70 (s, 2H), 6.95 (d, J = 8.2 Hz, 1H), 7.22
(t, J = 5.5 Hz, 1H), 7.34 (t, J = 5.5 Hz, 1H), 7.38 (m, 1H), 7.54
(m, 2H), 7.63 (d, J = 9.2 Hz, 2H), 7.74 (d, J = 9.6 Hz, 2H), 7.85
(d, J = 9.2 Hz, 2H), 7.90 (d, J = 7.8 Hz, 1H), 8.18 (d, J = 7.8 Hz, 2H),
8.78 (d, J = 3.2 Hz, 1H). LCMS: 3.15 min, m/z (MH)+ = 460.1.
tert-Butyl 2,4-Dioxo-3-(biphenyl-4-yl)-1,3,8-triazaspiro[4.5]-
decane-8-carboxylate (9). A solution of 821 (23.8 mmol) and 4-
iodobiphenyl (22.6 mmol) in acetonitrile (80 mL) and DMF (80 mL)
was degassed with a stream of nitrogen for 15 min. N,N′-Dimethyl-
ethylenediamine (5.94 mmol), copper(I) iodide (5.94 mmol), and
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Table 7. SAR Optimization of the N-Heteroaryl Portion of Spirohydantoins 6/11 for Optimal Desired Primary MOPED PD and
Undesired Off-Target Elevation of Liver Enzymes (ALT)
a
b
11f rat PK: Clp = 51 min/mL/kg, Vdss = 2.2 L/kg, cmax = 100 nM, t1/2 = 1.1 h, %F = 18. 11i rat PK: Clp = 22 min/mL/kg, Vdss = 1.6 L/kg, cmax
=
c
d
390 nM, t1/2 = 1.5 h, %F = 29. 11k rat PK: Clp = 16 min/mL/kg, Vdss = 2.0 L/kg, cmax = 580 nM, t1/2 = 2.2 h, %F = 8. 11l rat PK: Clp = 34 min/
mL/kg, Vdss = 1.9 L/kg, cmax = 390 nM, t1/2 = 1.1 h, %F = 47.
potassium carbonate (71.3 mmol) were added sequentially. The
resulting mixture was heated to 85 °C for 15 h, cooled to room
temperature, and combined with ethyl acetate (300 mL) and water
(300 mL). The organic layer was separated and washed with water
(2 × 300 mL), dried over sodium sulfate, and concentrated. Purification
by column chromatography on silica gel, eluted with a gradient of 0−
100% hexanes in ethyl acetate, yielded 8.3 g (83%) of 9c as a white
solid. LCMS: 3.21 min, m/z (MH-Boc)+ = 321.0.
tert-Butyl 3-(Biphenyl-4-yl)-1-(6-methoxypyrimidin-4-yl)-2,4-
dioxo-1,3,8-triazaspiro[4.5]decane-8-carboxylate (10c, Ar = 4-methoxy-
pyrimidine-2-yl). A solution of 9 (10 mmol), 2-iodo-4-methoxypyrimidine
(30 mmol) in acetonitrile (20 mL), and DMF (20 mL) was degassed with
a stream of nitrogen for 15 min. 2,2,6,6-Tetramethyl-3,5-heptadion
(10 mmol), copper(I) iodide (10 mmol), and cesium carbonate
(50 mmol) were added sequentially, and the resulting mixture was
heated to 85 °C for 15 h and allowed to cool to room temperature. The
mixture was combined with ethyl acetate (300 mL) and water (300 mL).
The organic layer was separated and washed with water (2 × 300 mL),
dried over sodium sulfate, and concentrated. Purification by preparative
HPLC yielded 5.0 g (78%) of 10c as a white salt of triflouroacetic acid.
LCMS: 4.21 min, m/z (MH-Boc)+ = 430.2.
3-(Biphenyl-4-yl)-8-[(3-methylpyridin-2-yl)methyl]-1-(6-hydroxypyrimi-
din-4-yl)-1,3,8-triazaspiro[4.5]decane-2,4-dione (6c). Boc Deprotection.
A 4 M solution of hydrogen chloride in dioxane (60 mL, 240 mmol)
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In Vitro Assay for HIF-PHD2. Catalytic activity represents a
general procedure used for the determination of HIF-PHD catalytic
activity for each of the three subtypes, HIF-PHD1−3: To each well of
a 96-well plate was added 1 μL of test compound in DMSO and 20 μL
of assay buffer (50 mM Tris pH 7.4/0.01% Tween-20/0.1 mg/mL
bovine serum albumin/10 μM ferrous sulfate/1 mM sodium
ascorbate/20 μg/mL catalase) containing 0.15 μg/mL FLAG-tagged
full length PHD2 expressed in and purified from baculovirus-infected
Sf9 cells. After a 30 min preincubation at room temperature, the
enzymatic reactions were initiated by the addition of 4 μL of substrates
(final concentrations of 0.2 μM 2-oxoglutarate and 0.5 μM HIF-1α
peptide biotinyl-DLDLEMLAPYIPMDDDFQL). After 2 h at room
temperature, the reactions were terminated and signals were develo-
ped by the addition of a 25 μL quench/detection mix to a final
concentration of 1 mM ortho-phenanthroline, 0.1 mM EDTA, 0.5 nM
anti-(His)6 LANCE reagent (Perkin-Elmer Life Sciences), 100 nM
AF647-labeled streptavidin (Invitrogen), and 2 μg/mL (His)6−VHL
complex.22 The ratio of time-resolved fluorescence signals at 665 and
620 nm was determined, and percent inhibition was calculated relative
to an uninhibited control sample run in parallel.
MoPED (Mouse Plasma Erythropoietin Determination).
Compounds are formulated in cyclodextrin. Mice (C57Bl/6, n = 3)
were dosed iv in a volume of 0.2 mL. After 4 h, blood was obtained via
cardiac puncture upon euthanasia with CO2. Plasma was stored at −80
°C and assayed the following day for Epo/VEGF. Results were
compared to vehicle dosed controls.
Reticulocytes (Days 3 and 4 Post PO Dose). Compounds were
formulated in cyclodextrin. Mice (C57Bl/6, n = 10) were dosed PO in
a volume of 0.2 mL. On days 3 and 4 postdose, blood was obtained
from five mice via cardiac puncture upon euthanasia with CO2. Blood
was analyzed for reticulocytes by FACS. Results were compared to
vehicle dosed controls, and the assay was quality controlled by
inclusion of a positive control compound, dosed at 15 mg/kg.
Figure 5. Incubation of 11l with liver microsomes across species
correlates well rat PK and mouse PD profile to human in vitro.
was added to 10c (6 mmol) via syringe. The resulting mixture
was stirred at room temperature for 1 h, concentrated to an
approximate volume of 30 mL, and cooled to 10 °C.
Deprotected amine was isolated as a white solid by filtration,
rinsed with 10 mL of dioxane, and dried in desiccator and used
in the subsequent step without purification.
Reductive Amination. The deprotected amine (4 mmol) was
dissolved in methylene chloride (40 mL). 3-Methylpyridine-2-carbox-
aldehyde (5 mmol) and sodium triacetoxyborohydride (20 mmol) were
added to the solution sequentially. The resulting mixture was stirred at
room temperature for 2 h. Methanol (20 mL) was added, and the
resulting mixture was stirred at ambient temperature for 5 min and then
concentrated. The purification was accomplished by preparative reverse
phase HPLC yielding 2.4 g (93%) of O-methyl-6c as a white salt of
1
trifluoroacetic acid. HNMR (CDCl3): δ = 2.03−2.15 (m, 4H), 2.03 (s,
3H), 3.53−4.30 (m, 6H), 4.06 (s, 3H), 6.60−8.49 (m, 14H). LCMS: 1.71
min, m/z (MH)+ = 535.
ASSOCIATED CONTENT
Demethylation. The O-methyl-6c (3.6 mmol) was combined with
1 M aqueous solution of hydrochloric acid (20 mL), and the resulting
mixture was refluxed in a sealed tube for 8 h and concentrated. The
final purification was accomplished by preparative reverse phase
HPLC, yielding 2.0 g (89%) of 6c as a white salt of trifluoroacetic acid.
1H NMR (CDCl3): δ = 1.96 (s, 3H), 2.24 (m, 2H), 2.34 (m, 2H),
3.84 (m,2H), 3.92 (m, 2H), 4.48 (s, 2H), 6.18 (d,1H, J = 6.6 Hz),
7.24−7.87 (m, 12H), 8.43 (d,1H, J = 4.3 Hz). LCMS: 1.60 min, m/z
(MH)+ = 521.
■
S
* Supporting Information
Full description of biological assays, characterization of
compounds 3a−3kk, 4a−g, 6a−rr, 11a−11m, and synthetic
procedures thereof as well as experimental details for the HTE
optimization of C−N coupling (step c of Scheme 1). This
material is available free of charge via the Internet at http://
pubs.acs.org.
2-Methyl-4′-(8-((3-methylpyridin-2-yl)methyl)-2,4-dioxo-1-(pyrimi-
din-4-yl)-1,3,8-triazaspiro[4.5]decan-3-yl)-[1,1′-biphenyl]-4-carboxylic
acid (11l) was prepared according to the general procedure described
for 6c (using 4-iodopyrimidine as the ArI) and isolated as a white solid
of trifluoroacetic acid; 11l. 1H NMR (CD3OD): δ = 2.42 (s, 3H), 2.45
(m, 2H), 3.80 (m, 2H), 3.80−3.94 (m, 2H), 4.04−4.10 (m, 2H), 4.76
(s, 2H), 7.37 (m, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 7.6 Hz,
1H), 7.79 (d, J = 8.5 Hz, 2H), 7.86 (d, J = 8.5 Hz, 2H), 8.14 (d, J = 8.3
Hz, 2H), 8.46 (m, 1H), 8.54 (d, J = 4.7 Hz, 1H), 8.74 (d, J = 5.9 Hz,
1H), 9.06 (s, 1H). LCMS: 1.54 min, m/z (MH)+ = 549.3.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +1-732-594-6624. Fax: +1-732-594-9464. E-mail:
petr_vachal@merck.com. Address: Merck Reaseach Laboratories,
RY123-2309, Rahway, NJ 07065, USA.
Notes
The authors declare no competing financial interest.
Figure 6. Overview of the discovery of a novel class of PHDi inhibitors exemplified by highly efficacious, short-acting leading preclinical candidate 11l .
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Section and Supporting Information.
ABBREVIATIONS USED
■
PHD, prolyl hydroxylase; AS-MS, affinity selection mass
spectrometry; HT, high-throughput; HTE, high-throughput
experimentation; PK, pharmacokinetics; Kv11.1, potassium
̀
channel gene; hERG, human ether-a-go-go related gene;
ALT, alanine aminotransferese; EPO, erythropoietin; RBC,
red blood cell; CKD, chronic kidney disease; CIA, chemo-
therapy-induced anemia; ACD, anemia of chronic disease; iv,
intravenous; HIF, hypoxia-inducible factor; FIH, factor
inhibiting hypoxia-inducible factor; PD, pharmacodynamic;
SAR, structure−activity relationship; MOPED, mouse pharma-
codynamic erythropoietin determination; POC, proof-of-
concept; TMHD, 1,1,6,6-tetramethyl-3,5-heptadione; MED,
minimal efficacious dose; NOEL, no observable effect level;
PPB, plasma protein binding; LMs, liver microsomes; AE,
adverse effect; NMP, N-methymorpholine; DMF, N,N-
dimethylformamide; LiHMDS, lithium bis(trimethylsilyl)-
amide; DME, dimethoxyethane; PXR, pregnane X receptor;
CYP, cytochrome P450; Cav1.2, calcium channel 1.2; PPB,
plasma−protein binding; VEGF, vascular endothelial growth
factor; RP, reversed phase; HPLC, high-pressure liquid
chromatography
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Journal of Medicinal Chemistry
Featured Article
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= acetonitrile + 0.05% TFA. Gradient: time/%A/%B: 0.00/95/05,
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