Note: Descriptions are shown in the official language in which they were submitted.
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CRYSTALLINE FORM OF
2-(2,6-DICHLOROPHENYL)-1-1(1S,3R)-3-(HI'DROXYMETHYL)-5-(3-HYDROXY-3-
METHYLBUTYL)-1-METHYL-
3,4-DIHYDROISOQUINOLIN-2(1H)-YLIETHANONE FOR THE TREATMENT OF PARKINSON'S
DISEASE
The present invention provides a crystalline form of 2-(2,6-dichloropheny1)-1-
[(1S,3R)-3-(hydroxymethyl)-543-hydroxy-3-methylbuty1)-1-methyl-3,4-
dihydroisoquinolin-2(1H)-yl]ethanone, pharmaceutical compositions thereof,
methods of
using the same, and processes for preparing the same.
The dopaminergic pathway is associated with a number of disorders including
Parkinson's disease, schizophrenia and attention deficit-hyperactivity
disorder (ADHD).
Parkinson's disease is a chronic, progressive, neurodegenerative disorder
characterized by
the loss of dopaminergic neurons in the brain. Parkinson's disease manifests
in resting
tremor along with other motor syinptoms (e.g. akinesia and bradykinesia) and
non-motor
symptoms (e.g. cognitive impairment, sleep disorders, and depression). Current
therapy
for treatment of Parkinson's disease includes dopamine precursors such as
levodopa, and
dopamine agonists such pramipexole. Such direct acting dopamine therapies are
limited
in effectiveness due in part to high dose associated cognition impairment,
seizure risk (as
shown in rodents), and tolerance development. There remains a significant
unmet need
for safe and effective treatment of Parkinson's disease.
Allosterie modulators are agents that remotely alter the interactions of
ligands
with their receptors by modifying the ligand-binding environment. An example
of this
type of modulation is when the binding of a modulator to an allosteric
(secondary) site
produces a conformational change in the receptor protein that is transmitted
to the ligands
orthosteric (primary) binding site. The quality of the allosteric effect is
said to be positive
if the modulator facilitates or potentiates an interaction or negative if it
inhibits an
interaction of the ligand with the orthosteric binding site.
Compounds of the present invention are positive allosteric modulators (PAM) of
the dopamine 1 receptor (D1). As such, compounds of the present invention are
useful
for the treatment of conditions in which D1 plays a role such as Parkinson's
disease and
schizophrenia, including relief of associated symptoms such as mild cognitive
impairment. Compounds of the present invention are also believed to be useful
in
treating symptoms of Alzheimer's disease such as cognitive impairment.
Compounds of
the present invention are also useful in improving motor symptoms in
Parkinson's disease
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as a monotherapy. As well, compounds of present invention are believed useful
in
treating depression and attention deficit-hyperactivity disorder (ADHD).
The present invention provides a new crystalline form of the compound, 242,6-
dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-
methyl-
3,4-dihydroisoquinolin-2(1H)-yl]ethanone, which is a positive allosteric
modulator
(PAM) of the dopamine 1 receptor (D1) and, as such, is useful in treatment of
the
disorders discussed above. A crystalline form of 2-(2,6-dichloropheny1)-1-
[(1S,3R)-3-
(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-
2(1H)-
yflethanone is believed to provide advantages in manufacturing and development
of the
compound itself as well as related compounds such as cocrystalline form of a
composition comprising 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-
(3-
hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-yl]ethanone and
4-
hydroxybenzoic acid.
U.S. Patent No. 8962654 discloses certain 3,4-dihydroisoquinolin-2(1H)-y1
compounds as positive allosteric modulators (PAM) of the dopamine 1 receptor
(D1)
including 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-
methylbutyl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl]ethanone and a
cocrystalline
form of a composition comprising 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-
(hydroxymethyl)-
5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihyciroisoquinolin-2(111)-
yl]ethanone and 4-
hydroxybenzoic acid.
The present invention provides a crystalline form of 2-(2,6-dichloropheny0-1-
[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-
dihydroisoquinolin-2(1H)-yl]ethanone.
Further, the present invention provides a crystalline form of 2-(2,6-
dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-
methyl-
3,4-dihydroisoquinolin-2(1H)-yliethanone characterized by an X-ray powder
diffraction
pattern using CuKa radiation having a diffraction peak at diffraction angle 2-
theta of
9.15 in combination with one or more peaks selected from the group consisting
of 19.4 ,
16.1 , and 16.6 ; with a tolerance for the diffraction angles of 0.2 degrees.
Further, the present invention provides a pharmaceutical composition
comprising
a crystalline form of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-
hydroxy-
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3-methylbuty1)-1-methy1-3,4-dihydroisoquinolin-2(11-/)-yflethanone, and a
pharmaceutically acceptable carrier, diluent or excipient.
Further, the present invention provides a method of treating Parkinson's
disease
comprising administrating to a patient in need thereof an effective amount of
a crystalline
form of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-
methylbutyl)-1-methyl-3,4-dihydroisoquinolin-2(111)-yliethanone.
Further, the present invention provides a method of treating mild cognitive
impairment associated with Parkinson's disease comprising administrating to a
patient in
need thereof an effective amount of a crystalline form of 2-(2,6-
dichloropheny1)-1-
[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-
dihydroisoquinolin-2(1H)-yflethanone.
Further, the present invention provides a crystalline form of 242,6-
dichloropheny1)-1-[(1S,3R)-3-(1iydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-
methyl-
3,4-dihydroisoquinolin-2(111)-yflethanone for use in therapy.
Further, the present invention provides a crystalline form of 242,6-
dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-
methyl-
3,4-dihydroisoquinolin-2(111)-yflethanone characterized by an X-ray powder
diffraction
pattern using CuKa radiation having a diffraction peak at diffraction angle 2-
theta of
9.15 in combination with one or more peaks selected from the group consisting
of 19.4 ,
16.1 , and 16.6 ; with a tolerance for the diffraction angles of 0.2 degrees
for use in
therapy.
Further, the present invention provides a crystalline form of 242,6-
dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-
methyl-
3,4-dihydroisoquinolin-2(111)-yflethanone for use in the treatment of
Parkinson's disease.
Further, the present invention provides the use of a crystalline form of 2-
(2,6-
dichloropheriy1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)- I-
methyl -
3,4-dihydroisoquinolin-2(1H)-yflethanone for the manufacture of a medicament
for
Parkinson's disease.
Compound 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-
3-methylbuty1)-1-methy1-3,4-dihydroisoquinolin-2(111)-yflethanone is
represented by
formula la.
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H
OH
010 CI
0 CI lel
La
A compound of formula la may be used in combination with other drugs that are
used in the treatment/prevention/suppression or amelioration of the diseases
or conditions
for which compounds of formula la are useful including Parkinson's disease and
schizophrenia. Such other drug(s) may be administered, by a route and in an
amount
commonly used therefore, contemporaneously or sequentially with a compound of
formula Ia. When a compound of formula Ia is used contemporaneously with one
or
more other drugs, a pharmaceutical unit dosage form containing such other
drugs in
addition to the compound of formula Ia is preferred. Accordingly, the
pharmaceutical
compositions of the present invention include those that also contain one or
more other
active ingredients, in addition to a compound of formula la. Examples of other
active
ingredients effective in the treatment of Parkinson's disease that may be
combined with a
compound of formula Ia, either administered separately or in the same
pharmaceutical
composition include, but are not limited to:
(a) dopamine precursors such as levodopa; melevodopa, and etilevodopa; and
(b) dopamine agonists including pramipexole, ropinirole, apomorphine,
rotigotine,
bromocriptine, cabergoline, and pergolide.
It is understood that compounds of the present invention may exist as
stereoisomers. Embodiments of the present invention include all enantiomers,
diastereomers, and mixtures thereof. Preferred embodiments are single
diastereomers,
and more preferred embodiments are single enantiomers.
As used herein, the term "cocrystal" refers to a multiple component
crystalline
solid form comprising two compounds where the association of compounds is
primarily
through non-covalent and non-ionic chemical interactions such as hydrogen
bonding. In
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the pharmaceutical arts, a cocrystal typically comprises a first compound
which is an
active pharmaceutical ingredient and a second compound which is referred to as
a guest
compound or coformer. A cocrystal may be distinguished from a crystalline salt
form in
that the first compound remains essentially uncharged or neutral. A cocrystal
may be
distinguished from a crystalline hydrate or solvate form in that the guest
compound is not
exclusively water or a solvent. A preferred cocrystal is a stable form having
a suitable
melting point.
As used herein, the term "patient" refers to an animal such as a mammal and
includes a human. A human is a preferred patient.
It is also recognized that one skilled in the art may treat Parkinson's
disease by
administering to a patient presently displaying symptoms an effective amount
of the
compound of formula Ia. Thus, the terms "treatment" and "treating" are
intended to refer
to all processes wherein there may be a slowing, interrupting, arresting,
controlling, or
stopping of the progression of an existing disorder and/or symptoms thereof,
but does not
necessarily indicate a total elimination of all symptoms.
It is also recognized that one skilled in the art may treat Parkinson's
disease by
administering to a patient at risk of future symptoms an effective amount of
the
compound of formula la and is intended to include prophylactic treatment of
such.
As used herein, the term "effective amount" of a compound of formula Ia refers
to
an amount that is a dosage, which is effective in treating a disorder, such as
Parkinson 's
disease described herein. The attending diagnostician, as one skilled in the
art, can
readily determine an effective amount by the use of conventional techniques
and by
observing results obtained under analogous circumstances. In determining an
effective
amount or dose of a compound of formula la, a number of factors are
considered,
including, but not limited to the compound of formula la to be administered;
the co-
administration of other agents, if used; the species of mammal; its size, age,
and general
health; the degree of involvement or the severity of the disorder, such as
Parkinson's
disease; the response of the individual patient; the mode of administration;
the
bioavailability characteristics of the preparation administered; the dose
regimen selected;
the use of other concomitant medication; and other relevant circumstances.
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A compound of formula la can be administered alone or in the form of a
pharmaceutical composition with pharmaceutically acceptable carriers, diluents
or
excipients. Such pharmaceutical compositions and processes for making the same
are
known in the art (See, e.g., Remington: The Science and Practice of Phaxmacy,
D.B.
Troy, Editor, 21st Edition., Lippincott, Williams & Wilkins, 2006).
Human D1 Receptor Positive Allosteric Modulation Assay
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HEK293 cells stably expressing the human D1 receptor are generated via
retroviral gene transduction using the pBABE-bleo vector and the Phoenix
retroviral
system. The cells are gown in DMEM/F12 (Gibco) supplemented with 10% calf
serum,
20mM HEPES, 2mM glutamate, and 1504mlzeocin at 37C in 5% CO2. At
approximately 80% confluency, the cells are harvested using 0.25%
trypsin/EDTA,
suspended in FBS plus 8% DMSO, and stored in liquid nitrogen. On the day of
the assay,
cells are thawed and re-suspended in ST1M buffer (Hanks Balanced Salt Solution
supplemented with 0.1% BSA, 20mM HEPES, 20011M IBMX, and 1001.1,M ascorbic
acid). A test compound is serially diluted (1:3) in DMSO and then further
diluted 1:40
into STIM buffer containing 2X an EC20 concentration of dopamine. An EC20
concentration of dopamine is defined as the concentration that increases
cyclic AMP up
to 20% of the maximum amount that can be induced by dopamine; in this assay,
the
EC. is 5p,M, and the EC20 is generally 12nM. Twenty-five pl of this solution
is mixed
with 25111 of cell suspension (1,250 cells) and dispensed into each well of 96-
well, half-
area plates; the fmal DMSO concentration is 1.25%. Plates are incubated at
250C for 60
min. cAMP production is quantified using HTRF detection (CisbioTM) as per
vendor
instructions: lysis buffer containing anti-cAMP cryptate and D2-conjugate
(25111 of each)
are added to the wells, plates are incubated for another 60-90 min, and
fluorescence is
detected using an EnVision plate reader (PerkinElmerru). Data are converted to
cAMP
concentrations using a cAMP standard curve, and analyzed as an absolute EC50
using a
4-parameter nonlinear logistic equation (AbaseTM v5.3.1.22). The absolute EC50
for a
positive allosteric modulator is calculated as the concentration generating a
half-maximal
amount of cAMP, based on a window ranging from the dopamine EC20, which
defines the
minimum response, to the EC. response, defined by the addition of 5 M
dopamine.
In the above assay, the compound of Reference Compound 1 exhibits an absolute
EC50 ' of 4 59 + 0.96 nM (SEM; n=7) with a maximum response of 79.7 +
6.2 % at the
human D1 receptor. The cocrystal of Reference Compound 2 exhibits an absolute
EC50
of 1.11 -4- 0.11 nM (SEM; n=2) at the human D1 receptor. These data
demonstrate the
compound of Reference Compound 1 is a positive allosteric modulator of the
human Dl
receptor.
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Anti-Parkinson effects of compounds of the invention can be determined using
procedures well known in the art such as animal models of locomotor activity.
For
example, the compound of Reference Compound 1 shows effects on basal
(habituated)
locomotor activity and on reserpine-induced akinesia in humanized dopamine 1
receptor
(D1) knock-in mice.
Generation of Human 1)1 Receptor Knock-in Mouse
A transgenic mouse in which the inurine dopamine 1 (D1) receptor is replaced
by
its human counterpart may be generated by standard techniques. For example,
mouse
genomic fragments are subcloned from the RP23 bacterial artificial chromosome
library
and recloned into a PGK-neo targeting vector. The mouse open reading frame is
replaced
with the human DI receptor open reading frame in exon 2. A neo selection
marker
upstream of exon 2 is flanked by frt sites for later removal. The flanking of
exon 2 by
loxP selection sites allows for the option to generate D1 knock-out mice by
crossing with
mice expressing the cre nuclease gene.
The C57BL/6 N embryonic stem cell line B6-3 is grown on a mitotically
inactivated feeder layer of mouse embryonic fibroblasts in high-glucose DMEM
with
20% fetal bovine serum and 2x106 unit/1 leukemia inhibitory factor. Ten
million
embryonic stem cells plus 30 micrograms of linearized vector DNA are
electroporated
and subjected to 0418 selection (200 micrograms/ml). Clones are isolated and
analyzed
by Southern blotting.
A clone containing the expected size insert is inserted into blastocysts and
the
resulting mice are genotyped by PCR. A male chimera is crossed with a female
containing the Flp nuclease gene to eliminate the selection marker. Progeny
containing
the human D1 receptor without the selection marker are identified by PCR. A
male
heterozygote is mated with female C57BL/6 mice. Male and female progeny
containing
the human D1 receptor are mated and homozygotes are identified by PCR.
Behavior and
reproduction of the homozygotes is normal, and the colony is maintained in the
homozygote state for succeeding generations.
Basal (habituated) Locomotor Activity
Locomotor activity is measured using an automated system to track movement in
mice. Human D1 receptor knock-in mice are placed in chambers and allowed to
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habituate to the chambers for 60 mins. During this time, they show reduced
locomotion
over time. Following administration of a compound of the invention, animal
movement
is increased in a dose-dependent fashion.
More specifically, locomotor activity boxes are situated in rectangular frames
with
infrared beams for measurement of motor activity (horizontal and vertical
activity) called
ambulations. Locomotor activity is recorded between time of 7:30 and 15:00
hours.
Mice are randomly assigned to treatment groups as shown in Table 1. Each
mouse is placed individually into one of the locomotor activity boxes for 60
minutes
habituation. Mice are then dosed orally and total number of ambulations is
recorded per
10 minutes for each mouse over a 60 minutes period. In mice with reserpine
pretreatment,
no previous habituation period is included. Thus, immediately after dosing,
the total
number of ambulations is measured for 60 minutes. Data is transferred from the
software/computers to spreadsheets for further analysis. Statistical analysis
is carried out
using one-way ANOVA followed by post-hoc analysis using Fishers' LSD or
Dunnett's
test.
In the basal (habituated) locomotor assay, Reference Compound 1 facilitates
movement in mice in a dose responsive manner (Table 1). This demonstrates
Reference
Compound 1 is effective in locomotor activation of animals that are habituated
to the
environment.
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Table 1
Basal Locomotor Activity
(Total Ambulations for 60
Test Compound
min.)
(Means, SEM, %SE)
Means 542
vehicle SEM 111
%SE 30
Reference Compound 1 Means 542
SEM 52
(1 mg/kg)
%SE 10
Reference Compound 1 Means 1118 *
SEM 289
(3 mg/kg)
%SE 26
Reference Compound 1 Means 1818 ** @
SEM 392
(6 mg/kg)
%SE 22
Reference Compound 1 Means 3047 *** @
SEM 306
(10 mg/kg)
%SE 10
Reference Compound 1 Means 4623 *** @
SEM 486
(30 mg/kg)
%SE 11
**p<0.01, ***p<0.001 compared to vehicle
(unpaired t-test)
@ p<0.0I compared to vehicle
One-way ANOVA Dunnett's Multiple Comparison Test
Reversal of Reserpine-Induced Akinesia
Reserpine is a catecholamine depleting agent (depletes dopamine and
noradrenalin) and 18-24 hours after treatment mice become akinetic and have
reduced
locomotor activity counts. Reserpine-induced akinesia is assessed by measuring
the
effect of compounds on locomotor activity approximately! 8-24 hours after a
single dose
of 0.15 or 0.3 mg/kg reserpine subcutaneous. The equipment used is the same as
that
used for habituated locomotor activity as previously discussed.
Male humanized dopamine DI receptor knock-in mice are randomly assigned to
treatment groups as shown in Table 2. Each mouse is placed individually into
one of the
locomotor activity boxes. Ambulations per 10 minutes for each mouse is
measured for up
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to 60 mins after dosing. Thus, effects on reserpine-induced exploratory
behavior is
assessed for a total of 60 minutes. Data is transferred from the
software/computers to
spreadsheets for further analysis. Statistical analysis is carried out using
one-way
ANOVA followed by post-hoc analysis using t-test.
In the above assay, the compound of Reference Compound 1 reverses the effects
of reserpine treatment and restores movement in mice in a dose responsive
manner (Table
2). This demonstrates that the compound of Reference Compound 1 is effective
in an in
vivo model of Parkinson's disease.
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Table 2
Locomotor Activity
(Total Ambulations for 60
Test Compound
min.)
(Means, SEM, %SE)
Vehicle Control Means 1629
SEM 188
(no reserpine)
%SE 12
Vehicle + Reserpine Means 1336
SEM 191
(0.15 mg/kg)
%SE 14
Vehicle + Reserpine Means 640
SEM 61
(0.3 mg/kg)
%SE 9
Reference Compound 1
Means 4623 ***
(10 mg/kg) + Reserpine SEM 486
(0.15 mg/kg) %SE 11
Reference Compound 1
Means 6222 ***
(30 mg/kg) + Reserpine SEM 659
(0.15 mg/kg) %SE 11
Reference Compound 1
Means 4056 ***
(30 mg/kg) + Reserpine SEM 548
(0.3 mg/kg) %SE 13
*p<0.05, **p<0.01, ***p<0.001 compared to vehicle
(unpaired t-test)
@ p<0.01 compared to vehicle
One-way ANOVA Dunnett's Multiple Comparison Test
Compounds of formula la may be prepared by processes known in the chemical
art or by a novel process described herein. A process for the preparation of a
compound
of formula Ia and novel intermediates for the manufacture of a compound of
formula Ia,
provide further features of the invention and are illustrated by the following
procedures.
Generally, a compound of formula Ia may be prepared from a compound of
formula H where Pgl represents a suitable hydroxyl protecting group (Scheme
1).
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Particular values of Pgl include tert-butyl(dimethypsily1 and tert-
butyl(diphenyl)silyl.
More specifically, a compound of formula II where Pgl is tert-
butyl(dimethyl)sily1 is
reacted with tetrabutylanunonium fluoride in a solvent such as
tetrahydrofitran to provide
a compound of formula Ia. A compound of formula II where Pgl is
tert-butyl(dimethyl)sily1 may be prepared by reacting a compound of formula
III with a
methyl lithium in a suitable solvent. Suitable solvents include
tetrahydrofuran. A
compound of formula III where Pgl is tert-butyl(dimethypsily1 may be prepared
by
reacting a compound of formula IV with hydrogen in the presence of a suitable
transition
metal catalyst such as 1,1'-bis(di-i-propylphosphino)ferrocene(1,5-
cyclooctadiene)rhodium(1) tetrafluoroborate. The reaction is conveniently
carried out in a
solvent such as methanol. A compound of formula IV where Pgl is
tert-butyl(dimethyl)sily1 may be prepared by reacting a compound of formula V
with
ethyl acrylate in the presence of a transition metal coupling catalyst such as
palladium
acetate, a ligand such as tri-o-tolylphosphine and a base such as
triethyamine. The
reaction is conveniently carried out in a solvent such as acetonitrile. A
compound of
formula V may be prepared by acylating a compound of formula VIII with 2,6-
dichlorophenylacetic acid in the presence of an activating agent such as
benzotriazol- 1 -yl-
oxytripyrrolidinophosphonium hexafluorophosphate and a base such as
triethylamine.
The reaction is conveniently carried out in a solvent such as ditnethyl form
amide.
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Scheme I
OH OH
0"Pg1 OH
.õ1
41i ; CI deprotection
õ.
41I N;*s CI
0 I *
CI 0CI I401
II la
MeLi
TIE
00 00
0-Pgi catalyst 0-Pg1
H2, Me011 õ1
,õ
Ott ; 010 N.. CI
0
ci 0CI 00
III Iv
A
ethyl acrylate
catalyst
Et3N, CH3CN
Br O'Pg1 Br 0-Pg1
2,6-diClphenylacetic acid
..= =activating reagent, DMF c,
NH
o
Ci
VI II V
Alternatively, a compound of formula 11 may be prepared from a compound of
formula VI where Pgl is a suitable hydroxyl protecting group (Scheme 2). More
specifically, a compound of formula VI where PgI is tert-butyl(dirnethypsily1
is acylated
with 2,6-dichlorophenylacetic acid in the presence of an activating agent such
as
1,1'-carbonyldiimidazole to provide a compound of formula II. The reaction is
conveniently carried out in a solvent such as tetrahydrofuran. A compound of
formula VI
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may be prepared by reducing a compound of formula VII with hydrogen in the
presence
of a catalyst such as palladium. The reaction is conveniently carried out in a
solvent such
as ethanol. A compound of formula VII may be prepared by coupling a compound
of
formula VIII with 2-methylbut-3-en-2-ol in the presence of a catalyst such as
palladium
acetate, a ligand such as 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl and
a base
such potassium carbonate. The reaction is conveniently carried out in a
solvent such as
dimethylformamide. A compound of formula VIII made be prepared in a multistep
fashion from a compound of formula X through an intermediate compound of
formula TX
as described in the Preparations and Examples. A compound of formula X may be
prepared as described in the Preparations and Examples.
Scheme 2
OH OH
o-Pgi 5% Pd/C Pgi
Et0H I 2,6-diClphenylacetic acid
1,1'-carbonyldiimidazole, THF
NH SI NH
VII VI
2-inethylbut-2-en-3-ol
catalyst
DMF
Pgi
Br Br Br 0
õI
OH
00 NH op NH miti NH
VIII ix X
As used herein, "DMSO" refers to dimethylsulfoxide; "Tris" refers to
trishydrox ymethylaminomethane; "DTT" refers to dithiothreitol; "HEC" refers
to
hydroxycthyl cellulose; "DMEM" refers to Dulbecco's Modified Eagle's Medium;
"MS"
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refers to mass spectrum; and "1-IF,PS" refers to 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid.
Preparation 1
Synthesis of methyl 2-bromo-D-phenylalaninate hydrochloride.
Br 0
4110 = ''"
N H2
HCI
Dissolve 2-bromo-D-phenylalanine (22.4 g, 91.8 mmol) in methanol (459 mL).
Add acetyl chloride (65.3 mL, 917.7 mmol) at room temperature. Stir for 36
hours.
Concentrate under reduced pressure to give the title compound (27.2 g, 92.3
mmol). MS
(nth): 258 (M+1).
Alternative synthesis of methyl 2-bromo-D-phenyialaninate hydrochloride.
Add acetyl chloride (562.79 g, 7.17 mol) to methanol (10.00 L) at 0 C in an
appropriate vessel. Heat the mixture to 17.5 C and stir. After 30 minutes add
2-bromo-
D-phenylalanine (500.00 g, 2.05 moles) and heat to reflux. After 4 hours, cool
to 20 C
and remove the solvent under reduced pressure to give the title compound (589
g, 1.96
mol) as an off-white solid. MS (m/z): 258(M-C1 (79Br)), 260(M-C1(81Br)).
Preparation 2
Synthesis of methyl 2-bromo-N-(nethoxycarbony1)-D-phenylalaninate.
Br .10 0
Y
0
Dissolve methyl 2-bromo-D-phenylalaninate hydrochloride (27.2 g, 92.3 mmol) in
dichloromethane (923 mL) and water (185 mL). Add sodium bicarbonate (31.0 g,
369.4
mmol) and methyl chloroformate (7.86 mL, 101.6 mmol) at room temperature. Stir
the
mixture 2.5 hours. Dilute with water and extract with dichloromethane. Dry the
dichloromethane extracts over sodium sulfate, filter, and concentrate under
reduced
pressure. Purify the residue by silica gel chromatography eluting with ethyl
acetate:
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hexanes (10-75% gradient) to give the title compound (29.1 g, 92.1 mmol). MS
(m/z):
316 (M+1).
Alternative synthesis of methyl 2-bromo-N-(methoxycarbony1)-D-phenylalaninate.
Add water (2.94 L) and sodium hydrogen carbonate (648.25 g, 7.64 mol) to
methyl 2-bromo-D-phenylalaninate hydrochloride (580 g, 1.91 mol) in
dichloromethane
(9.86 L) at 10 C in an appropriate vessel. After 5 minutes add methyl
chloroformate
(198.53 g, 2.10 mol) and stir the mixture at 20 C. After 3 hours add water
(2.5 L) and
separate the layers. Extract the aqueous with dichloromethane, dry the
combined organic
extracts over sodium sulfate and concentrate under reduced pressure to give
the title
compound (556 g, 1.74 mol). MS (m/z): 315.8(M+1 ("Br)), 317.8(M+1 (8IBr)).
Preparation 3
Synthesis of dimethyl (3R)-5-bromo-3,4-dihydro-1H-isoquinoline-2,3-
dicarboxylate.
Br 0
N 0
Y
0
Stir a mixture of methyl 2-bromo-N-(methoxycarbony1)-D-phenylalaninate (29.1
g, 92.10 mmol) and paxaformaldehyde (9.13 g, 101.3 mmol) in glacial acetic
acid (115
mL, 2.01 mol) and concentrated sulfuric acid (38.4 mL, 719.9 mmol) at room
temperature
for 7 hours. Partition between water and ethyl acetate. Separate the layers
and extract the
aqueous layer with ethyl acetate. Combine the ethyl acetate extracts and dry
over sodium
sulfate, filter, and concentrate under reduced pressure. Purify the residue by
silica gel
chromatography eluting with ethyl acetate: hexanes (5-40% gradient) to give
the title
compound (27.6 g, 84.0 mmol). MS (m/z): 328 (M+1).
Preparation 3a
Synthesis of dimethyl (3R)-5-bromo-3,4-dihydro-1H-isoquinoline-2,3-
dicarboxylate and
(3R)-5-bromo-2-methoxycarbony1-3,4-dihydro-1H-isoquinoline-3-carboxylic acid.
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Br 0 Br 0
CY- OH
N 0 N 0
Y Y
0 0
To acetic acid (4.29 L) at 10 C in an appropriate vessel, add 2-bromo-N-
(methoxycarbony1)-D-phenylalaninate (572 g, 1.81 mol) and paraformaldehyde
(205.86
g, 2.17 mol). After 10 minutes slowly add concentrated sulfuric acid (2.63 kg,
26.83 mol)
and then stir at 35 C. After 12 hours, cool to 15 C and add water (7.5 L)
and ethyl
acetate (6 L). Separate the layers and re-extract the aqueous with ethyl
acetate (2.5 L).
Dry the combined organic extracts over sodium sulfate, filter and concentrate
under
reduced pressure to give a mixture of the title compounds with acetic acid
(640 g, 1.69
moles). Mass spectrum (m/z): 3a: 327.95(M+1 ("Br)), 330.05(M+1 (81Br)). 3b:
314(M+1 (79Br)), 315.95(M+1 (81Br)).
Preparation 4
Br0
40 0,
NH
HCI
Synthesis of methyl (3R)-5-bromo-1,2,3,4-tetrahydroisoquinoline-3-carboxylate
hydrochloride.
Dissolve dimethyl (3R)-5-bromo-3,4-dihydro-1H-isoquinoline-2,3-dicarboxylate
(27.55 g, 84.0 mmol) in 5N hydrochloric acid (330.6 mL, 1.65 mol) and heat to
reflux for
three days. Concentrate under reduced pressure to give a white solid. Wash the
solid
with diethyl ether and dry under vacuum at 40 C overnight to give (3R)-5-bromo-
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid hydrochloride (1:1) (20.8 g,
71.1 mmol).
Add acetyl chloride (50.6 mL, 711.0 mmol) to a 0 C mixture of (3R)-5-bromo-
1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid hydrochloride (1:1) (20.8 g, 71.1
mmol) in
methanol (474 mL). Warm to room temperature and stir for 36 hours. Concentrate
under
reduced pressure and dry to give the title compound (21.9 g, 71.4 mmol). MS
(m/z): 270
(M+1).
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Preparation 5
Synthesis of (3R)-5-bromo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
hydrochloride.
Br 0
HC
H
Add water (1.3 L) and 36.5% hydrochloric acid (9.07 Kg, 90.81 moles) to a
mixture of dimethyl (3R)-5-bromo-3,4-dihydro-1H-isoquinoline-2,3-dicarboxylate
and
(3R)-5-bromo-2-methoxycarbony1-3,4-dihydro-1H-isoquinoline-3-carboxylic acid
(Preparation 3a) (520 g, 1.27 moles) in an appropriate vessel and stir the
mixture at 95 C.
After 12 hours cool the mixture to 10 C and stir for 15 minutes. Filter the
mixture and
dry the solid under vacuum at 40 C give the title compound (332 g, 1.13
moles). MS
(m/z): 256.1(M-C1 ("BO), 258(M-C1(81Br)).
Preparation 6
Synthesis of 2-tert-buty1-3-methyl-(3R)-5-bromo-3,4-dihydro-1H-isoquinoline-
2,3-
dicarboxylate.
Br 0
14.
".
0
Dissolve methyl (3R)-5-bromo-1,2,3,4-tetrahydroisoquinoline-3-carboxylate
hydrochloride (21.0 g, 68.5 mmol) in 1,4-dioxane (685 mL). Add saturated
sodium
bicarbonate solution (685 mL, 17.5 mol) and di-tert-butyldicarbonate (29.9 g,
137.0
mmol) at room temperature. Stir the biphasic mixture for 90 min. Extract with
ethyl
acetate. Dry the ethyl acetate over sodium sulfate, filter, and concentrate
under reduced
pressure. Purify the residue by silica gel chromatography eluting with ethyl
acetate:
hexanes (5-50% gradient) to give the title compound (19.5 g, 52.7 mmol). MS
(m/z): 270
(M4B0C+1).
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Preparation 7
Synthesis of [(3R)-5-bromo-1,2,3,4-tctrahydroisoquinolin-3-yl]methanol.
Br OH
*NH
Add lithium aluminium hydride (2 L, 2.00 mol, 1M in THF) to (3R)-5-bromo-
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid hydrochloride (325.4 g, 1.11
mol) in
tetrahydrofuran (4.88 L) at -35 'V in an appropriate vessel, then warm to 25
C over 60
minutes and stir. After 3 hours, cool the mixture to -5 C then add water (76
mL), 15%
w/w aqueous sodium hydroxide (76 mL) and water (228 mL). Heat the mixture to
25 C,
add anhydrous magnesium sulfate (750 g) and stir. Filter the mixture and
concentrate
under reduced pressure to give a solid. Add dichloromethane (690 mL) to the
solid and
slurry for 30 minutes before filtration to give a solid. Dry the solid under
vacuum at 35
C to give the title compound (148.9 g, 0.55 mol). MS (nth): 242(M+1 ("Br)),
244(M+1
(81Br)).
Preparation 8
Synthesis of tert-butyl (3R)-5-bromo-3-(hydroxymethyl)-3.4-dihydro-1H-
isoquinoline-2-
carboxylate.
Br OH
NyOl<
0
Add methanol (10.1 mL, 248.5 mmol) and lithium borohydride (99.4 mL, 198.8
mmol, 2 M in THF) to a solution of 2-tert-buty1-3-methyl-(3R)-5-bromo-3,4-
dihydro-1H-
isoquinoline-2,3-dicarboxylate (18.4 g, 49.7 mmol) in tetrahydrofuran (497 mL)
at room
temperature on a water bath. Stir 40 min and quench the reaction with water.
Extract
with ethyl acetate. Dry the ethyl acetate extracts over sodium sulfate,
filter, and
concentrate under reduced pressure. Purify the residue by silica gel
chromatography
eluting with ethyl acetate: hexanes (5-80% gradient). Dry under high vacuum
overnight
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to give the title compound as a white solid (19.1 g. 55.8 mmol). MS (m/z): 286
(M-
tBu+1).
Preparation 9
Synthesis of (3R)-5-bromo-3-({ [tert-butyl(dimethyl)silyl]oxy}methyl)-1,2,3,4-
tetrahydroisoquinoline.
I j<
Si
Br= 0' '`=
N H
Add trifluoroazetic acid (75.5 mL, 998.3 mmol) to solution of tert-butyl (3R)-
5-
bromo-3-(hydroxymethyl)-3,4-dihydro-1H-isoquitioline-2-carboxylate (15.5 g,
45.3
mmol) in dichloromethane (226 mL) at room temperature. Stir 30 min and
concentrate
under reduced pressure. Dry under vacuum to give [(3R)-5-bromo-1,2,3,4-
tetrahydroisoquinolin-3-yl]nethanol; 2,2,2-trifluoroacetic acid as a wet
solid. Dissolve
[(3R)-5-bromo-1,2,3,4-tetrahydroisoquinolin-3-yl]methanol; 2,2,2-
trifluoroacetic acid in
dichloromethane (753 mL). Add 1H-imidazole (51.3 g, 753 mmol), N,N-Dimethy1-4-
pyridinamine (460 mg, 3.77 mmol), and t-butyldimethylchlorosilane (13.6 g,
90.4 mmol).
Stir at room temperature overnight. Add saturated ammonium chloride solution
and
extract with dichloromethane. Dry the dichloromethane extracts over sodium
sulfate,
filter, and concentrate under reduced pressure. Combine with the crude product
from a
substantially same reaction run with 19.4 mmol of tert-butyl (3R)-5-bromo-3-
(hydroxymethyl)-3,4-dihydro-1H-isoquinoline-2-carboxylate. Purify the residue
by silica
gel chromatography eluting with ethyl acetate: hexanes (5-40% gradient) to
give the title
compound (14.3 g, 40.1 mmol). MS (m/z): 356 (M+1).
Alternative synthesis of (3R)-5-bromo-3-({ [tert-butyl(dimethyl)silyl]oxy)
methyl)-
1,2,3,4-tetrahydroisoquinoline.
Add tert-butyldimethylchlorosilane (193.7 g, 1.29 mol) to a mixture of [(3R)-5-
bromo-1,2,3,4-tetrahydroisoquinolin-3-yl]nethanol (148.9 g, 0.58 mol), 1H-
imidazole
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(202.9 g, 2.92 mol), 4-dimethylatninopyridine (0.72 g, 5.84 rnmol) and N,N-
dimethylformamide (1.04 L) in dichlormethane (2.61 L) at 20 C and stir in an
appropriate vessel. After 3 hours, cool the mixture to 10 C and add saturated
aqueous
ammonium chloride solution (1.3 L). Extract the aqueous with dichloromethane
and
wash the combined organic extracts with brine (2 x 2L), dry over anhydrous
sodium
sulfate and concentrate under reduced pressure to give a residue. Dissolve the
residue in
methyl tert-butyl ether (1.5 L) and wash with brine (2 x 1 L). Dilute the
organic phase
with toluene (5 L) and concentrate under reduced pressure to give a residue.
Add toluene
(2.6 L) to the residue and concentrate under reduced pressure to give the
title compound
(210 g, 0.53 mol). MS (m/z). 356(M+1 ("Br)), 358(M+1 (81Br)).
Preparation 10
Synthesis of (3R)-5-bromo-3-({[tert-butyl(dimethyl)silyl]oxy}methyl)-3,4
dihydroisoquinolirte.
I ,<"
Br 0'
Dissolve (3R)-5-bromo-3-(fitert-butyl(dimethypsilyl]oxy}methyl)-1,2,3,4-
tetrahydroisoquinoline (4.2 g, 11.8 mmol) in diethyl ether (118 mL). Add
N-chlorosuccinimide (2.36 g, 17.7 mmol). Stir 30 min at room temperature and
concentrate under reduced pressure. Dissolve the residue in potassium
hydroxide (42.0
mL, 30.3 mmol, 5% in Me0H) and stir for 30 min at room temperature. Pour into
water
and extract with dichloromethane. Dry the dichloromethane extracts over sodium
sulfate,
filter, and concentrate under reduced pressure. Purify the residue by silica
gel
chromatography eluting with ethyl acetate: hexanes (5-100% gradient) to give
the title
compound (3.40 g, 9.59 mmol). MS (tn/z): 354 (M+1).
Alternative synthesis of (3R)-5 -bromo-3-( [tert-
butyl(dimethypsilyl]oxy}methyl)-3,4
dihydroisoquinoline.
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Add N-chlorosuccinimide (106.7 g, 0.79 mol) to a solution of (3R)-5-bromo-3-
(f[tert-butyl(dimethypsilyfloxy}methyl)-1,2,3,4-tetrahydroisoquinoline (220 g,
0.52 mol)
in tetrahydrofuran (3.85 L) at 20 C in an appropriate vessel and stir. After
30 minutes
concentrate the mixture under reduced pressure and dissolve the residues in 5%
w/w
potassium hydroxide in methanol (2.2 L, 1.69 moles) and stir at 20 C. After
30 minutes,
add the mixture to water (3 L) and extract three times with dichloromethane (3
xl L).
Dry the combined organic extracts over anhydrous magnesium sulfate and
concentrate
under reduced pressure to give the title compound (210 g, 0.50 mol). MS (m/z):
354(M+1 ("Br)), 356(M+1 ("Br)).
Preparation 11
Synthesis of (1S,3R)-5-bromo-3-( fitert-butyl(dimethypsilyl]oxylmethyl)-1-
methyl-
1,2,3,4-tetrahydroisoquinoline.
I
si-
Br
Dissolve (3R)-5-bromo-3-(fitert-butyl(dimethyl)silyl]oxy}methyl)-3,4
dihydroisoquinoline (3.4 g, 9.59 mmol) in diethyl ether (160 mL). Cool to -78
C on a
dry ice ¨ acetone bath. Add methylmagnesium chloride (26.9 mL, 80.6 mmol, 3M
in
THF) dropwise. Warm the reaction mixture slowly to room temperature and stir
overnight. Quench with saturated ammonium chloride solution slowly. Extract
with
dichloromethane and dry over sodium sulfate, filter, and concentrate under
reduced
pressure. Combine with the crude product from a substantially same reaction
run with
1.73 mmol of (3R)-5-bromo-3-({[tert-butyl(dimethypsilyfloxy}methyl)-3,4
dihydroisoquinoline. Purify the combined residues by silica gel chromatography
eluting
with ethyl acetate: hexanes (5-65% gradient) to give the title compound (3.78
g, 10.2
mmol): MS (m/z): 370 (M+1).
The relative configuration of compound (1S,3R)-5-bromo-3-({[tert-
butyl(dimethyl)silyl]oxylmethyl)-1-methyl-1,2,3,4-tetrahydroisoquinoline is
determined
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by NMR spectroscopy using one-dimensional NOE experiments (1D-NOESY).
Selective
excitation of the methyl group at 1.30 ppm gives rise to a NOE for Ha at 3.11
ppm. This
NOE enhancement is only consistent with a configuration in which the methyl
and Ha are
on the same side of the ring (trans isomer) because in the cis isomer the
methyl protons
are too far away from Ha to show an NOE. Since the absolute chemistry for
position 3 is
known to be R, then the absolute chemistry at position 1 is deduced to be S.
\ ________________________________________
Br
401 Ha
NH
Hc He. me
Alternative synthesis of (1 S,3R)-5-bromo-34 [tert-butyl (dimethypsil yl] oxy
methyl)-1 -
methyl- 1,2,3,4-tetrahydroisoquino line.
1 k
=
Si
Br 0"
..õ
N H
Add methylmagnesium chloride (0.66 L, 1.99 mol, 3M in THF) to a solution of
(3R)-5-bromo-3-({ [tert-butyl(dimethypsilyl]oxy}methyl)-3,4
dihydroisoquinoline (93.5
g, 0.24 mol) in diethyl ether (2.8 L) at -65 C in an appropriate vessel. Then
heat the
reaction mixture to 20 C over 2 hours and stir. After 16 hours, cool the
mixture to 0 C
and quench the reaction with saturated aqueous ammonium chloride solution (2.5
L) and
extract with ethyl acetate (2.5 L) and filter the mixture. Wash the combined
organic
extracts with brine (1 L), dry over anhydrous magnesium sulfate and
concentrate under
reduced pressure to give the crude title compound as an oil. Combine the oil
with crude
products from substantially same reactions run with 48 mmol and 229 mmol of
[(3R)-5-
bromo-3,4-dihydroisoquinolin-3-yl]methoxy-tert-butyl-dimethyl-silane and
purify them
by silica gel chromatography eluting with ethyl acetate in hexanes (gradient 5-
65% ethyl
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acetate) to give the title compound (151 g, 0.41 mol). MS (m/z): 370.1 (M+1
(79Br)),
372.1(M+1 (8IBr)).
Preparation 12
Synthesis of 14(1S,3R)-5-bromo-3-(fitert-butyl(dimethyl)silyl]oxy}methyl)-1-
methyl-
3,4-dihydroisoquinolin-2(11-/)-y1]-2-(2,6-dichlorophenypethanone.
I ks,
Br 0' "=-
j
N CI
0CI I*
Add benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate (7.97
g, 15.3 mmol) to a mixture of (1S,3R)-5-bromo-3-({[tert-
butyl(dimethyl)silyl]oxy}methyl)-1-methyl-1,2,3,4-tetrahydroisoquinoline (3.78
g, 10.2
mmol) and 2,6-dichlorophenylacetic acid (2.30g. 11.2 mmol) in
dimethylformamide
(51.0 mL). Add triethylamine (2.13 mL, 15.3 mmol) and stir at room temperature
3
hours. Dilute with water and extract with dichloromethane. Dry the
dichloromethane
extracts over sodium sulfate, filter and concentrate under reduced pressure.
Purify the
residue by silica gel chromatography eluting with ethyl acetate: hexanes (5-
50% gradient)
to give the title compound (4.70 g, 8.43 mmol). MS (m/z: 556 (M+1).
Preparation 13
Synthesis of ethyl (2E)-3-{(1S,3R)-3-(fitert-butyl(dimethypsilyfloxy}methyl)-2-
[(2,6-
dichlorophenyl)acetyl]-1-methyl-1,2,3,4-tetrahydroisoquinolin-5-y1}prop-2-
enoate.
0
k
si
1410 N CI
0CI el
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Bubble nitrogen through acetonitrile. Place tri-o-tolylphosphine (72.3 mg,
0.23
mmol), palladium (II) acetate (11.8 mg, 0.052 mmol) and acetonitrile (0.96 mL)
in a
microwave vessel. Stir 10 min. Add ethyl acrylate ( 0.31 mL, 2.88 mmol). Add 1-
[(1S,3R)-5-bromo-3-({[tert-butyl(dimethyl)silyfloxy}methyl)-1-methyl-3,4-
dihydroisoquinolin-2(1.H)-y1]-2-(2,6-dichlorophenypethanone (0.54 g, 0.96
mmol). Add
triethylamine (0.40 mL, 2.88 mmol) and stir vigorously. Blow nitrogen across
the surface
of the reaction. Seal the vessel and heat to 160 C for 35 mm i in a
microwave. Cool to
room temperature and dilute with ethyl acetate. Filter the precipitate and
wash with ethyl
acetate. Concentrate the filtrate under reduced pressure to give a brown oil.
Combine
with the crude product from a substantially same reaction run with 0.27 mmol
of
1-[(1S,3R)-5-bromo-3-({[tert-butyl(dimethypsilyl]oxy}methyl)-1-methyl-3,4-
dihydroisoquinolin-2(1.H)-y1]-2-(2,6-dichlorophenypethanone. Purify the
combined
residues by silica gel chromatography, eluting with ethyl acetate: hexanes
(gradient, 0-
10%) to give the title compound (0.54 g, 0.93 nunol). MS (m/z): 576 (M+1).
Preparation 14
Synthesis of ethyl 3-1(1S,3R)-3-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-
[(2,6-
dichlorophenypacetyl]-1-methyl-1,2,3,4-tetrahydroisoquinolin-5-y1}propanoate.
0 0
s,
140 N CI
0CI II
In a dry box to a 85 ml Parr autoclave with stir bar and glass liner, add 1,1'-
bis(di-
i-propylphosphino)ferrocene(1,5-cyclooctadiene)rhodium(1) tetrafluoroborate (7
mg,
0.010 mmol). Add ethyl (2E)-3-1(1S,3R)-3-({[tert-
butyl(dimethyl)silyl]oxy}methyl)-2-
[(2,6-dichlorophenypacetyl]-1-methyl-1,2,3,4-tetrahydroisoquinolin-5-yllprop-2-
enoate
(133 mg, 0.23 mmol) as a solution in anhydrous methanol (5 mL). Seal the
autoclave and
remove from the dry box. Purge the vessel with hydrogen and pressurize to 690
kPa of
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hydrogen. Stir at room temperature overnight. Vent and open the vessel.
Concentrate the
reaction mixture under reduced pressure. Purify the residue by silica gel
chromatography,
eluting with 25% methyl t-butyl ether: hexanes to give the title compound as a
clear
colorless oil (121 mg, 0.21 nunol). MS (m/z): 578 (M+1).
Preparation 15
Synthesis of (1S,3R)-5-bromo-3-({[tert-butyl(dimethypsilyl]oxy) meth yl )- 1 -
meth yl-
1,2,3,4-tetrahydroisoquinoline hydrochloride.
Si
Br 0' I `'s=
N H
HCI
Add hydrogen chloride (267.24 g, 1.02 mol, 4M in 1,4-dioxane) to (1S,3R)-5-
bromo-3-({[tert-butykdimethypsilyfloxy}methyl)-1-methyl-1,2,3,4-
tetrahydroisoquinoline (419 g, 1.02 mol) in isopropyl acetate (4.19 L) at 10
C and stir for
minutes in an appropriate vessel. Filter the mixture and wash the filter cake
with
isopropyl acetate (2.5 L), dry on the filter for 30 minutes, then under vacuum
in an oven
at 40 C for 16 hours to give the title compound (380 g, 0.89 mol). MS (m/z):
370(M-C1
15 (79Br)), 372(M-C1(81Br)).
Preparation 16
Synthesis of (E)-4-[(1S,3R)-3-Rtert-butyl(dimethypsi lylioxymethy1]-1 -methyl-
1 .2,3.4-
tetrahydroisoquinolin-5-y1]-2-methyl-but-3-en-2-ol.
HO
0
N H
Add (1 S,3R)-5-bromo-3-( [tert-butyl(dimethyl)silyl]oxy} meth y1)-1-methyl -
1,2,3,4-tetrahydroisoquinoline hydrochloride (310 g, 723.83 nunol) and 2-
methylbut-3-
en-2-ol (508.95 g, 5.79 mol) to N,N-dimethylformamide (1.08 L) in an
appropriate vessel
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and degas by bubbling nitrogen through the solution for 10 minutes. Add
potassium
carbonate (315.12 g, 2.28 mol), 2-dicyclohexylphosphino-2',6'-
dimethoxybiphenyl (15.32
g, 36.19 mmol) and palladium (II) acetate (8.29 g, 36.19 mmol) and degas by
bubbling
nitrogen through the mixture for 15 minutes then heat to 125 C. After 16
hours, cool the
mixture to 20 C and dilute with ethyl acetate (1.5L) and water (2.5 L). Wash
the ethyl
acetate layer with brine (2.5 L), then dry over sodium sulfate and concentrate
under
reduced pressure to give a residue. Purify the residue by flash
chromatography, eluting
with 0-50% ethyl acetate in isohexanes to give the title compound (193 g,
459.1 mmol).
MS (m/z): 376(M+1).
Preparation 17
Synthesis of 4-[(1S,3R)-3-Rtert-butyl(dimethypsilylloxyrnethyl] -1-methy1-
1,2,3,4-
tetrahydroisoquinolin-5-y1]-2-methyl-butan-2-ol.
HO
Si
=
I
N H
Add a solution of (E)-4-[(1S,3R)-3-[Etert-butyl(dimethyl)silyl]oxymethyl]-1-
methyl-1,2,3,4-tetrahydroisoquinolin-5-y1]-2-methyl-but-3-en-2-ol (68 g,
168.83 mmol)
in ethanol (816 mL) to a pressure hydrogenation vessel and add 5% palladium on
activated carbon (35.83 g, 16.84 mmol). Purge the vessel with hydrogen gas,
pressurise
to 470 kPa of hydrogen gas and stir at 25 C. After 16 hours, vent the vessel
and filter the
reaction mixture through diatomaceous earth. Wash the diatomaceous earth with
ethyl
acetate and concentrate the filtrate under reduced pressure to give an oil.
Dissolve the oil
in ethyl acetate (1 L) and concentrate under reduced pressure to give the
title compound
(64 g, 161 mmol). MS (m/z): 378.2 (M+1).
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Preparation 18
Synthesis of 1-[(1S,3R)-3-({[tert-butyl(dimethyl)silyl]oxy}methyl)-5-(3-
hydroxy-3-
methylbutyl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-y1]-2-(2,6-
dichlorophenyl)ethanone.
OH
s,
CI
0
CI 411
Dissolve 4-[(1S,3R)-3-Rtert-butyl(dimethypsilyl]oxymethyl]-1-methyl-1,2,3,4-
tetrahydroisoquinolin-5-y1]-2-methyl-butan-2-ol (0.21 g, 0.36 mmol) in THF
(2.0 mL).
Cool on a dry ice ¨ acetone bath. Add methyl lithium (0.67 mL, 1.07 mmol, 1.6
M in
diethyl ether) slowly and stir on the dry ice-acetone bath for 4 hours. Add
saturated
ammonium chloride solution (2 mL). Remove the dry ice-acetone bath and allow
the
mixture to warm to room temperature. Extract with ethyl acetate. Combine the
ethyl
acetate extracts; wash with brine, dry over sodium sulfate, filter, and
concentrate under
reduced pressure to give a clear colorless oil. Purify the residue by silica
gel
chromatography, eluting with ethyl acetate: hexanes (gradient, 0-30%) to give
the title
compound as a clear, colorless oil (0.16 g, 0.29 mmol). MS (m/z): 564 (M+1).
Alternative synthesis of 1-[(1S,3R)-3-({[tert-butyl(dimethypsilyl]oxy}methyl)-
5-(3-
hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(110-y1]-2-(2,6-
dichlorophenypethanone.
Add 1,1'-carbonyldiimidazole (112.9 g, 682.36 mmol) to a mixture of 2,6-
dichlorophenylacetic acid (173.09 g, 818.83 mmol) in tetrahydrofuran (1.63 L)
in an
appropriate vessel and stir at 25 C. After 1 hour add a solution of 4-
[(1S,3R)-3-Htert-
butyl(dimethypsilyfloxymethyl]-1-methyl-1,2,3,4-tetrahydroisoquinolin-5-y1]-2-
methyl-
butan-2-ol (217 g, 545.89 mmol) in tetrahydrofuran (1.63 L) to the mixture,
heat the
mixture to 45 C and stir. After 24 hours, cool the mixture to 20 C, remove 2
L of
tetrahydrofuran by concentrating under reduced pressure and dilute the
residues with
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ethyl acetate (2.5 1.). Wash the ethyl acetate solution with saturated aqueous
ammonium
chloride (1.5 L), 1M aqueous sodium hydroxide (1 L), water (1 L) and brine
(1.5 L). Dry
the organics over anhydrous sodium sulfate and concentrate under reduced
pressure to
give the title compound (376 g, 532.70 mmol). MS (m/z): 564.2(M+1 (35C1)),
566.2
(M+1 (37C1)).
Reference Compound 1
Synthesis of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-
3-
methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-ylieihanone.
OH
0 H
CI
0CI
Dissolve 1-[(1S,3R)-3-({[tert-butyl(dimethypsilyl]oxylmethyl)-5-(3-hydroxy-3-
methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-y1]-2-(2,6-
dichlorophenypethanone
(0.16 g, 0.28 mmol) in THF (2.8 mL). Add tetrabutylammonitun fluoride (0.30
mL, 0.30
mmol, 1M in THF). Stir 40 min. Add saturated ammonium chloride solution and
extract
with ethyl acetate. Combine the ethyl acetate extracts; wash with water and
brine, dry
over sodium sulfate, filter, and concentrate under reduced pressure to give a
residue.
Purify the residue by silica gel chromatography, eluting with ethyl acetate:
hexanes
(gradient, 0-60%) to give the title compound as a white foam (0.12 g, 0.26
mmol). MS
(m/z): 450 (M+1).
Alternative synthesis of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-
(3-
hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yflethanone.
Add tetra-n-butylanunonium fluoride (651.71 mL, 651.71 nunol, 1M in THF) to a
solution of 1-[(1S,3R)-3-Rtert-butyl(dimethyl)silyl]oxymethyl]-5-(3-hydroxy-3-
methyl-
buty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-y1]-2-(2,6-dichlorophenypethanone
(400
g, 566.71 mmol) in tetrahydrofuran (4 L) at 5 *C in an appropriate vessel.
Heat the
mixture to 20 C and stir. After 3 hours, remove 3 L of tetrahydrofttran by
concentrating
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under reduced pressure and dilute the residues with ethyl acetate (2.5 L).
Wash the
organics with saturated aqueous ammonium chloride (2 L), water (2 L) and brine
(2 x 2
L). Dry the ethyl acetate solution over anhydrous sodium sulfate and
concentrate under
reduced pressure to give an oil. Dissolve this oil in 2-propanol (2.5 L) and
concentrate
under reduced pressure to give an oil. Purify by chiral SFC using AS-H column
(50 x
250mm, 5 micron particle size) eluting with 80 % supercritical carbon dioxide
and 20%
of a 0.2% solution of diethylmethylamine in isopropyl alcohol at 280 g/min. to
give the
title compound (182.8 g, 389.62 mmol). MS (m/z): 450.2(M+1 ("CI)), 452.2 (M+1
("CD). Optical rotation: [a] 20D -39.4 (c = 0.95, Me0H).
Reference Compound 2
Cocrystallization of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-
hydroxy-
3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-yl]ethanone and
4-hydroxybenzoic acid.
OH
OH
0 H
N
0 CI 1411) 0 OH
Compounds 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-
3-mcthylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-Aethanone (402.56 mg) and
4-hydroxybenzoic acid (144.7 mg) are placed in a 40 mL vial along with a
stirbar. The
vial is filled to the brim with water (39 mL). The sample is stirred at 1200
rpm at 50 C
(stirplate setting). Silicon oil is dripped around the base of the vial to
ensure good
thermal transfer with the hotplate. A thick white slurry results with chunks
of off-white
solid. After an hour of slurrying, a thermometer inserted through the septum
of the vial
read 40.5 C, and the sample had turned into a homogenous slurry of bright
white solid.
After overnight slurry, the sample is a homogenous slurry of flocculent white
solid. The
thermometer reads 43.1 C. Polarized light microscopy shows full birefringence.
The
bright white solid is isolated by vacuum filtration and dried in place under
air stream for
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minutes. The sample is placed in the 75 C vacuum oven for two hours to provide
the
title composition as a white crystalline solid (484 mg, 94.9% yield).
Melting point onset = 160.00C (differential scanning calorimetry).
5 X-Ray Powder Diffraction
The X-ray diffraction (XRD) patterns of crystalline solids are obtained on a
Bruker D4 Endeavor X-ray powder diffractometer, equipped with a CuKa source A.
=
1.54060 A) and a Vantec detector, operating at 35 kV and 50 mA. The sample is
scanned
between 4 and 40 in 20, with a step size of 0.009 in 20 and a scan rate of
0.5
10 seconds/step, and with 0.6 mm divergence, 5.28 fixed anti-scatter, and
9.5 mm detector
slits. The dry powder is packed on a quartz sample holder and a smooth surface
is
obtained using a glass slide. The crystal form diffraction patterns are
collected at ambient
temperature and relative humidity. It is well known in the crystallography art
that, for
any given crystal form, the relative intensities of the diffraction peaks may
vary due to
preferred orientation resulting from factors such as crystal morphology or
habit. Where
the effects of preferred orientation are present, peak intensities are
altered, but the
characteristic peak positions of the polymorph are unchanged. Furthermore, it
is also
well known in the crystallography art that for any given crystal form the
angular peak
positions may vary slightly. For example, peak positions can shift due to a
variation in
the temperature or humidity at which a sample is analyzed, sample
displacement, or the
presence or absence of an internal standard. In the present case, a peak
position
variability of 0.2 in 20 will take into account these potential variations
without hindering
the unequivocal identification of the indicated crystal form. Confirmation of
a crystal
form may be made based on any unique combination of distinguishing peaks (in
units of
20), typically the more prominent peaks. (United States Pharmacopeia #35,
National
Formulary #30, Chapter <941>, pages 427-432, 2012). The crystal form
diffraction
patterns, collected at ambient temperature and relative humidity, are adjusted
based on
NBS standard reference material 675 (mica) with peaks at 8.853 and 26.774
degrees 2-
theta.
A prepared sample of the co-crystal of Reference Compound 2 is characterized
by
an X-ray diffraction pattern using CuKa radiation as having diffraction peaks
(2-theta
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values) as described in Table 3 below, and in particular having peaks at 18.2
in
combination with one or more peaks selected from the group consisting of
16.0,25.4 ,
and 7.0 ; with a tolerance for the diffraction angles of 0.2 degrees.
Table 3
Reference Compound 2
I Angle ( 2-Theta) Relative Intensity
Peak +1- 0.2 (% of most intense peak)
1 7.0 74.00
2 15.0 53.70
3 16.0 87.60
4 17.4 66.20
18.2 100.00
6 19.7 63.00
7 20.2 54.80
8 21.0 63.60
9 23.4 29.40
25.4 74.20
5
Alternative cocrystallization of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-
(hydroxymethyl)-5-
(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yflethanone
and
4-hydroxybenzoic acid.
To a 20 mL vial is added 2-(2,6-dichloropheny1)-141S,3R)-3-(hydroxymethyl)-
10 5-(3-hydroxy-3-methylbuty1)-1-methy1-3,4-dihydroisoquinolin-2(111)-
yflethanone (2.00
g, 1.00 equiv; 4.44 rrunoles). Acetone (4 mL) is added while stirring at room
temperature. A clear solution is formed. 4-Hydroxybenzoic acid (0.756 g; 1.23
equiv;
5.47 mmoles) is added while stirring at room temperature. A slight suspension
is formed
then a thick suspension. The mixture is heated on hot plate to 60 C. Acetone
in 1 mL
aliquots is added until a nice mixing suspension is observed at 60 C. Total
acetone added
is 9.00 mL (122.43 mmoles, 7.11 g). The temperature is held at -600C for
several hours.
The mixture is cooled to room temperature and placed in refrigerator to
improve
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recovery. The resulting solid is collected by vacuum filtration, rinsed with 2
mL of
acetone and dried in a vacuum oven overnight at 400C to provide the title
composition as
a white crystalline solid. HPLC analysis demonstrates the molar ratio of 242,6-
dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-methylbutyl)-1-
methyl-
3,4-dihydroisoquinolin-2(1./)-yliethanone to 4-hydroxybenzoic acid in the
cocrystal is
one to one.
HPLC Analysis
Column: Agilent ZORBAX Bonus-RP, Rapid Resolution, 4.6x75mm, 3.511
Column temperature: 30 C
Injection volume: 2 L
Detection: UV
2-(2,6-dichloropheny1)-14(1 S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-
methylbuty1)-1-methy1-3,4-dihydroisoquinolin-2(1H)-yl]ethanone (Reference
Compound
1) @ 219nm
4-hydroxybenzoic acid @ 256nm
Flow rate: 1.5 mi./min.
Mobile phase: A) 0.1% TFA in water
B) 0.1% TFA in acetonitrile
Gradient Table
Time, minutes %A %13
0 95 5
9.5 23 77
12.1 23 77
13.0 5 95
16.0 5 95
16.1 95 5
20.0 95 5
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Relative Ratio
Theoretical
Molecular
Compound Potency for 1 Assayed Potency (n=3)
Weight
to 1
Ref. Compound
450.4 76.5 % 81.06% + 0.15%
1
4-hydroxy-
138.1 23.5% 23.74% + 0.20%
benzoic acid
Second alternative cocrystallization of 2-(2,6-dichlorophenyI)-1-[(1S,3R)-3-
(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-
2(111)-
yljethanone and 4-hydroxybenzoic acid.
Compounds 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-
3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-yflethanone (45.06 g,
0.1 mol)
and 4-hydroxybenzoic acid (14.5 g, 1.05 mol eq) are slurried at 23 *C in 53:47
isopropyl
alcohol:heptane (236 mL, 4 volumes) and heated to 65 C. The resulting
solution is
seeded with cocystals of 2-(2,6-dichloropherty1)-1-[(1S,3R)-3-(hydroxymethyl)-
5-(3-
hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yflethanone and
4-hydroxybenzoic acid (553 mg, 1.0 wt. % seed load) and stirred at 65 C for
30 minutes.
Heptane (943 mL, 16 volumes3) is added at 65 C over 4.6 hours. The slurry is
stirred at
65 C for a further 30 minutes, cooled to 23 C over 2 hours, stirred
overnight at 23 C,
and vacuum filtered. The product solids are rinsed with 10:90 isopropyl
alcohol:heptane
(2 x 50 mL) and heptane (50 mL) then dried in a vacuum oven at 40 C for 2
hours to
yield the title composition as a white crystalline product (51.3 g, 86.3 wt %
yield).
Melting point onset = 162.2 0C (differential scanning calorimetry).
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Example 1
Crystallization of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-
hydroxy-3-
methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yflethanone.
OH
OH
CI
0CI lei
Approximately lOg of cocrystal 242,6-dichloropheny1)-1-[(1S,3R)-3-
(hydroxymethyl)-5-(3-hydroxy-3-methylbutyl)-1-methyl-3,4-dihydroisoquinolin-
2(1H)-
yliethanone and 4-hydroxybenzoic acid is taken in a 500mL beaker and 100mL of
deionized water added at room temperature. A solution of 50mL of 1N NaOH is
slowly
added. A suspension is formed. 100mL of chloroform is added gradually. The
solids
dissolved and a bi layer solution is formed. The organic layer is separated
using a
separatory funnel. The organic layer is evaporated at room temperature. To the
resulting
gummy material is added 20mL of heptane and stirred. The resulting solid is
isolated
under vacuum and air dried at room temperature in open dish to provide
amorphous
2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-5-(3-hydroxy-3-
methylbuty1)-1-
methyl-3,4-dihydroisoquinolin-2(111)-yflethanone. ). MS (m/z): 450.2(M+1
(35C1)).
Amorphous 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-(hydroxymethyl)-543-hydroxy-
3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-yl]ethanone (251.8mg) is
taken in
a 20m1, vial and 4mL of toluene is added. The resulting suspension is stirred
overnight at
room temperature. The crystalline solid is filtered under vacuum and air
dried. Melting
point onset ¨ 146.3 0C (differential scanning calorimetry).
Alternative crystallization of 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-
(hydroxymethyl)-5-(3-
hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-2(111)-yflethanone.
Approximately 3g of amorphous 2-(2,6-dichloropheny1)-1-[(1S,3R)-3-
(hydroxymethyl)-5-(3-hydroxy-3-methylbuty1)-1-methyl-3,4-dihydroisoquinolin-
2(1H)-
yflethanone is taken in a 20mL vial and 10mL of acetone is added. A thick
precipitate is
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formed immediately upon stirring at room temperature. Another 5m1., of acetone
is added
to dilute the slurry and stirred at room temperature for overnight. The
resulting
crystalline solid is isolated under vacuum and air dried.
X-Ray Powder Diffraction
X-ray powder diffraction (XRPD) pattern is collected on a Bniker D8 Advance X-
ray powder diffractometer equipped with a CuKa source (X=1.54056 A) and a
Linxeye
detector, and operating at 40 kV and 40 mA, with a 0.2 mm divergence slit.
Sample is
scanned from 2 to 40 in 0.02 20 steps at a rate of 0.2 seconds per step. The
crystal form
diffraction patterns are collected at ambient temperature and relative
humidity and peak
position is adjusted based on NBS standard reference material 675 (mica) with
peaks at
8.853 and 26.774 degrees 2-theta. It is well known in the crystallography art
that, for any
given crystal form, the relative intensities of the diffraction peaks may vary
due to
preferred orientation resulting from factors such as crystal morphology and
habit. Where
the effects of preferred orientation are present, peak intensities are
altered, but the
characteristic peak positions of the polymorph are unchanged. Furthermore, it
is also
well known in the crystallography art that for any given crystal form the
angular peak
positions may vary slightly. For example, peak positions can shift due to a
variation in
the temperature or humidity at which a sample is analyzed, sample
displacement, or the
presence or absence of an internal standard. In the present case, a peak
position
variability of 0.2 in 20 will take into account these potential variations
without hindering
the unequivocal identification of the indicated crystal form. Confirmation of
a crystal
form may be made based on any unique combination of distinguishing peaks (in
units of
20), typically the more prominent peaks. (United States Pharmacopeia #35,
National
Formulary #30, Chapter <941>, pages 427-432, 2012).
A prepared sample of the crystal of Example 1 is characterized by an X-ray
diffraction pattern using CuKa radiation as having diffraction peaks (2-theta
values) as
described in Table 4 below, and, in particular, by having peaks at 9.150 in
combination
with one or more of the peaks selected from the group consisting of 19.4 ,
16.1 , and
16.6 ; with a tolerance for the diffraction angles of 0.2 degrees.
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Table 4
Example 1 Peak Positions
Relative
Angle ( 20) Intensity (% of
Peakd value (A)
0.2' most intense
peak)
1 9.15 100.0 9.65
2 11.8 16.1 7.51
3 12.5 18.0 7.06
4 14.6 10.6 6.06
15.6 18.7 5.67
6 16.1 27.7 5.52
7 16.6 24.5 5.33
8 17.4 16.2 5.09
9 19.4 63.2 4.57
20.2 21.7 4.40
11 21.0 15.2 4.23
12 23.0 23.0 3.86
13 23.5 11.0 3.78
14 23.9 11.6 3.72
25.2 22.5 3.54
