Discovery of N-[Bis(4-methoxyphenyl)methyl]-4-hydroxy-2-(pyridazin-3-yl)pyrimidine-5-carboxamide (MK-8617), an Orally Active Pan-Inhibitor of Hypoxia-Inducible Factor Prolyl Hydroxylase 1-3 (HIF PHD1-3) for the Treatment of Anemia

Article abstract of DOI:10.1021/acs.jmedchem.6b01242

The discovery of novel 4-hydroxy-2-(heterocyclic)pyrimidine-5-carboxamide inhibitors of hypoxia-inducible factor (HIF) prolyl hydroxylases (PHD) is described. These are potent, selective, orally bioavailable across several species, and active in stimulati

Full text of DOI:10.1021/acs.jmedchem.6b01242

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Article
Discovery of N-[bis(4-methoxyphenyl)methyl]-4-hydroxy-2-
(pyridazin-3-yl)pyrimidine-5-carboxamide (MK-8617) an
Orally Active Pan-Inhibitor of Hypoxia-Inducible Factor Prolyl
Hydroxylase 1-3 (HIF PHD1-3) for the Treatment of Anemia.
John S Debenham, Christina B. Madsen-Duggan, Matthew J. Clements, Thomas F. Walsh, Jeffrey
T Kuethe, Mikhail Reibarkh, Scott P. Salowe, Lisa M. Sonatore, Richard Hajdu, James A. Milligan,
Denise M. Visco, Dan Zhou, Russell B. Lingham, Dominique Stickens, Julie A. DeMartino, Xinchun
Tong, Michael Wolff, Jianmei Pang, Randy R. Miller, Edward C. Sherer, and Jeffrey J. Hale
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01242 • Publication Date (Web): 09 Nov 2016
Downloaded from http://pubs.acs.org on November 10, 2016
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Page 1 of 36
Journal of Medicinal Chemistry
Discovery of Nꢀ[bis(4ꢀmethoxyphenyl)methyl]ꢀ4ꢀhydroxyꢀ2ꢀ(pyridazinꢀ3ꢀ
yl)pyrimidineꢀ5ꢀcarboxamide (MKꢀ8617) an Orally Active PanꢀInhibitor of
HypoxiaꢀInducible Factor Prolyl Hydroxylase 1−3 (HIF PHD1−3) for the
Treatment of Anemia.
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John S. Debenham*, Christina MadsenꢀDuggan, Matthew J. Clements, Thomas F. Walsh, Jeffrey T.
Kuethe, Mikhail Reibarkh, Scott P. Salowe, Lisa M. Sonatore, Richard Hajdu, James A. Milligan, Denꢀ
ise M. Visco, Dan Zhou, Russell B. Lingham, Dominique Stickens, Julie A. DeMartino, Xinchun Tong,
Michael Wolff, Jianmei Pang, Randy R. Miller, Edward C. Sherer, and Jeffrey J. Hale.
Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, United States.
ABSTRACT: The discovery of novel 4ꢀhydroxyꢀ2ꢀ(heterocyclic)pyrimidineꢀ5ꢀcarboxamide inhibitors
of hypoxiaꢀinducible factor (HIF) prolyl hydroxylases (PHD) are described. These are potent, selective,
orally bioavailable across several species and are active in stimulating erythropoiesis. Mouse and rat
studies showed hematological changes with elevations of plasma EPO and circulating reticulocytes folꢀ
lowing single oral dose administration while 4 week QD po administration in rat elevated hemoglobin
levels. A major focus of the optimization process was to decrease the long halfꢀlife observed in higher
species with early compounds. These efforts led to the identification of 28 (MKꢀ8617) which has adꢀ
vanced to human clinical trials for anemia.
Key Words: PHD inhibitors, Prolyl hydroxylase inhibitors, HIF, hypoxia inducible factor, erythropoietin,
epo, erythropoietin stimulating agents, reticulocytes, anemia.
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INTRODUCTION
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Anemia is a condition of insufficient red blood cells (RBCs) or hemoglobin (Hb) levels that result in
reduced functional capability, fatigue, and shortness of breath. The incidence of anemia increases with
the severity of renal insufficiency progressing to most patients with stage 5 chronic kidney disease
(CKD).1 Cancer patients also suffer high prevalence of anemia at >40% with this approaching 90% in
patients on chemotherapy.2 The current standards of care include RBC/blood transfusions for immediate
interventions and the parenteral administration of recombinant human erythropoietin (rhEPO) agents
such as epoetin alfa or darbepoetin alfa that stimulates the division, differentiation and maturation of
erythroid progenitors in bone marrow over time.3 A small molecule stimulator of endogenous erythroꢀ
poietin production would be less costly and obviate the complexity of parenteral administration of bioꢀ
logics.
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Hypoxiaꢀinducible factor (HIF) is a α/β heterodimeric gene transcription factor involved in erythroꢀ
poiesis, angiogenesis, glycolytic energy metabolism and apoptosis.4 Levels of HIF are regulated metaꢀ
bolically via hydroxylation of proline residues on the αꢀsubunit by a family of hydroxylases known as
prolyl hydroxylases (HIFꢀPHDs). HIF is shortꢀlived under conditions of normoxia due to oxidative
degradation of the HIFα subunit via the action of HIF PHD which require oxygen as a substrate. Hyꢀ
poxia or inhibitors of the three isoforms of HIFꢀPHDs (1ꢀ3) stabilize HIF and stimulate the production
of red blood cells (RBC's) through the modulation of erythropoietin (EPO), the EPO receptor, and proꢀ
teins responsible for iron handling and transport.5 The foundational work of William G. Kaelin, Jr., Peꢀ
ter J. Ratcliffe and Gregg L. Semenza for unraveling the molecular basis of oxygen sensing and regulaꢀ
tion through the HIF pathway was recently recognized with the 2016 Albert Lasker Basic Medical Reꢀ
search Award.6 The use of inhibitors of HIF PHDs to stimulate erythropoiesis in both animal and cliniꢀ
cal studies has been previously described7 and reviewed.8 Several compounds are in clinical developꢀ
ment with FibroGen’s PHD inhibitor FGꢀ4592 (roxadustat) currently in phase III (Figure 1).9,10
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Journal of Medicinal Chemistry
Our research operating plan, a hierarchy of biological assays utilized for SAR optimization was deꢀ
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tailed previously.11 After assessment of compounds in assays for PHD catalytic activity, compounds of
interest were selected for pharmacodynamic (PD) evaluation in mouse pharmacodynamic erythropoietin
determination assay (MoPED) to assess for serum EPO elevation 4 hr post iv dose.11 Downstream of
EPO generation, reticulocyte elevations could be measured 72 hours post po dose and RBC increases
enumerated 1ꢀ2 weeks following po dose. In general the compounds described herein displayed little
selectivity for PHD subtypes (usually within 20 fold or less) and discussion will therefore highlight
PHD2 activity. PHD1 & 3 IC50s are available in the supporting information.
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Figure 1. Clinical compound 1 and uHTS screening hit 2.
RESULTS AND DISCUSSION
Our efforts started with a screen of the Merck compound collection identifying 2 as a fairly potent
PHD2 inhibitor at 110 nM IC50 and MW of 324 (Figure 1). The core 4ꢀhydroxyꢀ2ꢀ(heterocyclic) pyrimꢀ
idineꢀ5ꢀcarboxamide scaffold 3 was represented by many members with a range of potency building
confidence in the quality of the hit. Small molecule inhibitors of PHD2 (such as PDB complex 2G19
which contains an inhibitor similar to 1)15 form directed interactions to protein side chains of the active
site and chelate the bound Fe. The Fe is hexaꢀcoordinated to two histidines (H313 & H374), one asparꢀ
tic acid (D315), and one water molecule which leaves open two coordination sites for heteroaryl interacꢀ
tions. Crystal structures of small bicyclic inhibitors demonstrate a tight salt bridge formed between the
acid tail of the inhibitor and Arg383 at the base of a narrow and deep binding pocket. Noticeably absent
in the chemical series described here is the acid tail which eliminates this salt bridge to arginine.
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Molecular modeling (details in Methods) of the screening hit led us to develop a hypothesis that the
hetero biaryl chelated Fe with the pyridine ring filling the space normally occupied by a carboxylic acid
substituent of Fibrogenꢀlike PHD inhibitors. The benzyl tail of the hit series can adopt three possible
conformations interacting with the outer regions of the binding site (Figure 2). A water molecule forms
a hydrogen bonded bridge between the oxyanion of the hydroxypryimidine and the phenolic hydrogen
of Tyr303. A stacking interaction between Tyr310 and the terminal heteroaryl of the inhibitor is present.
Amide substitution can display monoꢀ or diꢀsubstituted branched aromatic rings which project out of the
primary binding pocket and interact with both solvent and several aromatic residues: Trp258, Trp389,
Arg322. Taken together, these observations supported further optimization of the hit to recruit additionꢀ
al directed interactions and the saltꢀbridge to Arg383.
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When comparing rotamers of the branched biaryl, docking scores for the three rotamers of the fluoroꢀ
phenol were roughly equivalent and the ligand would be expected to sample multiple conformations
when bound. This observation led us to consider diꢀ and triꢀsubstitution on the methylene linker.
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Journal of Medicinal Chemistry
Figure 2: Molecular models of the core scaffold to human PHD2 crystal structure 2G1915. Where
multiple rotamers are possible for the branched aromatics, only one pose per structure has been
displayed.
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Methylation of the methylene linker of 2 improved potency 4ꢀfold for 10 (Table 1) . The substitution
does not pick up directed interactions other than van der Waals contact to the protein so it is hypotheꢀ
sized that the gemꢀdimethyl preferentially adopts a conformation of the phenyl ring shown in Figure 2 to
pick up an edge to face stacking interaction with Trp258. Building from 10, the affinity again improves
when the terminal heteroaryl is changed from pyridine to pyrazole 7. There is a slight steric interaction
between the top of the pyridine ring and Tyr303 which may influence an improvement in affinity when
moving to smaller 5ꢀmembered heterocycles. While variation in chelation strength was expected for 5ꢀ
and 6ꢀmembered biaryls, quantum mechanical calculations quantifying variation in Fe chelation enerꢀ
getics did not improve interpretation of SAR and are not described further. When the methylene linkage
is disubstituted with phenyl rings affinity is improved as evidenced by 25, 26, and 28 (Tables 3ꢀ4).
Docking models for these ligands always favor one aryl ring stacked over Arg322 owing to cationꢀpi
interactions; however, substitution of the terminal phenyls can lead to functionality which more favoraꢀ
bly picks up directed interactions to other residues influencing rotameric variation in binding of the
branched aromatics.
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With the observation that substitution of the terminal phenyl rings could provide additional directed
interactions, we suggest that methoxy substitution 26 specifically led to hydrogen bonds to each methꢀ
oxy oxygen, one from the indole NH of Trp258, and one from the Nδ of Asn318. Further aromatic
substitution of 10 is unfavorable, as the trajectory of the last open position of the methylene linkage is
directed at a suboptimal angle towards the Tyr389 and a third aromatic ring would clash with the proꢀ
tein.
On the carboxamide portion of scaffold 3 wide structural diversity was tolerated in the hit class. The
hits were primarily represented by large amides with significant structural flexibility. The early optimiꢀ
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Page 6 of 36
zation strategy was to identify a reasonably efficient right hand side amide and then further refine the
war head interaction with the heterocycle at R1 (Table 1). This could then be followed by another round
of potency tuning for amide R2 (Table 3). After initial benchmarking of the hit, confidence continued to
grow in the scaffold as 2 showed >30 ꢀM offꢀtarget hERG activity which contrasts sharply with our laꢀ
boratory’s previous HIF PHD inhibitor scaffold.11 Rat pharmacokinetic (PK) assessment showed a reaꢀ
sonable starting point with %F 67, Clp 2.0 ml/min/kg, Vdss 0.35 L/kg and t½ 3.3 hr. In terms of mouse
PD, 2 showed modest activity with a minimum effective dose (MED) of 100 mg/kg (mpk) in MoPED
resulting in an EPO concentration of 1200 pg/mL.
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Table 1. Heterocyclic 2ꢀposition SAR
MoPED
pg/mL
(mpk)
Rat PK
%F,
t1/2
PHD2
IC50 (nM)
Compd
4
R1
24
110 (100 iv)
Nd
61
2.9 hr
5
6
7
16
210
3.7
2100 (100 iv)
Nd
Nd
460 (5 iv)
5000 (15 iv)
24000 (50 iv)
99
4.2 hr
3
2.1 hr
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9
2.1
1900 (15 iv)
Nd
730
Nd
10
11
28
Nd
Nd
Nd
Nd
>2700
5
0.23 hr
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13
6.0
220 (15 iv)
Nd
390
Nd
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Journal of Medicinal Chemistry
14
12
Nd
Nd
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7
A quick survey of amines (data not shown) demonstrated that the cumyl amide 10 provided a 4ꢀfold
improvement in potency vs the screening hit to 28 nM PHD2 IC50. This enabled a starting point to anꢀ
chor the carboxamide thereby allowing examination of the 2ꢀposition SAR. A general synthetic apꢀ
proach to the assembly of these compounds is exemplified for 28 in Scheme 1. Both 5 and 6 membered
heterocycles were tolerated at this position. Modeling had suggested the presence of an acid on this ring
system would make a strong interaction with Arg383. This indeed improved potency with both pyrazole
8 and pyridine 12 analogs showing improvement as low as 2 nM PHD2 IC50, but unfortunately was acꢀ
companied by very low rat bioavailability for both compounds. However as MoPED is IV dosed, imꢀ
provement was apparent in 12 with the MED dropping to 15 mpk. For the 6 membered ring system,
CF3 substitution of the pyridine ring 11 resulted in a large drop in activity. Diaza rings showed a wide
range of activity. Pyrimidine 13 was particularly ill favored, but pyrazole 7 and pyridazine 14 delivered
large potency improvements to 3.7 and 12 nM PHD2 IC50 respectively. The methylated pyrazole isomer
6 lost over 50ꢀfold of activity. The thiazole analogs 4 and 5 had improved potency to 2, but still reꢀ
quired the maximum feasible dose of 100 mpk to show an EPO elevation in MoPED. In contrast to the
pyrazole acid, the unsubstituted pyrazole 7 displayed high bioavailability and a 30ꢀfold improvement in
potency relative to the screening lead 2. This resulted in a greatly improved PD response with MoPED
MED dropping to 5 mpk.
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Table 2. Pharmacokinetic profile of 7
mouse*
98
30
Rat*
99
49
dog**
61
11
rhesus**
71
F (%)
cmax (ꢁM)
22
Cl (ml/min/kg)
Vdss (L/kg)
t1/2 (hr)
1.0
0.3
3.7
9.3
0.2
0.06
4.2
1.2
0.004
0.1
>100
2.7
0.001
0.13
95
PPB (% unbound )
0.4
*2 mg/kg po, 1 mg/kg iv; **1 mg/kg po, 0.5 mg/kg iv. PPB is from 100% serum; Mouse = Clb, rat, dog, rhesus Clp. Mouse: IV and P.O.:
DMSO: PEG400: water (5:40:55, v/v/v). Rats: IV and P.O.: DMSO: PEG400: water (10:50:40, v/v/v). Dog and Monkey: IV:
PEG200:12%HPCD: water (30:12:58 v/v/v); P.O.: Imwitor:Tween (1:1 w/w).
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PK studies with 7 demonstrated robust oral absorption in all preclinical species tested (Table 2).
However, in higher species, an exceptionally long t1/2 of >95 hours was observed in monkeys and dogs.
In vitro incubations with both liver microsomes and hepatocytes from rat, dog, monkey and human disꢀ
played very little turnover. These data suggested the compound could have a very long human t1/2
which was not in keeping with our desire for a QD dosing paradigm. It was therefore decided that
compounds would need to have shorter residence times in higher species in order to advance. Since 7
was low molecular weight (323) and not readily metabolized (as evident by little turnover in both miꢀ
crosome and hepatocyte incubations) the team anticipated that by increasing MW and adding structural
features amenable to phase I or II metabolism would result in compounds with more appropriate ADME
properties. As a first in vivo read out of shortened residence time in higher species, compounds of interꢀ
est were screened for PK via dog cassette IV dosing.
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As the 2ꢀpyrazole of 7 provided both potency and PD, attention focused towards 5ꢀcarboxamide optiꢀ
mization (Table 3). In order to get a better sense of the minimum pharmacophore the benzamide was
replaced with methyl cyclopropyl amide 15 which resulted in a 40ꢀfold loss of PHD2 activity. Returnꢀ
ing to benzamides, 3ꢀ6 nM PHD2 IC50s could be achieved without the need for alpha methyl groups (16
or 17). Adding back one methyl group generated optically active amides 18ꢀ21 that showed a slight
preference in potency for the Sꢀisomer. While the potency difference was small there was a noted drop
in PD activity for the R isomers 19 and 21. Regardless of the reason for decreased in vivo activity, the
compounds, represented by 20, had a very long t1/2 in dog of 60 hours. Building in a metabolic softꢀspot
with the 4ꢀmethoxy group of 23 provided further improved potency and efficacy but no improvement in
reducing dog t1/2. While adding in an extra phenyl ring as in biphenyl 24 and benzhydryl 25 amides
provided 1ꢀ2 nM PHD2 potency, the dog t t1/2 moved in the wrong direction as exemplified by 24 with a
143hr t1/2. In an attempt to really leverage phase I metabolism the dimethoxybenzhydryl 26 was preꢀ
pared. This compound had the lowest MED tested with significant EPO stimulation at a 1.5 mpk dose
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in mice. Offꢀtarget hERG counterꢀscreening revealed that 26 was active at 900 nM IC50. This was a
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3
departure from the smaller amides that were generally inactive on hERG.
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6
Table 3. 5ꢀCarboxamide SAR
7
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9
10
11
12
13
14
15
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17
18
19
20
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24
25
26
27
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PHD2
IC50
(nM)
MoPED
pg/mL
(mpk)
Dog
PK
t1/2 hr
Compd
15
R2
150
Nd
Nd
16
17
2.9
5.6
1100 (15 iv)
Nd
Nd
180 (15 iv)
8900 (50 iv)
690 (5 iv)
10000 (15 iv)
18
5.0
Nd
19
20
21
8.1
1.9
10
322 (15 iv)
16000 (15 iv)
350 (15 iv)
Nd
60
Nd
22
4000
Nd
Nd
23
24
1.4
1.3
5300 (15 iv)
Nd
94
143
25
26
2.0
1.1
2900 (15 iv)
Nd
Nd
190 (1.5 iv)
740 (5 iv)
31000 (15 iv)
Table 4. Pyridazine warhead leading to 28.
PHD2
IC50
MoPED
pg/mL (mpk)
hERG
IC50
Compd
R3
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(nM)
1
2
380 (5 iv)
1600 (15 iv)
360 (1.5 iv)
6200 (5 iv)
27
H
2.5
>30 ꢁM
>30 ꢁM
3
4
5
28*
OCH3
1.0
12000 (15 iv)
18000 (50 iv)
27000 (100 iv)
6
7
8
9
*28 PHD1 and 3 IC50 are 1.0 and 14 nM respectively.
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To evaluate if the choice of warhead could impact hERG activity and PK, the ring sizes were changed
from 5 to 6 with an expansion of pyrazole to pyridazine (Table 4). There was no change in PHD2 poꢀ
tency, but the hERG activity was eliminated in both 27 and 28. The robust PD response noted for 26
was further enhanced with 28 utilizing the pyridazine warhead resulting in a MoPED MED of 1.5 mpk
for EPO elevation. Even with maximal EPO responses (EPO concentration >10,000 pg/mL), no
changes in plasma VEGF were observed.
Off target activity of 28 was evaluated to establish specificity. It was not a significant inhibitor of the
cytochrome p450 enzymes in vitro (IC50): CYP1A2, 3A4, 2B6, 2C9, 2C19, or 2D6 >60 ꢀM and was a
moderate reversible inhibitor of CYP2C8 at 1.6 ꢀM in vitro. Probing more broadly, 28 was inactive
12
when screened at 10 ꢁM against a general panel of 171 radioligand binding and enzymatic assays.
Additionally in terms of a related enzyme in the same pathway, the IC50 of 28 was determined for factor
inhibiting HIF (FIH) to be 18 uM suggesting good selectivity of 28 for the target. Also of note was
that, in mouse, there were no elevations in plasma ALT levels below 200 mg/kg which had been obꢀ
served in our previously disclosed scaffold.11
Tritiated 28 exhibited minimal metabolic turnover in liver microsomes (+NADPH) from rat, dog and
monkey (<10% turover), but significant turnover in human liver microsomes (34% turnover) after 60
min (10 ꢁM compound, 1 mg/mL microsomal protein). As anticipated, the use of the two methoxy
groups to build in phase I metabolism delivered, as an Oꢀdemethylated metabolite was detected in liver
microsomal incubations of all species.
Table 5. Pharmacokinetic profile of 28.
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mouse* rat**
dog*
71
18
rhesus*
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0.15
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F (%)
Cmax (ꢀM)
Clb or Clp
(ml/min/kg)
Vdss (L/kg)
t1/2 (hr)
63
8.4
36
11
0.13
0.20
0.07
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0.16
9.3
0.12
19
1
0.10
10
0.5
PPB % unbound
0.6
*1 mg/kg po, 0.5 mg/kg iv; **2 mg/kg po, 1 mg/kg iv. PPB is from 100% serum; Mouse=blood clearance Clb; rat, dog, rhesus=plasma
clearance Clp. Mouse: IV and P.O.: DMSO: PEG400: water (5:40:55, v/v/v). Rats: IV and P.O.: DMSO: PEG400: water (10:50:40, v/v/v).
Dog and Monkey: IV: PEG200:12%HPCD: water (30:12:58 v/v/v pH 8.2); P.O.: 0.5% Methylcellulose, pH8.
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In terms of its pharmacokinetic profile (Table 5), 28 showed good oral bioavailability across species
(36ꢀ71%), with low clearance and volume of distribution. While the compound still had a relatively
long elimination halfꢀlife across preclinical species, it was greatly reduced compared to 7 and combined
with the observed turnover in human microsomal incubations there was no expectation of overly long
human t1/2.
Tritiated 28 was prepared and dosed to bileꢀduct cannulated rats (Table 6). After 48 hours, post dose
recovery of the radioactivity was about 26% bile, 12% urine and 38% in feces indicating that ~38% of
the dose was absorbed and eliminated into bile and urine which is consistent with the oral bioavailability
(~36%) observed in the rat PK study (Table 4). The major radioactive component detected in feces was
parent compound (~90% of the radioactivity, ~34% of the dose), while parent compound represented
<10% of the total radioactivity in bile and urine, respectively. In both bile and urine the major observed
radioactive component was a [parent +16] oxidation product.
Table 6. Percent of Radioactive Dose Recovered in the Bile, Urine, and Feces of Rats Following a
po Dose of [3H]ꢀ28*
Species
0ꢀ48 hr
Rat
Dose/Route
5 mg/kg po
Percent of Radioactive Dose
Urine
Bile
Feces
Total
26 ± 8
12 ± 2 39 ± 15 78 ± 8
(BDC)
*Fasted male bile ductꢀcannulated (BDC) SpragueꢀDawley rats were dosed orally (5 mg/kg) with a mixture of 28 and [3H]ꢀ28 formulated in
0.5% methylcellulose. Values shown are means ± standard deviation (n = 3).
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Figure 3. Single dose 28 mediated change in reticulocytes in mouse. * p < 0.05 2ꢀway ANOVA
As shown in Table 4, 28 elicited an increase in EPO levels with a mouse MED of 1.5 mpk when dosed
iv. The compound was then evaluated for its ability to stimulate EPO production and/or reticulocytes
dosed orally to both mice and rats (Figures 3ꢀ6). In mice (C57Bl/6), single doses of 5 and 15 mpk po
(n=3) caused increases in circulating reticulocytes measured on both 3 and 4 days postꢀcompound chalꢀ
lenge (Figure 3). In rat (SpragueꢀDawley), a single dose titration of 1.5, 5 and 15 mpk po (n=5) caused
a large increase in serum EPO levels ranging from 1.7, 8 and 204ꢀfold relative to vehicle, respectively
(Figure 4). Increases in circulating reticulocytes were observed at 5 and 15 mg/kg three days post chalꢀ
lenge and, with the 15 mg dose, at 4 days post challenge (Figure 5). A plot of 28 Cp levels over time for
the rat study is shown in Figure 6. Cmax values (ꢀM) of 6.8+0.5 (6hr, 1.5 mpk), 14+1.1(6hr, 5 mpk),
and 30+2.1 (6hr, 15 mpk) were observed. The effect of 4 week QD po administration of 28 on
haemoglobin levels in SpragueꢀDawley rats is shown in Figure 7. Dosing at 1.5 and 15 mpk daily
resulted in 1.0 and a more extreme 7.6 g/dL change in Hb at week 4 relative to vehicle treated animals.
The rate of Hb increase for the 1.5 mpk dose group is consistent with what would be desired in a clinical
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Figure 4. Single dose 28 mediated EPO response in rats.
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Figure 5. Single dose 28 mediated change in reticulocytes in rat. * p < 0.05 2ꢀway ANOVA
Figure 6. Single dose 28 plasma concentration in rat.
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Figure 7. Changes in Hb following 28 day once daily oral dosing of 28 in rats. Mean +/ꢀ SEM
The synthesis of 28 is shown in Scheme 1 and it represents the general method of preparation of the 4ꢀ
hydroxyꢀ2ꢀ(heterocyclic)pyrimidineꢀ5ꢀcarboxamides presented herein. The key step in the formation of
the pyrimidinone core is condensation of the of the amidine 32 bearing heterocycle R1 with diethyl ethꢀ
oxymethylenemalonate. Following saponification of the ester, optimal amide coupling was found to be
with CDI activation of the acid 33. Other methods of activation provided very inconsistent yields and
were often problematic even at the small scale for SAR exploration.
While the bis(4ꢀ
methoxyphenyl)methanamine 35 was commercially available at gram scale, it could be readily obtained
by conversion of ketone 34 to the oxime followed by reduction with Pd/C to obtain the amine in bulk.
The below convergent route was used to prepare 28 in 54% overall yield. This route, with optimizations
to accommodate larger scale, allowed preparation of 2.7 kg of 28 in a similar overall yield to support
human clinical evaluation.
Scheme 1. Synthesis of 28a
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aReagents and conditions: (a) TMSCN, Cat AlCl3, pꢀTosCl, DCM, rt, 85%; (b) DBU, THF, rt, 97%; (c) NaOMe, NH4Cl, MeOH, rt; (d)
NaOEt, EtOH, diethyl 2ꢀ(ethoxymethylene)malonate, reflux; (e) KOH, H2O, reflux, 75%; (f) CDI, NMP, NEt3 35, 70°C, 87%; (g)
NH2OHꢂHCl, EtOH, pyridine, 70ꢀ80°C, 95%; (h) 50% Pd/C, H2, HCl, MeOH, 30ꢀ40°C 65%.
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CONCLUSION
In summary, optimization of the 4ꢀhydroxyꢀ2ꢀ(heterocyclic)pyrimidineꢀ5ꢀcarboxamide scaffold 3
identified through a high throughput screen of the Merck compound collection afforded high potency
pan HIFꢀPHD inhibitors with promising PK profiles across species. From early lead 7, the key optimiꢀ
zation strategy was to increase metabolic intrinsic clearance in higher species by introducing moieties
amenable to phase I or II metabolism. On the basis of its overall pharmacological, pharmacokinetic,
and early safety profile (data not shown), 28 was advanced to human clinical studies as an oral treatment
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for anemia and designated as MKꢀ8617.
EXPERIMENTAL SECTION
15
Modeling. Modeling predicted poses began from the crystal structure of PDB code 2G19. We
removed the ligand and maintained protein coordination to Fe along with water molecules observed in
the active site, one of which was coordinating the Fe. Ligands were docked into the active site using
16
Schrödinger’s Glide SP. Restrained docking was employed to maintain the location of the hydroxypyꢀ
17
rimidine. The pKa (ACD pKa DB v12.0 predicts pKa of 4.6) of the hydroxypyrimidine supports this
group existing in a deprotonated (anionic) state.
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Chemistry. All reactions were carried out in ovenꢀdried roundꢀbottom flasks or Biotage microꢀ
wave vials under an atmosphere of nitrogen with magnetic stirring unless otherwise noted. Airꢀ and
moistureꢀsensitive liquids and solutions were transferred via syringe or stainlessꢀsteel cannula. Organic
solutions were concentrated via rotary evaporation on a variable pressure Buchi rotovap. Normal phase
and reverse phase chromatography were performed on a Biotage Isolera or ISCO Combiflash Rf chroꢀ
matography system using Biotage silica cartridges, ISCO silica cartridges, or Biotage C18 reverse phase
cartridges. Thin layer chromatography was performed using EMD Millipore TLC Silica Gel 60_F254
2.5 x 7.5 cm silica gel plates. Thin layer chromatography plates were visualized using 254 nm ultravioꢀ
let light and/or exposure to cerium ammonium molybdate, phosphomolybdic acid, or silica gel impregꢀ
nated with iodine. All purchased materials were used as received unless otherwise noted. Anhydrous
solvents were purchase from Aldrich and Across and used as received. For air sensitive reactions solꢀ
vents were purged by bubbling nitrogen through the solvent prior to the reaction. Proton nuclear magꢀ
netic resonance (1H NMR) spectra and carbon nuclear magnetic resonance (13C NMR) spectra were recꢀ
orded with Varian Oxford 500 MHz spectrometer unless otherwise noted. Chemical shifts for protons
are reported in a parts per million scale downfield from tetramethylsilane and are referenced to residual
protium in the solvent used for the experiment (e.g. for spectra recorded in CDCl3, CHCl3 proton was
used as reference: δ7.26). Chemical shifts for carbons are reported in a parts per million scale (δ scale)
downfield from tetramethylsilane and are referenced to carbon resonances in the solvent used for the
experiment (e.g. for spectra recorded in CDCl3, CDCl3 carbon was used as reference: δ77.00). Data are
represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, br = broad, o = overlapped), coupling constant(s), and integration (where mixtures of diaꢀ
stereomers are reported some integrals may be reported as less than one proton). NMR data for 28 and
22 were acquired using 600 MHz Agilent spectrometer equipped with 3mm Cold Probe. Solutions of 28
and 22 in any solvent showed significant degree of lineꢀbroadening, which complicated NMR analysis.
The exchange between two or more tautomeric forms was hypothesized to be the cause. 0.5% (v/v) TFA
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was added to NMR samples in order to suppress tautomeric exchange by protonating all basic nitrogens
of the molecules. This lead to improvement in NMR spectra quality, and subsequent NMR analyses
were performed on samples with TFA. Structures of 28 and 22 were confirmed using 1H, 13C, 1Hꢀ1H 2D
COSY, 1Hꢀ13C 2D HSQC and 1Hꢀ13C 2D HMBC experiments. All chemical shifts and heteronuclear corꢀ
relations were consistent with the structures. While line broadening was observed with other similar
compounds no additional NMR optimization was carried out. Analytical HPLC/MS ꢀ Standard Method:
Mass analysis was performed on a Waters Micromass® ZQTM with electrospray ionization in positive
ion detection mode. High performance liquid chromatography (HPLC) was conducted on an Agilent
1100 series HPLC on Waters C18 XTerra 3.5 ꢀm 3.0 x50 mm column with gradient 10:90ꢀ100 v/v
CH3CN/H2O + v 0.05 % TFA over 3.75 min then hold at 100 CH3CN + v 0.05 % TFA for 1.75 min;
flow rate 1.0 mL/min, UV wavelength 254 nm (all HPLC/MS data was generated with this method unꢀ
less indicated otherwise). Analytical HPLC/MS ꢀ Basic Method: Mass analysis was performed on a
Waters Micromass® ZQTM with electrospray ionization in positive ion detection mode. High perforꢀ
mance liquid chromatography (HPLC) was conducted on an Agilent 1100 series HPLC on Waters C18
XBridge 3.5 ꢀm 3.0 x 50 mm column with gradient 10:90ꢀ98:2 v/v CH3CN/H2O + v 0.025 % NH4OH
over 3.25 min then hold at 98:2 CH3CN + v 0.025 % NH4OH for 2.25 min; flow rate 1.0 mL/min, UV
wavelength 254 nm. All compounds reported are of at least 95% purity, as judged by LCAP.
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A representative approach toward the general preparation of 4ꢀhydroxyꢀ2ꢀ(heterocyclic)pyrimidineꢀ5ꢀ
carboxamides is described with the procedure for the synthesis of compound 2, below.
Nꢀ(4ꢀFluorobenzyl)ꢀ4ꢀhydroxyꢀ2ꢀ(pyridinꢀ2ꢀyl)pyrimidineꢀ5ꢀcarboxamide (2). 4ꢀHydroxyꢀ2ꢀ
pyridinꢀ2ꢀylpyrimidineꢀ5ꢀcarboxylic acid (compound prepared in a manner similar to that described for
corresponding intermediate obtained in the preparation of compound 28, see below (0.100 g, 0.460
mmol) was dissolved in DMF (2.0 mL) and HATU (0.280 g, 0.737 mmol), N,Nꢀdiisopropylethylamine
(0.322 mL, 1.842 mmol) and 4ꢀfluorobenzylamine (0.105 mL, 0.921 mmol) were added. The reaction
mixture was stirred at RT for 2 hrs.
Additional HATU (0.245 g, 0.645 mmol), N,Nꢀ
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diisopropylethylamine (0.080 mL, 0.460 mmol) and 4ꢀfluorobenzylamine (0.105 mL, 0.921 mmol) were
added to the reaction and the mixture was stirred at room temperature for an additional 1 hr. The crude
reaction was partitioned between EtOAc and 10% NaHSO4 (aq.). The organic layer was separated and
washed with water, followed by brine. The organic layer was separated and evaporated. The residue
was purified by reverse chromatography eluting with MeCN/H2O/NH3OH to afford the title compound.
1H NMR (500 MHz, DMSOꢀd6) δ 8.76 (d, J = 4.8 Hz, 1H), 8.65 (s, 1H), 8.36 (d, J = 7.9 Hz, 1H), 8.05
(td, J = 7.8, 1.7 Hz, 1H), 7.65 (dd, J = 7.6, 4.8 Hz, 1H), 7.45 – 7.29 (m, 2H), 7.14 (t, J = 8.8 Hz, 2H),
4.50 (s, 2H). LCMS m/z = 325.1 (M+H)+; Rt = 2.56 min
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A less preferred approach toward the general preparation of 4ꢀhydroxyꢀ2ꢀ(heterocyclic)pyrimidineꢀ5ꢀ
carboxamides is described with the procedure for the synthesis of compound 4 below.
4ꢀHydroxyꢀ2ꢀ(2ꢀmethylthiazolꢀ4ꢀyl)ꢀNꢀ(2ꢀphenylpropanꢀ2ꢀyl)pyrimidineꢀ5ꢀcarboxamide (4).
Methyl 3ꢀoxoꢀ3ꢀ((2ꢀphenylpropanꢀ2ꢀyl)amino)propanoate. 2ꢀPhenylpropanꢀ2ꢀamine (0.9 g, 6.66
mmol), methyl 3ꢀchloroꢀ3ꢀoxopropanoate (1.14 g, 8.32 mmol) and triethylamine (1.16 mL, 8.32 mmol)
were added to MeCN (10 mL) at RT. Much precipitation was observed. After 50 min additional methyl
3ꢀchloroꢀ3ꢀoxopropanoate (0.20 mL, 1.87 mmol) and triethylamine (0.30 mL, 2.15 mmol) were added.
The reaction was stirred an additional 1 h 30 min and the reaction was diluted with EtOAc and washed
with 2 M HCl/ saturated NaCl (aq.), Na2CO3/saturated NaCl (aq.), 2 M HCl/saturated NaCl (aq.) and
saturated NaCl (aq.). The product was dried with Na2SO4, filtered and concentrated. The solid was
washed with Et2O and hexanes and filtered. Material used as is (1.03 g, 66.0%).
Methyl (Z)ꢀ3ꢀ(dimethylamino)ꢀ2ꢀ((2ꢀphenylpropanꢀ2ꢀyl)carbamoyl)acrylate. Methyl 3ꢀoxoꢀ3ꢀ((2ꢀ
phenylpropanꢀ2ꢀyl) amino)propanoate (0.60 g, 2.55 mmol) was dissolved in DMF (6 mL) and 1,1ꢀ
dimethoxyꢀN,Nꢀdimethylmethanamine (0.72 mL, 5.10 mmol) was added. The mixture was heated at 90
oC for 10 min. The reaction was cooled and additional 1,1ꢀdimethoxyꢀN,Nꢀdimethylmethanamine (0.36
mL, 2.55 mmol) was added. The reaction was heated an additional 1 hr 15 min and the mixture was
cooled and concentrated. The mixture was diluted with DMA and used without further purification.
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4ꢀHydroxyꢀ2ꢀ(2ꢀmethylthiazolꢀ4ꢀyl)ꢀNꢀ(2ꢀphenylpropanꢀ2ꢀyl)pyrimidineꢀ5ꢀcarboxamide
4.
2ꢀ
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Methylthiazoleꢀ4ꢀcarboximidamide hydrochloride (compound prepared in a manner similar to that deꢀ
scribed for corresponding intermediate obtained in the preparation of compound 28, see below (0.129 g,
0.728 mmol) and methyl (Z)ꢀ3ꢀ(dimethylamino)ꢀ2ꢀ((2ꢀphenylpropanꢀ2ꢀyl)carbamoyl)acrylate (0.106 g,
0.364 mmol) were added to a CEM microwave flask and DMA (1.5 mL) was added, followed by DBU
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o
(0.110 mL, 0.728 mmol). The reaction was heated in a microwave for 10 min at 115 C at 30 watts.
The solution was diluted with MeOH (1 mL) and EtOAc and washed with brine and then 2 M HCl.
The aqueous portion was back extracted with EtOAc (2x) and the combined organics were concentratꢀ
ed. The residue was purified by reverse phase chromatography eluting with MeCN/H2O/TFA to afford
the titled compound (0.356 g, 27.6%). 1H NMR (500 MHz, DMSOꢀd6) δ 8.59 (s, 1H), 8.45 (s, 1H),
7.43 – 7.36 (m, 2H), 7.32 (t, J = 7.8 Hz, 2H), 7.25 – 7.17 (m, 1H), 2.79 (s, 3H), 1.70 (s, 6H). LCMS
m/z = 355.1 (M+H)+; Rt = 2.75 min
4ꢀHydroxyꢀNꢀ(2ꢀphenylpropanꢀ2ꢀyl)ꢀ2ꢀ(thiazolꢀ4ꢀyl)pyrimidineꢀ5ꢀcarboxamide (5). Compound 5
(0.048 g, 29%) was prepared in a manner similar to that described for compound 4. 1H NMR (500
MHz, DMSOꢀd6) δ 9.37 (d, J = 1.9 Hz, 1H), 8.79 (d, J = 2.0 Hz, 1H), 8.48 (s, 1H), 7.45 – 7.36 (m, 2H),
7.31 (t, J = 7.7 Hz, 2H), 7.26 – 7.14 (m, 1H), 1.69 (s, 6H). LCMS m/z = 341.2 (M+H)+; Rt = 2.55 min
4ꢀHydroxyꢀ2ꢀ(1ꢀmethylꢀ1Hꢀpyrazolꢀ3ꢀyl)ꢀNꢀ(2ꢀphenylpropanꢀ2ꢀyl)pyrimidineꢀ5ꢀcarboxamide
(6). Compound 6 (0.042 g, 26%) was prepared in a manner similar to that described for compound 4.
1H NMR (500 MHz, DMSOꢀd6) δ 9.92 (s, 1H), 8.46 (s, 1H), 7.95 (d, J = 2.3 Hz, 1H), 7.44 – 7.36 (m,
2H), 7.32 (dd, J = 8.5, 7.0 Hz, 2H), 7.24 – 7.16 (m, 1H), 6.99 (d, J = 2.4 Hz, 1H), 4.01 (s, 3H), 1.69 (s,
6H). LCMS m/z = 338.1 (M+H)+; Rt = 2.53 min
4ꢀHydroxyꢀNꢀ(2ꢀphenylpropanꢀ2ꢀyl)ꢀ2ꢀ(1Hꢀpyrazolꢀ1ꢀyl)pyrimidineꢀ5ꢀcarboxamide (7). Comꢀ
pound 7 (0.421 g, 54%) was prepared in a manner similar to that described for compound 28, see below.
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1H NMR (500 MHz, DMSOꢀd6) δ 10.01 (s, 1H), 8.61 (d, J = 2.7 Hz, 1H), 8.36 (s, 1H), 8.01 (d, J = 1.5
Hz, 1H), 7.46 – 7.35 (m, 2H), 7.31 (t, J = 7.7 Hz, 2H), 7.25 – 7.16 (m, 1H), 6.69 (dd, J = 2.7, 1.6 Hz,
1H), 1.68 (s, 6H). LCMS m/z = 342.1 (M+H)+; Rt = 2.66 min
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id (8).
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4ꢀHydroxyꢀ2ꢀ(4ꢀiodoꢀ1Hꢀpyrazolꢀ1ꢀyl)ꢀNꢀ(1ꢀmethylꢀ1ꢀphenylethyl)pyrimidineꢀ5ꢀcarboxamide.
Compound 7 (0.75 g, 2.32 mmol) was dissolved in MeCN (30 mL) and I2 (0.38 g, 1.5 mmol) and CAN
(1.78 g, 3.25 mmol) were added and the reaction was heated at 85 °C for 6 h. The solution was cooled,
diluted with EtOAc and washed with water containing a few chunks of sodium thiosulfate. The organic
layer was washed with NaHSO4 (5 % aq.) and brine, the solution was dried with Na2SO4, filtered and
concentrated affording the title product which was used without further purification.
1ꢀ(4ꢀHydroxyꢀ5ꢀ((2ꢀphenylpropanꢀ2ꢀyl)carbamoyl)pyrimidinꢀ2ꢀyl)ꢀ1Hꢀpyrazoleꢀ4ꢀcarboxylic acid
8. 4ꢀHydroxyꢀ2ꢀ(4ꢀiodoꢀ1Hꢀpyrazolꢀ1ꢀyl)ꢀNꢀ(1ꢀmethylꢀ1ꢀphenylethyl)pyrimidineꢀ5ꢀcarboxamide (0.100
g, 0.223 mmol) was dissolved in DMSO (2 mL) and dppf (25 mg, 0.0.045 mmol) and Pd(OAc)2 (2.5
mg, 0.011 mmol) were added. The reaction mixture was purged, backfilled with carbon monoxide 3
times and stirred under a carbon monoxide balloon at 80 °C for 4 h. The reaction was diluted with
EtOAc, washed with 2 M HCl, dried with Na2SO4, filtered and concentrated. The residue was purified
on a reverse phase Cꢀ18 column eluting with 0 to 90 % MeCN/H2O/NH3OH. The desired fractions were
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concentrated affording the title compound (11 mg, 13% yield, 2 steps). H NMR (500 MHz, DMSOꢀd6)
δ 13.07 (s, 1H), 9.79 (s, 1H), 8.89 (d, J = 1.6 Hz, 1H), 8.49 – 8.20 (m, 2H), 7.39 (d, J = 8.2 Hz, 2H),
7.32 (td, J = 7.8, 1.7 Hz, 2H), 7.21 (dd, J = 8.1, 6.4 Hz, 1H), 1.69 (d, J = 1.6 Hz, 6H). LCMS m/z =
368.0 (M+H)+; Rt = 2.49 min.
2ꢀ(4ꢀCarbamoylꢀ1Hꢀpyrazolꢀ1ꢀyl)ꢀ4ꢀhydroxyꢀNꢀ(2ꢀphenylpropanꢀ2ꢀyl)pyrimidineꢀ5ꢀcarboxamide
(9). Compound 8 (40 mg, 0.109 mmol) was dissolved in DMF (1 mL) and PyBOP (85 mg, 0.183
mmol), ammonium chloride (11.65 mg, 0.218 mmol) and DIPEA (0.057 mL, 0.327 mmol) were added
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Journal of Medicinal Chemistry
and the reaction was stirred for 1 h. The residue was purified on a reverse phase Cꢀ18 column eluting
with 0 to 80 % MeCN/H2O/NH3OH. The desired fractions were concentrated affording the title comꢀ
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pound (38 mg, 95% yield). H NMR (500 MHz, DMSOꢀd6) δ 9.16 (s, 1H), 8.37 (s, 1H), 8.28 (s, 1H),
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7.91 (s, 1H), 7.39 (d, J = 7.5 Hz, 3H), 7.31 (t, J = 7.7 Hz, 2H), 7.21 (d, J = 7.3 Hz, 1H), 1.69 (s, 6H).
LCMS m/z = 389.3 (M+H)+; Rt = 2.50 min.
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4ꢀHydroxyꢀNꢀ(2ꢀphenylpropanꢀ2ꢀyl)ꢀ2ꢀ(pyridinꢀ2ꢀyl)pyrimidineꢀ5ꢀcarboxamide (10). Compound
10 (13 mg, 6 %) was prepared in a manner similar to that described for compound 28, see below.
LCMS m/z = 335.2 (M+H)+; Rt = 2.72 min
4ꢀHydroxyꢀNꢀ(2ꢀphenylpropanꢀ2ꢀyl)ꢀ2ꢀ(5ꢀ(trifluoromethyl)pyridinꢀ2ꢀyl)pyrimidineꢀ5ꢀ
carboxamide (11). Compound 11 (0.038 g, 27%) was prepared in a manner similar to that described for
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compound 28, see below. H NMR (500 MHz, DMSOꢀd6) δ 9.21 (s, 1H), 8.59 – 8.49 (m, 2H), 7.40 (d,
J = 7.8 Hz, 2H), 7.32 (t, J = 7.7 Hz, 2H), 7.21 (t, J = 7.3 Hz, 1H), 1.70 (d, J = 3.1 Hz, 6H). LCMS m/z =
403.1 (M+H)+; Rt = 3.08 min
2ꢀ(4ꢀHydroxyꢀ5ꢀ((2ꢀphenylpropanꢀ2ꢀyl)carbamoyl)pyrimidinꢀ2ꢀyl)isonicotinic acid (12).
2ꢀ(4ꢀBromopyridinꢀ2ꢀyl)ꢀ4ꢀhydroxyꢀNꢀ(1ꢀmethylꢀ1ꢀphenylethyl)pyrimidineꢀ5ꢀcarboxamide.
The
titled compound (0.251 g, 82%) was prepared in a manner similar to that described for compound 28,
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see below. H NMR (500 MHz, DMSOꢀd6) δ 8.70 (d, J = 5.3 Hz, 1H), 8.51 (s, 1H), 8.03 (dd, J = 5.2,
2.0 Hz, 1H), 7.40 (d, J = 7.7 Hz, 2H), 7.32 (t, J = 7.7 Hz, 2H), 7.22 (t, J = 7.3 Hz, 1H), 1.70 (s, 6H).
LCMS m/z = 414.9 (M+H)+; Rt = 3.05 min
2ꢀ(4ꢀHydroxyꢀ5ꢀ((2ꢀphenylpropanꢀ2ꢀyl)carbamoyl)pyrimidinꢀ2ꢀyl)isonicotinic acid 12. To 2ꢀ(4ꢀ
bromopyridinꢀ2ꢀyl)ꢀ4ꢀhydroxyꢀNꢀ(1ꢀmethylꢀ1ꢀphenylethyl)pyrimidineꢀ5ꢀcarboxamide (0.198 g, 0.479
mmol) in DMSO (5.0 mL) was added potassium acetate (0.188 g, 1.916 mmol), palladium(II) acetate
(5.38 mg, 0.024 mmol), and dppf (0.053 g, 0.096 mmol). The reaction mixture was purged, backfilled
with carbon monoxide 3 times, and stirred under a carbon monoxide balloon 120 °C for 30 min. The
reaction was diluted with EtOAc, washed with 1 M HCl and concentrated. The residue was purified by
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preparative HPLC (NonꢀPolar Method). The material was further purified by flash chromatography on
silica gel eluted with EtOH to afford the title compound (50 mg, 27.6% yield). 1H NMR (500 MHz,
DMSOꢀd6) δ 8.95 (d, J = 4.9 Hz, 1H), 8.74 (s, 1H), 8.53 (s, 1H), 8.09 (dd, J = 4.9, 1.7 Hz, 1H), 7.39 (dt,
J = 8.4, 1.9 Hz, 2H), 7.31 (t, J = 7.8 Hz, 2H), 7.25 – 7.17 (m, 1H), 1.69 (s, 6H). LCMS m/z = 379.0
(M+H)+; Rt = 2.62 min
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4ꢀHydroxyꢀNꢀ(2ꢀphenylpropanꢀ2ꢀyl)ꢀ[2,2'ꢀbipyrimidine]ꢀ5ꢀcarboxamide (13). Compound 13
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(0.020 g, 13%) was prepared in a manner similar to that described for compound 28, see below. H
NMR (500 MHz, DMSOꢀd6) δ 9.98 (s, 1H), 9.10 (d, J = 4.9 Hz, 1H), 8.57 (s, 1H), 7.79 (t, J = 4.9 Hz,
1H), 7.40 (d, J = 7.8 Hz, 2H), 7.32 (t, J = 7.6 Hz, 2H), 7.21 (t, J = 7.3 Hz, 1H), 1.71 (s, 6H). LCMS m/z
= 336.2 (M+H)+; Rt = 2.38 min
4ꢀHydroxyꢀNꢀ(2ꢀphenylpropanꢀ2ꢀyl)ꢀ2ꢀ(pyridazinꢀ3ꢀyl)pyrimidineꢀ5ꢀcarboxamide (14). Comꢀ
pound 14 (0.082 g, 69%) was prepared in a manner similar to that described for compound 28, see beꢀ
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low. H NMR (500 MHz, DMSOꢀd6) δ 9.50 (dd, J = 5.0, 1.6 Hz, 1H), 8.51 (dd, J = 8.6, 1.7 Hz, 1H),
8.01 (dd, J = 8.6, 5.0 Hz, 1H), 7.39 (d, J = 7.8 Hz, 2H), 7.31 (t, J = 7.6 Hz, 2H), 7.20 (t, J = 7.3 Hz, 1H),
1.70 (s, 6H). LCMS m/z = 336.0 (M+H)+; Rt = 2.45 min
Nꢀ(Cyclopropylmethyl)ꢀ4ꢀhydroxyꢀ2ꢀ(1Hꢀpyrazolꢀ1ꢀyl)pyrimidineꢀ5ꢀcarboxamide (15). Comꢀ
pound 15 (0.056 g, 41.8%) was prepared in a manner similar to that described for compound 28, see
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below. H NMR (500 MHz, DMSOꢀd6) δ 9.40 (s, 1H), 8.63 (d, J = 2.8 Hz, 1H), 8.44 (s, 1H), 8.06 (s,
1H), 6.72 (t, J = 2.2 Hz, 1H), 3.18 (t, J = 6.3 Hz, 2H), 1.02 (dd, J = 9.5, 4.9 Hz, 1H), 0.67 – 0.30 (m,
2H), 0.23 (t, J = 4.8 Hz, 2H). LCMS m/z = 260.2 (M+H)+; Rt = 1.80 min
Nꢀ(4ꢀChlorobenzyl)ꢀ4ꢀhydroxyꢀ2ꢀ(1Hꢀpyrazolꢀ1ꢀyl)pyrimidineꢀ5ꢀcarboxamide (16). Compound
16 (0.108 g, 67.7%) was prepared in a manner similar to that described for compound 28, see below.
1H NMR (500 MHz, DMSOꢀd6) δ 9.75 (s, 1H), 8.63 (d, J = 2.8 Hz, 1H), 8.46 (s, 1H), 8.06 (d, J = 1.5
Hz, 1H), 7.40 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.3 Hz, 2H), 6.72 (t, J = 2.1 Hz, 1H), 4.51 (d, J = 6.0 Hz,
2H). LCMS m/z = 330.0 (M+H)+; Rt = 2.63 min
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4ꢀHydroxyꢀ2ꢀ(1Hꢀpyrazolꢀ1ꢀyl)ꢀNꢀ(4ꢀ(trifluoromethoxy)benzyl)pyrimidineꢀ5ꢀcarboxamide (17).
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Compound 17 (0.012 g, 9%) was prepared in a manner similar to that described for compound 4. H
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NMR (500 MHz, DMSOꢀd6) δ 9.79 (s, 1H), 8.63 (d, J = 2.7 Hz, 1H), 8.46 (s, 1H), 8.06 (d, J = 1.5 Hz,
1H), 7.44 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 8.3 Hz, 2H), 6.72 (dd, J = 2.8, 1.5 Hz, 1H), 4.55 (d, J = 6.1
Hz, 2H). LCMS m/z = 380.1 (M+H)+; Rt = 2.87 min
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(S)ꢀ4ꢀHydroxyꢀNꢀ(1ꢀphenylethyl)ꢀ2ꢀ(1Hꢀpyrazolꢀ1ꢀyl)pyrimidineꢀ5ꢀcarboxamide (18). Comꢀ
pound 18 (0.094 g, 62.8%) was prepared in a manner similar to that described for compound 28, see
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below. H NMR (500 MHz, DMSOꢀd6) δ 8.62 (d, J = 2.8 Hz, 1H), 8.42 (s, 1H), 8.05 (d, J = 1.5 Hz,
1H), 7.39 – 7.29 (m, 4H), 7.25 (tt, J = 5.7, 2.8 Hz, 1H), 6.71 (dd, J = 2.8, 1.6 Hz, 1H), 5.12 (p, J = 7.1
Hz, 1H), 1.47 (d, J = 6.9 Hz, 3H). LCMS m/z = 310.1 (M+H)+; Rt = 2.47 min
(R)ꢀ4ꢀHydroxyꢀNꢀ(1ꢀphenylethyl)ꢀ2ꢀ(1Hꢀpyrazolꢀ1ꢀyl)pyrimidineꢀ5ꢀcarboxamide (19). Compound
19 (0.075 g, 50.0%) was prepared in a manner similar to that described for compound 28, see below.
1H NMR (500 MHz, DMSOꢀd6) δ 9.77 (s, 1H), 8.62 (d, J = 2.7 Hz, 1H), 8.43 (s, 1H), 8.06 (d, J = 1.5
Hz, 1H), 7.47 – 7.28 (m, 4H), 7.25 (tt, J = 5.7, 2.8 Hz, 1H), 6.72 (dd, J = 2.9, 1.6 Hz, 1H), 5.12 (p, J =
7.1 Hz, 1H), 1.47 (d, J = 6.9 Hz, 3H). LCMS m/z = 310.1 (M+H)+; Rt = 2.50 min
(S)ꢀNꢀ(1ꢀ(4ꢀFluorophenyl)ethyl)ꢀ4ꢀhydroxyꢀ2ꢀ(1Hꢀpyrazolꢀ1ꢀyl)pyrimidineꢀ5ꢀcarboxamide (20).
Compound 20 (0.092 g, 57.6%) was prepared in a manner similar to that described for compound 28,
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see below. H NMR (500 MHz, DMSOꢀd6) δ 9.76 (s, 1H), 8.63 (d, J = 2.8 Hz, 1H), 8.43 (s, 1H), 8.07
(d, J = 1.4 Hz, 1H), 7.45 – 7.34 (m, 2H), 7.30 – 7.09 (m, 2H), 6.73 (dd, J = 2.8, 1.6 Hz, 1H), 5.13 (p, J =
7.1 Hz, 1H), 1.47 (d, J = 7.0 Hz, 3H). LCMS m/z = 328.1 (M+H)+; Rt = 2.56 min
(R)ꢀNꢀ(1ꢀ(4ꢀFluorophenyl)ethyl)ꢀ4ꢀhydroxyꢀ2ꢀ(1Hꢀpyrazolꢀ1ꢀyl)pyrimidineꢀ5ꢀcarboxamide (21).
Compound 21 (0.092 g, 57.9%) was prepared in a manner similar to that described for compound 28,
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see below. H NMR (500 MHz, DMSOꢀd6) δ 9.75 (s, 1H), 8.63 (d, J = 2.7 Hz, 1H), 8.43 (s, 1H), 8.06
(d, J = 1.5 Hz, 1H), 7.53 – 7.28 (m, 2H), 7.17 (t, J = 8.8 Hz, 2H), 6.72 (t, J = 2.1 Hz, 1H), 5.12 (t, J =
7.2 Hz, 1H), 1.47 (d, J = 6.9 Hz, 3H). LCMS m/z = 328.1 (M+H)+; Rt = 2.56 min
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(S)ꢀ4ꢀHydroxyꢀNꢀmethylꢀNꢀ(1ꢀphenylethyl)ꢀ2ꢀ(1Hꢀpyrazolꢀ1ꢀyl)pyrimidineꢀ5ꢀcarboxamide (22).
Compound 22 (0.277 g, 70.6%) was prepared in a manner similar to that described for compound 28,
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see below. H NMR (600 MHz, DMSOꢀd6) δ 8.56 (d, J = 2.8 Hz, 0.5H), 8.54 (d, J = 2.8 Hz, 0.5H), 8.14
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(s, 0.5H), 8.12 (s, 0.5H), 7.93 (d, J = 1.6 Hz, 0.5H), 7.92 (d, J = 1.6 Hz, 0.5H), 7.39 – 7.31 (om, 4H),
7.26 (m, 1H), 6.63 (m, 1H), 5.85 (q, J = 7.1 Hz, 0.5H), 4.93 (q, J = 6.9 Hz, 0.5H), 2.58 (bs, 3H), 1.52 (d,
J = 7.1 Hz, 1.5H), 1.50 (d, J = 6.9 Hz, 1.5H). Note: Restricted amide bond rotation gives rise to two sets
of rotamers. Integrals of 0.5H for single protons and of 1.5H for methyl groups reflect that. Note 2:
0.5% TFA was added to improve NMR line shape. LCMS m/z = 324.0 (M+H)+; Rt = 2.51 min
(S)ꢀ4ꢀHydroxyꢀNꢀ(1ꢀ(4ꢀmethoxyphenyl)ethyl)ꢀ2ꢀ(1Hꢀpyrazolꢀ1ꢀyl)pyrimidineꢀ5ꢀcarboxamide
(23). Compound 23 (0.220 g, 66.4%) was prepared in a manner similar to that described for compound
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28, see below. H NMR (500 MHz, DMSOꢀd6) δ 8.63 (d, J = 2.8 Hz, 1H), 8.43 (s, 1H), 8.06 (d, J = 1.6
Hz, 1H), 7.32 – 7.25 (m, 2H), 6.95 – 6.86 (m, 2H), 6.72 (dd, J = 2.8, 1.6 Hz, 1H), 5.07 (p, J = 7.0 Hz,
1H), 3.73 (s, 3H), 1.45 (d, J = 6.9 Hz, 3H). LCMS m/z = 340.1 (M+H)+; Rt = 2.64 min
Nꢀ([1,1'ꢀBiphenyl]ꢀ4ꢀylmethyl)ꢀ4ꢀhydroxyꢀ2ꢀ(1Hꢀpyrazolꢀ1ꢀyl)pyrimidineꢀ5ꢀcarboxamide (24).
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Compound 24 (0.088 g, 26.3%) was prepared in a manner similar to that described for compound 4. H
NMR (500 MHz, DMSOꢀd6) δ 9.79 (s, 1H), 8.49 (s, 1H), 8.07 (d, J = 1.5 Hz, 1H), 7.70 – 7.57 (m, 4H),
7.52 – 7.39 (m, 4H), 7.38 – 7.29 (m, 1H), 6.73 (dd, J = 2.8, 1.6 Hz, 1H), 4.58 (d, J = 6.0 Hz, 2H).
LCMS m/z = 372.1 (M+H)+; Rt = 3.01 min
NꢀBenzhydrylꢀ4ꢀhydroxyꢀ2ꢀ(1Hꢀpyrazolꢀ1ꢀyl)pyrimidineꢀ5ꢀcarboxamide (25). Compound 25
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(0.194 g, 77.0%) was prepared in a manner similar to that described for compound 4. H NMR (500
MHz, DMSOꢀd6) δ 8.64 (d, J = 2.8 Hz, 1H), 8.08 (d, J = 1.5 Hz, 1H), 7.52 – 7.13 (m, 11H), 6.74 (dd, J
= 2.8, 1.6 Hz, 1H), 6.28 (d, J = 8.1 Hz, 1H). LCMS m/z = 372.2 (M+H)+; Rt = 2.93 min
NꢀBenzhydrylꢀ4ꢀhydroxyꢀ2ꢀ(1Hꢀpyrazolꢀ1ꢀyl)pyrimidineꢀ5ꢀcarboxamide (26). Compound 26
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(0.108 g, 34.3%) was prepared in a manner similar to that described for compound 28, see below. H
NMR (500 MHz, DMSOꢀd6) δ 10.24 (s, 1H), 8.62 (d, J = 2.8 Hz, 1H), 8.44 (s, 1H), 8.06 (d, J = 1.6 Hz,
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Journal of Medicinal Chemistry
1H), 7.22 – 7.14 (m, 4H), 6.95 – 6.86 (m, 4H), 6.72 (dd, J = 2.8, 1.6 Hz, 1H), 6.17 (d, J = 8.1 Hz, 1H),
3.73 (s, 6H). LCMS m/z = 431.9 (M+H)+; Rt = 3.07 min
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NꢀBenzhydrylꢀ4ꢀhydroxyꢀ2ꢀ(pyridazinꢀ3ꢀyl)pyrimidineꢀ5ꢀcarboxamide (27). Compound 27 (0.074
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g, 50.1%) was prepared in a manner similar to that described for compound 28, see below. H NMR
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(500 MHz, DMSOꢀd6) δ 10.93 (s, 1H), 9.43 (dd, J = 5.0, 1.7 Hz, 1H), 8.64 (s, 1H), 8.46 (dd, J = 8.6, 1.7
Hz, 1H), 7.94 (dd, J = 8.5, 5.0 Hz, 1H), 7.47 – 7.30 (m, 8H), 7.31 – 7.19 (m, 2H), 6.30 (d, J = 8.1 Hz,
1H). LCMS m/z = 384.2 (M+H)+; Rt = 2.79 min
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The preferred approach toward the general preparation of 4ꢀhydroxyꢀ2ꢀ(heterocyclic)pyrimidineꢀ5ꢀ
carboxamides is described with the procedure for the synthesis of compound 28, exemplified below.
Nꢀ[Bis(4ꢀmethoxyphenyl)methyl]ꢀ4ꢀhydroxyꢀ2ꢀ(pyridazinꢀ3ꢀyl)pyrimidineꢀ5ꢀcarboxamide (28).
To a suspension of 4ꢀhydroxyꢀ2ꢀpyridazinꢀ3ꢀylpyrimidineꢀ5ꢀcarboxylic acid 33 (1.02 g, 4.68 mmol) in
NMP (5 mL) was added TEA (1.303 mL, 9.35 mmol). The reaction was aged for 10 min at RT before
adding CDI (0.834 g, 5.14 mmol). The reaction was heated at 70°C for 1 h followed by the addition of
4,4'ꢀdimethoxybenzhydrlamine (1.133 g, 4.68 mmol) in NMP (2.0 mL). The reaction was aged 30 min
and then cooled to RT. The reaction was diluted with water (12.0 mL) and washed with IPAc (5 mL) 2
times. The aqueous layer (pH ~10) was then slowly neutralized with 5 N HCl (2.7 mL) to a final pH of
7.0ꢀ7.5. Product began to crystallize from solution druing the HCl addition. Control of the pH is imꢀ
portant to reject residual 33. Care should be taken not to lower the pH below 7 so that 33 does not crysꢀ
tallize from solution. The mixture was filtered and then washed with 2 bed volumes of water and 2 bed
volumes of MTBE. The solid was dried under vacuum/N2 sweep at RT for 2 h and then in a vacuum at
50 °C under a sweep of nitrogen for 12 h to afford the titled compound (1.81 g , 87%, 99.6 LCAP).
HPLC Conditions: Zorbax Eclipse Plus C18 50 x 4.6 mm, 1.8 um, 1.5 mL/min, 210 nm, 25 °C, Eluꢀ
ents: Water 0.1% H3PO4 (A), Acetonitrile (B). 90% A 0 min, 5% A 5 min, 5% A 6 min, 90% A 8 min.
1H NMR (600 MHz, CDCl3): δ 9.26 (dd, J = 5.0, 1.7 Hz, 1H), 8.85 (bs, 1H), 8.57 (dd, J = 8.5, 1.7 Hz,
1H), 7.75 (dd, J = 8.6, 5.0 Hz, 1H), 7.15 (d, J = 8.6 Hz, 4H), 6.77 (d, J = 8.8 Hz, 4H), 6.22 (s, 1H), 3.69
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(s, 6H). Note: 10% CD3OD and 0.5% TFA were added to improve NMR line shape. HPLC/MS:
444.18 (M+H)+; Rt = 2.90 min.
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2ꢀ[(4ꢀMethylphenyl)sulfonyl]ꢀ2,3ꢀdihydropyridazineꢀ3ꢀcarbonitrile (30). To pyridazine (264 mL,
3646 mmol), DCM (3 L) and trimethylsilyl cyanide (814 mL, 6068 mmol) was added aluminum chloꢀ
ride (1.00 g, 7.50 mmol). The reaction was aged at RT for 45 minutes before addition of pꢀ
toluenesulfonyl chloride (1230 g, 6452 mmol) in DCM (5.00 L). The reaction was aged at RT for a total
of 65 h. The reaction was washed with saturated NaHCO3 (10 L, aq.), back extracting the aqueous porꢀ
tion with DCM. The combined organic portion was washed with NaHCO3 (10 L, aq.). The organic porꢀ
tion was concentrated almost to dryness before adding EtOH (3.00 L). The organic portion was concenꢀ
trated almost to dryness again before adding EtOH (3.00 L). The mixture was partially concentrated
and filtered washing with EtOH until the filtrate was colorless. The material was air dried for 1 hr prior
to trituration with heptane. The product was dried overnight under vacuum affording the title compound
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as a white solid (810.2 g, 85 %). H NMR (500 MHz, DMSOꢀd6) δ 8.04 ꢀ 7.83 (m, 1H), 7.68 (dd, J =
3.5, 1.7 Hz, 2H), 7.57 (d, J = 7.8 Hz, 2H), 6.52 (ddt, J = 9.1, 7.0, 1.9 Hz, 1H), 6.40 (dd, J = 9.2, 3.4 Hz,
1H), 6.25 (d, J = 6.7 Hz, 1H), 2.66 ꢀ 2.53 (m, 3H). HPLC/MS: 262.1 (M+H)+; Rt = 2.51 min.
Pyridazineꢀ3ꢀcarbonitrile
(31).
To
2ꢀ[(4ꢀmethylphenyl)sulfonyl]ꢀ2,3ꢀdihydropyridazineꢀ3ꢀ
carbonitrile 30 (807.7 g, 3091 mmol) in THF (4.00 L) was added DBU (520 mL, 3450 mmol) while
maintaining the temperature between 18ꢀ25 °C. The reaction was aged at RT under a nitrogen atmosꢀ
phere for 2 h. The reaction was combined with EtOAc (2L), saturated NH4Cl (4.00 L, aq.) and water
(4.00 L). The layers were separated and the aqueous portion was attempted to be extracted with DCM
(2L); but solids were present. An additional portion of EtOAc (4 L) was added and the layers were sepꢀ
arated. The aqueous portion was extracted with EtOAc 4 times and the combined organic portions were
concentrated to a solid. This material was dissolved in EtOAc and washed with brine. The combined
aqueous portions were extracted with EtOAc 3 times and DCM. The combined extracts were dried
over Na2SO4, filtered and concentrated. The solid was dissolved in DCM and passed through a 1.8 kg
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Journal of Medicinal Chemistry
silica gel column eluting with a gradient of 5ꢀ50% EtOAc in DCM affording the product as a solid (314
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2
3
1
g, 97%). H NMR (500 MHz, DMSOꢀd6) δ 9.72 – 9.29 (m, 1H), 8.38 (ddd, J = 8.4, 2.6, 1.3 Hz, 1H),
4
5
6
8.01 (ddd, J = 8.5, 5.2, 1.2 Hz, 1H). HPLC/MS: 106.2 (M+H)+; Rt = 0.38 min.
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Amino(pyridazinꢀ3ꢀyl)methaniminium chloride (32). To pyridazineꢀ3ꢀcarbonitrile 31 (313.8 g,
2986 mmol) in MeOH (2.00 L) was added sodium methoxide (68.3 mL, 299 mmol, 25 wt% in MeOH).
The reaction was stirred at RT overnight and then diluted with ammonium chloride (176 g, 3284 mmol).
The reaction was refluxed for 5 h and cooled to RT overnight. The reaction was partially concentrated
and diluted with EtOAc, filtered, washed with EtOAc and heptane to afford the title compound as a solꢀ
id (491 g, 104 % yield) that was used in the next step without additional processing. 1H NMR (500
MHz, DMSOꢀd6) δ 9.88 (s, 4H), 9.56 (dt, J = 4.2, 2.0 Hz, 1H), 8.60 (dt, J = 8.6, 2.2 Hz, 1H), 8.09 (ddd,
J = 7.7, 5.1, 2.6 Hz, 1H), 3.34 (d, J = 2.7 Hz, 1H).HPLC/MS: 123.1 (M+H)+; Rt = 0.34 min.
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4ꢀHydroxyꢀ2ꢀpyridazinꢀ3ꢀylpyrimidineꢀ5ꢀcarboxylic
acid
(33).
To
amino(pyridazinꢀ3ꢀ
yl)methaniminium chloride 32 (490.6 g, 3094 mmol) in EtOH (9.00 L) was added sodium ethoxide (222
g, 3094 mmol, 95 %) followed by diethyl ethoxymethylenemalonate (940 mL, 4652 mmol). The reacꢀ
tion mixture was heated to reflux for about 28 h. Potassium hydroxide (605 g, 9281 mmol, 86%) in waꢀ
ter (5 L) was added and the reaction was refluxed for 6 h. After cooling with an ice bath, a solid precipiꢀ
tated. The reaction was diluted with water until the solids dissolved. The reaction was acidified to pH 2
using conc. HCl and aged overnight. The mixture was filtered, and washed with water 2 times. The solꢀ
id was treated with water and mechanically stirred and then filtered and washed with EtOH and trituratꢀ
ed with heptane 3 times. The solid was dried in a vacuum oven at 40 °C overnight affording the titled
compound (505 g, 74.8% yield). 1H NMR (500 MHz, DMSOꢀd6) δ 9.53 (dd, J = 5.1, 1.6 Hz, 1H), 8.60
(s, 1H), 8.55 (dd, J = 8.5, 1.7 Hz, 1H), 8.04 (dd, J = 8.6, 5.0 Hz, 1H). HPLC/MS: 219.0 (M+H)+; Rt =
0.28 min (basic method).
In Vitro Assay for HIFꢀPHD. The catalytic activity assays for the HIFꢀPHD isoforms were performed
at subsaturating levels of 2ꢀoxoglutarate. To each well of a 96ꢀwell plate was added 1 ꢁL of test comꢀ
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Page 28 of 36
pound in DMSO and 20 ꢁL of assay buffer (50 mM Tris pH 7.4/0.01% Tweenꢀ20/0.1 mg/mL bovine
serum albumin/10 ꢁM ferrous sulfate/1 mM sodium ascorbate/20 ꢁg/mL catalase) containing 0.15
ꢁg/mL FLAGꢀtagged full length HIFꢀPHD isoform expressed in and purified from baculovirusꢀinfected
Sf9 cells. After a 30 min preincubation at room temperature, the enzymatic reactions were initiated by
the addition of 4 ꢁL of substrates (reaction concentrations of 0.2 ꢁM (PHD1/PHD2) or 4 ꢁM (PHD3) 2ꢀ
oxoglutarate and 0.5 ꢁM HIFꢀ1α peptide biotinylꢀDLDLEMLAPYIPMDDDFQL). After 2 h at room
temperature, the reactions were terminated and signals were developed by the addition of a 25 ꢁL
quench/detection mix to a final concentration of 1 mM orthoꢀphenanthroline, 0.1 mM EDTA, 0.5 nM
antiꢀ(His)6 LANCE reagent (PerkinꢀElmer Life Sciences), 100 nM AF647ꢀlabeled streptavidin (Invitroꢀ
gen), and 2 ꢁg/mL (His)6−VHL complex18. The ratio of timeꢀresolved fluorescence signals at 665 and
620 nm was determined, and percent inhibition was calculated relative to an uninhibited control sample
run in parallel.
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In Vivo. All animal related procedures were conducted under a Merck IACUC approved protocol in
compliance with Animal Welfare Act regulations and the Guide for the Care and Use of Laboratory Anꢀ
imals.
Primary in vivo assay: MoPED (Mouse Plasma Erythropoietin Determination). Compounds are formuꢀ
lated 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.
Secondary in vivo assay: 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.
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Journal of Medicinal Chemistry
Mouse Assay (Reticulocytes only): Compound 28 is formulated in cyclodextrin. Mice (C57Bl/6, n =
10 per dose level) are dosed po with a volume of 0.2 mL of the compound solution. On days 3 and 4
postꢀchallenge, blood is obtained from the mice (5 on each day) via cardiac puncture upon euthanasia
with CO2. Blood is analyzed for reticulocytes by FACS. Compound pharmacokinetics are determined
in a parallel experiment in which C57BL/6J mice (n = 3 per dose level) are dosed po with 28 in cyꢀ
clodextrin. Results are compared to vehicle dosed controls and the assay quality is assessed by incluꢀ
sion of a positive control compound dosed at 15 mg/kg po.
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Rat Assay (EPO and Reticulocytes): Compound 28 is formulated in PEG200/water. Rats (Spragueꢀ
Dawley, n = 5 per dose level) are dosed po with 2.5 mL/kg of the dosing solution so as to administer
1.5, 5 and 15 mg/kg of compound. Blood samples are obtained by jugular venipuncture at 6, 24, 48, 72,
and 96 hours postꢀdose. Plasma is prepared from these samples by centrifugation and stored at ꢀ80° C.
Plasmas from the first 72 hours of sampling are assayed for EPO and VEGF by ELISA. Blood from the
72 and 96 hour samples are analyzed for reticulocytes by FACS. All plasmas are analyzed to determine
compound levels. EPO, reticulocyte and pharmacokinetic data are used to provide target Cmax and exꢀ
posure (AUCs) targets required to stimulate a pharmacodynamic response.
Four week QD po study with 28 in rat: Male SpragueꢀDawley rats (approximately 300g each, n =
15/arm) were dosed once daily for 28 days with vehicle (25:75 v/v PEG200/water + 1 molar equivalent
of NaOH) or 28 (1.5 or 15 mg/kg in vehicle). A group of ageꢀmatched, untreated controls (n = 15) were
included in the experiment. On study days ꢀ3, 14 and 28 blood samples (~0.25 mL) were obtained via
jugular venipuncture and on study day 36 by cardiocentesis for hematological and compound level analꢀ
yses.
General Analytical Methods: Epo/VEGF; ELISA: Measurements are performed using serum or
plasma with the MesoScale Discovery Mouse/Rat Hypoxia Serum/Plasma Kit, cat# K11123C as per the
manufacturer's instructions.
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