Identification of Morpholino-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-ones as Nonsteroidal Mineralocorticoid Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.7b01515

A novel series of morpholine-based nonsteroidal mineralocorticoid receptor antagonists is reported. Starting from a pyrrolidine HTS hit 9 that possessed modest potency but excellect selectivity versus related nuclear hormone receptors, a series of libraries led to identification of morpholine lead 10. After further optimization, cis disubstituted morpholine 22 was discovered, which showed a 45-fold boost in binding affinity and corresponding functional potency compared to 13. While 22 had high clearance in rat, it provided sufficient exposure at high doses to favorably assess in vivo efficacy (increased urinary Na⁺/K⁺ ratio) and safety. In contrast to rat, the dog and human MetID and PK profiles of 22 were adequate, suggesting that it could be suitable as a potential clinical asset.

Full text of DOI:10.1021/acs.jmedchem.7b01515

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Article
Identification of Morpholino-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-
ones as Non-steroidal Mineralocorticoid Antagonists
David W. Piotrowski, Kentaro Futatsugi, Agustin Casimiro-Garcia, Liuqing Wei, Matthew
Sammons, Michael Herr, Wenhua Jiao, Sophie Y Lavergne, Steven B. Coffey, Stephen W Wright,
Kun Song, Paula M. Loria, Mary Ellen Banker, Donna N. Petersen, and Jonathan Bauman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01515 • Publication Date (Web): 04 Jan 2018
Downloaded from http://pubs.acs.org on January 4, 2018
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Identification of Morpholinoꢀ2Hꢀpyrido[3,2ꢀb]
[1,4]oxazinꢀ3(4H)ꢀones as Nonꢀsteroidal
Mineralocorticoid Antagonists
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David W. Piotrowski,* Kentaro Futatsugi, Augstin CasimiroꢀGarcia, Liuqing Wei, Matthew F.
Sammons, Michael Herr, Wenhua Jiao, Sophie Y. Lavergne, Steven B. Coffey, Stephen W.
Wright, Kun Song, Paula M. Loria, Mary Ellen Banker, Donna N. Petersen, Jonathan Bauman
Pfizer Research and Development, Groton, Connecticut 06340, United States
KEYWORDS: nuclear hormone receptor, mineralocorticoid, nonꢀsteroidal
Abstract: A novel series of morpholineꢀbased nonꢀsteroidal mineralocorticoid receptor
antagonists is reported. Starting from a pyrrolidine HTS hit 9 that possessed modest potency but
excellect selectivity versus related nuclear hormone receptors, a series of libraries led to
identification of morpholine lead 10. After further optimization, cis disubstituted morpholine 22
was discovered, which showed a 45ꢀfold boost in binding affinity and corresponding functional
potency compared to 13. While 22 had high clearance in rat, it provided sufficient exposure at
high doses to favorably assess in vivo efficacy (increased urinary Na+/K+ ratio) and safety. In
contrast to rat, the dog and human MetID and PK profiles of 22 were adequate, suggesting that it
could be suitable as a potential clinical asset.
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Introduction
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The steroid hormone, aldosterone, discovered over 50 years ago, is the primary endogenous
agonist for the mineralocorticoid receptor (MR). This receptor is a ligandꢀdependent
transcription factor that belongs to the nuclear hormone receptor (NHR) superꢀfamily and is a
regulator of sodium reabsorption in the kidney.1 MR shares structural similarities with other
NHRs that also recognize steroidal ligands including the progesterone receptor (PR), androgen
receptor (AR), glucocorticoid receptor (GR), and estrogen receptor (ER). In the disease state,
excessive levels of aldosterone activate the MR contributing to conditions such as congestive
heart failure, hypertension and chronic kidney disease. Intervention with MR antagonists
represents an attractive therapeutic option for the treatment of such diseases.2 However,
treatment with MR antagonists can also lead to elevation of circulating aldosterone levels, which
can trigger subsequent genomic and nonꢀgenomic effects.3 Nonetheless, the steroidal MR
antagonists, spironolactone (1) and eplerenone (2)4 (Figure 1), have proven beneficial in
reducing the risk of death and hospitalization in patients with severe heart failure5,6,7 and aid in
blood pressure control and antiproteinuric effects in patients with diabetic nephropathy.8,9,10,11
However, the broader use of these agents has been limited by their sex hormone related adverse
effects. Compound 1 is associated with impotence, gynecomastia, and menstrual irregularities.
These effects are commonly attributed to the low selectivity against other NHRs such as AR and
PR.12 Both of these steroidal agents are contraindicated for use in diabetic patients because of
the potential risk of hyperkalemia. The accumulated body of data suggests that the limitations
associated with steroidal agents can likely be mitigated with nonꢀsteroidal MR inhibitors.13
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Figure 1. Marketed Steroidal MR Antagonist Drugs
Several selective nonꢀsteroidal agents have been or are currently being evaluated in human
clinical trials (Figure 2). Preclinical and clinical data from a few of these MR antagonists
suggests that mitigation of the risk of hyperkalemia might be possible. Among these compounds
are finerenone (3),14 esaxerenone (4)15 and apararenone (5).16 Compound 3 is the most advanced
in clinical trials.17,18 The first report of phase 2 study results showed that 3 had a reduction in
hyperkalaemia and deterioration of renal function versus treatment with 1. Biomarker data from
this study showed a lower incidence of side effects that did not come at a cost of lower efficacy.
This may, in part, be explained by differences in tissue distribution that could lead to reduction
of observed hyperkalaemia: 3 distributes equally to kidney and heart in rats,19 but 1 had at least
sixꢀfold higher concentration in kidney relative to cardiac tissue.20,21 Taken together, these
clinical results support the continued search for alternative structural classes of nonꢀsteroidal MR
antagonists that have improved profiles.
Figure 2. MR Antagonists, Phase 2 and Beyond
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Previous reports from Pfizer have disclosed novel nonꢀsteroidal MR antagonists,22 including
the pyrazolines PFꢀ03882845 (6) and 7 and the sulfonamide 8 (Figure 3). Of note, the
conformationally restricted 6 was found to be a potent and selective antagonist with favorable
pharmacokinetic profile that allowed for advancement to phase 1 clinical trials. However, 6 and
related pyrazolines had the drawback of low solubility and empirically derived selectivity
profiles.22e,f
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Figure 3. Pfizer MR Antagonists
Results and Discussion
The evaluation of the physicochemical property space, potency and selectivity data from all
known MR antagonists at the time this work was conducted23 suggested that 2 is an outlier (MW
414.5, logD 0.5, ~50% plasma protein binding) and could be used as an aspirational guide for
discovery of potent and more selective compounds with modest dose requirements. Furthermore,
knowledge gained from the coꢀcrystal structure of the marginally selective 124 showed that three
main regions of interaction with MR exist: (1) Hꢀbonding of the C3ꢀketo group with Gln776
from H3 helix and Arg817 from H5 helix of MR, (2) occupancy of a lipophilic pocket which is
lined with several lipophilic residues including Leu814, Leu938, Met845, Met 852, Cys849,
Phe829 and Phe835, by the C7 thioacetyl group, (3) Hꢀbonding of Thr945 or alternatively
Asn770 to the lactone carbonyl (Figure 4). Furthermore, a variety of NHR modulators (agonists
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or antagonists) make equivalent polar interactions in the region occupied by the A ring ketone of
steroids (Figure 4, region 1). For example, in coꢀcrystal structures both the 3ꢀketone of
progesterone25 or the nitrile of tanaproget26 show interactions with Gln725/Arg766 (PR
numbering) and the 3ꢀketone of testosterone27 bound to AR shows interactions with
Gln711/Arg752 (AR numbering). In principle, truncated analogs that are unable to make the
polar interactions in region 1 and have enhanced interactions in regions 2 and 3 could lead to MR
selective compounds. Thus, potency improvements could come from enhanced lipophilic
interactions in region 2 while region 3 would serve to lock the ligand in place through Hꢀbonding
to Asn770 (Figure 5). Mindful of this possible binding mode, the discovery of a nonꢀsteroidal
MR chemotype that had predictable selectivity over other NHRs was targeted. An in vitro
screening cascade identical to that used for discovery of the previous Pfizer clinical candidate 6
and its backꢀup 7 was implemented to facilitate identification of a new orally bioavailable
compound suitable for use in preclinical studies and beyond.22e,f New analogs were assessed in a
panel of functional Gal4ꢀbased cellular assays to assess the transcriptional antagonist activity
against MR, PR, GR and AR. Selected analogs were subsequently assessed for their ability to
inhibit [3H]ꢀaldosterone in a lowerꢀthroughput MR filter binding assay.22d,f
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Figure 4. Crystal structure of 1 (2ab2) with key areas of interaction
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Figure 5. Depiction of the hypothetical binding mode of a generalized structure related to hit 9
A suitable starting point for the aforementioned truncated series was generated by
identification of a 2ꢀarylpyrrolidine high throughput screening (HTS) hit 9 that was further
refined with targeted libraries. Cyclic amines with small lipophilic groups alpha to the nitrogen
were combined with (het)aryls containing a Hꢀbond donor/acceptor motif capable of interacting
with Asn770. Libraryꢀenabled chemistry suited for such CꢀN bond forming reactions included
BuchwaldꢀHartwig and ChanꢀLam couplings (Scheme 1). The libraries produced pyrrolidine and
morpholine hits such as I and II (data not shown) and highlighted the importance of a
benzo/pyridoꢀfused oxazinone moiety. Morpholine lead 10 was identified by this process (Table
1).
Scheme 1. Exploration of 9 by Parallel Chemistry
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Conditions for Buchwald-Hartwig and Chan-Lam libraries: a) (het)aryl bromide, cyclic amine,
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NaOtBu, Pd2dba3, BINAP, DMSO, 60 oC b) arylboronic acid, CH2Cl2, pyridine, Cu(OAc)2.
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MR potency, selectivity, MW, eLogD and topological polar surface area (TPSA) indicated
that 10 was a suitable lead (Table 1). In addition, the ADME (permeability, human liver
microsomal (HLM) stability) and in vitro safety ([3H]ꢀdofetilide binding, cytochrome P450
(CYP) inhibition) assessments were adequate. However, the high intrinsic clearance in rat liver
microsomes (RLM) highlighted an area for improvement to allow for potential selection of a
representative analog for in vivo rat studies.
Table 1. In vitro Pharmacological, Absorption, Metabolism, and Safety Properties of 10
10b
MW 310.4, elogD 2.7, TPSA 51
MR functional IC50 (nM)
MR binding IC50 (nM)
PR functional IC50 (nM)
GR functional IC50 (nM)
AR functional IC50 (nM)
RRCK (Papp, 10ꢀ6 cm/sec)a
RLM Clint (ꢁL/min/mg)
HLM Clint (ꢁL/min/mg)
583
401
>10000
>10000
>10000
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CYP (pct inh at 3 ꢁM)
[3H]ꢀdofetilide (pct inh at 10 ꢁM)
1A2, 2D6, 2C9, 3A4 < 10 %
5 %
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aRRCK = Modified MadinꢀDarby Permeability Assay. The corresponding (S)ꢀenantiomer had
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an order of magnitude lower affinity and functional activity and was not pursued further.
With lead 10 in hand, we targeted specific modifications to three regions for optimization:
(1) addition of small lipophilic substituents to the morpholine to fill the space occupied by C1 of
the steroid Aꢀring and/or the C19 methyl of 1, (2) insertion of Nꢀatoms into the electron rich
benzoxazinone moiety to adjust pKa and further reduce logD, and (3) addition of small lipophilic
substituents to pendant phenyl ring to optimize nonꢀpolar interactions.
For analog work, a modular approach was desired so that the synthesis of more complex
morpholine and halo benzo/pyridoꢀoxazinone fragments could be addressed separately. Despite
the successful use of the BuchwaldꢀHartwig conditions for the libraries, further optimization was
required for application of this methodology to singleton synthesis. A number of ligand/base
combinations were screened to identify suitable conditions for broader use. Of note was the use
of the electronꢀrich phosphine ligand iPrBiPPyPhos, along with LiOtBu as base and HMPA as an
additive. The latter two reagents were critical to keep the reactants in solution. For example, the
unoptimized 8% yield (condition a, Scheme 2) for the coupling of morpholine 11 and bromide 12
was improved under these optimized conditions to provide an 86% yield of 13 (condition b,
Scheme 2). With these improved CꢀN bond forming conditions in hand, attention was turned
toward an efficient morpholine synthesis.
Scheme 2. Optimization of CꢀN Coupling
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Reagents and conditions: (a) 2.5 mol% Pd2(dba)3, 5 mol% BiPPyPhos, 2 equiv. KOtBu,
o
tAmylOH, 100 C, 8%. (b) 2.5 mol% Pd2(dba)3, 5 mol% iPrꢀBiPPyPhos, 6 equiv. LiOtBu, 5
equiv HMPA, tAmylOH, 60 oC, 86%.
Several routes to access morpholinones and morpholines have been reported and were used
to synthesize analogs to explore the early SAR.28 Route improvements were made, as we have
reported previously (Scheme 3),29 which allowed for installation of the defined C5 stereocenter
from commercially available (R)ꢀ2ꢀphenylglycinols 14, which could be acylated to provide
amide 15. Base promoted intramolecular Williamson ether synthesis on amide 15 provided
lactam intermediate 16 that could be separated (cis/trans isomers) and further manipulated as
required to provide morpholines with a range of C2 substituents. For example, the use of racemic
2ꢀchloropropanoyl chloride for the acylation of (R)ꢀ2ꢀphenylglycinol and cyclization provided a
mixture of morpholinone diastereomers 16 that could be controlled by judicious choice of
conditions.29 Reduction of the morpholinone provided the required morpholines 17. The trans
morpholine isomer 18a could be accessed from morpholinone 16 (R1 = H) by an Nꢀprotection,
methylation, deprotection and reduction sequence. The gemꢀdimethyl morpholine 18b could be
accessed using a similar sequence with the addition of a second methylation step (Scheme 3).
The final analogs 22ꢀ24 and 28ꢀ34 were synthesized from morpholines 17aꢀd and 18 a,b by the
previously described CꢀN coupling procedures (vide supra).
Scheme 3. Synthesis of Morpholine Intermediates
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Reagents and conditions: a) Et3N, 2ꢀMeTHF, b) KOtBu, tBuOH, c) LiAlH4 or Vitride®, toluene,
d) pꢀmethoxybenzyl chloride, NaH, DMF, e) LDA, CH3I, THF, f) CAN, aq. CH3CN
Compounds 25ꢀ27, containing elaborated substituents at the 2ꢀposition of the morpholines,
were synthesized starting from known morpholine intermediate 19.30 The hydroxymethyl group
of 19 was manipulated either before or after the CꢀN coupling procedure (Scheme 4).
Scheme 4. Synthesis 2ꢀElaborated Morpholine Analogs
Reagents and conditions: a) 10% Pd/C, MeOH, H2/50 psi, b) CH3I, NaH, DMF, c) general CꢀN
coupling procedure from Scheme 2, d) Et3N, 1,2ꢀdichloroethane, (CH3SO2)2O, e) NaCN, DMF,
120 oC.
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Table 2. Potency and Properties of Selected Analogs
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MR
MR binding
IC50 (nM)a
HLM
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H
R2
H
R3
H
core
functional
IC50 (nM)a
eLogDc
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Clint
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CH3
H
H
CH3
CH3
H
H
H
H
H
H
H
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727
44
1217
407
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24.3
15.2
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8.0
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2.7
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2.3
2.5
1.8
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27
A
A
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A
A
A
CH3
86
CH2CN
CH2OCH3
CH2OH
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98
H
276
358
H
>10000
>10000
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CH3
CH3
CH3
H
H
H
H
H
H
109
2225
113
252
2992
266
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13.9
8.0
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2.7
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CH3
CH3
CH3
H
H
H
2ꢀF
3ꢀF
4ꢀF
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19
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14.0
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3.1
3.2
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8.0
aMost values are reflective of n ≥ 3 experiments. Please see supporting information for the
b
c
number of experiments and associated error. intrinsic clearance ꢀL/min/mg. measured
eLogD.31
We hypothesized that addition of substituents to the morpholine C2 position could increase
potency through improved lipophilic interactions. We therefore prepared the cisꢀ22 and transꢀ23
monoꢀmethyl analogs as well as dimethyl analog 24. Of significant note was the 45ꢀfold boost in
binding affinity of cisꢀ22 versus its unsubstituted counterpart 13, while the dimethyl 24 and
transꢀ23 analogs had more modest affinity increases. The greater than 3ꢀfold boost in binding
affinity that might be expected from the increased lipophilicity suggested that the conformation
of cisꢀ2ꢀmethylꢀ5ꢀphenylmorpholine enforced burial of both the methyl and phenyl groups in
hydrophobic regions of MR; a soꢀcalled “magic methyl” effect.32,33 This supposition was
supported with computational assessments using ConfGen34 and Jaguar35: cisꢀ22 revealed a
strong axial phenyl preference (>5 kcal/mol), while transꢀ23 preferred the equatorial phenyl (1.6
kcal/mol) over the axial phenyl conformer.36 Furthermore, a small molecule crystal structure of
22 was obtained. The structure clearly displays the axial 5ꢀphenyl group and the equatorial 2ꢀ
methyl group on the morpholine.37 Thus, subsequent analogs incorporated the cisꢀ2,5ꢀ
disubstituted morpholine moiety as a key feature. Attempts to independently increase or decrease
bulk at the 2ꢀ or 5ꢀpositions of the morpholine led to compounds with reduced MR binding
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affinity and/or increased HLM Clint (data not shown). Some small substituents (CN 25, OMe 26)
were tolerated on the C2 methyl but increased polarity (OH 27) led to a reduction in binding
affinity. However, the additional synthetic complexity introduced by these changes did not
warrant further exploration of these analogs.
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Next, we examined a number of core changes. The other pyridooxazinone isomers 28 and 29
and pyrimidinooxazinone 30 led to a 6ꢀ to 68ꢀfold reduction in functional potency. The
benzoxazinone 31 was made for comparative purposes. While 31 was slightly more potent than
22, the electronꢀrich benzoxazinone moiety presented concerns about possible lateꢀstage
idiosyncratic toxicity38 and minimal profiling was conducted on this compound. Other changes
to the benzo/pyridooxazine core were tolerated (small substituents on aromatic or aliphatic
portion, replacement of the ether oxygen with carbon or other heteroatoms) but lactam Nꢀ
methylation essentially abolished binding affinity (data not shown). Again, the additional level of
synthetic complexity combined with the lack of significant improvement precluded these analogs
from further consideration.
Finally, using SAR data generated from the library compounds depicted in Scheme 1, a select
number of substituted 5ꢀaryl groups were examined. For illustrative purposes, the data for oꢀF
32, mꢀF 33, pꢀF 34 substituted analogs are shown in Table 2. Nearly equivalent functional
potency was noted for these substituted analogs versus their unsubstituted counterpart 22.
Similar trends were noted for other monoꢀsubstituted and some diꢀsubstituted analogs (data not
shown).39 Taken together, 22 emerged as the compound with best balance of potency, selectivity
and ADME properties.
We had previously established22e,f that a few different rodent models (Kagawa assay, blood
pressure) could be used to support translation of in vitro potency to in vivo efficacy. Several
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compounds in Table 2 were potent and selective, of modest elogD and had low intrinsic
clearance in HLM but nevertheless suffered from high intrinsic clearance in RLM (selected data
shown in Table 3). To better understand this disparity and explore a possible in vitroꢀin vivo PK
correlation in rat, a group of analogs that spanned a range of elogD and RLM Clint were selected
for iv rat PK assessment regardless of their potency. In general, the rat PK profile of these
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compounds was characterized by highꢀtoꢀveryꢀhigh clearance, short t , and moderateꢀtoꢀhigh
½
volume of distribution. In order to further understand the high RLM Clint findings, 22 was
subjected to a metabolite identification (MetID) study using RLM, dog liver microsomes (DLM)
and HLM. The results indicated a facile oxidation of the morpholine ring was the primary
contributor to microsomal instability in rodents, while unchanged parent remained for DLM and
HLM.40 These data strongly suggested that the metabolic liabilities in rat would limit our ability
to use 22 in many of the established in vivo models. The favorable MetID study in DLM
prompted a dog pharmacokinetics study, where 22 exhibited moderate clearance, moderate
volume of distribution, long half life and good bioavailability. Thus, there was a solid in vitroꢀin
vivo PK correlation in dog. The similar MetID and microsomal stability profiles between dog
and human suggested that the dog PK parameters were best suited for use in human PK
projections.
Table 3. RLM Clint and Rat Cl for Selected Analogs
a
Cmpd eLogD RLM Clint Rat Clb
3.1
2.8
2.7
2.7
377
287
54
141
95
31
22
30
23
146
>564
77
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1.8
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52
27
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aꢀL/min/mg; bmL/min/kg after 1 mg/kg iv dose
Before commencing with the rat toxicology study, 22 was further assessed in an advanced
battery of in vitro assays (Table 4). Of note was the high selectivity over CYP enzymes,
selectivity over a wide panel of enzymes, receptors and ion channels and high selectivity over
most other NHRs.
9
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22
23
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Table 4. Pharmacological, ADME and Safety Properties of 22
MW
325.4
2.80
64
eLogD
TPSA (Å2)
hFunctional
IC50 (nM)a
44b
hBinding
IC50 (nM)a
25c
MR
AR
GR
PR
>10000
>10000
>10000
n.d.d
>10000
>10000
>10000
>10000
ERα
microsomal intrinsic Clint (ꢁL/min/mg)
rat
287
dog
38.3
human (fu, mic 0.80) 6.9
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cytochrome P450 % inh. at 3 ꢁM
(1A2, 2C9, 2D6, 3A4) all < 20 %
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thermodynamic solubility
pH 6.5, phosphate buffered saline, 5.5 ꢁg/mL (17 ꢁM)
plasma protein binding (% free, fu)
rat
15.0
13.6
15.0
dog
human
permeability (Papp (cm/s))
RRCK
AB 45.9 × 10−6
AB 24.9 × 10−6
BA 24.7 × 10−6
MDR1ꢀMDCK
Genetox
Ames assay negative
hERG inhibition (patch clamp)
IC50 > 100 ꢁM
CEREP panel (81 assays)
IC50 > 10 ꢁM except GR binding in IMꢀ9 cells (IC50 = 597 nM)
in vivo pharmacokinetics
Cl (mL/min/kg)
Vss (L/kg)
t
1/2 (h)
F (%)
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rat
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6.15
2.37
2.37
0.65
17.7
4ꢀ16
65
dog
5.37
3.75
projected humane
40
aValues are means of >3 experiments ± standard deviation. bIC50 = 33 nM in a serum free version
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c
of the human MR assay and IC50 = 3.2 nM in the rat MR assay. Ki = 6.3 nM (derived from
ChengꢀPrusoff equation). dn.d. = not determined. eprojected human PK scaled from dog.
Working under the assumption that sufficient exposure would be obtained at high doses by
possible saturation of the clearance mechanisms, 22 was advanced into a 14ꢀday rat toxicology
study using both male and female rats with doses ranging from 30, 100, and 500 mg/kg.41 The
highlights from the study are briefly described. Based on AUC (ng h/mL), free drug exposures at
•
day 14 were between 79ꢀ3166x functional IC50 (serum free, rat MR) in male rats and 1227ꢀ8250x
IC50 in female rats.42 The expected compensatory increase in plasma aldosterone was noted at all
doses. No reproductive tract findings were observed in the female rats.43
The higher exposure in female rats in the 14ꢀday safety study provided the impetus to test 22
in an acute measure of urinary excretion of sodium and potassium.44 Female rats were randomly
assigned to treatment groups (n = 7/group) to receive a single oral dose of vehicle, 22 (30, 100,
or 500 mg/kg) or 2 (100 mg/kg), which was used as a positive control. Urine samples were
collected at intervals of 0ꢀ2, 2ꢀ4, and 4ꢀ6 h postꢀdose for measurement of urinary sodium and
potassium concentration. Treatment with 500 mg/kg of 22 and the positive control 2 resulted in a
statistically significant increase in urinary Na+/K+ ratio at 2ꢀ4 h and 4ꢀ6 h postꢀdose (Figure 6).
Average AUC concentrations at the doses of 30, 100 and 500 mg/kg were 2850, 9410 and 13500
ng•
h/mL, respectively. These results confirmed that 22 acted as a MR antagonist.
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Kagawa Index (4-6 hr)
1.5
1.0
0.5
0.0
*
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*P< 0.05 vs. vehicle
Figure 6: Na+/K+ ratio (Kagawa index) for oral administration of 22 to female rats at 30, 100 and
500 mg/kg doses.
After this work had been completed,45 the first Xꢀray coꢀcrystal structure of a nonꢀsteriodal
MR antagonist was disclosed.46,47 Working under the assumption that our small molecule Xꢀray
(ground state) structure approximates the bound conformation, the pyridooxazinone portion of 22
was overlaid with the benzoxazinone portion of the MR coꢀcrystal structure (3vhv, Figure 7).
Gratifyingly, the superimposed molecules show good overlap throughout the ligand binding
domain with the cisꢀoriented 2ꢀmethyl and 5ꢀphenyl moieties occupying key lipophilic pockets.48
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Asn770
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N
N
N
F
N
H
N
S
O
O
Compound 1d
from reference 46a
Figure 7: Overlay of 22 (magenta) with the coꢀcrystal structure 3vhv (orange, Compound 1d
from reference 46a) with hydrogen bonds to Asn770 noted.
Conclusions
In conclusion, a potent and orally available MR antagonist 22 was identified starting from
lead 10. The cis disubstituted morpholine 22 showed a 45ꢀfold boost in binding affinity and
commensurate functional potency versus the desꢀmethyl morpholine 13. While 22 had high
clearance in rat, it provided sufficient exposure at high doses to favorably assess in vivo efficacy
and safety. The dog and human MetID and PK profiles of 22 suggest that it could be suitable as a
potential clinical asset.
Experimental Section
General
All chemicals, reagents and solvents were purchased from commercial sources when
available and used without further purification. Nuclear magnetic resonance spectroscopy
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(NMR) was recorded at 400 MHz (1H) and 101 MHz (13C) on Varian spectrometers unless
otherwise noted. Chemical shifts are expressed in parts per million downfield from
tetramethylsilane. The peak shapes are denoted as follows: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; br s, broad singlet. Mass spectrometry (MS) was performed via
atmospheric pressure chemical ionization (APCI) or electron scatter (ES) ionization sources.
Silica gel chromatography was performed primarily using a medium pressure Biotage or ISCO
systems using columns preꢀpackaged by various commercial vendors including Biotage and
ISCO. Microanalyses were performed by Quantitative Technologies Inc. and were within 0.4%
of the calculated values. Purity of final compounds was assessed by reversedꢀphase HPLC with
UV detection at 215 nM; all tested compounds were >95% purity, unless otherwise noted. The
terms “concentrated” and “evaporated” refer to the removal of solvent at reduced pressure on a
rotary evaporator with a bath temperature less than 60 °C. The abbreviation “min” and “h” stand
for “minutes” and “hours” respectively.
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(R)ꢀ6ꢀ(3ꢀPhenylmorpholino)ꢀ2Hꢀbenzo[b][1,4]oxazinꢀ3(4H)ꢀone (10)
A
mixture
of
(R)ꢀ3ꢀphenylmorpholine
(292
mg,
1.8
mmol),
tris(dibenzylideneacetone)dipalladium (0) (1.5 mg, 0.018 mmol), 6ꢀbromoꢀ2Hꢀ
benzo[b][1,4]oxazinꢀ3(4H)ꢀone (342 mg, 1.5 mmol), 2ꢀ(2ꢀdicyclohexylphosphanylphenyl)ꢀN,Nꢀ
dimethylaniline (1.5 mg, 0.36 mmol), lithium bis(trimethylsilyl)amide (1 M solution in hexanes,
3.3 mL) and THF (6 mL) in was stirred at 70 °C overnight. The reaction mixture was diluted
with EtOAc and extracted with saturated aq. NH4Cl. The aqueous layer was extracted with
EtOAc. The combined organic layers were washed with saturated aq. NaCl, dried over MgSO4,
filtered, and concentrated. The crude material was purified by silica gel column chromatography
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(gradient: 0–80 % EtOAc/heptanes) to afford 75 mg (16%) of a light yellow solid. 1H NMR
(CDCl3) δ 7.60 (br s, 1H), 7.30–7.14 (m, 5H), 6.75 (d, J = 8.6 Hz, 1H), 6.60 (dd, J = 8.8, 2.5 Hz,
1H), 6.37 (d, J = 2.5 Hz, 1H), 4.51 (s, 2H), 4.15 (dd, J = 9.0, 3.5 Hz, 1H), 4.00–3.89 (m, 3H),
3.55 (dd, J = 11.5, 9.0 Hz, 1H), 3.32 (dt, J = 12.1, 2.7 Hz, 1H), 3.04 (ddd, J = 12.1, 9.3, 4.5 Hz,
1H).
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(R)ꢀ6ꢀ(3ꢀPhenylmorpholino)ꢀ2Hꢀpyrido[3,2ꢀb][1,4]oxazinꢀ3(4H)ꢀone (13)
Prepared from (R)ꢀ3ꢀphenylmorpholine and 6ꢀbromoꢀ2Hꢀpyrido[3,2ꢀb][1,4]oxazinꢀ3(4H)ꢀone
1
according to the general CꢀN coupling procedure to afford 125 mg (66%) of a solid. H NMR
(DMSO-d6) δ 10.86 (br s, 1H), 7.36–7.25 (m, 4H), 7.22–7.17 (m, 1H), 7.15 (d, J = 8.8 Hz, 1H),
6.20 (d, J = 8.8 Hz, 1H), 5.17 (t, J = 3.1 Hz, 1H), 4.47 (s, 2H), 4.16 (dd, J = 11.7, 2.0 Hz, 1H),
3.94 (dt, J = 10.7, 2.5 Hz, 1H), 3.87 (dd, J = 11.7, 3.7 Hz, 1H), 3.79 (dt, J = 13.1, 2.9 Hz, 1H),
3.63 (td, J = 11.1, 3.4 Hz, 1H), 3.37–3.29 (m, 2H).
(2R,5R)ꢀ2ꢀMethylꢀ5ꢀphenylmorpholine (17a).31 General procedure for the synthesis of cisꢀ
(2R,5R)ꢀ2ꢀmethylꢀ5ꢀarylmorpholines.
Step 1: A solution of 2ꢀchloroꢀNꢀ((R)ꢀ2ꢀhydroxyꢀ1ꢀphenylethyl)propanamide (US 7629338, 60
g, 260 mmol) in tꢀBuOH (540 mL) was added to a stirred suspension of KOtꢀBu (59.1 g, 527
mmol) in tꢀBuOH (920 mL) at rt. The reaction mixture was stirred for 1 h. The pH of the
reaction mixture was adjusted to pH 4 by adding aq. HCl (1 N, 140 mL). The mixture was
concentrated to remove the tꢀBuOH. EtOAc (1000 mL) and H2O (500 mL) were added. After
the layers were separated, the organic layer was washed with saturated aq. NaCl (250 mL), dried
over Na2SO4, filtered and concentrated to provide a solid. The solid was completely dissolved in
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hot heptanes/EtOAc. The product precipitated upon cooling to rt overnight. The solid was
filtered and dried to yield 33.75 g (67%). 1H NMR (CDCl3) δ 7.42–7.29 (m, 5H), 6.75 (br s, 1H),
4.61 (q, J = 3.7 Hz, 1H), 4.34 (q, J = 7.0 Hz, 1H), 4.00 (dd, J = 11.9, 4.1 Hz, 1H), 3.84 (ddd, J =
11.9, 4.5, 0.8 Hz, 1H), 1.51 (d, J = 7.0 Hz, 3H).
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Step 2: A solution of (2R,5R)ꢀ2ꢀmethylꢀ5ꢀphenylmorpholinꢀ3ꢀone (32 g, 167.3 mmol) in toluene
(600 mL) was added to an ice cooled solution of sodium bis(2ꢀmethoxyethoxy)aluminum
o
hydride (65% wt in toluene, 300 mL, 1000 mmol). The reaction mixture was stirred at 5 C for 1
h and stirred at rt overnight. Aq. NaOH (2 M, 700 mL, 1390 mmol) was added to the reaction
o
mixture, allowing the temperature to rise to 45 C. The solution was diluted with toluene (100
mL) and the layers were separated. The organic layer was washed with aq. K2CO3 (10%, 100
1
mL), dried over Na2SO4, filtered, and concentrated to afford 31.0 g (100%) of an oil. H NMR
(CDCl3) δ 7.52 (d, J = 7.4 Hz, 2H), 7.43–7.34 (m, 2H), 7.33–7.27 (m, 1H), 4.14–3.72 (m, 4H),
2.86–2.71 (dd, J = 12.0, 6.0 Hz, 1H), 1.88 (br s, 1H), 1.34 (d, J = 6.4 Hz, 3H); 13C NMR
(CDCl3) δ 141.5, 128.4, 127.7, 127.3, 70.7, 68.0, 57.3, 48.4, 17.4; FTIR (cmꢀ1) 3317 (w); HRMS
Calcd. for C11H15NO (M+H)+ 178.1226; Found 178.1232; [α]D20 3.1 (c 1.01, MeOH).
(2R,5R)ꢀ5ꢀ(4ꢀFluorophenyl)ꢀ2ꢀmethylmorpholine (17b)
Prepared from (R)ꢀ2ꢀaminoꢀ2ꢀ(4ꢀfluorophenyl)ethanol according to the general procedure for the
1
synthesis of cisꢀ(2R,5R)ꢀ2ꢀmethylꢀ5ꢀarylmorpholines (54.6 g, 90%) as a yellow oil. H NMR
(CDCl3) δ 7.52–7.45 (m, 2H), 7.03 (t, J = 8.8 Hz, 2H), 4.01 (dd, J = 11.3, 5.3 Hz, 1H), 3.89–
3.83 (m, 2H), 3.78–3.83 (m, 1H), 2.92 (dd, J = 12.0, 3.2 Hz, 1H), 2.72 (dd, J = 12.0, 6.0 Hz,
1H), 1.30 (d, J = 6.4 Hz, 3H).
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(±)-cisꢀ5ꢀ(3ꢀFluorophenyl)ꢀ2ꢀmethylmorpholine (17c)
5
6
Prepared from racemic 2ꢀaminoꢀ2ꢀ(3ꢀfluorophenyl)ethanol according to the general procedure
7
8
1
for the synthesis of cisꢀ(2R,5R)ꢀ2ꢀmethylꢀ5ꢀarylmorpholines. H NMR (CDCl3) δ 7.33–7.22 (m,
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3H), 6.99–6.92 (m, 1H), 4.08ꢀ3.97 (m, 1H), 3.89–3.79 (m, 3H), 2.89 (d, J = 11.7 Hz, 1H), 2.71
(dd, J = 11.7, 6.4 Hz 1H), 1.28 (d, J = 6.4 Hz, 3H).
(±)-cisꢀ5ꢀ(2ꢀFluorophenyl)ꢀ2ꢀmethylmorpholine (17d)
Prepared from racemic 2ꢀaminoꢀ2ꢀ(2ꢀfluorophenyl)ethanol according to the general procedure
1
for the synthesis of cisꢀ(2R,5R)ꢀ2ꢀmethylꢀ5ꢀarylmorpholines. H NMR (CDCl3) δ 7.82–7.75 (m,
1H), 7.28–7.20 (m, 1H), 7.15–7.08 (m, 1H), 7.05–6.98 (m, 1H), 4.22–4.17 (m, 1H), 4.02 (ABq, J
= 3.7 Hz, 2H), 3.90–3.75 (m, 1H), 2.82 (d, J = 11.7 Hz, 1H), 2.64 (dd, J = 11.7, 7.4 Hz, 1H),
1.77 (br s, 1H), 1.22 (d, J = 11.9 Hz, 3H).
(2S,5R)ꢀ2ꢀMethylꢀ5ꢀphenylmorpholine (18a)
o
Step 1: To a 0 C solution of (R)ꢀ5ꢀphenylmorpholinꢀ3ꢀone (US 7629338, 1 g, 5.64 mmol) in
anhydrous DMF (5 mL) was added sodium hydride (60% dispersion in oil, 239 mg, 5.98 mmol).
o
The mixture was stirred at rt for 15 min and then cooled to 0 C before pꢀmethoxybenzyl
chloride (0.830 mL, 5.98 mmol) was added. The reaction mixture was stirred at rt for 4 h, diluted
with EtOAc and washed with H2O. The aqueous layer was extracted with EtOAc. The combined
organic layers were washed with saturated aq. NaCl, dried over MgSO4, filtered, and
concentrated. The crude residue was purified by silica gel column chromatography (gradient:
1
20–50% EtOAc/heptanes) to provide 1.4 g (83%) of a white solid. H NMR (DMSO-d6) δ 7.42–
7.31 (m, 3H), 7.30–7.25 (m, 2H), 7.10–7.04 (m, 2H), 6.91–6.85 (m, 2H), 5.19 (d, J = 14.8 Hz,
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1H), 4.47–4.38 (m, 1H), 4.28 (ABq, J = 16.4 Hz, 2H), 3.96 (dd, J = 11.9, 3.7 Hz, 1H), 3.74 (d, J
= 11.3, 3.3 Hz, 1H), 3.73 (s, 3H), 3.36 (d, J = 14.8 Hz, 1H).
7
8
9
Step 2: To a solution of diisopropylamine (1.1 mL, 7.7 mmol) in THF (10 mL) at ꢀ78 °C was
added nꢀBuLi (2.5 M in hexanes, 3 mL, 7.7 mmol). The solution was stirred at 0 °C for 15 min
and then cooled to ꢀ78 °C. A solution of (R)ꢀ4ꢀ(4ꢀmethoxybenzyl)ꢀ5ꢀphenylmorpholinꢀ3ꢀone
(1.84 g, 6.2 mmol) in THF (10 mL) was added. After stirring at ꢀ78 °C for 30 min, methyl
iodide (0.56 mL, 8.67 mmol) was added. The reaction mixture was warmed to rt overnight. The
reaction mixture was poured into aq. HCl (1 N) and the mixture was extracted 3 x EtOAc. The
combined organic layers were dried over Na2SO4, filtered and concentrated. The residue was
purified by silica gel column chromatography (gradient: 0–60 % EtOAc in heptanes) to provide
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1.69 g (87%) of the desired compound containing 15% of the cis diastereoisomer. H NMR
(CDCl3) δ 7.44–7.35 (m, 3H), 7.22–7.17 (m, 2H), 7.02–6.96 (m, 2H), 6.84–6.80 (m, 2H), 5.44
(d, J = 14.4 Hz, 1H), 4.48–4.41 (m, 2H), 4.04 (dd, J = 12.2, 4.6 Hz, 1H), 3.81 (s, 3H), 3.67 (dd,
J = 12.2, 7.9 Hz, 1H), 3.39 (d, J = 14.4 Hz, 1H), 1.59 (d, J = 7.4 Hz, 3H).
Step 3: To a solution of (2S,5R)ꢀ4ꢀ(4ꢀmethoxybenzyl)ꢀ2ꢀmethylꢀ5ꢀphenylmorpholinꢀ3ꢀone (1.69
g, 1.57 mmol) in 50% acetonitrile/water (48 mL) was added ammonium cerium (IV) nitrate (6.04
g, 10.9 mmol). The reaction mixture was stirred at rt for 4 h, poured into aq. HCl (1 N)
and extracted with EtOAc (2 x 100 mL). The combined organic layers were dried over Na2SO4,
filtered and concentrated. The residue was purified by silica gel column chromatography
(eluent: 50% EtOAc/heptanes) to provide 502 mg (48%) of a solid. 1H NMR (CDCl3) δ 7.44–
7.30 (m, 5H), 6.04 (br s, 1H), 4.82 (dd, J = 10.0, 4.5 Hz, 1H), 4.31–4.24 (m, 1H), 4.09–4.03 (m,
1H), 3.53 (dd, J = 11.9, 10.0 Hz, 1H), 1.54 (d, J = 6.8 Hz, 3H).
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Step 4: Prepared by reduction of (2S,5R)ꢀ2ꢀmethylꢀ5ꢀphenylmorpholinꢀ3ꢀone to give 382 mg
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(82%) of an oil. H NMR (CDCl3) δ 7.42–7.25 (m, 5H), 3.92–3.84 (m, 2H), 3.78–3.73 (m, 1H),
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3.73–3.67 (m, 1H), 3.52–3.43 (m, 1H), 3.06 (dd, J = 11.5, 2.3 Hz, 1H), 2.75 (dd, J = 11.5, 10.2
Hz, 1H), 1.20 (d, J = 6.3 Hz, 3H).
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(R)ꢀ2,2ꢀDimethylꢀ5ꢀphenylmorpholine (18b)
Prepared from (2R,5R)ꢀ2ꢀmethylꢀ5ꢀphenylmorpholinꢀ3ꢀone using the methods described above.
1H NMR (CDCl3) δ 7.44–7.38 (m, 2H), 7.34–7.22 (m, 3H), 3.67 (dd, J = 10.2, 3.7 Hz 1H), 3.45
(dd, J = 11.1, 3.7 Hz, 1H), 3.40 (ABq, J = 10.5 Hz, 1H), 2.68 (Abq, J = 11.7 Hz, 1H), 1.33 (s,
3H), 1.10 (s, 3H).
6ꢀ((2R,5R)ꢀ2ꢀMethylꢀ5ꢀphenylmorpholino)ꢀ2Hꢀpyrido[3,2ꢀb][1,4]oxazinꢀ3(4H)ꢀone
(22).
General CꢀN coupling procedure.
A mixture of tris(dibenzylideneacetone)dipalladium(0) (12.8 mg, 0.014 mmol) and 5ꢀ
(diisopropylphosphino)ꢀ1',3',5'ꢀtriphenylꢀ1'Hꢀ1,4'ꢀbipyrazole (prepared using the method
described in Org. Process Res. Dev., 2008, 12, 480ꢀ489, 13.4 mg, 0.028 mmol) in tꢀamyl alcohol
(0.7 mL) in a sealed reaction vessel was stirred at rt under nitrogen for 30 min. (2R,5R)ꢀ2ꢀ
methylꢀ5ꢀphenylmorpholine (100 mg, 0.564 mmol), 6ꢀbromoꢀ2Hꢀpyrido[3,2ꢀb][1,4]oxazinꢀ
3(4H)ꢀone (129 mg, 0.564 mmol) and HMPA (0.516 g, 2.82 mmol) or DMSO (0.48 mL, 6.8
mmol) were added to the mixture followed by solid LiOtBu (91.2 mg, 1.13 mmol) and a solution
of LiOtBu in tꢀamyl alcohol (1 M, 2.26 mL, 2.26 mmol). The reaction mixture was stirred at 60
oC overnight. The solution was diluted with EtOAc and washed with saturated aq. NH4Cl. The
aqueous layer was extracted with EtOAc. The combined organic layers were washed with
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saturated aq. NaCl, dried over MgSO4, filtered, and concentrated. The crude material was
purified by column chromatography on silica gel (gradient: 5–50% EtOAc/ heptanes). The
resulting solid was triturated with acetonitrile to afford 31 mg (17%) from the HMPA reaction;
72 mg (39%) from the DMSO reaction. mp 203.8–204.7 oC; 1H NMR (CDCl3) δ 7.73 (br s, 1H),
7.36–7.20 (m, 5H), 7.10 (d, J = 8.6 Hz, 1H), 6.13 (d, J = 8.8 Hz, 1H), 5.19 (d, J = 3.1 Hz, 1H),
4.55 (s, 2H), 4.41 (dd, J = 11.7, 1.6 Hz, 1H), 4.06 (dd, J = 11.8, 3.8 Hz, 1H), 3.91 (dd, J = 13.1,
3.1 Hz, 1H), 3.80–3.71 (m, 1H), 2.99 (dd, J = 13.1, 10.7 Hz, 1H), 1.27 (d, J = 6.2 Hz, 3H); 13C
NMR (125 MHz, CDCl3) δ 166.4, 153.3, 139.8, 138.2, 130.8, 128.7, 128.0, 127.2, 126.7, 101.3,
72.3, 70.5, 67.8, 54.0, 47.4, 19.3; FTIR (cmꢀ1) 1701.4 (s); HRMS Calcd. for C18H19N3O3 (M+H)+
326.1505; Found 326.1498; HPLC: tR = 3.937 min, 99.75%; Anal. Calcd. for C18H19N3O3: C;
66.45, H; 5.89, N; 12.91. Found, C; 66.26, H; 5.62, N; 12.75. [α]D20 ꢀ224.5 (c 0.392, MeOH).
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6ꢀ((2S,5R)ꢀ2ꢀMethylꢀ5ꢀphenylmorpholino)ꢀ2Hꢀpyrido[3,2ꢀb][1,4]oxazinꢀ3(4H)ꢀone (23)
Prepared from (2S,5R)ꢀ2ꢀmethylꢀ5ꢀphenylmorpholine and from 6ꢀbromoꢀ2Hꢀpyrido[3,2ꢀ
b][1,4]oxazinꢀ3(4H)ꢀone according to the general CꢀN coupling procedure to afford 47 mg
(13%). 1H NMR (CDCl3) δ 7.61 (br s, 1H), 7.34–7.17 (m, 5H), 6.94 (d, J = 8.6 Hz, 1H), 6.09 (d,
J = 8.6 Hz, 1H), 4.53 (s, 2H), 4.27 (dd, J = 9.9, 4.2 Hz, 1H), 4.04–3.95 (m, 2H), 3.56 (dd, J =
11.8, 9.9 Hz, 1H), 2.81 (dd, J = 12.9, 10.1 Hz, 1H), 1.28 (d, J = 6.2 Hz, 3H).
(R)ꢀ6ꢀ(2,2ꢀDimethylꢀ5ꢀphenylmorpholino)ꢀ2Hꢀpyrido[3,2ꢀb][1,4]oxazinꢀ3(4H)ꢀone (24)
Prepared from (R)ꢀ2,2ꢀdimethylꢀ5ꢀphenylmorpholine and 6ꢀbromoꢀ2Hꢀpyrido[3,2ꢀb][1,4]oxazinꢀ
1
3(4H)ꢀone according to the general CꢀN coupling procedure to afford 9.3 mg (2.3%). H NMR
(CDCl3) δ 7.60 (br s, 1H), 7.35–7.25 (m, 5H), 7.03 (d, J = 8.8 Hz, 1H), 6.00 (d, J = 8.8 Hz, 1H),
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4.98 (dd, J = 5.7, 5.7 Hz, 1H), 4.54 (s, 2H), 4.09–3.95 (m, 3H), 3.29 (d, J = 13.7 Hz, 1H), 1.29
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(s, 3H), 1.27 (s, 3H). LCꢀMS (Method B): tR = 2.50 min. MS (ES+): 340.5 (M+H)+.
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2ꢀ((2R,5R)ꢀ4ꢀ(3ꢀOxoꢀ3,4ꢀdihydroꢀ2Hꢀpyrido[3,2ꢀb][1,4]oxazinꢀ6ꢀyl)ꢀ5ꢀphenylmorpholinꢀ2ꢀ
yl)acetonitrile (25)
A mixture of 6ꢀ((2S,5R)ꢀ2ꢀ(hydroxymethyl)ꢀ5ꢀphenylmorpholino)ꢀ2Hꢀpyrido[3,2ꢀb][1,4]oxazinꢀ
3(4H)ꢀone (57 mg, 0.17 mmol), DCE (5 mL), Et3N (38.6 ꢁL, 0.273 mmol) and methanesulfonic
anhydride (43.1 mg, 1.2 mmol) was stirred at 0 ºC for 2 h and at rt for 6 h. The reaction mixture
was then partitioned between DCM (20 mL) and aq. NaOH (1 N, 20 mL). The organic layer was
separated, washed with saturated aq. NaCl, dried over MgSO4, filtered and concentrated to afford
((2S,5R)ꢀ4ꢀ(3ꢀoxoꢀ3,4ꢀdihydroꢀ2Hꢀpyrido[3,2ꢀb][1,4]oxazinꢀ6ꢀyl)ꢀ5ꢀphenylmorpholinꢀ2ꢀ
yl)methyl methanesulfonate 35 mg (50%). The crude methanesulfonate (35 mg, 0.083mmol) was
dissolved in DMF (1 mL) and treated with sodium cyanide (82 mg, 1.7 mmol). After heating at
120 ºC for 4 h, the mixture was partitioned between EtOAc (10 mL) and aq. NaOH (1 N, 10
mL). The aqueous layer was extracted with EtOAc (2 x 50 mL). The combined organic layers
were washed with saturated aq. NaCl (10 mL), dried over MgSO4, filtered and concentrated. The
residue was purified by silica gel column chromatography (gradient: 0–100% EtOAc in
1
heptanes) to provide 4.5 mg (15%) of a white solid. H NMR (CDCl3) δ 7.73 (br s, 1H), 7.36–
7.24 (m, 5H), 7.13 (d, J = 8.8 Hz, 1H), 6.17 (d, J = 8.8 Hz, 1H), 5.31 (m, 2H), 5.19 (d, J = 2.2
Hz, 1H), 4.57 (s, 2H), 4.47 (dd, J = 11.7, 1.5Hz, 1H), 4.18–4.07 (m, 2H), 3.99–3.94 (m, 1H),
3.12 (dd, J = 13.2,11.0 Hz, 1H), 2.64 (dd, J = 6.1,2.0 Hz, 2H).
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6ꢀ((2S,5R)ꢀ2ꢀ(Methoxymethyl)ꢀ5ꢀphenylmorpholino)ꢀ2Hꢀpyrido[3,2ꢀb][1,4]oxazinꢀ3(4H)ꢀ
one (26)
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Step 1: To a solution of ((2S,5R)ꢀ4ꢀbenzylꢀ5ꢀphenylmorpholinꢀ2ꢀyl)methanol (19) (100 mg,
0.353 mmol) in DMF (2 mL) at 0 °C was added sodium hydride (17 mg, 60% dispersion in oil,
0.424 mmol). The solution was stirred at 0 °C for 30 min. Methyl iodide (0.068 mL, 1.06 mmol)
was added. The solution was stirred overnight at rt. To the reaction mixture was added EtOAc.
The mixture was extracted with saturated aq. NH4Cl and saturated aq. NaCl. The organic layer
was dried over Na2SO4, filtered, concentrated and purified by column chromatography to afford
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62 mg (59%). H NMR (CDCl3) δ 7.49–7.44 (m, 2H), 7.37–7.18 (m, 8H), 4.05–3.95 (m, 2H),
3.83 (dd, J = 11.7, 8.4 Hz, 1H), 3.73 (dd, J = 11.7, 3.7 Hz, 1H), 3.67 (d, J = 13.5 Hz, 1H), 3.52–
3.46 (m, 2H), 3.39 (s, 3H), 2.98 (d, J = 13.7 Hz, 1H), 2.73 (dd, J = 12.1, 3.1 Hz, 1H), 2.39 (dd, J
= 12.1, 3.7 Hz, 1H).
Step 2: A mixture of (2S,5R)ꢀ4ꢀbenzylꢀ2ꢀ(methoxymethyl)ꢀ5ꢀphenylmorpholine (350 mg, 1.18
mmol), MeOH (10mL), pꢀtoluenesulphonic acid (452 mg, 2.35 mmol) and 10% PdꢀC (50%
water wet, 251 mg, 0.118 mmol) was hydrogenated in a Parr shaker for 1 h at 50 psi hydrogen.
The mixture was filtered through Celite® and concentrated. The residue was dissolved in DCM
and extracted with 4.3% aq. NaHCO3. The layers were separated and the organic layer was
washed with saturated aq. NaCl, dried over Na2SO4, filtered, and concentrated to provide 193 mg
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(79%) of (2S,5R)ꢀ2ꢀ(methoxymethyl)ꢀ5ꢀphenylmorpholine (21) as a light yellow solid. H NMR
(DMSO-d6) δ 7.46–7.42 (m, 2H), 7.35–7.29 (m, 2H), 7.26–7.21 (m, 1H), 3.79–3.66 (m, 3H),
3.65–3.57 (m, 2H), 3.51 (dd, J = 10.2, 5.7 Hz, 1H), 3.26 (s, 3H), 2.83 (dd, J = 12.3, 3.5 Hz, 1H),
2.71 (dd, J = 12.2, 4.4 Hz, 2H).
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Step 3: Prepared from (2S,5R)ꢀ2ꢀ(methoxymethyl)ꢀ5ꢀphenylmorpholine (21) and from 6ꢀbromoꢀ
2Hꢀpyrido[3,2ꢀb][1,4]oxazinꢀ3(4H)ꢀone according to the general CꢀN coupling procedure. The
residue was dissolved in DMSO and purified by preparative HPLC Method B. Gradient: 75%
water/ acetonitrile linear gradient to 100% acetonitrile in 8.5 min. LCꢀMS (Method A): tR = 2.78
min. MS (ES+): 361.11 (M+H)+.
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6ꢀ((2S,5R)ꢀ2ꢀ(Hydroxymethyl)ꢀ5ꢀphenylmorpholino)ꢀ2Hꢀpyrido[3,2ꢀb][1,4]oxazinꢀ3(4H)ꢀ
one (27)
Step 1: ((2S,5R)ꢀ5ꢀPhenylmorpholinꢀ2ꢀyl)methanol (20) was prepared by debenzylation of
((2S,5R)ꢀ4ꢀbenzylꢀ5ꢀphenylmorpholinꢀ2ꢀyl)methanol. 1H NMR (METHANOL-d4) δ 7.53–7.47
(m, 2H), 7.37–7.30 (m, 2H), 7.29–7.23 (m, 1H), 4.85 (s, 2H), 4.08–4.00 (m, 1H), 3.88ꢀ3.77 (m,
3H), 3.75–3.68 (m, 1H), 3.64 (dd, J = 10.9, 4.9 Hz, 1H), 2.87 (d, J = 4.9 Hz, 2H).
Step 2: Prepared from ((2S,5R)ꢀ5ꢀphenylmorpholinꢀ2ꢀyl)methanol (20) and 6ꢀbromoꢀ2Hꢀ
pyrido[3,2ꢀb][1,4]oxazinꢀ3(4H)ꢀone according to the general CꢀN coupling procedure to afford
230 mg (67%). 1H NMR (CDCl3) δ 8.84 (br s, 1H), 7.53–7.47 (m, 2H), 7.39–7.25 (m, 3H), 7.17
(d, J = 8.6 Hz, 1H), 6.39 (d, J = 8.6 Hz, 1H), 4.64–4.55 (m, 2H), 4.53 (d, J = 2.2 Hz, 2H), 4.21–
4.14 (m, 1H), 4.01–3.91 (m, 2H), 3.82 (dd, J = 11.0, 2.9 Hz, 1H), 3.16 (d, J = 3.9 Hz, 2H).
7ꢀ((2R,5R)ꢀ2ꢀMethylꢀ5ꢀphenylmorpholino)ꢀ1Hꢀpyrido[3,4ꢀb][1,4]oxazinꢀ2(3H)ꢀone (28)
Prepared
from
(2R,5R)ꢀ2ꢀmethylꢀ5ꢀphenylmorpholine
and
7ꢀchloroꢀ1Hꢀpyrido[3,4ꢀ
b][1,4]oxazinꢀ2(3H)ꢀone according to the general CꢀN coupling procedure. The residue was
dissolved in DMSO and purified by preparative HPLC Method A. Gradient: 90% water/
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