Design, Synthesis, and Evaluation of a Novel Series of Oxadiazine Gamma Secretase Modulators for Familial Alzheimer’s Disease

Article abstract of DOI:10.1021/acs.jmedchem.6b01620

Herein we describe the design, synthesis, and evaluation of a novel series of oxadiazine-based gamma secretase modulators obtained via isosteric amide replacement and critical consideration of conformational restriction. Oxadiazine lead 47 possesses good in vitro potency with excellent predicted CNS drug-like properties and desirable ADME/PK profile. This lead compound demonstrated robust Aβ₄₂ reductions and subsequent Aβ₃₇ increases in both rodent brain and CSF at 30 mg/kg dosed orally.

Full text of DOI:10.1021/acs.jmedchem.6b01620

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Article
Design, Synthesis and Evaluation of a Novel Series of Oxadiazine
Gamma Secretase Modulators for Familial Alzheimer’s Disease
Matthew G Bursavich, Bryce A. Harrison, Raksha Acharya, Donald E. Costa, Emily A. Freeman, Hilliary
E. Hodgdon, Lori A. Hrdlicka, Hong Jin, Sudarshan Kapadnis, Jeffrey S. Moffit, Deirdre A. Murphy, Scott
Nolan, Holger Patzke, Cuyue Tang, Melody Wen, Gerhard Koenig, Jean-François Blain, and Duane A Burnett
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01620 • Publication Date (Web): 23 Feb 2017
Downloaded from http://pubs.acs.org on February 24, 2017
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Design, Synthesis and Evaluation of a Novel Series of
Oxadiazine Gamma Secretase Modulators for Famil-
ial Alzheimer’s Disease
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†
,
Matthew G. Bursavich, * Bryce A. Harrison, Raksha Acharya, Donald E. Costa, Emily A. Freeman,
Hilliary E. Hodgdon, Lori A. Hrdlicka, Hong Jin, Sudarshan Kapadnis, Jeffrey S. Moffit, Deirdre A.
Murphy, Scott Nolan, Holger Patzke, Cuyue Tang, Melody Wen, Gerhard Koenig, JeanꢀFrançois Blain,
Duane A. Burnett
†
FORUM Pharmaceuticals, 225 Second Avenue, Waltham, MA 02451, USA
ABSTRACT: Herein we describe the design, synthesis and evaluation of a novel series of oxadiazineꢀ
based gamma secretase modulators obtained via isosteric amide replacement and critical consideration
of conformational restriction. Oxadiazine lead 47 possesses good in vitro potency with excellent preꢀ
dicted CNS drugꢀlike properties and desirable ADME/PK profile. This lead compound demonstrated
robust Aβ reductions and subsequent Aβ increases in both rodent brain and CSF at 30 mg/kg dosed
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37
orally.
Introduction.
In the 100+ years since the German physician Alois Alzheimer first described Alzheimer’s disꢀ
ease (AD), a vast amount of pharmaceutical research has been undertaken to address this devastating
neurodegenerative disorder that leads to loss of cognition, loss of motor function and ultimately death.
Currently, AD affects approximately 5.3 million people in the United States with estimated costs exꢀ
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th
pected to grow to $1.1 trillion by 2050. AD is the 6 leading cause of death in America and a signifiꢀ
cant burden to patients, caregivers and society as a whole. While current therapies including NMDA reꢀ
ceptor antagonists and acetylcholinesterase inhibitors demonstrate temporary symptomatic treatment,
they do not have an effect on disease progression. There is an urgent need for new therapies that will
affect the course of the disease to prevent, or at least delay, the onset of cognitive decline and ultimate
death due to this disease.
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The amyloid hypothesis, formulated more than 20 years ago, ascribes a central role for betaꢀ
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,3
amyloid (Aβ) AD. Several mutations in the amyloid precursor protein (APP) have been reported to be
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5
associated with familial AD (FAD) while a specific mutation appears to protect against the disease ,
further supporting the central role of Aβ in the disease pathology. The Aβ peptides are produced by the
sequential proteolysis of APP by beta amyloid cleavage enzyme 1 (BACE1) and gamma secretase (GS),
resulting in peptides of various lengths of which the longer 42 amino acid Aβ is believed to be the
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most toxic. Gamma secretase is an enzyme complex consisting of four different proteins, one of which is
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,7
presenilin (PS). Presenilin has been described as the catalytic subunit of GS and interestingly, mutaꢀ
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tions in the two presenilin genes have been associated with FAD. The consequence of many FADꢀ
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0–13
causing PS mutations is a decreased catalytic activity of GS
with partial lossꢀofꢀfunction contribꢀ
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uting to the increased Aβ :Aβ ratio observed in FAD.
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Compounds that could modify GS activity to produce less Aβ without affecting total Aβ levels
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6
were first described in 2001 by Weggen et al. These compounds, termed gamma secretase modulators
GSMs), offered a new, potentially safer alternative to gamma secretase inhibitors (GSIs) to reduce toxic
(
1
3
Aβ . Moreover, they have the opposite effect on GS kinetics than PS mutations, suggesting they
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might have a profound impact on disease development in PS mutation carriers. Much research has been
devoted to GSMs over the past 15 years to afford many examples of very potent compounds targeting
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this unique intramembrane cleaving protease.
Unfortunately these very potent compounds typically
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possess poor CNS drugꢀlike properties as measured by fraction sp character (Fsp values), molecular
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weight (MW), lipophilicity (cLogP) and CNS multiꢀparameter optimization (MPO) scores.
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In our effort to discover novel, potent and efficacious GSMs that possess good CNS drugꢀlike
properties for proofꢀofꢀmechanism studies in human clinical trials, we focused our initial design efforts
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on E2012 (1) , the early GSM from the Eisai group (EC = 83 nM) that demonstrated doseꢀdependent
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plasma reductions of Aβ and Aβ in Phase 1 studies. Based on a survey of the GSM literature, we
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hypothesized the importance of the central amide hydrogen bond acceptor, but also wanted to remove
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the cinnamide group, a potential Michael acceptor and undesirable structural feature. These initial hyꢀ
potheses led to our amide designs as shown in Figure 1. The key hydrogen bond acceptor is attached diꢀ
rectly to the methoxyphenyl ring and the aminoether linker places the pendent phenyl in a similar locaꢀ
tion as the p-fluoro phenyl of 1. Similar hypotheses and designs have been communicated independently
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from the Pfizer, Janssen, Amgen and Dainippon Sumitomo groups. From our initial amides I, we
further investigated 1) heteroaryl replacements for the imidazole, 2) nonꢀaromatic replacements for the
methoxyphenyl ring and 3) cyclic amines to induce a conformational restriction into the amino ether
linkage. Focused libraries afforded compound II which demonstrated good in vitro potency (EC = 35
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0
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nM) with greatly reduced aromaticity (increased Fsp value to 0.50). Unfortunately, this compound also
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possesses high molecular weight (497), high cLogP (4.6) and a low calculated CNS MPO score (3.2).
Due to the nonꢀoptimal predicted CNS drugꢀlike properties, as well as potential for amide hydrolysis, we
designed a series of novel oxadiazines as described in Figure 2. The oxadiazine moiety mimics the reꢀ
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quired hydrogen bond acceptor of the amide
while also imparting a key conformational constraint
into the pendant alkyl chain to potentially lock the compound into its putative bioactive conformation.
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Figure 1. Initial amide design strategy
Figure 2. Initial oxadiazine design strategy
Results and discussion.
The synthesis of the initial oxadiazines IV began from commercially available tert-butyl (S)ꢀ4ꢀ
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3
(hydroxymethyl)ꢀ2,2ꢀdimethyloxazolidineꢀ3ꢀcarboxylate as described in Scheme 1. We initially preꢀ
pared both oxadiazine enantiomers starting from both R and S alcohols 1 (synthesis not shown with the
Rꢀalcohol). The commercially available primary alcohol derived from Garner’s aldehyde was reacted
with either 4ꢀfluoroꢀ2ꢀmethylphenol or 4ꢀchloroꢀ2ꢀ(trifluoromethyl)phenol in a Mitsunobu reaction to
afford aryl ether 2 in good yields (70 and 77% respectively). The amino alcohol protecting groups were
removed with trifluoroacetic acid in dichloromethane and the amine reprotected with the p-methoxyl
benzyl group to afford 3 in good yields over two steps. Amine coupling with the in situ generated acid
chlorides of 4ꢀbromoꢀ3ꢀmethoxybenzoic acid provided amides 4 in 59 and 95% yield respectively. Conꢀ
version of the primary hydroxyl to the alkoxylamines 5 was achieved via Mitsunobu reaction with N-
hydroxyphthalimide and subsequent deprotection with hydrazine in high yields over two steps. The key
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cyclization step in the synthesis to successfully form the oxadiazine was realized in moderate yields (45
and 55% respectively) with dehydrative P O conditions in iꢀPrOH to afford oxadiazine 6. Suzuki or
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Buchwald conditions successfully installed the R heteroaryl groups to afford 7 and the p-
methoxybenzyl protecting groups were removed with acidic conditions in a microwave reactor to afford
the desired oxadiazines 8-11.
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To investigate the importance of the oxadiazine NꢀH we embarked on the related synthesis in
Scheme 2. Here ring opening of aryl ether 2 under pꢀtoluenesulfonic acid conditions provided the Bocꢀ
protected amino alcohol which was protected with TBDMSꢀCl and alkylated with methyl iodide to proꢀ
vide 12 in good yields over 3 steps. Compound 13 was realized in high yields via deprotection of the
TBDMS group, Mitsunobu reaction with N-hydroxyphthalimide and subsequent NꢀBoc deprotection.
Amine coupling with the in situ generated acid chloride of 4ꢀbromoꢀ3ꢀmethoxybenzoic acid followed by
hydroxyphthalimide deprotection with hydrazine provided 14 in high yields over two steps. Dehydrative
cyclization to successfully form the oxadiazine heterocycle 15 was accomplished in low yield (29%)
with P O in iꢀPrOH. Buchwald reaction conditions successfully installed the 4ꢀmethylimidazole to afꢀ
2
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ford NꢀMethyl oxadiazine (S)-16.
Scheme 1. Synthesis of (S)-aryl ether oxadiazine GSMs
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OH
HO
PMBHN
O
R3
O
R3
R3
O
a
b
c
O
d
O
N
O
N
OH
N
Boc
PMB
Boc
R4
Br
R4
R4
OMe
2
2
a: R3 = Me; R4 = F
b: R3 = CF
3a: R3 = Me; R4 = F
3b: R3 = CF ; R4 =Cl
4a: R3 = Me; R4 = F
; R4 =Cl
4b: R3 = CF ; R4 =Cl
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ONH
2
O
O
O
N
R3
N
R3
N
R3
O
R3
O
e
O
f or g
R4 R1
O
h
O
N
N
N
N
H
PMB
PMB
PMB
R1
R4 Br
R4
R1
R4
OMe
OMe
OMe
OMe
8
-11
a: R3 = Me; R = F
b: R3 = CF
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6a: R3 = Me; R = F
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7a1: R = 4-Me-Imid; R = Me; R4 = F
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3
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; R4 =Cl
6b: R3 = CF ; R4 =Cl
7a2: R = 5-Me-Pyridine; R = Me; R = F
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3
1
3
4
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b1: R = 4-Me-Imid: R = CF ; R =Cl
7b2: R = 5-Me-Pyridine; R = CF
3
; R4 =Cl
3
1
3
(
a) 4ꢀfluoroꢀ2ꢀmethylphenol or 4ꢀchloroꢀ2ꢀ(trifluoromethyl)phenol, DIAD, PPh , THF, 70% and 77%; (b) a. TFA, DCM; b.
3
TEA, MgSO , pꢀanisaldehyde, then NaBH , 71 and 68% over two steps (c) 4ꢀbromoꢀ3ꢀmethoxybenzoic acid, DMF/oxalyl
4
4
chloride, DCM, 59% and 95%; (d) a. N-hydroxyphthalimide, DIAD, PPh , THF; b. hydrazine hydrate, EtOH, 85% and 91%
3
over two steps; (e) P O , iꢀPrOH, 45% and 55%; (f) 4ꢀmethylimidazole, K PO Pd (dba) , Me ꢀdiꢀtꢀBuXPhos, toluꢀ
2
5
3
4,
2
3
4
ene/dioxane, 67% and 57%; (g) (2ꢀmethylpyridinꢀ4ꢀyl)boronic acid, Cs CO PdCl (dppf), dioxane/H O, 68% and 60%; (h)
2
3,
2
2
TFA, DCE, 59ꢀ84%.
Scheme 2. Synthesis of N-methyl (S)-aryl ether oxadiazine analogs
O
TBSO
PthNO
MeHN
CF3
CF3
CF3
a
b
c
O
O
N
O
Boc
BocMeN
Cl
Cl
12
Cl
2
b
13
ONH
2
O
O
O
CF
3
N
CF
3
N
CF3
O
d
O
e
O
N
N
N
Me
Me
Me
Br
Cl
Br
Cl
N
Cl
OMe
14
OMe
OMe
N
15
(
S)-16
(
a) a. pꢀTsOHꢀH O, MeOH; b. TBSꢀCl, imidazole, DCM; c. MeI, KHMDS, THF/DMF, 63% over three steps; (b) a. HCl,
2
MeOH/THF; b. N-hydroxyphthalimide, DIAD, PPh , THF; c. 4N HCl in dioxane, DCM, 72% over three steps; (c) a. 4ꢀ
3
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bromoꢀ3ꢀmethoxybenzoic acid DCM/DMF/oxalyl chloride, b. hydrazine hydrate, EtOH, 40% over two steps; (d) P O , iꢀ
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5
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9
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PrOH, 29%; (e) 4ꢀmethylimidazole, K PO Pd (dba) , Me ꢀdiꢀtꢀBuXPhos, toluene/dioxane, 53%.
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Table 1. In vitro activity of aryl ether oxadiazine GSMs
a
1
2
3
4
EC (nM)
R
R
R
R
50
Cmpd
R)-8
(
H
H
Me
Me
Me
Me
F
F
2,888
230
(
S)-8
(
R)-9
H
F
6,446
109
(
S)-9
H
F
(S)-10
(S)-11
H
CF
CF
CF
3
Cl
Cl
Cl
109
H
249
3
3
(
S)-16
Me
113
a
Aβ EC values were determined in H4 cells over expressing WT human APP751. Aβ EC values shown here are the
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50
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50
average of n > 2 assays run in duplicate.
Next we determined the Aβ lowering potency (EC ) in H4 cells over expressing WT human
4
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APP751. The initial compound (R)-8 demonstrated only low micromolar potency, but its (S)ꢀenantiomer
(S)-8 showed a 12ꢀfold improvement (Aβ EC = 230 nM) demonstrating a high eudysmic ratio. Reꢀ
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2
50
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placement of the 4ꢀmethylꢀimidazolꢀ1ꢀyl with 2ꢀmethylpyridinꢀ4ꢀyl demonstrated a similar preference
for the Sꢀenantiomer comparing (R)-9 with (S)-9 (Aβ EC 6,446 vs. 109 nM). Incorporation of the
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more lipophilic 2ꢀCF , 4ꢀCl phenyl in compound (S)-10 improved the potency 2ꢀfold relative to comꢀ
3
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pound (S)-8. Replacing the 4ꢀmethylꢀimidazolꢀ1ꢀyl with 2ꢀmethylpyridinꢀ4ꢀyl to afford compound (S)-
11 demonstrated a 2ꢀfold reduction in potency relative to (S)-10. Addition of an NꢀMe to the more lipoꢀ
philic (S)-10 afforded an equipotent analog in (S)-16 (Aβ EC = 109 nM and 113 nM, respectively)
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albeit at an even higher cLogP (4.3 vs. 4.6). Unfortunately, none of these initial compounds demonstratꢀ
ed reduction of brain Aβ in mice when dosed orally at 30 mg/kg.
4
2
In a strategic effort to lower the cLogP, restrict the rotation about the left hand side of the moleꢀ
cule via an intramolecular hydrogen bond and potentially improve the potency, we initiated the chemisꢀ
try to replace the methoxyphenyl with a 2ꢀmethoxypyridine group first disclosed by the Eisai group
34
(
Scheme 3). Here we only focused on the more potent (S)ꢀoxadiazines. PMBꢀprotected amino alcohols
3
were coupled with the in situ generated acid chloride of the commercially available 5ꢀbromoꢀ6ꢀ
methoxypicolinic acid to provide amides 17 in moderate yields (78 and 59% respectively). Conversion
of the primary hydroxyl to oxadiazine 18 was accomplished via Mitsunobu reaction with N-
hydroxyphthalimide, deprotection with hydrazine and dehydration with P O in iꢀPrOH over 3 steps
2
5
with low yields again in the key cyclization step (17 and 21% respectively). Suzuki or Buchwald condiꢀ
1
tions successfully installed the R heteroaryl groups and removal of the p-methoxybenzyl protecting
group with TFA provided the desired oxadiazines 19-25 in moderate yields.
Scheme 3. Synthesis of (S)-2-methoxypyridine aryl ether oxadiazine GSMs
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5
OH
O
O
O
R3
N
R3
N
R3
HO
PMBHN
R3
O
b
O
c or d
R4
O
a
N
N
N
H
O
N
PMB
N
PMB
N
Br
R4
Br
R1
R4
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
R4
OMe
OMe
OMe
1
9-25
a: R = Me; R4 = F
3
17a: R = Me; R4 = F
3
18a: R = Me; R4 = F
3
3
3
4
3
4
3
4
3b: R = CF ; R =Cl
17b: R = CF ; R =Cl
18b: R = CF ; R =Cl
3
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
a) 5ꢀbromoꢀ6ꢀmethoxypicolinic acid, DCM/DMF/oxalyl chloride, 78 and 59%; (b) a. N-hydroxyphthalimide, DIAD, PPh3,
THF; b. hydrazine hydrate, EtOH; c. P O , iꢀPrOH, 12 and 15% over 3 steps; (c) a. 4ꢀmethylimidazole or 4ꢀchloroimidazole,
2
5
K PO Pd (dba) , Me ꢀdiꢀtꢀBuXPhos, toluene/dioxane; b. TFA/DCE, 77ꢀ47% over two steps; (d) a. heteroarylboronic acid,
3
4,
2
3
4
Cs CO PdCl (dppf), dioxane/H O; b. TFA/DCE, 86ꢀ31% over two steps.
2
3,
2
2
Rewardingly, the initial methoxypyridine compound 19 demonstrated in a 5ꢀfold potency imꢀ
provement (Aβ EC = 51 nM) at a lower cLogP (3.2) relative to (S)-8. The SAR of the left hand side
4
2
50
heterocycle was further explored with compounds 20-24. Both compound 20 and 21 provided moderateꢀ
ly potent analogs (Aβ EC = 98 and 72 nM, respectively) but at the expense of significantly higher
4
2
50
cLogP (3.9). All three of these potent analogs were further profiled for stability in rat hepatocytes and
demonstrated very high turnover rates which appeared to correlate with increasing cLogP. Replacement
of the methyl imidazole with other 5ꢀmember heterocycles provided compounds 22-24 which all
demonstrated reduced cellular potency. Turning our attention back to the aryl ether, combining the more
lipophilic 2ꢀCF , 4ꢀCl phenyl with the more potent 4ꢀmethylimidazole provided compound 25 which
3
demonstrated an approximate 10ꢀfold potency improvement relative to compound (S)-8 (Aβ EC = 27
4
2
50
and 230 nM, respectively). This potency improvement comes with the expense of a higher cLogP (4.1)
and demonstrated the expected high clearance in rat hepatocytes. Not surprisingly, orally dosing this relꢀ
ꢀ1
ꢀ1
atively rat unstable compound (rat CLint, u = 3,830 ml min kg ) at 30 mg/kg afforded only minimal exꢀ
posure and demonstrated no brain Aβ reduction in vivo.
4
2
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. In vitro activity of aryl ether pyrimidine oxadiazine GSMs
O
N
R3
O
N
H
N
R1
R4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OMe
rat CLint, u
EC50
1
3
4
R
R
R
cLogP
3.2
-1
-1
Cmpd
a
(mL min kg )
(nM)
1
9
Me
Me
Me
Me
F
F
F
F
51
1906
20
3.9
3.9
3.4
98
72
3259
4105
ꢀ
2
2
1
2
604
2
2
2
3
4
5
Me
Me
F
F
3.8
3.0
4.1
1,271
1,364
27
ꢀ
ꢀ
CF
Cl
3830
3
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Aβ EC values were determined in H4 cells over expressing WT human APP751. Aβ EC values shown here are the
42
50
42
50
average of n > 2 assays run in duplicate.
In continuing to explore the alkylꢀsubstituted aryl ether oxadiazines we engineered another conꢀ
formational constraint to further restrict the rotation of the pendant aryl group as shown below in Figure
3. To mimic the potentially bioactive 3ꢀdimentional presentation of the aryl motif in V we designed a
series of piperidine aryl ethers VI. In this design there are only 5 rotatable bonds in the scaffold comꢀ
pared to the 7 rotatable bonds found in the initial amide designs. To mimic another possible bioactive 3ꢀ
dimentional presentation of the aryl motif in VII we also designed a series of Nꢀaryl substituted piperaꢀ
zines VIII. With the piperazine scaffold there are now only 4 rotatable bonds compared to the initial amꢀ
ides.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Design strategies incorporated into fused piperidine and piperazine scaffolds.
The racemic synthesis of piperidine ether oxadiazine GSMs began from commercially available
2ꢀ(hydroxymethyl)pyridinꢀ3ꢀol is described in Scheme 4. The primary alcohol of pyrimidine 26 was proꢀ
tected with TBDMSꢀCl, reduced to the piperidine with 60 psi of H gas over PtO then protected with
2
2,
Boc O to afford piperidinol 27 as a mixture of cis enantiomers. Piperidinol 27 was reacted with either 4ꢀ
2
fluoroꢀ2ꢀmethylphenol or 4ꢀchloroꢀ2ꢀ(trifluoromethyl)phenol in a Mitsunobu reaction to afford aryl
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ether 28. The silyl protecting group was removed with TBAF, the amine protecting group removed with
TFA in DCM and the resulting amine coupled with the in situ generated acid chloride of 5ꢀbromoꢀ6ꢀ
methoxypicolinic acid to provide amides 29. Conversion of the primary hydroxyl to the alkoxyamine 30
was achieved via Mitsunobu reaction with N-hydroxyphthalimide and subsequent deprotection with hyꢀ
drazine. Successful oxadiazine formation was realized with dehydrative P O conditions in i-PrOH and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
5
further purification via preparative chiral HPLC afforded 31a(+), 31a(-), 31b(+) and 31b(-). Suzuki or
1
Buchwald coupling conditions with each enantiomer successfully installed the R heteroaryl groups to
afford the desired oxadiazines 32-34(+) and 32-34(-).
Scheme 4. Synthesis of piperidine oxadiazine GSMs
OH
HO
N
OTBDMS
OH
R2
OTBDMS2
O
R
d
e
O
f
OH a-c
O
N
BocN
BocN
N
R3
Br
R3
26
27(+/-)
OMe
2
3
2
3
2
2
8a(+/-): R = Me; R = F
29a(+/-): R = Me; R = F
2
3
2
3
8b(+/-): R = CF ; R =Cl
29b(+/-): R = CF ; R =Cl
3
3
ONH2
O
O
N
O
O
N
R2
N
R2
N
R2
O
O
g
h or i
N
N
N
N
R3
R1
R3
Br
R3
Br
OMe
OMe
OMe
2
3
2
3
32-34(+)
32-34(-)
30a(+/-): R = Me; R = F
31a(+/-): R = Me; R = F
2
3
2
3
3
0b(+/-): R = CF ; R =Cl
31b(+/-): R = CF ; R =Cl
3
3
(
a) TBDMSꢀCl, imidazole, DMF, 48%; (b) H , PtO , EtOH, 87%; (c) Boc O, TEA, DCM, 63%; (d) 4ꢀfluoroꢀ2ꢀ
2 2 2
methylphenol or 4ꢀchloroꢀ2ꢀ(trifluoromethyl)phenol, DIAD, PPh , DCM, 78 or 85% (e) a. TBAF, THF; b. TFA,
3
DCM; c. 5ꢀbromoꢀ6ꢀmethoxypicolinic acid, DMF/oxalyl chloride, DCM then TEA, THF, 32 and 57% over three
steps; (f) a. N-hydroxyphthalimide, DIAD, PPh , toluene; b. hydrazine hydrate, EtOH, 69 and 87% over two steps
3
(
g) POCl , i-PrOH, 35 and 56% (h) 4ꢀmethyl imidazole, K PO Pd (dba) , Me ꢀdiꢀtꢀBuXPhos, toluene/dioxane, 74ꢀ
3 3 4, 2 3 4
6
5% (i) (2ꢀmethylpyridinꢀ4ꢀyl)boronic acid, Cs CO PdCl (dppf), dioxane/H O, 61ꢀ56%.
2 3, 2 2
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4
5
The racemic synthesis of Nꢀaryl piperazine oxadiazine GSMs began from commercially availaꢀ
ble tert-butyl 2ꢀ(hydroxymethyl)piperazineꢀ1ꢀcarboxylate as described in Scheme 5. The primary alcoꢀ
hol of piperazine 35 was protected with TBDMSꢀCl then coupled with 2ꢀbromoꢀ5ꢀ
chlorobenzotrifluoride using Pd (dba) and DavePhos to afford Nꢀaryl piperazine 36. Both the amine
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
and primary alcohol protecting groups were removed with HCl, and the resulting liberated amine was
coupled with 5ꢀbromoꢀ6ꢀmethoxypicolinic acid under HATU conditions to provided amide 37. Converꢀ
sion of the primary hydroxyl to the alkoxyamine 38 was achieved via Mitsunobu reaction with N-
hydroxyphthalimide and subsequent hydrazine deprotection. Successful oxadiazine formation was realꢀ
ized with POCl in EtOH with catalytic addition of DMAP to afford 39. Buchwald coupling successfully
3
installed the 4ꢀmethyl imidazole to afford the desired oxadiazines which were purified via preparative
chiral HPLC to provide both 40(+) and 40(-).
Scheme 5. Synthesis of piperazine oxadiazine GSMs
OTBDMS
ONH2
CF3
OH
N
O
HO
O
BocN
CF3
N
b
c
N
d
a
N
CF3
BocN
N
N
N
Br
NH
5(+/-)
Br
OMe
OMe
Cl
Cl
3
36(+/-)
37(+/-)
Cl
38(+/-)
O
O
N
N
N
N
CF3
CF3
e
N
N
N
N
Br
N
Me
OMe
OMe
40(+)
0(-)
Cl
N
Cl
39(+/-)
4
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
a) a. TBDMSꢀCl, imidazole, DCM; b. NaOtBu, Pd (dba) , DavePhos, 2ꢀbromoꢀ5ꢀchlorobenzotrifluoride, toluene, 70%
2 3
over two steps; (b) a. HCl, MeOH/dioxane; b. 5ꢀbromoꢀ6ꢀmethoxypicolinic acid, DIEA, HATU, DMF; c. TBAF,
DCM, 80% over three steps; (c) a. N-hydroxyphthalimide, DIAD, PPh , toluene; b. Hydrazine hydrate, EtOH; (d)
3
POCl , DIEA, DMAP, EtOH; (f) 4ꢀmethyl imidazole, K PO Pd (dba) , Me ꢀdiꢀtꢀBuXPhos, toluene/dioxane
3
3
4,
2
3
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. In vitro activity of piperidine and piperazine oxadiazine GSMs
O
O
N
N
R2
O
N
Cl
N
N
N
N
R1
R3
N
Me
OMe
OMe
N
CF3
32-34
40
a
1
2
3
EC (nM)
R
R
R
cLogP
4.0
50
Cmpd
32(+)
Me
Me
Me
F
F
197
>3,000
240
3
2(-)
4.0
4.6
4.8
4.4
3
3(+)
F
34(+)
CF
3
Cl
Cl
15
4
0(+)
CF
3
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Journal of Medicinal Chemistry
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2
3
4
4
0(-)
CF
3
Cl
4.4
307
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Aβ EC values were determined in H4 cells over expressing WT human APP751. Aβ EC values shown here are the
42
50
42
50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
average of n > 2 assays run in duplicate.
Rewardingly the initial piperidine aryl ether afforded a modestly potent analog 32(+) (Aβ EC
50
4
2
=
197 nM). Here again a high eudysmic ratio was observed as the enantiomeric 32(-) demonstrated an
Aβ EC > 3,000 nM. Replacement of the methyl imidazole with methylpyridine provided compound
4
2
50
3
3(+). Once again the methylpyridine demonstrated less potency (Aβ EC = 240 nM) with a signifiꢀ
42 50
cantly higher and less desirable cLogP (4.6). Incorporation of the more lipophilic 2ꢀCF , 4ꢀCl phenyl in
3
compound 34(+) significantly improved the potency (Aβ EC = 15 nM) relative to compound 32(+)
4
2
50
albeit at an even higher cLogP (4.8). Given this drastic improvement in potency we further profiled
compound 34(+) in both rat and human hepatocytes. While the stability in rat hepatocytes was considerꢀ
ꢀ1
ꢀ1
ably low (rat CLint,u= 4251 mL min kg ) the predicted human CL
was much better (human hep
int,u
ꢀ1
ꢀ1
CLint,u = 167 mL min kg ). We decided to determine the reduction of brain Aβ in mice with this highꢀ
42
ly potent compound with promising human CLint,u. Here, dosing compound 34(+) at 90 mg/kg in mice
provides a 32% Aβ reduction in the brain at 6 hours postꢀdose with a total concentration of 36 µM of
4
2
the compound observed in the brain and a B:P of 0.6. Unfortunately, the desired potency and modest efꢀ
ficacy comes at the expense of increasing the cLogP (4.8). The related Nꢀaryl piperazine afforded modꢀ
estly potent 40(+) (Aβ EC = 64 nM) which demonstrated a eudysmic ratio of 5 (40(-): Aβ EC =
4
2
50
42
50
3
07 nM), but further work on this high cLogP analog (4.4) was deprioritized due to the exciting results
from our next set of designs.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
To further explore the alkylꢀsubstituted aryl ether pyrimidine oxadiazine GSMs, we engineered a
different conformational constraint from the alkyl group to the aryl group restricting the rotation of the
pendant aryl group as shown below in Figure 4. In this fashion the alkyl ether V was converted to hetꢀ
eroaryls IX. Here there are only 4 rotatable bonds in the newly designed scaffold compared to the 7 roꢀ
tatable bonds found in the initial amide designs.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
N
N
Novel
Benzofuran
O
X
N
H
N
H
R
N
R
N
Designs
N
N
Me
Me
OMe
V
OMe
IX
N
N
Conformational
restriction
Figure 4. Design strategies incorporated into heteroaryl oxadiazine scaffold.
The heteroaryl oxadiazines were obtained via the stereoselective synthesis as described below in
Scheme 6. The boronic acid 41 was converted into amino alcohol 42 via an asymmetric Petasis Borono–
3
5
Mannich multicomponent reaction and reduction of the resulting amino acid. Protecting group swap
from the sulfinamide to the PMBꢀprotected amine afforded 43. The secondary amine was coupled with
5
ꢀbromoꢀ6ꢀmethoxypicolinic acid to provided amide 44. Mitsunobu reaction with Nꢀhydroxyphthalimide
and subsequent deprotection with hydrazine afforded the alkoxyamine 45. Treatment with TEA in DCE
effected acyl migration to the alkoxyamine which was then cyclized with POCl in EtOH and catalytic
3
addition of DMAP to provided oxadiazine 46. Subsequent 4ꢀmethyl imidazole incorporation via Buchꢀ
wald coupling afforded the key oxadiazines 47-48.
Scheme 6. Asymmetric synthesis of heterocyclic oxadiazine GSMs
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5
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7
8
9
HO
OH
HO
O
X
a
b
c
d
X
X
X
B(OH)2
HN
S
N
PMBHN
N
PMB
tBu
O
Br
4
4
1a: X = 0
1b: X = S
OMe
4
4
2a: X = 0
2b: X = S
43a: X = 0
43b: X = S
44a: X = 0
44b: X = S
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ONH2
X
O
N
O
N
O
N
e
f
X
g
X
N
N
H
N
N
PMB
N
PMB
Br
Br
N
OMe
OMe
OMe
N
45a: X = 0
5b: X = S
46a: X = 0
46b: X = S
47-48
4
(
a) a. glyoxylic acid monohydrate, (S)ꢀ2ꢀmethylpropaneꢀ2ꢀsulfinamide, InBr DCM; b. LiAlH , THF, 21 and 16% over
3, 4
two steps; (b) a. 4N HCl, dioxane; b. TEA, MgSO , pꢀanisaldehyde, then NaBH MeOH,68 and 84% over two steps
4
4,
(
c) NaOMe, 5ꢀbromoꢀ6ꢀmethoxypicolinic acid, MeOH, 78 and 82%; (e) a. N-hydroxyphthalimide, DIAD, PPh , tolꢀ
3
uene; b. Hydrazine hydrate, EtOH, 68 and 51% over two steps (f) a. TFA, DCE; b. POCl , DIEA, DMAP, EtOH, 37
3
and 32% over two steps; (g) a. 4ꢀmethyl imidazole, K PO Pd (dba) , Me ꢀdiꢀtꢀBuXPhos, toluene/dioxane; b. TFA,
3
4,
2
3
4
DCE, 51 and 43% over two steps.
Table 5. In vitro activity of heterocyclic oxadiazines
O
N
X
N
N
H
N
Me
OMe
N
rat CLint, u
Brain
Aβ42
lowꢀ
human
[
Brain]
Total
[Brain]
Free
ꢀ
1
cLogP/
MPO
LLE/
(mL min
CLint, u
a
X
EC (nM)
50
Cmpd
ꢀ
1
LELP
kg )
ꢀ1
(
mL min
(
uM)
(nM)
b
ering
ꢀ
1
kg )
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1
2
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9
1
1
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2
2
2
2
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3
4
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4
4
4
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5
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5
5
5
5
5
5
5
6
4
4
7
8
O
S
35
8
2.8/5.0
3.7/4.1
4.7/7.6
4.4/9.4
84
44
29.8
30.2
427
76
43
34
430
322
a
Aβ EC values were determined in H4 cells over expressing WT human APP751. Aβ EC values shown here are the
42
50
42
50
b
average of n > 2 assays run in duplicate. Brain Aβ lowering was measured 6 hours postꢀdose.
42
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Rewardingly the initial benzofuran afforded potent analog 47 (Aβ EC = 35 nM) now with
4
2
50
great predicted CNS drugꢀlike properties based on a low cLogP (2.8) and high CNSꢀMPO score (5.0).
The potency of this compound is achieved with very high efficiency as measured by both LLE (4.7) and
19
LELP (7.6) scores. Further profiling demonstrated greatly improved stability in rat hepatocytes (CL
int,u
ꢀ1
ꢀ1
ꢀ1
ꢀ1
=
84 mL min kg ) and predicted human stability (human hep CL
= 44 mL min kg ). We decided
int,u
to immediately determine the reduction of brain Aβ in mice with this potent, promising compound.
4
2
Dosing compound 47 orally at 30 mg/kg in mice provides a 43% reduction of brain Aβ at 6 hours with
4
2
30 µM (427 nM free) compound observed in the brain and a B:P of 1.5. Here, the free drug concentraꢀ
tion in the brain exceeded the EC in the cellular assays, which predicates good in vivo activity in the
9
0
3
6
CNS. For the first time we had combined good potency and great predicted CNS drugꢀlike properties
with in vivo efficacy. Replacement of the benzofuran with the benzothiophene provided compound 48, a
more active analog (Aβ EC = 8 nM) albeit with a higher cLogP (3.7) and lower CNSꢀMPO score
4
2
50
(
4.1). This analog is also highly efficient as measured by both LLE (4.4) and LELP (9.4) scores. Here
the increase in lipophilicity leads to a reduction in both the rat and human hepatocyte stability (rat CLint,u
ꢀ1
ꢀ1
ꢀ1
ꢀ1
=
430 mL min kg and human hep CL = 322 mL min kg ). We proceeded to determine the reducꢀ
int,u
tion of mouse brain Aβ with this potent, promising compound. Dosing compound 48 at 30 mg/kg in
4
2
our mouse model provided a 34% reduction of brain Aβ at 6 hours with 30 µM total compound (76 nM
4
2
free) observed in the brain and a B:P of 2.8.
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Journal of Medicinal Chemistry
Further profiling compound 47 demonstrated good aqueous solubility (84 µM) in a pH 7.4 buffꢀ
1
2
3
7
er. Permeability and PgꢀP efflux potential were assessed via Cacoꢀ2 assay with compound 47 demonꢀ
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ꢀ
6
strating good permeability (PA
= 10 x 10 cm/s) and minimal PgꢀP efflux potential (Efflux ratio =
B
ꢀ
1
.1). Compound 47 also demonstrated minimal cytotoxicity (TC > 72 µM) in HepG2 cells measuring
50
ATP to assess viability as well as minimal hERG liability (IC = 15 µM) in a functional patch clamp
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
assay. The cytochrome P450 profiling of compound 47 demonstrated minimal inhibition against CYP
3
A4, 2C19 and 2C8 (IC s of 25, 17 and >25 µM respectively) and moderate inhibition of CYP 2C9,
50
2D6 and 1A2 (IC s of 8, 2 and 3 µM respectively). Profiling for CYP3A4 time dependent inhibition
5
0
was shown to be negative as inhibition concentrations from 5 to 25 µM were consistent with vehicle. No
genotoxicity with or without S9 metabolic activation was observed for compound 47 in a GreenScreen
3
8
assessment. Compound 47 was also shown to possess an excellent pharmacokinetic profile in both rat
and monkey (Table 6).
Table 6. PK parameters of 47 in rat and monkey
b
b
AUClast
µMꢀh)
67.8
Cmax
a
c
d
d
ꢀ1
ꢀ1 d
Species
%F
93
T1/2 (h)
V (L/kg)
CL (mL min kg )
ss
(
(µM)
7.3
Wistar Rat
Cynomolgus
monkey
2.2
1.5
1.0
0.9
5.8
5.6
8
.1
11
136+34
a
b
1
0 mg/kg for po; 1 mg/kg for iv. AUC (area under the curve) and Cmax (maximal drug concentration) obtained with p.o.
c
d
administration. %F: bioavailability. T (halfꢀlife), V (volume of distribution), CL (in vivo clearance) obtained with i.v.
1
/2
ss
administration.
Compound 47 demonstrates robust in vivo reduction of Aβ and subsequent increase of Aβ in
4
2
37
3
6
rodents (Table 7). Dosing 47 at 30 mg/kg in mice provides a 43% reduction of Aβ and increases Aβ
37
4
2
3
.2ꢀfold in mouse brain at 6 h. In order to assess these effects in both brain and CSF we dosed 47 at 30
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mg/kg in rats. Here a 30% Aβ reduction and 2.5ꢀfold increase in Aβ was observed in the rat brain at
4
2
37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
hr. A greater effect size was noted in CSF, where compound 47 demonstrated a 58% reduction in Aβ42
and a 20ꢀfold increase in Aβ also at 6 hr. To further characterize this compound a time course efficacy
3
7
study (Figure 7) was conducted in mice dosed at 30 mg/kg. This study led to peak Aβ reductions of
4
2
4
5% with a calculated Aβ lowering AUC
of 34%. Peak increases of 3.1ꢀfold were observed for
24
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
42
1
ꢀ
Aβ37.
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Journal of Medicinal Chemistry
Table 7. In vivo efficacy profile of 47 in rodents
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
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5
5
5
5
5
5
5
6
[
Brain]
CSF Aβ37
Brain Aβ37
CSF Aβ42
[
Brain]
Brain Aβ42
Free (nM)
Species
a
a
a
increase
a
increase
reduction
Total (uM)
reduction
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Mouse
Rat
29.8
13.7
427 nM
156 nM
43%
30%
3.2ꢀfold
2.5ꢀfold
ꢀ
ꢀ
58%
20ꢀfold
aExpressed as % vehicle in CSF and brain 6 h postꢀdose
Figure 7. In vivo time course profile of 47 in Mouse Brain. Aβ (left) and Aβ (right) in the brain of
37
42
mice treated acutely with a single oral dose of 47 (30 mg/kg). Data represent the mean ± SEM of 5ꢀ8
animals per group, expressed as % vehicle.
Conclusions.
From initial amide designs based on 1, we have pursued molecules with the potential to alter the
course of FAD in an effort to prevent, or at least delay, the impending cognitive decline. We have foꢀ
cused our efforts with a keen eye to overcome the shortcoming of previous GSMs ꢀ combining the deꢀ
20
sired in vitro potency and in vivo efficacy with good CNS drugꢀlike properties. Through the use of an
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isosteric amide replacement and critical consideration of conformational restriction we have discovered
a very exciting series of oxadiazines that address these critical issues. Oxadiazine 47 possesses very
good potency and efficacy with excellent predicted CNS drugꢀlike properties as measured by low MW
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(389), low lipophilicity (cLogP = 2.8), high CNSꢀMPO score (5.0) and high LLE (4.7) and LELP (7.6)
binding efficiencies. This compound also demonstrates a desirable profile across various measurements
including solubility, permeability, metabolic stability, CYP inhibition, hERG liability, Pꢀgp efflux, genꢀ
otoxicity and PK assays. This class of compounds has provided a new avenue to further develop the
molecules required for proofꢀofꢀmechanism studies in the clinic. Our additional efforts to further imꢀ
prove potency and efficacy while maintaining this excellent CNS drugꢀlike profile will be the subject of
further discussion in due course.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Experimental Section.
General Methods
All reactions were carried out using commercial materials and reagents without further purification unꢀ
less otherwise noted. All reactions were monitored by thin layer chromatography (TLC) on silica gel
plates (Keiselgel 60 F254, Merck), highꢀperformance liquid chromatography (HPLC), liquid chromaꢀ
tography mass spectrometry (LCMS) or ultraꢀperformance liquid chromatography (UPLC). All products
1
1
were characterised by H NMR and mass spectrometry. H NMR data was recorded on a JEOL ECX400
MHz or Bruker Avance II Ultra shield 500 MHz spectrometer. Chemical shifts are expressed in parts
per million values (ppm) and are designated as s (singlet); br s (broad singlet); d (doublet); t (triplet); q
(quartet); quint (quintet) or m (multiplet). Coupling constants (J) are expressed as values in Hertz (Hz).
Optical rotations were taken on a Rudolph Research Analytical; Autopol III automatic polarimeter;
Model A21101 AIII/2W. Flash column chromatography was performed on silica gel using Fluorochem
silica gel LC60A 40ꢀ63 micron and reagent grade solvents as eluent. All compounds submitted for in
vitro testing were >95% purity (HPLC) and those for in vivo testing were >98% purity (HPLC). LCMS
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Journal of Medicinal Chemistry
analysis using LCMS A, standard conditions: XTerra RP18 column, 3.0 x 50 mm, 3.5 ꢁm; Mobile phase
A: H O, mobile phase B: CH CN with 0.1% formic acid (FA). 0ꢀ1 min. isocratic (5% B), 1ꢀ6 min. graꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
3
dient (5ꢀ95% B), 6ꢀ7 min. isocratic (95% B); flow rate: 1 mL/min; LCMS analysis using LCMS B,
standard conditions: Ascentis Express Cꢀ18 column, 50 × 3.0 mm, 2.7 µm; mobile phase: 0.025% Aq
TFA+5% ACN: ACN+5% 0.025% Aq TFA; T/B% 0.01/5, 0.5/5, 3/100, 5/100: flow rate: 1.2 mL/min.
LCMS analysis using LCMS C, standard conditions: XBridge C18 column, 4.6 x 30 mm, 3.5 ꢁm; Moꢀ
bile phase A: H O (10.0 mM NH HCO ), mobile phase B: CH CN. 0.0ꢀ0.2 min. isocratic (5% B), 0.2ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
2
3
2.0 min. gradient (5ꢀ100% B), 3.0ꢀ3.0 min. isocratic (100% B); flow rate: 3.0 mL/min.
(S)-tert-Butyl 4-((4-fluoro-2-methylphenoxy)methyl)-2,2-dimethyloxazolidine-3-carboxy-late (2a):
DIAD (1.70 mL, 8.68 mmol, 1.2 equiv) was added to a solution of triphenylphosphine (2.28 g, 8.68
mmol, 1.2 equiv), (S)ꢀtert-butyl 4ꢀ(hydroxymethyl)ꢀ2,2ꢀdimethyloxazolidineꢀ3ꢀcarboxylate (1.83 g, 7.89
mmol, 1.0 equiv) and 4ꢀfluoroꢀ2ꢀmethylphenol (1.09 g, 8.68 mmol, 1.2 equiv) in THF (20 mL) at RT.
The resulting mixture was then stirred for 18 h at 80 °C. The reaction mixture was diluted with EtOAc
and the organic layer was washed with 1 N aqueous NaOH and brine, dried over MgSO , filtered and
4
concentrated. The residue was purified by normal phase chromatography on silica (0ꢀ20% EtOAc / hexꢀ
anes) to afford the desired product (1.90 g, 70%) as a colourless oil. LCMS (ES+) [MꢀBoc+H]+: 240.4.
(S)-tert-Butyl
4-((4-chloro-2-(trifluoromethyl)phenoxy)methyl)-2,2-dimethyloxazolidine-3-
carboxylate (2b): DIAD (0.46 mL, 2.35 mmol, 1.1 equiv) was added to a solution of triꢀ
phenylphosphine (616 mg, 2.35 mmol, 1.1 equiv), (S)ꢀtert-butyl 4ꢀ(hydroxymethyl)ꢀ2,2ꢀ
dimethyloxazolidineꢀ3ꢀcarboxylate (495 mg, 2.14 mmol, 1.0 equiv) and 4ꢀchloroꢀ2ꢀ
(trifluoromethyl)phenol (462 g, 2.35 mmol, 1.1 equiv) in THF (8 mL) at RT. The resulting mixture was
then stirred for 18 h at 80 °C. The reaction mixture was diluted with EtOAc and the organic layer was
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washed with 1 N aqueous NaOH and brine, dried over MgSO , filtered and concentrated. The residue
4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was purified by normal phase chromatography on silica (0ꢀ20% EtOAc / hexanes) to afford the desired
product (673 mg, 77%) as a colourless oil. LCMS (ES+) [MꢀBoc+H]+: 310.6/312.6.
(R)-3-(4-Fluoro-2-methylphenoxy)-2-(4-methoxybenzylamino)propan-1-ol (3a): A solution of (S)ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
tert-butyl 4ꢀ((4ꢀfluoroꢀ2ꢀmethylphenoxy)methyl)ꢀ2,2ꢀdimethyloxazolidineꢀ3ꢀcarboxylate (2a) (787 mg,
2.32 mmol, 1.0 equiv) in DCM (10 mL) was treated with TFA (5 mL) at RT and the resulting mixture
was stirred for 1 h at the same temperature. The reaction mixture was then concentrated and the residue
was dissolved in THF (10 mL). 1 N aqueous HCl (10 mL) was added and the mixture was stirred for 18
h at RT. The reaction mixture was diluted with Et O (50 mL) and extracted twice with 1 N aqueous
2
HCl. The aqueous phases were combined, concentrated to dryness and coꢀevaporated twice with MeOH
to give the desired ammonium salt (516 mg, 96%) as a white solid. LCMS (ES+) [M+H]+:200.3. A soꢀ
lution of (R)ꢀ2ꢀaminoꢀ3ꢀ(4ꢀfluoroꢀ2ꢀmethylphenoxy)propanꢀ1ꢀol hydrochloride (515 mg, 2.18 mol, 1.0
equiv) in DCM (12 mL) at ambient temperature was treated successively with triethylamine (0.61 mL,
4.36 mmol, 2.0 equiv), MgSO (1.00 g) and pꢀanisaldehyde (0.27 mL, 2.18 mmol, 1.0 equiv). The reꢀ
4
sulting suspension was stirred for 16 h at ambient temperature and filtered through a pad of celite which
was washed with EtOAc. The filtrate was concentrated under vacuum to afford the corresponding imine
intermediate. This intermediate was dissolved in MeOH (15 mL) and cooled to 0 °C. NaBH (249 mg,
4
3
.54 mmol, 3.0 equiv) was added portion wise over 5 min. The resulting mixture was stirred for 30 min
at 0 °C and then quenched by the slow addition of a saturated aqueous solution of NaHCO . Water and
3
DCM were added. The layers were separated, and the aqueous layer was extracted with DCM twice.
The combined organic layers were dried over MgSO , filtered and concentrated to afforded a residue
4
that was purified by normal phase chromatography on silica (0ꢀ6% MeOH / DCM) to afford the desired
1
aminoꢀalcohol (516 mg, 74%) as an offꢀwhite solid. H NMR (500 MHz, CDCl )
δ = 7.27 – 7.24 (m,
3
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Journal of Medicinal Chemistry
2
H), 6.88 – 6.85 (m, 3H), 6.81 (td, J = 8.3, 2.9 Hz, 1H), 6.70 (dd, J = 8.9, 4.5 Hz, 1H), 4.01 (dd, J =
.4, 4.9 Hz, 1H), 3.95 (dd, J = 9.4, 6.1 Hz, 1H), 3.84 (s, 2H), 3.80 (s, 3H), 3.78 (dd, J = 10.9, 4.4 Hz,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
1H), 3.59 (dd, J = 10.9, 5.3 Hz, 1H), 3.14 (dt, J = 10.3, 5.0 Hz, 1H), 2.19 (s, 3H); LCMS (ES+)
[
M+H]+: 320.5.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(R)-3-(4-Chloro-2-(trifluoromethyl)phenoxy)-2-(4-methoxybenzylamino)propan-1-ol (3b): A soluꢀ
tion of (S)ꢀtert-butyl 4ꢀ((4ꢀchloroꢀ2ꢀ(trifluoromethyl)phenoxy)methyl)ꢀ2,2ꢀdimethyloxazolidineꢀ3ꢀ
carboxylate (2b) (528 mg, 1.29 mmol, 1.0 equiv) in DCM (4 mL) was treated with TFA (2 mL) at RT
and the resulting mixture was stirred for 1 h at the same temperature. The reaction mixture was then
concentrated and the residue was dissolved in THF (4 mL). 1 N aqueous HCl (4 mL) was added and the
mixture was stirred for 18 h at RT. The reaction mixture was diluted with Et O (30 mL) and extracted
2
twice with 1 N aqueous HCl. The aqueous phases were combined, concentrated to dryness and coꢀ
evaporated twice with MeOH to give the desired ammonium salt (341 mg, 86%) as a white solid. LCMS
(
ES+) [M+H]+:270.4/272.4. A solution of (R)ꢀ2ꢀaminoꢀ3ꢀ(4ꢀchloroꢀ2ꢀ(trifluoromethyl)phenoxy)propanꢀ
ꢀol hydrochloride (339 mg, 1.01 mol, 1.0 equiv) in DCM (10 mL) at ambient temperature was treated
successively with triethylamine (0.28 mL, 2.02 mmol, 2.0 equiv), MgSO (1.00 g) and pꢀanisaldehyde
1
4
(123 µL, 1.01 mmol, 1.0 equiv). The resulting suspension was stirred for 16 h at RT and filtered through
a pad of celite which was washed with EtOAc. The filtrate was concentrated to afford the corresponding
imine intermediate. This intermediate was dissolved in MeOH (8 mL) and cooled to 0 °C. NaBH4
(112mg, 3.03 mmol, 3.0 equiv) was added portion wise over 5 min. The resulting mixture was stirred
for 30 min at 0 °C and then quenched by the slow addition of a saturated aqueous solution of NaHCO3.
Water and EtOAc were added. The layers were separated, and the aqueous layer was extracted with
EtOAc twice. The combined organic layers were dried over MgSO , filtered and concentrated to affordꢀ
4
ed a residue that was purified by normal phase chromatography on silica (0ꢀ6% MeOH / DCM) to afꢀ
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Page 26 of 63
1
ford the desired aminoꢀalcohol (312 mg, 79%) as an offꢀwhite solid. H NMR (500 MHz, CDCl )
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
6
δ
1
3
= 7.55 (d, J = 2.6 Hz, 1H), 7.43 (dd, J = 8.9, 2.6 Hz, 1H), 7.27 – 7.22 (m, 2H), 6.90 (d, J = 8.8 Hz,
H), 6.88 – 6.84 (m, 2H), 4.11 (dd, J = 9.2, 4.6 Hz, 1H), 4.04 (dd, J = 9.2, 6.3 Hz, 1H), 3.82 (s, 2H),
.80 (s, 3H), 3.77 (dd, J = 11.1, 4.5 Hz, 1H), 3.59 (dd, J = 11.1, 4.9 Hz, 1H), 3.15 (dq, J = 6.1, 4.7 Hz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1H); LCMS (ES+) [M+H]+: 390.5/392.5.
(R)-4-Bromo-N-(1-(4-fluoro-2-methylphenoxy)-3-hydroxypropan-2-yl)-3-methoxy-N-(4-
methoxybenzyl)benzamide (4a): A solution of 4ꢀbromoꢀ3ꢀmethoxybenzoic acid (411 mg, 1.78 mmol,
1.1 equiv) in DCM (8 mL) at ambient temperature was treated with a catalytic amount of DMF (1 drop)
and oxalyl chloride (0.53 mL, 4.86 mmol, 3.0 equiv). The resulting mixture was stirred at ambient temꢀ
perature for 1 h at which point LCMS monitoring showed completion of the reaction. The mixture was
concentrated to dryness, diluted with anhydrous THF (8 mL), concentrated again and dried under high
vacuum for 1 h. The residue was diluted with anhydrous THF (8 ml), triethylamine (0.45 mL,
3
.23mmol, 2.0 equiv) was added and the resulting mixture was cooled to 0 °C. A solution of (R)ꢀ3ꢀ(4ꢀ
fluoroꢀ2ꢀmethylphenoxy)ꢀ2ꢀ(4ꢀmethoxybenzylamino)propanꢀ1ꢀol (3a) (516 mg, 1.62 mmol, 1.0 equiv)
in THF (6 ml) was quickly added and the resulting mixture was stirred at 0 °C for 30 min. A saturated
aqueous solution of NaHCO and EtOAc were then successively added. The layers were separated and
3
the aqueous layer was extracted with EtOAc twice. The combined organic layers were dried over
MgSO , filtered and concentrated. The residue was purified by normal phase chromatography on silica
4
(0ꢀ100% EtOAc / hexanes) to afford the desired amide (640 mg, 59%) as a white foam. LCMS (ES+)
[
M+H]+: 532.6/534.6.
(R)-4-Bromo-N-(1-(4-chloro-2-(trifluoromethyl)phenoxy)-3-hydroxypropan-2-yl)-3-methoxy-N-(4-
methoxybenzyl)benzamide (4b): A solution of 4ꢀbromoꢀ3ꢀmethoxybenzoic acid (1.52 g, 6.58 mmol,
1
.1 equiv) in DCM (30 mL) at RT was treated with a catalytic amount of DMF (1 drop) and oxalyl chloꢀ
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Page 27 of 63
Journal of Medicinal Chemistry
ride (1.70 mL, 19.7 mmol, 3.3 equiv). The resulting mixture was stirred at RT for 1 h at which point
LCMS monitoring showed completion of the reaction. The mixture was concentrated to dryness, diluted
with anhydrous THF (15 mL), concentrated again and dried under high vacuum for 1 h. The residue was
diluted with anhydrous THF (30 ml), triethylamine (2.50 mL, 17.9 mmol, 3.0 equiv) was added and the
resulting mixture was cooled to 0 °C. A solution of (R)ꢀ3ꢀ(4ꢀchloroꢀ2ꢀ(trifluoromethyl)phenoxy)ꢀ2ꢀ(4ꢀ
methoxybenzylamiꢀno)propanꢀ1ꢀol (3b) (2.33 g, 5.98 mmol, 1.0 equiv) in THF (15 ml) was quickly
added and the resulting mixture was stirred at 0 °C for 30 min. A saturated aqueous solution of NaHCO3
and EtOAc were then successively added. The layers were separated and the aqueous layer was extractꢀ
1
2
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7
8
9
1
1
1
1
1
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4
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5
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6
0
1
2
3
4
5
6
7
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9
0
1
2
3
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8
9
0
1
2
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4
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9
0
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8
9
0
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2
3
4
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7
8
9
0
ed with EtOAc twice. The combined organic layers were dried over MgSO , filtered and concentrated.
4
The residue was purified by normal phase chromatography on silica (0ꢀ100% EtOAc / hexanes) to afꢀ
ford the desired amide (3.44 g, 95%) as a white foam. LCMS (ES+) [M+H]+: 602.4/604.4/606.4.
(S)-N-(1-(Aminooxy)-3-(4-fluoro-2-methylphenoxy)propan-2-yl)-4-bromo-3-methoxy-N-(4-
methoxybenzyl)benzamide (5a): DIAD (0.28 mL, 1.44 mmol, 1.2 equiv) was added to a solution of
triphenylphosphine (377 mg, 1.44 mmol, 1.2 equiv), (R)ꢀ4ꢀbromoꢀNꢀ(1ꢀ(4ꢀfluoroꢀ2ꢀmethylphenoxy)ꢀ3ꢀ
hydroxypropanꢀ2ꢀyl)ꢀ3ꢀmethoxyꢀNꢀ(4ꢀmethoxybenzyl)benꢀzamide (4a) (640 mg, 1.20 mmol, 1.0 equiv)
and N-hydroxyphtalimide (235 mg, 1.44 mmol, 1.2 equiv) in THF (10 mL) at 0 °C. The resulting mixꢀ
ture was kept at 0 °C for 1 h, then warmed to ambient temperature and stirred for 16 h. The reaction
mixture was diluted with EtOAc and the organic layer was washed with 1 N aqueous NaOH (twice),
water, and brine, dried over MgSO , filtered, and concentrated. The residue was purified by normal
4
phase chromatography on silica (0ꢀ50% EtOAc / hexanes) to afford the desired product (778 mg, 96%)
as a white solid. LCMS (ES+) [M+H]+: 677.6/679.6. A suspension of (S)ꢀ4ꢀbromoꢀNꢀ(1ꢀ(1,3ꢀ
dioxoisoindolinꢀ2ꢀyloxy)ꢀ3ꢀ(4ꢀfluoroꢀ2ꢀmethylphenoxy)propanꢀ2ꢀyl)ꢀ3ꢀmethoxyꢀNꢀ(4ꢀ
methoxybenzyl)benzamide (778 mg, 1.15mol, 1.0 equiv) in ethanol (90%, 12 mL) and THF (6 mL) at
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Page 28 of 63
ambient temperature was treated with hydrazine hydrate (50ꢀ60%, 1.2 mL). The resulting mixture was
stirred for 30 min, then water was added. The mixture was extracted with EtOAc (three times) and the
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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6
combined organic layers were washed with water and brine, dried over MgSO , filtered, and concentratꢀ
4
ed. The residue was purified by normal phase chromatography on silica (0ꢀ100% EtOAc / DCM) to afꢀ
ford the desired product (560 mg, 89%) as a white solid. LCMS (ES+) [M+H]+: 547.6/549.6.
0
1
2
3
4
5
6
7
8
9
0
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2
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0
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0
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9
0
(
S)-N-(1-(Aminooxy)-3-(4-chloro-2-(trifluoromethyl)phenoxy)propan-2-yl)-4-bromo-3-methoxy-N-
4-methoxybenzyl)benzamide (5b): DIAD (1.35 mL, 6.85 mmol, 1.2 equiv) was added to a solution of
(
triphenylphosphine
(1.80
trifluoromethyl)phenoxy)ꢀ3ꢀhydroxypropanꢀ2ꢀyl)ꢀ3ꢀmethoxyꢀNꢀ(4ꢀmethoxybenzyl)benzamide
3.44 g, 5.71 mmol, 1.0 equiv) and N-hydroxyphtalimide (1.12 g, 6.85 mmol, 1.2 equiv) in THF (30mL)
g,
6.85
mmol,
1.2
equiv),
(R)ꢀ4ꢀbromoꢀNꢀ(1ꢀ(4ꢀchloroꢀ2ꢀ
(
(
(4b)
at 0 °C. The resulting mixture was kept at 0 °C for 1 h, then warmed to RT and stirred for 16 h. The reꢀ
action mixture was diluted with EtOAc and the organic layer was washed with 1 N aqueous NaOH
(
twice), water and brine, dried over MgSO , filtered and concentrated. The residue was purified by norꢀ
4
mal phase chromatography on silica (0ꢀ60% EtOAc / hexanes) to afford the desired product (3.94 g,
92%) as a white solid. LCMS (ES+) [M+H]+: 747.4/749.4/751.3. A suspension of (S)ꢀ4ꢀbromoꢀNꢀ(1ꢀ(4ꢀ
chloroꢀ2ꢀ(trifluoromethyl)phenoxy)ꢀ3ꢀ(1,3ꢀdioxoisoindolinꢀ2ꢀyloxy)propanꢀ2ꢀyl)ꢀ3ꢀmethoxyꢀNꢀ(4ꢀ
methoxyꢀbenzyl)benzamide (3.94 g, 5.27 mol, 1.0 equiv) in ethanol (90%, 20 mL) and THF (10 mL) at
RT was treated with hydrazine hydrate (50ꢀ60%, 4.0 mL). The resulting mixture was stirred for 30 min
and then water was added. The mixture was extracted with EtOAc (three times), the combined organic
layers were washed with water and brine, dried over MgSO , filtered and concentrated to afford the deꢀ
4
sired product (3.25 g, 99%) as a white solid. LCMS (ES+) [M+H]+: 617.5/619.5/621.5.
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Journal of Medicinal Chemistry
1
2
3
4
5
(
S)-3-(4-Bromo-3-methoxyphenyl)-5-((4-fluoro-2-methylphenoxy)methyl)-4-(4-methoxy-benzyl)-
,6-dihydro-4H-1,2,4-oxadiazine (6a): A sealable vial was charged with (S)ꢀNꢀ(1ꢀ(aminooxy)ꢀ3ꢀ(4ꢀ
fluoroꢀ2ꢀmethylphenoxy)propanꢀ2ꢀyl)ꢀ4ꢀbromoꢀ3ꢀmethoxyꢀNꢀ(4ꢀmethoxybenꢀzyl)benzamide (5a)
560 mg, 1.02 mmol, 1.0 equiv) and iPrOH (10.0 ml). P O (1.45 g, 10.2 mmol, 10.0 equiv) was added
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
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0
1
2
3
4
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6
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8
9
0
1
2
3
4
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6
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8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
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6
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8
9
0
1
2
3
4
5
6
7
8
9
0
(
2
5
at ambient temperature. The vial was sealed and the reaction was stirred at 80 °C for 16 h. The reaction
mixture was cooled to ambient temperature and diluted with EtOAc. A saturated aqueous solution of
NaHCO was added, the layers were separated and the aqueous phase was extracted with EtOAc. The
3
organic phases were combined, washed with brine, dried over MgSO , filtered and concentrated. The
4
residue was purified by normal phase chromatography on silica (0ꢀ50% EtOAc / hexanes) to afford the
1
desired product (241 mg, 45%) as a white solid. H NMR (500 MHz, CDCl )
δ = 7.58 (d, J = 8.1 Hz,
3
1H), 7.11 – 7.08 (m, 2H), 7.07 (d, J = 1.8 Hz, 1H), 7.03 (dd, J = 8.1, 1.9 Hz, 1H), 6.89 – 6.85 (m, 3H),
6
4
2
.79 (td, J = 8.3, 2.7 Hz, 1H), 6.65 (dd, J = 8.9, 4.5 Hz, 1H), 4.39 – 4.35 (m, 2H), 4.23 – 4.20 (m, 1H),
.09 – 4.00 (m , 2H), 3.89 (s, 3H), 3.81 (s, 3H), 3.74 – 3.70 (m, 1H), 3.58 (dd, J = 10.9, 2.5 Hz, 1H),
.21 (s, 3H); LCMS (ES+) [M+H]+: 529.5/531.5.
(S)-3-(4-Bromo-3-methoxyphenyl)-5-((4-chloro-2-(trifluoromethyl)phenoxy)methyl)-4-(4-
methoxybenzyl)-5,6-dihydro-4H-1,2,4-oxadiazine (6b): A sealable vial was charged with (S)ꢀNꢀ(1ꢀ
(aminooxy)ꢀ3ꢀ(4ꢀchloroꢀ2ꢀ(trifluoromethyl)phenoxy)propanꢀ2ꢀyl)ꢀ4ꢀbromoꢀ3ꢀmethoxyꢀNꢀ(4ꢀ
methoxybenzyl)benzamide (5b) (1.00 g, 1.62 mmol, 1.0 equiv) and IPA (15 ml). P O (2.30 g,
2
5
1
6.2 mmol, 10.0 equiv) was added at RT. The vial was sealed and the reaction was stirred at 100 °C for
30 h. The reaction mixture was cooled to RT and diluted with EtOAc. A saturated aqueous solution of
NaHCO was added, the layers were separated and the aqueous phase was extracted with EtOAc. The
3
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