Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
SPIROPIPERIDINE DERIVATIVES' AS MELANOCORTIN RECEPTOR
AGONISTS
CROSS REFERENCE TC) RELATED APPLICATIONS
This application is based on, and claims priority from, provisional
applications 60/08890 filed June i 1, 1998, and 60/I2326 filed March 8, 1999,
which
are hereby incorporated bar reference :in their entireties.
SUMMARY OF THE INVENTION
Spiropiperidine derivatives are meianocortin receptor agonists, and as
such are useful in the treatment of disorders responsive to the activation of
melanocortin receptors, such as obesity, diabetes as well as male and/or
female sexual
dysfunction.
1~
BACKGROUND OF THI:, INVENTION
Pro-opiomc;lanocortin (POMC) derived peptides are known to affect
food intake. Several lines of evidence support the notion that the G-protein
coupled
receptors (GPCRs} of the ;melanocortin receptor (MC-R) family, several of
which are
expressed in the brain, are the targets of POMC derived peptides involved in
the
control of food intake and metabolism. A specific single MC-R that may be
targeted
for the control of obesity has not yet been identified.
Evidence for the involvement of MC-Rs in obesity includes: i) the
agouti (A"j') mouse which ectopically expresses an antagonist of the MC-1R, MC-
3R
and -4R is obese, indicating that blocking the action of these three MC-Rs can
lead to
hyperphagia and metabolic disorders;. ii) MC-4R knockout mice (Huszar et al.,
Cell,
88, 13I-I41, 1997) recapitulate the phenotype of the agouti mouse and these
mice are
obese; iii) the cyclic hepta.peptide M7."-II (MC-1R, -3R, -4R, -SR, agonist)
injected
intracerebroventricularly (ICV) in rodents, reduces food intake in several
animal
feeding models (NPY, oblob, agouti, fasted) while ICV injected SHU-9119 (MC-
3R,
-4R antagonist; MC-IR and -SR agor~ist) reverses this effect and can induce
hyperphagia; iv) chronic intraperitoneal treatment of Zucker fatty rats with
an a -
NDP-MSH derivative (HF'228) has been reported to activate MC-1R, -3R, -4R and -
SR and to attenuate food intake and body weight gain over a I2 week period.
SUBS'3CITUTF: SHEET t rule 26 )
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Five MC-R~ have thus far been identif ed, and these are expressed in
different tissues. MC-IR was initially characterized by dominant gain of
function
mutations at the Extension locus, affecting coat color by controlling
phaeomelanin to
eumelanin conversion through control of tyrosinase. MC-1R is mainly expressed
in
melanocytes. MC-2R is ea;pressed in the adrenal gland and represents the ACTH
receptor. MC-3R is expressed in the brain, gut and placenta and may be
involved in
the control of food intake ~u~.d thermogenesis. MC-4R is uniquely expressed in
the
brain and its inactivation was shown to cause obesity. MC-5R is expressed in
many
tissues including white fat.. placenta and exocrine glands. A low level of
expression is
also observed in the brain. MC-5R knock out mice reveal reduced sebaceous
gland
lipid production (Chen et al., Cell, 1997, 91, 789-798).
Intrarnuscu'.(ar administration of the MC-1R, -3R, -4R, -5R agonist,
melanotan - .II {MT-II; O.OC~S - 0.03 mg/kg; Dorr et al., Life Sciences, vol.
58, # 20,
1777-1784. 1996) caused intermittent non-painful penile erections in three
normal
male volunteers for a period of 1-5 hours after dosing. Intramuscular
administration of
MT-II (0.025 mg/kg and O.I mg/kg) to 10 non-organic impotent patients caused
transient erections (8 responders) with onset from 50-180 minutes; penile
erections
subsided after ejaculation (15th American Peptide Symposium 6/14-19, I997,
Nashville, TN, .study now published in J. Urology, 160, 389-393. 1998).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compounds having the formula I:
R~~ N URbRb)m'-Q
0 0
,N.
Y
cy2~ ~
Y ~--X
R2
I
wherein
-2-
SUBS'~TITUTE SHEET ( rule 26 )
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Cy2 is . a six-membered aromatic ring containing 0 or 1 N atom or
cyclohexane;
Q is
Rc
f ,
p CY Rc
HN
a
X is O, CH2, SO2, CHC02Rb, CHS02Ra, CHC{O)N(Rb)2, NRb,
NSO2Ra, NS02N(Rb)2, NCORa, NCON(Rb)2, CHN(Rb)CORa, CHN(Rb)S02Ra,
CHCH20Rb, or CH(CH2)-heteroary:l;
Y is (CH2)r, CH-C 1 _galkyl, O, C=O or S02, with the proviso that when Y
is O, the ring atom of X i:~ carbon;
Rl is H, CI_galk:yl, CH(Rb)-aryl, CH(Rb)-heteroaryl, (CH2)n-
CS_~cycloalkyl in which aryl and heteroaryl are optionally substituted by one
or two
Rc groups;
R2 is H or halo;
Ra is Rb~ (CH2)nN{Rb)2~ {CH2)nN{Rb)C{=~d)NRb~ {CH2)n~-2_
pyridyl, (CH2)nNH-2-imiidazolyl, (CH2)nNH-2-thiazolyl, (CH2)nNH-2-pyrimidinyl,
Rd
N
-NON-Rb CH -N O (CH2)n-N
(CH2)n ~ ~, 2)n ,~ H
,
Rb is H, C~_galkyl, {CH2)naryl, (CH2)nheteroaryl, C3_~cycloalkyl; or 2 Rb
together with the nitrogen atom to which they are attached form a ~- or 6-
membered
ring optionally containing; an additional heteroatom selected from O, S, and
NR1;
Rc is Rb, halo, ORb, NHSO2Rb, N(Rb)2, CN, N02, S02N(Rb)2, SO2Rb,
CF3, OCF3; or two Rc groups attached to adjacent carbon atoms together form
methylenedioxy;
Rd is H, N02, or CN;
Cy is aryl, 5- or 6-membered heteroaryl, 5- or 6-membered heterocyclyl, or
5-or 6-membered carboc;~ciyl;
-3-
SUBSTITUTE SHEET ( ruie 26
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nis Oto3;
m, p and q are independently 0, 1 or 2;
ris l,2or3;
or a pharmaceutically acceptable salt thereof.
In one subset of compounds of formula I are compounds wherein Cy2
is benzene or cvclohexane.
In another subset of compounds of formula I are compounds wherein
X is CHC02Rb, CHC(O)N(Rb)2, NS02Ra, CHN(Rb)CORa, CHN(Rb)S02Ra,
CHCH20Rb or CHCH2-heteroaryl.
In another subset of compounds of formula I are compounds wherein
Q is
Rb
CY R~
HN~
Rb and Rc are as defined under formula I, and Cy is aryl, 5- or 6-membered
heteroaryl, or ~-or 6-merribered carbocyclyl. Preferably Cy is benzene or
cyclohexane.
In another aubset of compounds of formula I are compounds wherein
R 1 is CH2-aryl in which aryl is optionally substituted by Rc.
In a prefewed embodiment there are provided compounds of formula
Ia:
HN C~Rc
Rc ~. \ w* ~R~
O 0
R
Ia
-4-
SUBSTITUTE SHEET ( rule 2b )
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wherein
X is CHC02Rb, CHC(O)N(Rb)2, NS02Ra, CHN(Rb)CORa, or
CHN(Rb)S02Ra;
R2 is H or halo;
Ra is Rb, (CH2)ruN(Rb)2~ (CH2)n~-2-PYridyl, (CH?}nNH-2-imidazolyl,
(CHZ)n'N~N'Rb
(CH2)nNH-2-thiazolyl, (CH2)nNH-2-pyrimidinyl, ~ , or
(CH2)n-
Rb is H, C1_galkyl, (CH?)naryl, (CH2)nheteroaryl, or C;_6cycloalkyi;
Rc is H, halo, RF~, ORb, CF;;, OCF3;
Cy is benzene, pyridine, imidazole or cyclohexane;
nis Oto3;
or a pharmaceutically accf;ptable salt thereof.
In another ;preferred embodiment are compounds of the forrn:ula Ib:
H HN CY ~Rc
* N
Rb
/ O O
N
-X
Ib
wherein
X is CHC02Rt~, CHC(O)N(Rb)2, CHCH20Rb or CHCH2-heteroaryl;
Rb is H, C1_gall;yl, {CH2}naryl, (CH2)nheteroaryl, or C3_6cycloalkyl;
Rc is H, halo, R'~, ORb, CF'3, OCF3;
Cy is benzene, pyridine, imidazole or cyclohexane;
nis Oto3;
or a pharmaceutically acceptable salt thereof.
SUBSTITUTE SHEET ( rule 26 )
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In a more preferred embodiment of compounds of formulas Ia and Ib,
the carbon atom marked v~ith * has the R configuration. In another mare
preferred
embodiment of formulas l:a and Ib Cy is benzene or cyclohexane.
Representative compounds of formula I are as follows:
HN ~' ~ H HN
/ ~ N
~O O
H CO I ''~ O O Cl /
N N
/ /
N, .~
'.,.. N,
o' s,o o s,o
H
HN OH
H HN
/ O O H w N /
CI N I ~ / ~O O
C
N
NS02Me
-._ F
Ms
-6-
SUBSTITUTE SHEET { ruie 26 )
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H I-IN I ~ H HN I w
N / ~ N /
I O I / OO
Br / Br
N N
N I s~ N I \~
Ms ~~ Ms
H HN I ~ F H HN I ~ N
N
( \ N~ / F
Cl / ~O O Cl / ~O O
N
F
N I
Ms ~ Ms
H HN ( N H HN ~ F
N ~ ~. N ~ I
w
I /~ ~ ~, I li
CI N CI / O O
N
F I \
N
Ms ~-
-7-
SUBSTITUTE SHEET ( rule 26 )
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HN I
w N ~ F OCF3
I 0 0 H HN I
CI / N
N I W
CI ~- ° O
N
N i ~)
Ms
N
Ms
w
H t-I N I ~ ~ H N
~ N I I .~ N I W f. Ci
I I / ~O O CI / N O O
C
N
CI
Ms
Ms
HN ~ HN
NH~ I / ~ NH I /
I / O O I ,,-- ~ O O
CI ~~ CI N
C) N~
O O
_g_
SUBSTITUTE SHEET ( rude 2C )
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I-IN l ~ _ HN
NH~ ''~ ~ NH
~o IoI l
CI N CI '~ ~ O
N
1
0
~~ N
EtN
~ NH
v
~O O
CI
N
Another aspect of the present invention provides a method for the
treatment or prevention of obesity or diabetes in a mammal which comprises
administering to said mammal an effective amount of a compound of formula I.
Another aspect of the present invention provides a method fox the
treatment or prevention of rnaie or female sexual dysfunction including
erectile
dysfunction which comprisEa administering to a patient in need of such
treatment or
prevention an effective amount of a compound of formula I.
Another aspa~ct of the present invention provides a method for the
treatment or prevention of male or female sexual dysfunction including
erectile
dysfunction which comprises administering to a patient in need of such
treatment or
prevention an effective amount of an agonist of melanocortin-4 receptor.
-9-
SUBSTITUTE SHEET ( rule 2b )
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Yet another aspect of the present invention provides a pharmaceutical
composition comprising a compound of formula I and a pharmaceutically
acceptable
carrier.
Throughout: the instant application, the following terms have the
indicated meanings:
The alkyl groups specified above are intended to include those alkyl
groups of the designated ie,ngth in either a straight or branched conf
guration.
Exemplary of such alkyl groups are methyl, ethyl, propyl, isoprop~-1, butyl,
sec-butyl,
tertiary butyl, penryl, isopentyl, hexyl, i.sohexyl, and the like.
The term "halogen" is intended to include the halogen atoms fluorine,
chlorine, bromine and iodine.
The term "aryl" includes phenyl and naphthyl.
The term ''l'neteroaryl" includes mono- and bicyclic aromatic rings
containing from 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur.
"5- or
6-membered heteroaryl" are monocyclic heteroaromatic rings, examples thereof
include thiazole, oxazole, thiophene, furan, pyrrole, imidazole, isoxazole,
pyrazoie,
triazole, thiadiazole, tetraa:ole, oxadiazole, pyridine, pyridazine,
pyTimidine, pyrazine,
and the like. Bicyclic hetc:roaromatic rings include, but are not limited to,
benzothiadiazole, indole, 'benzothiophene, ben~ofuran, benzimidazole,
benzisoxazole,
benzothiazole, quinoline, benzotriazole, benzoxazole, isoquinoline, purine,
furopyridine and thienopyridine.
The term "5- or 6-mernbered carbocyclyl" is intended to include non-
aromatic rings containing only carbon atoms such as cyclopenty 1 and
cyclohexyl.
The term "5 and 6-membered heterocyclyl" is intended to include non-
aromatic heterocycles cor.~taining one to four heteroatoms selected from
nitrogen,
oxygen and sulfur. Examples of a 5 or 6-membered heterocyclyl include
piperidine,
morpholine, thiamorpholine, pyrrolicline, imidazolidine, tetrahydrafuran,
piperazine,
and the like.
Certain of the above defined terms may occur more than once in the
above formula and upon :>uch occurrence each term shall be defined
independently of
the other; thus for exampl~,e, NRbRb may represent NH2, NHCH3, N(CH3)CH2CH3~
and the like.
The term "'composition", as in pharmaceutical composition, is intended
to encompass a product comprising the active 'ingredient(s), and the inert
ingredients)
-10-
SUBSTITUTE SHEET ( rule 26 )
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that make up the carrier, as well as any product which results, directly or
indirectly,
from combination, complex:ation or aggregation of any two or more of the
ingredients,
or from dissociation of one or more of the ingredients, or from other types of
reactions
or interactions of one or more of the ingredients. Accordingly, the
pharmaceutical
compositions of the present: invention encompass any composition made by
admixing
a compound of the present :invention and a pharmaceutically acceptable
carrier.
"Erectile dysfunction" is a disorder involving the failure of a male
mammal to achieve erection, ejaculation, or both. Symptoms of erectile
dysfunction
include an inability to achif;ve or maintain an erection, ejaculatory failure,
premature
ejaculation, inability to achieve an orgasm. An increase in erectile
dysfunction is often
associated with age and is ~;enerally caused by a physical disease or as a
side-effect of
drug treatment.
"Female sexual .dysfunction" encompasses, without limitatin,
conditions usch as a lack o~C sexual desire and related arousal disorders,
inhibited
orgasm, lubrication difficulties, and vaginismus.
Abbreviations Used
9-BBN 9-borabicyclo[3.3.1 jnonane
Bn b enzyl
BOC (boc) t-butyloxycarbonyl
BOP benzotriazol-1-yloxy tris(dimethylamino)
phosphonium
hexafluorophosphate
Bu butyl
colt. calculated
CBZ (Cbz) benzyloxycarbonyl
DCC dicyclohexylcarbodiimide
DCM dichloromethane
DIEA diisopropyiethylaxnine
DMAP 4-{N,N-dimethylamino)pyridine
DMF d.imethyiformamide
DPPA d.iphenylphosphoryl azide
EDC 1-(3-dimethylaminopropyl)3-ethylcarbodiimide
HCl
eq. equivalent(s)
ESI-MS electron ion-mass spectroscopy
-I1-
SUBSTITUTE SHEET { rule 26 )
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EtOAc ~ evthyl acetate
FAB-MS fast atom bombardment-mass spectroscopy
HOBt 1-hydroxybenzotxiazole hydrate
HPLC high pressure liquid chromatography
KHDMS potassium bis(trimethylsilyl)amide
LAH lithium aluminum hydride
LHMDS lithium bis{trimethylsilyl)amide
MC-xR melanocortin receptor {x being a nurnbei)
Me methyl
MF molecular farmuia
MHz megahertz
MPLC nnedium pressure liquid chromatography
Ms rnethanesulfonyl
NMM rJ-methyimorpholine
NMR nuclear magnetic resonance
PCC pyridiurn chlorochromate
Ph phenyl
Pr I>ropyl
prep. prepared
PyBrop bromo-tris-pyrrolidino-phosphonium
hexafluorophosphate
TFA trifluoroacetic acid
THF tetrahydrofuran
Tic ;C,2,3,4-tetrahydroisoquinoline-3- carboxylic
acid
TLC thin-layer chromatography
TMS t:etramethylsilane
Optical Isomers - Diastere;omers - Geometric Isomers - Tautomers
Compounds of Formula I contain one or more asymmetric centers and
can thus occur as racemates and racemic mixtures, single enantiomers,
diastereomeric
mixtures and individual diastereomers. The present invention is meant to
comprehend ali such isomeric forms of the compounds of Formula I.
_ ~2 _
SUBSTITITTE SHEET ( rude 26 )
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Some of thE; compounds described herein contain olefinic double
bonds, and unless specified otherwise, are meant to include both E and Z
geometric
isomers.
Some of thc~ compounds described herein may exist as tautomers such
as keto-enol tautomers. Tlae individual tautomers as well as mixture thereof
are
encompassed with compounds of Formula I.
Compounds of the Formula I may be separated into diastereoisomeric
pairs of enantiomers by, for example, fractional crystallization from a
suitable solvent,
for example methanol or ethyl acetate or a mixture thereof. The pair of
enantiomers
thus obtained may be separated into individual stereoisomers by conventional
means,
for example by the use of an optically active acid as a resolving agent.
Alternatively, any enantiomer of a compound of the general Formula I
or Ia may be obtained by ;>tereospecific synthesis using optically pure
starting
materials or reagents of known configuration.
1S
Salts
The term "pharmaceutically acceptable salts" refers to salts prepared
from pharmaceutically acceptable non-toxic bases or acids including inorganic
or
organic bases and inorganic or organic acids. Salts derived from inorganic
bases
include aluminum, ammonium, calcium, copper, fernc, ferrous, lithium,
magnesium,
manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly
preferred are the ammoni~.un, calcium, lithium, magnesium, potassium, and
sodium
salts. Salts derived from ;pharmaceutically acceptable organic non-toxic bases
include
salts of primary, secondary, and tertiary amines, substituted amines including
2S naturally occurring substituted amines, cyclic amines, and basic ion
exchange resins,
such as arginine, betaine, caffeine, clzoline, N,N'-dibenzylethylenediamine,
diethylamine, 2-diethyianainoethanol, 2-dimethylaminoethanol, ethanolamine,
ethylenediamine; N-ethyl-morpholin.e, N-ethylpiperidine, glucamine,
glucosamine,
histidine, hydra.bamine, isopropylarnine, lysine, rnethylglucamine,
morpholine,
pipera~zine, piperidine, polyamine resins, procaine, purines, theobromine,
triethylamine, trimethylamine, tripropylamine, trornethamine, and the like.
When the compound of the present invention is basic, salts may be
prepared from phazmaceutically acceptable non-toxic acids, including inorganic
and
organic acids. Such acids include acetic, benzenesulfonic, benzoic,
camphorsulfonic,
-13-
SUBSTITUTE SHEET ( rule ZG )
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citric, ethanesulfonic, formic, fumaric, gluconic, glutamic, hydrobromic,
hydrochloric, ,isethionic, lactic, malefic, malic, mandelic, methanesulfonic,
malonic,
mucic; nitric, pamoic, pantothenic, phosphoric, propionic, succinic, sulfuric,
tartaric,
p-toluenesulfonic acid, trifluoroacetic acid, and the like. Particularly
preferred are
citric, fumaric, hydrobromic, hydrochloric, malefic, phosphoric, sulfuric, and
tartaric
acids.
It will be understood that, as used herein, references to the compounds
of Formula I are meant to aso include the pharmaceutically acceptable salts.
Utili
Compounds of formula I are melanocortin receptor agonists and as
such are useful in the treatanent, control or prevention of diseases,
disorders or
conditions responsive to th.e activation of one or more of the melanocortin
receptors
including, but are not limited to, MC-l, MC-2, MC-3, MC-4, or MC-~. Such
diseases, disorders or conditions include, but are not limited to, obesity (by
reducing
appetite, increasing metabolic rate, reducing fat intake or reducing
carbohydrate
craving), diabetes mellitus (by enhancing glucose tolerance, decreasing
insulin
resistance), hypertension, hyperlipidemia, osteoarthritis, cancer, gall
bladder disease,
sleep apnea, depression, anxiety, compulsion, neuroses, insomnia/sleep
disorder,
substance abuse, pain, male and female sexual dysfunction (including
impotence, loss
of libido and erectile dysfunction), fever, inflammation, immune modulation,
rheumatoid arthritis, skin i:anning, acne and other skin disorders,
neuroprotective and
cognitive and memory enhancement including the treatment of Alzheimer's
disease.
Some compounds of formula I show highly specific activity toward the
melanocortin-
2~ 4 receptor which makes them especially useful in the prevention and
treatment of
obesity, as well as male arid female sexual dysfunction.
Administration and Dose :(tan es
Any suitable route of administration may be employed for providing a
mammal, especially a human with an effective dosage of a compound of the
present
invention. For example, oral, rectal, topical, parenterai, ocular, pulmonary,
nasal, and
the like may be employed. Dosage forms include tablets, taroches, dispersions,
suspensions, solutions, capsules, creams, ointments, aerosols, and the like.
Preferably
compounds of Formula I ~~re administered orally.
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SUB~~TITUTE SHEET { rule 26 )
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The effective dosage of active ingredient employed may vary
depending on the particular compound employed, the mode of administration, the
condition being treated and the severity of the condition being treated. Such
dosage
may be ascertained readily by a person. skilled in the art.
When treating obesity, in conjunction with diabetes and/or
hyperglycemia, or alone, generally satisfactory results are obtained when the
compounds of the present invention are administered at a daily dosage of from
0.01
milligram to about 100 millligrams per kilogram of animal body weight,
preferably
given in a single dose or in divided doses two to six times a day, or in
sustained
release form. In the case oil a 70 kg adult human, the total daily dose will
generally be
from about 0.7 milligrams t:o about 3500 milligrams. This dosage regimen may
be
adjusted to provide the optimal therapeutic response.
When treating diabetes mellitus and/or hyperglycemia. as well as other
diseases or disorders for which compounds of formula T are useful. generally
satisfactory results are obtained when the compounds of the present invention
are
administered at a daily dosage of from about 0.00I milligram to about 100
milligram
per kilogram of animal body weight, preferably given in a single dose or in
divided
doses two to six times a da;y, ar in sustained release form. In the case of a
70 kg adult
human, the total daily dose will generally be from about 0.07 milligrams to
about 350
milligrams. This dosage regimen may be adjusted to provide the optimal
therapeutic
response.
For the treatment of sexual dysfunction compounds of the present
invention are given in a dose range of 0.001 milligram to about I 00 milligram
per
kilogram of body weight, preferably as a single dose orally or as a nasal
spray.
Pharmaceutical Compositions
.Another aspect of the present invention provides pharmaceutical
compositions which comprises a compound of Formula I and a pharmaceutically
acceptable carrier. The pharmaceutical compositions of the present invention
comprise a compound of Formula I as an active ingredient or a pharmaceutically
acceptable salt thereof, and may also contain a pharmaceutically acceptable
carrier
and optionally other therapeutic ingredients. The term "pharrnaceuticalIy
acceptable
salts" refers to salts prepared from pharmaceutically acceptable non-toxic
bases or
acids including inorganic bases or acids and organic bases or acids.
-15-
SUBSTITUTE SHEET ( rule 26 )
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The compositions include compositions suitable for oral, rectal,
topical; parenterai (including subcutaneous, intramuscular, and intravenous),
ocular
(ophthalmic), pulmonary (nasal or buccal inhalation), or nasal administration,
although the most suitable route in any given case will depend on the nature
and
severity of the conditions being treated and on the nature of the active
ingredient.
They may be conveniently presented in unit dosage form and prepared by any of
the
methods well-known in thE; art of pharmacy.
In practical use, the compounds of Formula I can be combined as the
active ingredient in intimate admixture with a pharmaceutical carrier
according to
I O conventional pharmaceutical compounding techniques. The carrier may take a
wide
variety of forms depending; on the form of preparation desired for
administration, e.g.,
oral or parenteral (including intravenous). In preparing the compositions for
oral
dosage form. any of the usual pharmaceutical media may be employed, such as,
for
example, water, glycols, oils, alcohols, flavoring agents, preservatives,
coloring
agents and the like in the case of oral liquid preparations, such as, for
example,
suspensions, elixirs and solutions; or carriers such as starches, sugars,
micracrystalline cellulose, diluents, granulating agents, lubricants, binders,
disintegrating agents and the like in the case of oral solid preparations such
as, for
example, powders, hard and soft capsules and tablets, with the solid oral
preparations
being preferred over the liquid preparations.
Because of their ease of administration, tablets and capsules represent
the most advantageous orG~l dosage unit form in which case solid
pharmaceutical
corners are obviously employed. If desired, tablets may be coated by standard
aqueous or nonaqueous techniques. such compositions and preparations should
contain at least 0. i percent of active compound. The percentage of active
compound
in these compositions may, of course, be varied and may conveniently be
between
about 2 percent to about 60 percent of the weight of the unit. The amount of
active
compound in such therapeutically useful compositions is such that an effective
dosage
will be obtained. The active compounds can also be administered intranasally
as, for
example, liquid drops or spray.
The tablets, pills, capsules, and the like may also contain a binder such
as gum tragacanth, acacia, corn starch or gelatin; excipients such as
dicalcium
phosphate; a disintegrating agent such as corn starch, potato starch, alginic
acid; a
lubricant such as magnesium stearate; and a sweetening agent such as sucrose,
lactose
-16-
SUBSTITUTE SHEET ( rule 26 )
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or saccharin. When a dosage unit form is a capsule, it may contain, in
addition to
materials of the above type, a liquid carrier such as a fatty oil.
Various other materials may be present as coatings or to modify the
physical form of the dosage unit. For instance, tablets may be coated with
shellac,
sugar or both. A syrup or elixir may contain, in addition to the active
ingredient,
sucrose as a sweetening agent, methyl and propyiparabens as preservatives, a
dye and
a flavoring such as cherry or orange flavor.
Compounds of formula I may also be administered parenterally.
Solutions or suspensions of these active compounds can be prepared in water
suitably
mixed with a surfactant such as hydroxy-propylcellulose. Dispersions can also
be
prepared in glycerol, liquid polyethylene glycols and mixtures thereof in
oils. Under
ordinary conditions of storage and use, these preparations contain a
preservative to
prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile
1 ~ aqueous solutions or dispersions and sterile powders for the
extemporaneous
preparation of sterile inject;able solutions or dispersions. In all cases, the
form must
be sterile and must be fluid to the extent that easy syringability exists. It
must be
stable under the conditions of manufacture and storage and must be preserved
against
the contaminating action o~~ microorganisms such as bacteria and fungi. The
carrier
can be a solvent or dispersion medium containing, for example, water, ethanol,
polyol
(e.g. glycerol, propylene glycol and liquid polyethylene glycol), suitable
mixtures
thereof, and vegetable oils.
Combination Therapy
Compound:. of Formula I may be used in combination with other drugs
that are used in the treatmewt/prevention/suppression or amelioration of the
diseases
or conditions for which compounds of Formula I are useful. Such other drugs
may be
administered, by a route arid in an amount commonly used therefor,
contemporaneously or sequentially with a compound of Formula I. When a
compound of Formula I is used contemporaneously with one or more other drugs,
a
pharmaceutical composition containing such other drugs in addition to the
compound
of Formula I 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 I. Examples of other active ingredients
that
SUBSTITUTE SHEET ( rule 26 )
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WO 99/64002 PCT/US99/13252
may be combined with a c;ompound of Formula I, either administered separately
or in
the same pharmaceutical compositions, include, but are not limited to:
{a) insulin sensitizers including (i) PPARy agonists such as the
glitazones (e.g. troglitazone, piogiitazone, englitazone, MCC-555. BRL49653
and the
like), and compounds disclosed in W097/27857, 97/28115, 97128137 and 97/27847;
(ii) biguanides such as mE;tformin and phenforrnin;
(b) insulin or insulin rnimetics;
(c) sulfonylureas such as tolbutamide and glipizide;
(d) a-glucosidase inhibitors (such as acarbose),
(e) cholesterol lowering agents such as (i) HMG-CoA reductase
inhibitors (Iovastatin, .simvastatin and pravastatin, fluvastatin,
atorvastatin, and other
statins), (ii} sequestrants ( cholestyramine, colestipol and a
diaikylaminoalkyl
derivatives of a cross-Iinls;ed dextran), (ii) nicotinyl alcohol nicotinic
acid or a salt
thereof, (iii) proliferator-activates receptor a agonists such as fenof brie
acid
derivatives (gemfibrozil, clofibrat, fenofibrate and benzafibrate), (iv)
inhibitors of
cholesterol absorption for- example beta-sitosterol and (acyl CoA:cholesterol
acyltransferase) .inhibitor;s for example meiinamide, (v) probucol, (vij
vitamin E, and
{vii) thyromimetics;
(f) PPARFi agonists such as those disclosed in WO97/28149;
(g) antiobesity compounds such as fenfluramine, dexfenfluramine,
phentermine, sibutramine, oriistat, or X33 adrenergic receptor agonists;
(h) feeding behavior modifying agents such as neuropeptide Y
antagonists (e.g. neuropeptide Y5) such as those disclosed in w'O 97/19682, WO
97/20820, WO 97r'20821, WO 97/2t)822 and WO 97120823;
(i') -PPARa agonists such as described in WO 97,'36579 by Glaxo;
(j) PPAR~r antagonists as described in W097/10813;
(k) serotonin reuptake inhibitors such as fluoxetine and sertraline;
(1) growth hormone secretagogues such as MK-0677; and
(m) agents useful in the treatment of male and/or female sexual
dysfunction such as phosphodiester V inhibitors such as sildenafil, and a-2
adrenergic
receptor antagonists.
-i8_
SUBSTITUTE SHEET ( rule 26 )
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Biological Assays
A. Binding Assay. The membrane binding assay is used to identify
competitive inhibitors of l:zsl-a-NDP-MSH binding to cloned human MCRs
expressed
in L- or CHO- cells.
Cell lines expressing melanocortin receptors are grown in T-180 flasks
containing selective mediiun of the composiiton: 1 L Dulbecco's modified
Eagles
Medium (DMEM) with 4.5 g L-glucose, 25 mM Hepes, without sodium pyruvate,
(GibcoBRl); 100 mI 10% heat-inactivated fetal bovine serum (Sigma); IO rnl
10,000
unit/ml penicillin & 10,000 ug/mi streptomycin {GibcoBRl); 10 mi 200 mM
L-glutamine (GibcoBRl); 1 mg/ml Geneticin (G4I8) (GibcoBRl). The cells are
grown at 37°C with C02 and humidity control until the desired cell
density and cell
number is obtained
The medium is poured off and 10 mls/monolayer of enzyme-free
dissociation media (Specialty Media Ine.) is added. The cells are incubated at
37°C
I ~ for 10 minutes or until cells slough off when flask is banged against
hand.
The cells are harvested into 200 ml centrifuge tubes and spun at 1000
rpm, 4° C, for 10 min. 'Tl~e supernatant is discarded and the cells are
resuspended in 5
mls/monolayer membrane: preparation buffer having the composition: 10 mM Tris
pH
7.2-7.4; 4 ug/ml Leupepti:n (Sigma); i 0 uM Phosphoramidon {Boehringer
Mannheim); 40 ug/ml Bacitracin (Sigma); 5 ug/ml Aprotinin (Sigma); IO mM
Pefabloc (Boehringer Mannheim). The cells are homogenized with motor-driven
dounce {Talboy setting 4()), using IO strokes and the homogenate centrifuged
at 6,000
rpm, 4 C, for I5 minutes.
The pellets are resuspended in 0.2 mls/monolayer membrane prep
buffer and aliquots are placed in tubes (500-1000 uUtube) and quick frozen in
liquid
nitrogen and then store at -80 ° C.
Test compounds or unlabelled NDP-a-MSH is added to 100 p.L of
membrane binding buffer to a final concentration of 1 p.M. The membrane
binding
buffer has the composition: 50 mM Tris pH 7.2; 2 mM CaCl2; 1 mM MgCl2; 5 mM
KCI; 0.2% BSA; 4 ug/ml Leupeptin (SIGMA); 10 uM Phosphoramidon {Boehringer
Mannheim); 40 uglml Bacitracin (SIGMA); 5 ug/ml Aprotinin (SIGMA); and 10 mM
Pefabloc {Boehringer Mannheim). One hundred ~l of membrane binding bufifer
containing 10-40 ug membrane protein is added, followed by 100 ~M i25I-NDP-a-
-I9-
SUBSTITUTE S~IEET ( rude 26 )
CA 02334551 2000-12-05
WO 99164002 PCT/US99/13252
MSH to final. concentration of 100 pM. The resulting mixture is vortexed
briefly and
incubated for 90-I20 min at room temp while shaking.
The mixture is filtered with Packard Microplate 196 filter apparatus
using Packard Unifilter 96-well GF/C filter with 0.1% polyethyleneimine
(Sigma).
The f lter is washed (5 times with a total of 10 ml per well) with room
temperature of
filtez wash having the composition: 50mM Tris-HCl pH 7.2 and 20 mM NaCI. The
filter is dried, and the bottom sealed and 50 ul of Packard Microscint-20 is
added to
each well. The top is sealed and the radioactivity quantitated in a Packard
Topcount
Microplate Scintillation counter.
IO
B. Functional assay. Functional cell based assays are developed to
discriminate agonists and antagonists.
Cells (for example, CHO- or L-cells or other eukaryotic cells)
expressing a human melaruocortin receptor (see e.g. Yang-YK; Ollmann-MM;
15 Wilson-BD; Dickinson-C; Yamada-T; Barsh-GS; Gantz-I; Mol-Endocrinoi. 1997
Mar; 11(3): 274-80) are dissociated from tissue culture flasks by rinsing with
Ca and
Mg free phosphate buffered saline (14190-136, Life Technologies, Gaithersbwrg,
MD)
and detached following 5 :minutes incubation at 37°C with enzyme free
dissociation
buffer (S-014-B, Specialt3~ Media, Lavellette, N~. Cells are collected by
20 centrifugation and resuspe,nded in Earle's Balanced Salt Solution (14015-
069, Life
Technologies. Gaithersbw~g, MD) with additions of I O mM HEPES pH 7.5, 5 mM
MgCl2, 1 mM glutamine and I mg/ml bovine serum albumin. Cells are counted and
diluted to 1 to 5 x 106/mi. The phosphodiesterase inhibitor 3-isobutyl-1-
methylxanthine is added to cells to 0.6 mM.
25 Test compounds are diluted in dimethylsulfoxide (DMSO) (10'5 to
10-'° M) and 0.1 volume of compound solution is added to 0.9 volumes of
cell
suspension; the final DMSO concentration is 1%. After room temperature
incubation
for 45 min., cells are lysed by incubation at 100 C for 5 min. to release
accumulated
cAMP.
30 CAMP is measured in an aliquot of the cell lysate with the Amersharn
(Arlington Heights, IL) c~AMP detection assay (RPA556). The amount of CAMP
production which results :from an unknown compound is compared to that amount
of
cAMf produced in response to alpha-MSH which is defined as a I00 % agonist.
The
-20-
SUBSTITUTE SHEET ( ruie 26 )
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WO 99164002 PCT/US99l13252
EC50 is defined as the compound concentration which results in half maximal
stimulation, when comparf:d to its own maxim level of stimulation.
Antagonist assa : Antagonist activity is defined as the ability of a
compound to block CAMP production in response to alpha-MSH. Solution of test
compounds and suspension of receptor containing cells are prepared and mixed
as
described above; the mixt~~re is incubated for 15 min., and an EC50 dose
(approximately 10 nM alp:ha-MSH) is added to the cells. The assay is
terminated at
45 min. and cAMP quantii:ated as above. Percent inhibition is determined by
comparing the amount of CAMP produced in the presence to that produced in the
absence of test compound..
C. In vivo food intake models.
1) Overnil;ht food intake. Sprague Dawley rats are injected
intracerebroventricularly with a test compound in 400 nL of ~0% propylene
glycol/artificial cerebrospinal fluid one hour prior to onset of dark cycle
(12 hours).
Food intake is determined using a computerized system in which each rat's food
is
placed on a computer montored balance. Cumulative food intake for 16 hours
post
compound administration is measured.
2) Food ir,~take in diet induced obese mice. Male C~7B 16J mice
maintained on a high fat diet (60% fat calories) for 6.5 months from 4 weeks
of age
are are dosed intraperiton~~ally with test compound. Food intake and body
weight are
measured over an eight day period. Eiochemical parameters relating to obesity,
including leptin, insulin, triglyceride, free fatty acid, cholesterol and
serum glucose
levels are determined.
D. Rat Ex Copula Assay
Sexually mature male Caesarian Derived Sprague Dawley (CD) rats
(over 60 days old) are used with the suspensory ligament surgically removed to
prevent retraction of the penis back into the penile sheath during the ex
copula
evaluations. Animals receive food and water ad lib and are kept on a normal
lightldark cycle. Studies are conducted during the light cycle.
-21-
SUB~3TITUTE SHEET ( rule 2f )
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WO 99/64002 PCT/CTS99/13252
a) Conditioning to Supine Restraint for Ex Copula Reflex Tests.
This conditioning takes ~ 4 days. Da;y 1, the animals are placed in a darkened
restrainer and left for I5 - 30 minutes. Day 2, the animals are restrained in
a supine
position in the restrainer for 15 - 30 minutes. Day 3, the animals are
restrained in the
supine position with the penile sheath retracted for 15 - 30 minutes. Day 4,
the
animals are restrained in the supine position with the penile sheath retracted
until
penile responses are obse~.-ved. Some animals require additional days of
conditioning
before they are completely acclimated to the procedures; non-responders are
removed
from further evaluation. After any handling or evaluation animals are given a
treat to
ensure positive reinforcement.
b) Ex Copula Reflex Tests. Rats axe gently restrained in a supine
position with their anterior torso placed inside a cylinder of adequate size
to allow for
normal head and paw grooming. For a 404-500 gram rat, the diameter of the
cylinder
is approximately 8 cm. The lower torso and hind limbs are restrained with a
non-
adhesive material (vetrap). An additional piece of vetrap with a hole in it,
through
which the glares penis will be passed, is fastened over the animal to maintain
the
preputial sheath in a retracted position. Penile responses will be observed,
typically
termed ex copula genital reflex tests, Typically, a series of penile erections
will occur
spontaneously within a fe;w minutes after sheath retraction. The types of
normal
reflexogenic erectile responses include elongation, engorgement, cup and flip.
An
elongation is classified a:a an extension of the penile body. Engorgement is a
dilation
of the glares penis. A cup is defined as an intense erection where the distal
margin of
the glares penis momentarily flares open to form a cup. A flip is a
dorsiflexion of the
penile body.
Baseline and or vehicle evaluations are conducted to determine how
and if an animal will respond. Some animals have a long duration until the
first
response while others are; non-responders altogether. During this baseline
evaluation
latency to first response, number and type of responses are recorded. The
testing time
frame is I5 minutes after the fzrst response.
After a minimum of 1 day between evaluations, these same animals are
administered the test compound at 20 mg/kg and evaluated for penile reflexes.
All
evaluations are videotaped and scored later. Data are collected and analyzed
using
paired 2 tailed t-tests to compared baseline and/ or vehicle evaluations to
drug treated
-22-
SUBSTITUTE SHEET ( rude 2~ )
CA 02334551 2000-12-05
WO 99164002 PCT/US99/13252
evaluations for individual animals. Groups of a minimum of 4 animals are
utilized to
reduce variability.
Positive reference controls are included in each study to assure the
validity of the study. Animals can be dosed by a number of routes of
administration
depending on the nature of the study to be performed, The routes of
administration
includes intravenous (IV), intraperitoneal {IP), subcutaneous (SC) and
intracerebrai
ventricular (ICV).
E. Models of Female Sexual Dysfunctioin
Rodent assays relevant: to female sexual receptivity include the
behavioral model of lordosis and direct observations of copulatory activity.
There is
also a urethrogenital reflex: model in anesthetized spinally transected rats
for
measuring orgasm in both male and female rats. These and other established
animal
models of female sexual dysfunction are described in McKenna KE et al, A Model
I S For The Study Of Sexual I?unction In Anesthetized Male And Female Rats,
Am. J.
Physiol. {Regulatory Integrative Comp. Physiol 30): 81276-R128~, 1991; McKenna
KE et al, Modulation By Peripheral Serotonin Of The Threshold For Sexual
Reflexes
In Female Rats, Pharm. Bioch. Behav., 40:151-156, 1991; and Takahashi LK et
al,
Dual Estradiol Action In The Diencephalon And The Regulation Of Sociosexual
Behavior In Female Gold~;n Hamsters, Brain Res., 359:194-207, 1985.
Preparation of Compound of the Invention
The preparation of compounds of Formula I of the present invention
may be carried out in sequential or convergent synthetic routes. Syntheses
detailing
the preparation of the compounds of Formula I in a sequential manner are
presented in
the following reaction schemes. The instant compounds are generally isolated
in the
form of their pharmaceutically acceptable salts, such as those described
previously
hereinabove.
The phrase; "standard peptide coupling reaction conditions" is used
repeatedly here, and it means coupling a carboxylic acid with an amine using
an acid
activating agent such as EDC, DCC, and BOP in a inert solvent such as
dichloromethane in the presence of a. catalyst such as HOBT. The uses of
protective
groups for amine and carboxylic acid to facilitate the desired reaction and
minimize
undesired reactions are well documented. Conditions required to remove
protecting
-23-
SUBSTITUTE SHEET ( rule Zb )
CA 02334551 2000-12-05
WO 99/64002 PCT/US99113252
groups which may be present and can be found in Greene, T, and W uts, P. G.
M.,
Protective Groups in Organic Synthesis, John Wiley & Sons, inc.. New York, NY
1991. CBZ and BOC are used extensively in the synthesis, and their removal
conditions are known to those skilled i.n the art. For example, removal of CBZ
groups
can be achieved by a number of methods known in the art; for example,
catalytic
hydrogenation with hydrogen in the presence of a noble metal or its oxide such
as
palladium on activated carbon in a protic solvent such as ethanol. In cases
where
catalytic hydrogenation is contraindicated by the presence of other
potentially reactive
functionality, removal of CBZ groups can also be achieved by treatment with a
solution of hydrogen bromide in acetic acid, or by treatment with a mixture of
TFA
and dimethylsulfide. Removal of BOC protecting groups is carried out in a
solvent
such as methylene chloride; or methanol or ethyl acetate, with a strong acid,
such as
trifluoroacetic acid or hydrochloric acid or hydrogen chloride gas.
The protected amino arid derivatives 1 are, in many cases,
commercially available, wJhere the protecting group L is, for example, BOC or
CBZ
groups. Other protected amino acid derivatives 1 can be prepared by literature
methods (Williams, R. M. Synthesis ~f Optically Active a Amino Acids, Pergamon
Press: Oxford, 19$9). Mary of the piperidines of Formula 2 are either
commercially
available or known in the literature arid others can be prepared following
literature
methods described for analogous compounds. Some of these methods are
illustrated
in the subsequent schemes. The skills required in carrying out the reaction
and
purification of the resulting reaction products are known to those in the art.
Purification procedures include crystallization, normal phase or rev erse
phase
chromatography.
-24-
SUBSTITUTE SHEET ( rule 2~ )
CA 02334551 2000-12-05
WO 99164002 PCT/US99/1325Z
SCHEME I
H
R~-~--NH2
H H C~O
R~--~--N_L
C4 H
,) 2
RZ Removal of
L RZ
R2
2
Intermediates of Formula 3 may be synthesized as described in Scheme
l . Coupling of amine of hormula 2 to protected amino acids of Formula l,
wherein L
is a suitable protecting group {BOC, CBZ, etc), is conveniently carried out
under
standard peptide coupling, conditions, and the removal of the protecting group
may be
conducted using well-known methods..
Compounds of Formula I may be prepared as shown in Schemes 2 and
3. In Scheme 2 intermediates of Formula 3 are coupled to protected amino acids
of
Formula 4 (L = protecting; group such as Boc, CBZ, FMUC, etc.) under the
standard
peptide-type coupling reaction conditions. The amino acids 4 are either
commercially
available or can be synthesized by methods as described later.
In Scheme. 3, amino acid ester intermediates of Formula S, wherein L'
is a small alkyl such as methyl or ethyl or a benzyi or allyl unit, can be
synthesized by
1 S well documented methods in the literature. Coupling of intermediates 4 and
S under
standard peptide coupling conditions followed by removal of the ester group L'
yields
the intermediate 6. Compounds of formula I are obtained by coupling
intermediates
of Formula 6 to spiropipe;ridines of formula 2 under standard peptide coupling
reaction conditions, followed by the removal of the amino protecting group, L.
-25-
SUBSTITUTE SHEET ( rule 26 )
CA 02334551 2000-12-05
WO 99164002 PCTIUS99/13252
SCHEME 2
H
R~--~--NH2
c=o o Rb
i
N ~ FiC)--~.-(CRbRb)m p C ~~ Rc
L~ N
Rz q Rc 1. coupling
4
CY2 X 2. Removal
R2 3 H H O Rb of L
R~- ~ -N -C-(CRbRb)m p Cy Rc
C=O ,N
.N H 4 Rc
R2
Formula 1
R2 .."., x
-26-
SUBSTITUTE SHEET { ruie 26 )
CA 02334551 2000-12-05
WO 99!64002 PCT/US99/13252
SCHEME 3
O Rb
H H HO~"(CRbR~)m p ~ Rc
R -+--N~H
C=O -~ L~ N
R
OL'
4
H
N
R2
O Ftb
HO-~L.-(CRbRb)m"' p CY Rc R2 ''~X
2
L~ P~
6 q R~ removal of L
H ri O R~
R~----~-rJ -C- (CRbR~)m p CY Rc
CTt) H.N
q Rc
N
R2 Form ula I
CY2
RZ .,~ X
The compounds of the present invention may also be prepared from a
variety of substituted natuz~al and unnatural amino acids of formulas $.
H R
R~~N'H
C02H
8
The preparation of many o~f these acids is described in US Patent No.
5,246,237. The
preparation of these interniediates in racemic form is accomplished by
classical
-2?-
SUBS'~TITUTE SHEET ( rude 26 )
CA 02334551 2000-12-05
WO 99!64002 PCT/US99l13252
methods familiar to those :>killed in the art (Williams, R. M. "Synthesis of
Optically
Active a-Amino Acids" Pergamon Press: Oxford, 1989; Vol. 7). Several methods
exist
to resolve (DL)-amino acids. One of the common methods is to resolve amino or
carboxyl protected intermediates by crystallization of salts derived from
optically
active acids or amines. Alternatively, the amino group of carboxyl protected
intermediates may be coul>led to optically active acids by using chemistry
described
earlier. Separation of the individual diastereomers either by chromatographic
techniques or by crystallization followed by hydrolysis of the chiral amide
furnishes
resolved amino acids. Similarly, amino protected intermediates may be
converted to a
mixture of chiral diastereo~meric esters and amides. Separation of the mixture
using
methods described above and hydrolysis of the individual diastereomers
provides {D)
and {L) amino acids. Fina~.lly, an enzymatic method to resolve N-acetyl
derivatives of
(DL)-amino acids has been reported by Whitesides and coworkers in J. Am. Chem.
Soc. 1989, _l l 1. 6354-6364.
When it is desirable to synthesize these intermediates in optically pure
form, established methods include: {I) asymmetric electrophiiic amination of
chiral
enoiates (J. Am. Chem. So~c. 1986, _108, 6394-6395, 6395-6397, and 6397-6399),
(2)
asymmetric nucleophilic amination of optically active carbanyl derivatives,
(J. Am.
Chem. Soc. 1992, _l 14, 1906; Tetrahedron Lett. 1987, 28, 32), {3)
diastereoselective
alkylation of chiral glycine enolate synthons {.J. Am. Chem. Soc. 1991, 113,
9276; J.
Org. Chem. 1989, _~4, 3916), (4) diastereoselective nucleophiiic addition to a
chiral
electrophilic glycinate synthon (J. Am. Chem. Soc. 1986, 108, 1103), (5)
asymmetric
hydrogenation of prochiral dehydroarnino acid derivatives ("Asymmetric
Synthesis,
Chiral Catalysis; Morrison, J. D., Ed; Academic Press: Orlando, FL, 1985; Vol
5),
and .(6) enzymatic syntheses (Angew. Chem. Int. Ed. Engl. 1978,1; , 176).
_2g_
SUBSTITUTE SHEET ( rule 26 )
CA 02334551 2000-12-05
WO 99164002 PCT/LJS99/13252
SCHEME 4
Ph Ph
Ph.,, - NaN(TMS)2, Ph.,, -
._~ O >
~N~p~ p-OCF;~-benzyi t-Boc
t-Boc bromide
OCF3
'S ) TFA 1
2)PdCl2/Hz
NHS
/; p02H
F3CO
11
For exampla~, alkylation of the enolate of diphenyloxazinone 9 (J. Am.
Ghem. Soc. 1991, _113, 9276) with p-trifluorornethoxybenzyl bromide in the
presence
of sodium bis(trimethylsilyl)amide proceeds smoothly to afford 10 which is
converted
into the desired (D)-amino acid 11 by removing the N-t-butyloxycarbonyl group
with
trifluoroacetic acid and hydrogenation over a PdCl2 catalyst (Scheme 5).
The spiropi:peridines of formula 12 may be prepared by a number of
methods. including the syntheses described below. In cases where a sulfide is
present
in the molecule, it may be oxidized to a sulfoxide or to a sulfone with
oxidizing
agents such as sodium periodate, m-chioroperbenzoic acid or Oxone~~ in an
solvent
such as dichloromethane, acohoi or water or their mixtures.
H
N
R2
12 X = NRb, NS02R~, NS02N(Rb)2, NCORa, NCON(Rb)2
-29-
SUBSTITUTE SHEET ( rude 26 )
CA 02334551 2000-12-05
WO 99Ib4002 PCT/US99/13252
As shown in Scheme ~, the spiropiperidine of formula 13, wherein L is
a protecting group (such as methyl or benzyl), is synthesized by methods that
are
known in the literature (for example H. Ong et al J Med. Chew. 1983. 23, 981-
986).
The indoline nitrogen of 13 can be reacted by with a variety of electrophiles
to yield
spiro piperidines of formula I4, wherein the substitutent can be a variety of
functionalities, including Rb, S02Ra, S02N(Rb)2, CORa, CON(Rb)2. Compound 13
can be reacted with, for example, isocyanates in an inert solvent like
dichloromethane
to yield urea derivatives, cl~~loroformates in an inert solvent such as
dichloromethane
to yield carbamates, acid chlorides, anhydrides, or acyl imidazoles to
generate amides,
sulfonyl chlorides to generate sulfonamides, sulfamyl chlorides to yield
sulfamides.
Also, the indoline nitrogen of 13 can be reductively alkylated with
aldehydes with conditions known in the art. Aromatic units, including
substituted
heteroaryl groups, can be introduced by reacting 13 with a fluoro phenyl or
fluoro
I3 heteroaryl reagent. This chemistry is detailed by H. Ong et al J. s~Ied.
Chem. 1983,
23, 981-986.
SCHEME 5
L L
N N
Ht
14
X = NRb, NSOZRa, NSOzN(Rb)2,
NCORa, NCON(Rb)z
As shown i:n Scheme 6, the spiro piperidine intermediate 14 (L = Me
or Bn) can be dernethyiated or debenzylated' using a number of methods well
know to
those skilled in the art to produce 15. Demethylation of 14 be accomplished by
reacting it with cyanagen bromide and potassium carbonate in an inert solvent
solvent
such as dichloromethane to yield a cyanamide which can reduced to give 15 by
-30-
SUBSTITUTE SHEET t rule 26 }
CA 02334551 2000-12-05
WO 99/64002 PCTIUS991I3252
treatment with lithium alumtinum -hydride in refluxing tetrahydrofuran,
refluxing
strong acid like aqueous hydrochloric acid, or with Grignard reagents like
methyl
magnesium bromide. Alternatively, demethylation of 14 can be effected with the
ACE-Cl method as described in R. Olafson et al. J. Org. Chem. 1984, 49, 2795
and
references therein. Debenzylation can be accomplished by reductive methods
including hydrogenation in the presence of platinum or palladium catalyst in a
erotic
solvent like methanol. Alternatively, debenzylation of 14 can be effected with
the
ACE-Cl method as described in R. Olofson et al. .I. Org. Chem. 1984, 49, 2795
and
references therein.
X
SCHEME 6
H
N N
14
L = methyl o~r benzyl
X = NRb, N~~U2Ra, NSO2N(Rb)2, NCORa, NCON(R~')2
The spiro heterocyclic compounds of formula 15 can be prepared by a
15 number of methods, including the syntheses as.described in Scheme 7.
Allylic
oxidation of the protected piperidine 17 is accomplished by classical methods
familiar
to those skilled in the art (Rabjohn, N. Org. React. 1976, 24, 261). The
resulting
allylic alcohol is treated with thionyl chloride in an inert solvent such as
benzene to
provide the corresponding, chloride 18. When X=O or S, the alkylation is
carried out
in DMF or acetone as solvent with potassium carbonate as a base, and when X =
N or
derivativized with an aik~~l, aryl, acyl, sulfonyl, carbamate) the reaction is
carried out
with sodium hydride as aybase in an inert solvent such as THF to afford the
eyclizatian precursor 19. When L is a defined protecting group, compound 19
can be
cyclized by a number methods familiar to those skilled in the art. For
example,
cyciization of 19 can be accomplished by reaction with tributyltin hydride
(Curran ,
D. P. Synthesis 1988, 417 and 489) in an inert solvent such as benzene to
yield 16.
-3I-
SUBSTITUTE SHEET ( rule 2b )
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SCHEME 7
9 ) Se02 ~ v ~%--X-H
a>;~ocsz N --;
Y
base
17 Ci 18
L H
N N ~N
Bu3SnH
X Y X I / X
19 / 2~ 15
X = NRb, NS02Ra, NSO2N(Rb)2, NCORa, NCON(Rb)2
Y = halide, S~e or S
SCHEME 8
L
N N
Io1
X ~ /
X /
16 X=S
16 X = Suifoxide or sulfone
As shown in Scheme 8, when X=S, compound 16 can be oxidized to
the sulfoxide 16 (X = S(())) and the sulfone 16 (S02) by many oxidizing
agents. For
example, sodium periodate is often used for the synthesis of sulfoxides and
Oxone is
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SUBSTITUTE SHEET ( rule 26 )
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WO 99164002 PCTIUS99113252
used for the synthesis of sulfones. Removal of the protecting group provides
the
amine 16 which then can be elaborated to melanocortin agonists.
SCHEME 10
L
KN(TMS)2 Pd(Ac)2
2 > s
(F3cso2)2NPr co, MeOH
G 2~ Tf0
22
L L
I I
H2, Pd/C
2
cH3o cH3o
24
23
Hornologat:ion of the s;piroindanone 21 provides easy access to
spiroindanyl intermediates containing acid and ester groups. This chemistry is
described in Scheme 10. Treatment of 21 with a base in an inert solvent such
as THF
followed by the addition of a triflating agent provides the enol triflate.
Carboxylation
of the enol triflate according to the procedure of Cacchi, S. Tetrahedron
Letters, 1985,
1109-1112 provides the e;~ter 23. Hydrogenation of 23 using a palladium
catalyst in
an inert solvent provides the saturated ester 24. The protecting group can
then be
removed as described above and the resulting amine can be incorporated into
the
subject compound via the chemistry depicted in earlier schemes.
Sapanifacation of the ester of 24 provides an acid which can be
conveniently derivatized as for example reaction with an amine in the presence
of a
coupling agent such as EEC gives amides. which can then be incorporated into
final
compounds.
- 33 -
SUBSTITUTE SHEET ( rule 26 )
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WO 99164002 PCT/US99113252
. The ester 24 may also be reduced to a primary alcohol with LAH and
to a aldehyde with DIBALH. Reductive alkylation of the aldehyde ~i~ith
ammonium
acetate and sodium cyanoborohydride affords an amino methyl analog. These
aminomethyl analogs may then be further reacted with acylating and
suifonylating
agents to afford additional melanocortin compounds of the general formula I.
As illustrated in Scheme I I, spiroindanes can be hydrogenated with
Pt/C or Rh/alumina as cat<<lysts in solvents such as methanol, ethanol or
acetic acid to
afford corresponding perh;ydroindanes. High pressures are often required to
carry out
this saturation reaction. The L protecting group can be removed by standard
methods
I4 as discussed above.
SCHEME I1
L_ L
i
r~
H~lcatalyst
C02H
IS
Chiral acids are available by a variety of methods known to those
skilled in the art including asymmetric catalytic hydrogenation and resolution
of a
pair of diastereomeric salsa formed by reaction with a chiral amine such as D
or L oc-
methylbenzylamine. The absolute stereochemistry can be determined in a number
of
20 ways including X-ray crystallography of a suitable crystalline derivative.
Protected ~unino acids of formula 5, wherein L is a suitable protecting
group such as Boc or CB~~, can by conveniently synthesized by methods well
documented in the literattue.
- 34 -
SUBSTITUTE SHEET ~ ruse 26 )
CA 02334551 2000-12-05
WO 99/64002 PCT/US99I13252
O R~
n
Eio-c
N P CY Rc
L'
Rc
For example;, as shown in Scheme 11, a substituted phenyl alanine
derivative 2~ can be treated with aqueous formaldehyde in concentrated
hydrochloric
acid to afford, after protection of the amino functionality in a second step
by well
documented methods, the t~~trahydroisoquinoline compound 2b. This reaction can
also be effected with heterocyclic amino acids such as 2- and 3-thienyl Ala.
Since the
above chemistry works generally with retention of stereochemistry, D- and L-
amino
acids of general formula 5 can be prepared from D- and L- amino acids.
SCHEME 12
Rb O Rb
t!
HO-C ~ ~- F~~ '(. 37°/'° HCHO HOC N ~ ~ Rc
N Hz ~ L
conc. I-iC!
z5 2. Protection of 26
amine functionality
As shown in Scheme 12, a second method to prepare compounds of
formula 5 includes alkylation of a dihalide {L = Br, CI, I) of formula 27 with
dimethylacetamidomalonavte in the presence of a strong base such as NaH in DMF
to
afford alkylated material of formula 28. Treatment of esters of formula 28
with alkali
leads to formation of the corresponding mono carboxylic acid which can be
treated
with refluxing hydrochloric to affect hydrolysis of the acetamide derivative
to provide
amino acid of formula 29. Once again, standard protection of the amino
functionality
provides intermediates of i:orrnula 30.
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SUBSTITUTE SHEET ( rule 2C
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WO 99164002 PCT/US99II3252
SCHEME 13
1. Base,
Z -
Me00C~~NHAc O Cp2Me
~ R° Me0--C--~
/ COOMe i R~
Z Ac N
27 2$
Z = halide
O H
Me0-O C02Me \' 2. NaOH (aq) HO-C
> ~ ~R
A~ N ~ ~~ Rc 3. aq. HCI, heat H'N /
2$ 29
O H Protection of o H
HO-C y amine functionalityHO-C 1 c
N ~ -r R~ > ~~N ~ = R
H
29
S Saturated zunino side chains of formula 3 I can be prepared by
hydrogenating campaund;s of formula 30 in the presence of rhodium or platinum
catalysts.
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SUBSTITUTE SHEET ( rule 26 )
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O H
ii
HO-C
L~N
31
For example, following thc: method of Ornstein and coworkers (Ornstein, P. L.;
Arnold, M. B.; Augenstein, N. K.; Paschal, J. W. J. Org. Chem. 1991, .56,
4388),
compound 30 can be hydrogenated in the presence of 5% Rh on alumina to give
compound 31. Individual diastereomers of 31 can be resolved via classical
resolution
methods.
Schemes 14 illustrates one method for the preparation of
tetrahydroisoquinolineacetic acid of formula 33. This is carried out
conveniently
by the Arndt-Eistert reaction which proceeds with retention of
stereochemistry.
Other methods involve require reduction of the acid or its ester derivative to
an
alcohol, conversion of the alcohol to a leaving group such as a mesylate or
halide,
displacement of it with cyanide anion and hydrolysis of the nitrite to the
carboxylic acid by well documented literature methods.
SCHEME 14
O H Arndt-Eistert O H
HO 'w chemistry .J.L.CH
~"".. HO 2
L~ N w/~:~ ~ N
L
32 33
It is understood that in some cases the order of carrying out the
foregoing reaction scherne;s may be varied to facilitate the reaction or to
avoid
unwanted reaction products.
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SUBSTITUTE S~IEET ( rule 26
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Preparation of Intermediates
INTERMEDIATE 1
NHZ HCI
/,
CI
~1 N
S02CH3
To a solution of N-Boc-D-4-chlorophenylalanine (10.958; 36.55
mmol) in 166mL of dichloromethane was added 10.068 (33.27 mmol) of 1,2-dihydro-
1-methanesulfonylspiro[3H-indole-3,4'-piperidine] hydrochloride (see for
example
US Patent 5,536,716), llm.L ofN-mel;hylmorphoiine, 7.018 ofEDC and 4.948 of
HOBt and stirred at room temperature for 18h. The reaction mixture was diluted
with
dichioromethane and washed with 1N HCl and saturated aqueous sodium
bicarbonate
solution and brine. The organic layer was dried over MgS04 and evaporated to
give
an intermediate that was cl:~romatographed on silica gel using hexane-ethyl
acetate
(1:1) as the eluent to give the coupled product.
The product; above was dissolved in 140 mL of dichloromethane and
treated with 25 mL of 4.OM HCl in dioxane for 18h. The volatiles were removed
and
the sticky residue was dissolved in methanol and concentrated to dryness to
provide
the desired title compound.
1 H NMR (CD3OD, 400MHz) 7.26-7.I2 (m, 5 H); 4.90-4.37 (m, 1 H); 2.65-2.60 (rn,
2
H); 1.97 (s, 3 H); 1.87 -1.82 (m, 1 H); 1.73-1.65 (m, 3 H).
INTERMEDIATE 2-5
The following Intermediates were prepared from the appropriately
substituted phenylalanine (Phe) and spiroindoline in an analogous manner to
the one
described for the preparation of Intermediate 1.
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SUBSTITUTE SHEET ( rule 26 y
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NH2
HCI
Rrr ~~ C~O
x
SO2CH3
Intermediate Phe; Rii x
2 N-Boc-D-4-methoxy-Phe CH30 H
3 N-l3oc-U-4-bromo-xne x~r
4 N-l3oc-D-4-chloro-Phe Cl F*
N-l3oc-D-4-methyl-Phe CH3 H
*the starting material, 1,2-~dihydro-~-fluoro-I-methanesulfonylspiro[3H-indole-
3,4'-
piperidine] hydrochloride, may be prepared according to general method
disclosed in
5 US Patent 553671b.
INTERMEDIATE 6
Boc~
N
N
1
C=O O
CI OH
To a solution of of D-4-chlorophenylalanine methyl ester
hydrochloride in dichloromethane was added N-Boc-D-I,2,3,4-tetrahydroquinoline-
3-
carboxylic acid {N-Boc-D-Tic}, EDC, HOBT and NMM, and the mixture was stirred
at room temperature overnight. The crude product was isolated afrer standard
work-
I 5 up as described for the preparation of Intermediate I . The crude ester
was dissolved
in methanol-water {I:1) and hydrolyzed to the desired acid by treatment with
2.5 eq.
of NaOH. The reaction nuxture was concentrated to ~SO% of the volume,
acidified to
pH 2 with 1N HCl and extracted with dichloromethane. The combined organics
were
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SUB~~TITUTE SHEET ( rule 26 )
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WO 99!64002 PCT/US99l13252
washed with.brine, dried over anhydrous magnesium sulfate, filtered and
concentrated
to give the desired product: as a colorless solid.
INTERMEDIATE 7
Boc,,
N
\ N
t
H3CO / C=O O
OH
The title compound was synthesized in an analogous manner to
Intermediate 3 using D-4-(methoxy)phenylalanine methyl ester hydrochloride in
place
of D-4-chlorophenylalanine methyl ester hydrochloride.
INTERMEDIATE 8
Boc~
N
\ N I /
C1' ~ ~ O O
OH
The title compound was synthesized in an analogous manner to
Intermediate 3 using N-Boc-L-Tic in place of N-Boc-D-Tic.
INTERMEDIATE 9
Boc,
N
\ N
H3C0 ~ C=O O
OH
_4p_
SUBSTITUTE SHEET ( ruie 26 )
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The title corrtpound was synthesized in an analogous manner to
Intermediate 4 using t N-Boc-L-Tic in place of N-Boc-D-Tic.
INTERMEDIATE 10. 3R-:3-amino-1'-(t-butyloxycarbonyl)spiro[1H-indan-1,4'-
piperidine]
BOC
N
NH2
St- ep A: Preparation of 3-ors:o-1'-(t-butyloxycarbonyl)spiro[IH-indan-1.4'-
piperidine]
BOC
t
O
To a solution of 51.0 g (0.177 moI) of 1'-(t-butyloxy-
carbonyl)spiro[1H-indene-1,4'-piperidine] [prepared by the method of Chambers,
et
_aI, J. Med. Chem. , 1992, 35, 2036] in. 200 ml ofTHF was added 430 ml (0.5 M
in
THF, 0.213 mol) of 9-BBN. The reaction mixture was heated at 70°C
until TLC
analysis indicated that the ;starting material was consumed (18 hrs). The
solution was
concentrated to 300 ml and then cooled to 0°C and quenched with
methanol (10 ml).
4 N Sodium hydroxide (213 mI) and 30 % hydrogen peroxide (108 ml) were added
via an addition funnel over 45 minutes. The reaction mixture was stirred for
3.5 hours
and then solid sodium sulfite was added until starch paper indicated that no
more
peroxides were present. The reaction mixture was extracted with ethyl acetate
(4 X 1
vol). The ethyl acetate layer was dried over magnesium sulfate f ltered and
concentrated. The crude material was dissolved in dichloromethane (300 ml) and
the
solution was cooled to 0°t: then celite (25 g} and PCC (57 g) were
added in five
-4I-
SUB>TITUTE SHEET ( ruie 2G )
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portions over 20 minutes. 'The reaction mixture was warmed to room temperature
and
stirred overnight. The solution was then diluted with ether and f Itered
through a pad
of a mixture of celite and florisil. Purification by flash chromotgraphy
(silica gel,
hexane/ethyl acetate, S:I to 3:1) gave 58.6 g of the title compound. 1 H NMR
(200
MHz, CDCl3): 7.7~-7.60 (:m, 2H), 7.50-7.44 (m, 2H), 4.30-4.15 {m. 2H), 2.85
(dt,
2H), 2.63 {s, 2H), 1.98 (dt, 2H), 1.53-1.40 (m, 2H), 1.49 (s, 9H).
Step B: Preparation of 3-[(trifluoromethanesulfonyl)oxy]-1'-(t-
butyloxycanbonyl)spiro[ 1 H-indene-1,4'-pipendine]
BOC
N
O
\ n
O-S -CF3
Potasium bis(trimethylsilyi)amide (127.5 ml, 0.5 ~i ) was added to the
ketone of Step A (16.0 g, _'''>3 mmol) in THF (200 mL) at 0°C. The
reaction mixture
was stirred far one hour ar,~d then N-phenyltriflurornethanesulfonamide was
added.
The ice bath was allowed 1:o melt and the reaction mixture was stirred
overnight at
room temperature. Water was added and the aqueous layer was extracted with
ethyl
acetate (3 X 1 vol). The organic layer was washed with brine and then dried
over
magnesium sulfate, f ltered and then concentrated. The crude proauct was
purified by
flash chromatography (hexane/ethyl acetate 8 : 1) to give the title compound
(17.8 g )
as a waxy solid. 1HNMR (200 MHz, CDC13): 7.65-7.14 {m, 4 Hl. 6.66 (s, 1 H),
4.30-
4.1~ (m, 2 H}, 3.24-2.96 (m, ZH), 2.06 (dt, 2 H), 1.50 (s, 9 H), 1.49-1.38 (m,
2 H).
Ste~C: Preparation of 3-(ethoxycarbanyl)-1'-{t-butyloxycarbonyllspiro[1H-
indene-
1,4'-piperidineJ
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SUBSTITUTE SHEET ( rule 26 )
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WO 99164002 PCT/US99/13252
BOC
I
OEt
A solution of 17.4 g of the intermediate from Step B, 11.0 ml of
triethylamine, 634 mg of triphenylphosphine, and 240 mg of palladiurn acetate
in 72
ml of ethanol and 158.0 ml. of DMF was purged for 10 minutes with carbon
monoxide
and then stirred under a cwbon monoxide atmosphere for 24 hours. The ethanol
was
removed in vacuum and the reaction mixttue was diluted with water and
extracted
repeatedly with ethyl acetate. The ethyl acetate layer was washed with 1N HCI,
water, and brine and then dried over magnesium sulfate; filtered and
concentrated.
Purification by flash chromatography {hexane/ethyl acetate 8:1 ) provided 27.6
g of
the title compound as a colorless oil. 1HNMR (200 MHz, CDC13): 8.0-7.94
(m,lH),
7.7 (s, 1 H), 7.4-7.25 (m, 3H), 4.4 (q,2H), 4.25-4.15 (m, 2H), 3.13 (dt, 2H),
2.03 (dt,
ZH), 1.5 (s, 9H), 1.55-1.35 (m, 2H), 1.4 (t, 3H).
Step D: Preparation of 3-(c;arboxy)-1'-{t-butyloxycarbonyl)spiro[1H-indan-1,4'-
piperidine]
B
t
OH
O
To a suspension of Pd/C (1.7g) in ethanol (300 ml) was added the title
compound (27 g) from Step C. The reaction mixture was purged with hydrogen and
then shaken under a hydrogen atmosphere for 3 hours. The mixture was purged
with
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SUBSTITUTE SHEET ( rule 26
CA 02334551 2000-12-05
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nitrogen and filtered through celite and concentrated to give the title
compound (27
g). The crude product was dissolved in ethanol ( 200 ml ) and 2N sodium
hydroxide
(76 ml) was added. The reaction mixture was heated to 50 OC for three hours
then
cooled and the ethanol was removed under vacuum and the residue was dissloved
in
ethyl acetate. iN HCl was added anal the layers were separated and the aqueous
layer
was extracted with ethyl acetate {3 x I vol). The combined organic layers were
washed with saturated aquE;ous NaCI, dried over anhydrous sodium sulfate,
filtered
and concentrated to provide the title compound (23.8 g) as a white solid. I
HNMR
(200 MHz, CDCl3): 7.50-T.42 (m, 1 H), 7.34-7.I2 (m, 3 H), 4.22--1.04 (m, 3 H),
3.06-
2.84 (m, 2 H), 2.40 (d, 2 H), 1.88-I.6 (m, 4 H), 1.50 (s, 9 H).
St-ep E: Preparation of 3S-:S-(carboxy)-1'-(t-butyioxycarbonyl)spiro[1H-indan-
1,4'-
piperidineJ
Boc
N
/~
/ ~f~~H
The acid from Step D (23.5 g, 0.07 mol) was dissolved in toluene (150
ml) and R- methylbenzylaamine (9.02 ml) was added. The toluene solution was
heated
on a steam bath until everything was in solution. The solution was then seeded
with
crystals grown in the same. way on a much smaller scale. The solution was
allowed to
sit overnight and then the mixture was f ltered to give l 5.8 g of crystals.
The crystals
were recrystalized from toluene two more times. The crystals (12 g} were
dissolved
in ethyl acetate /1 N HCl amd the organic layer was washed with I N HCL (2 X I
vol)
and brine. The organic layer was dried over magnesium sulfate, filtered and
concentrated to give 8.9 g of the title compound. [a]D = -I6.9 (c= 0.84,
methanol)
Step F: Preparation of 3R-~3-(carboxy)-I'-(t-butyloxycarbonyl)spiro[IH-indan-
1,4'-
pipezzdine]
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SUBSTITUTE SHEET ( rule 26 )
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WO 99/64002 PCT/US99/13252
Boc
OH
O
The mother :liqueurs from Step E were washed with 1 N HCI (2 x 1 vol)
and brine dried over magnesium sulfate, filtered, and concentrated to give
recovered
acid ( 15.4 g). To this acid i,n toluene ( 100 mL} was added S-
rnethylbenzylamine
(5.95 mL). The crystals were recrystallized four times from toluene as above
to give
12.3 g of salt. The salt was dissolved in ethyl acetate / 1 N HCl and washed
with 1 N
HCl (2 X 1 vol) and brine. The organic layer was dried over magnesium sulfate
and
filtered and concentrated to give the title compound (9.0 g}. [oc]D = +17.1
(c= 1.06,
methanol).
Step G: Preparation of 3R-:3-[[(benzyloxy)carbonyl]amino]-1°-(t-
butyloxycarbonyl)spiro [ 1 fl -indan-1,4'-piperidine]
BOC
I
~ I O
N'~ow Pn
H
To a stirred solution of 3R-3-(carboxy)-1'-(t-
butyloxycarbonyl)spiro[IPk-indan-1,4°-piperidine] (3.56 gm, 10.76 mmol)
in dry
toluene (30 mL) was addeck triethylamine (1.52 gm, 15.06 mmol), DPPA {3.55 gm,
12.91 mmol) and the mixtcue heated to 85°C for four hours to form 3R-3-
{isocyanato)-I'-(t-butyloxycarbonyl)spiro[IH-indan-1,4'-piperidine]. The
mixture
was cooled to r.t. and benzyi alcohol {1.40 gm, I2.9i mmol) added and the
reaction
mixture stirred an additional 1.5 hr. The mixture was diluted with 50 ml of
ethyl
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SUBSTITUTE SHEET ( rule 2G )
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WO 99164002 PCTIUS99/13252
acetate and washed with 1 N HC1, brine and dried over MgSO~. Concentrate and
chromatograph (Si02, 1;1 EtOAc/hexane} to provide 4.1 grams of the clear,
colorless viscous oil that is the title compound. 1HNMR: (CDC13; 300 Mhz) .
ESI-MS calc. for C26H32IV2O4: 436; Found 454 (M+H+NH3).
Step H: Preparation of ?'~R-3-amino-1'-(t-butyloxycarbonyl)spiro[iH-indan-I,4'-
piperidineJ hydrochloride salt
B~C
i
N
/ ~ ~HC!
'' N H2
To a stirred solution of the product of Step G (4.1 am, 9.4 mmol) in
methanol (50 mL) was added HCi ~~on~.> (0.9 mh, 10.3 mmol) and Pd(OH)Z-C (0.5
gm). The mixture was stirred vigorously under an H2 atomosphere for 16 hr. The
reaction mixture was filtered through celite and the solvent removed in vacuo
to
provide 2.85 gm of the white solid. ESI-MS calc. for C 18H26N2O2: 302; Found
303
(M+H), 203 (M+H-Boc).
INTERMEDIATE 11. 3R-3-(acetylamino)-spiro[1H-indan-1,4'-piperidine] HCl
H ~HC!
N
/ ' O
N
H
To a stirred solution of Intermediate 10 (1.5 gm, 4.4 mmol) and DMA.P
(54 mg, 0.4 mmol) in dry dichloromethane (15 mL) was added triethylamine (1.3
gm,
13.3 rnmol), acetic anhydride (0.68 grn, 6.6 mmol) and the mixture stirred for
16 hr.
The reaction mixture was concentrated, diluted with 50 ml of ethy 1 acetate
and
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SUBSTITUTE SHEET ( rule 26 )
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washed with.NaHC03 (sat'd), brine and dried over MgSO:~. The organic phase was
oncentrated and chromatographed (Si02, 3:1:0.1 EtOAc/hexane/methanol) to
pxovide
1.6 grams (81%) of the N-Boc protected title compound as white solid. ESI-MS
calc.
for C20H28N203: 344; Found 345 (M+H).
To a stirred solution of N-Boc protected title compound ( 1.2 g, 3.6
mmol) in methanol (1.0 mL), HCl-EtOAc was added to the mixture (5 mL). The
reaction was stirred for 20 minutes and the solvent was removed in vacuo to
afford
0.95 g of the product. ESI-MS calc. for C 15H20N20: 244; Found 245 (M+H), 286
(M+H+CH3 CN).
INTERMEDIATE I2
H
N HC!
I /' H
N-a
N
H
The general procedure described in Step G of intermediate 10 was
followed using cyclopropylamine instead of benzyl alchol to react with the
isocyanato
compound to provide the N-Boc protected title compound. The iL'-Boc protecting
group was removed according to the general procedure described in Intermediate
11
to provide the title compound.
INTERMEDIATES 13-24
Following the general procedure described for Intermediate I 1, and
using Intermediate i 0 and the appropriate acylating agent, or following the
general
procedure described for Intermediate 12 using the appropriate amine to react
with the
isocyanato compound described in Intermediate 10, step G, Intermediates 13-24
were
prepared.
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SUB~~TITUTE SHEET ( rule 26 )
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WO 99!64002 PCTIUS99/13252
H
c
HCI
NH-R'
IntermediateAcylatirig Agent/Amine Igi
13 methane;sulfonyl chloride S02CH3
14 3-pyridinecarbonyl chlorideCO-3-pyridyl
1 S 4-pyridinecarbonyl chlorideCO-4-pyridyl
16 2-pyrazinecarbonyl chlorideCO-2-pyrazinyl
I7 2-aminopyrimidine CONH-2-pyrimidinyl
I 8 piperidine CO-N(CH2)S
19 morpholine CO-N(CH2)20{CH2)2
20 2-aminothiazole CONH-2-thiazolyl
21 2-pyridinecarbonyl chlorideCO-2-pyridyl
22 benzoyl chloride CO-Ph
23 benzene;sulfonyl chloride S02Ph
24 2-thiopllenecarboxylic CO-2-thienyl
acid
S INTERMEDIATE 25
NH2 ~ HCI
Cl
N
C
'' N'
H
To a stirred! solution of Intermediate 1 I (6S7 mg, 2.34 mmoi}, N-Boc-
D-4-chiorophenylalanine 1;737 mg, 2.5 mmol), PyBrop (1091 mg, 2.34 mmol) and
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SUBSTITUTE SHEET ( rule 26 )
CA 02334551 2000-12-05
WO 99164002 PCT/IJS99/I3252
DMAP ( 172 mg, 1.4 mmol) in dichloromethane, 6 mL was added DIEA (907 mg,
7.02 mmol). The solution was stirred 16 hr, concentrated and chromatographed
directly (Si02,19:1 EtOAcI methanol) to provide 1.08 gm of the N-Boc protected
title
compound as white solid. ESI-MS calc. for C29H36CIN3O4: 52~; Faund 526
(M+H), 426 (M+H-Boc).
To a stirred solution of the N-Boc protected title compound from the
previous step (1.1 g, 2.1 mmoi) in methanol (0.5 mL), HCI-EtOAc was added (5
mL).
The reaction was stirred for 20 minutes and the solvent was removed in vacuo
to
afford 0.95 g of the title compound. ESI-MS calc. for C24H28C1N302: 425; Faund
426 (M+H), 443 (M+H+N:H3).
INTERMEDIATE 26-38
Following the procedure described for Intermediate 2~ and unsing
Intermediates 12-24, the following compounds were prepared:
CI
N
'1'' NHR'
NH2 ~ HCf
O
Intermediate Ri
26 CONH-cyclopropyl
27 S02CH3
28 CO-3-pyridyl
29 CO-4-pyridyl
30 CO-2-pyrazanyl
31 CONH-2-pyrimidinyl
32 CO-N(CH2)S
33 CO-N(CH2)20(CHi2)2
34 CONH-2-thiazolyl
3 5 CO-2-pyridyl
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36 CO-Ph
37 SO'2Ph
38 CO-2-thienyl
INTERMEDIATE 39
O' /O
~'N
H
C02H
A heterogE:neous mixture of the product of Intermediate 10, Step F
(5.0 gm, 15.1 mmol), Rlv'A1~03 (0.85 gm) in ethanol (50 mL) was agitated under
an
atmosphere of hydrogen !;2000 psi, 100°C) for 18 hr. The mixture was f
itered
through celite and the solvent removed in vacuo to provide {3.8~ gm) (75 %} of
the
white solid which is the title compound. ESI-MS talc. for:; Found (M+H}.
INTERMEDIATE 40
HCI
r
N
o2cH3
To a stirred solution of HCl-MeOH ( mL) was added Intermediate 39
(350 mg, 104 mmol) and the mixture stirred 16 hr. The solvent was removed in
vacuo to provide (300 m;g) (100 %) of the white solid which is the title
compound.
ESI-MS calc. For C 1 SH26C1N02: 287; Found 288{M+H).
-50-
SUB~aTITUT'E SHEET ( rule 26 )
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INTERMEDIATE 41
N HCI
OH
H
Step A.
In a dry three-necked round-bottomed flask equipped with magnetic
stir bar and nitrogen purge, was added Intermediate 39 (2.5 gm, 7.41 mmol) and
tetrahydrofuran {7.5 mL, anhydrous). The mixture was stirred and cooled to -
10°C
and borane -dimethylsulfide (7.4 mL, 2M in THF, 2 eq.) was added dropwise over
a
period of 20 min. When the addition was complete, the mixture was warmed to
r.t.,
refluxed for one hour, and cooled to r.t. The reaction was quenched with the
addition
of 1 mL waterlacetic acid accompanied by vigorous stirring. The mixture was
concentrated under reduced pressure, diluted with ethyl acetate, washed with
saturated
NaHC03, brine and then dried over MgSO4. The salvent was removed in vacuo to
provide (2.32 gm) ( 97%) of the white solid which is the N-Boc protected title
compound. ESI-MS calc. For C19H33N03: 323; Found 324(M+H).
Step B.
To a stirred mixture of N-Boc protected title compound ( 180 mg, 0.4
mmol.} in a minimal amount of methanol {ca. 100 p.L) was added ~ mL of a
saturated
HCl-EtOAc solution. The mixture was stirred 20 min and the solvent removed in
vacuo to provide (154 m~;} of the wbite solid which is the title compound. ESI-
MS
calc. For C 14H26C1N0: 260; Found 261 (M+H).
_5~_
SUBSTITUTE SHEET ( rule 26 )
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INTERMEDIATE 42
N
N
To a stiired solution of Intermediate 39 (400 mg, 1.2 mmol), PyBrop
S (607 mg, 1.2 mmol) and I3MAP (92 mg, 0.7 mmol) in dichloromethane, 2.0 mL
was
added DIEA (459 mg, 3.6 mmol}. The reaction mixture was stirred 16 hr, diluted
with
dichloromethane, washed with 1N HCl and concentrated in vacuo. The residue was
purified via preparative HPLC to provide 43~ mg of the white solid that is the
N-Boc
protected title compound. ESI-MS calc. For C21H36N203: 364; Found 365{M+H).
The procedure described in Step B of Intermediate 41 was followed
using the N-Boc protected title compound to provide the title compound. ESI-MS
265 (M+H).
INTERMEDIATE 43
H
N
N
NON
To a solution of intermediate 41 (1.~ gm, 5.6 mmol) in dichloromethane (15 mL)
add
triethylamine ( 1.69 gm, :3.0 eq) stir and cool to 0°C, then add mesyl
chloride (1.0 gm,
1.5 eq) and continue stirring three hours. Concentrate, dilute with DMF ( 6
mL), stir
and add sodium triazole {I.0 gm, 3 eq.). dilute with ethyl acetate then wash
with
saturated NaHC03, brine. and dry over MgS04. Remove solvent in vacuo to
provide
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(154 mg) of the yellow soEid which is the N-Boc protected title compound. ESI-
MS
calc. For C21H32N4O3: ?~88; Found 389(M+H).
The procedure described in Step B of Intermediate 41 was followed
using the N-Boc protected. title compound to provide the title compound. ESI-
MS
289 (M+H).
INTERMEDIATE 44
H
N
0
To a solution of intermediate 4T (0.5 gm, 1.~ mmol) in tetrahydrofuran
(5 mL) was added imidazoie { 105 mg, 1.0 eq), and the mixture was stirred and
cooled
to 0°C, then sodium hydride (74 rng, 2.0 eq) was added thereto and
stirring continued
for 30 min. Methyl iodide {439 mg, 2 eq.) was added via syringe, and the
mixture
was warmed to r.t. and stirring was contined far 2 hr. The reaction mixture
was
1 S concentrated and partitioned between EtOAc / 1N HCI, washed with brine and
dried
over MgS04. The solvent was removed in vacuo to provide (250 mg) of the yellow
oil
which is the title N-protected title compound. ESI-MS calc. For C20H35N03:
337;
Found 338(M+H).
The procedure described in Step B of Intermediate 41 was followed
using the N-Boc protected title compound to provide the title compound. ESI-MS
23 8 {M+H).
The follov~ring Examples are provided to illustrate the invention, and
are not to be construed as limiting the scope of the invention in any manner.
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EXAMPLE 1
H HN
N
,~ O O
CI N
HCI
C
/ ~ -N
-''' ~$02CH3
St_-ep A: Preparation of 3S-~~1-Boc-3-decahydroisoquinolinecarboxylic acid
Boc-N
HO
O
A 50-mL, three-necked, round-bottomed flask equipped with an
nitrogen inlet adapter, glass stopper, and rubber septum was charged with
commercially available N Boc-(L)-Tic (1.00 g, 3.6 mmol) and 18 mL of DMF.
Potassium carbonate (0.597 g, 4.30 mmol) was then added followed by the
addition of
methyl iodide (1.1 mL, 18,0 mmol) vna syringe. The resulting mixture was
stirred at
room temperature for 20 h and then tr~ethylene chloride and water were added.
The
aqueous layer was separated and extracted with two portions of methylene
chloride,
and the combined organic phases were washed with saturated sodium chloride
solution, dried over sodium sulfate, filtered, and concentrated. The crude
product was
purified by flash chromatography {1:1 ethyl acetate-hexane) to give N Boc-(L)-
Tic
methyl ester ( 1.17 g) as a :yellow oil.
A solution ofN Boc-(T.,)-Tic methyl ester (1.05 g, 3.60 mmol) and 12
mL of methanol was char~;ed with 5% rhodium on alumina (0.53 g) and then
heated at
55-60 °C under 40 psi of hydrogen for 36 h. After cooling to room
temperature, the
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reaction mixture was filtered through Celite using methanol to rinse and
concentrated.
The crude oil was then filtered again tr~rough Celite using ethyl acetate as
the eluent
and concentrated to give meahyl 3S N Boc-3-decahydroisoquinoline-carboxylate
(0.733 g) as a clear oil. ESI-MS caled for C~6H27NO4: 297: Found: 298 (m + 1).
Hydrogenation of a similar compound gave exclusively the cis-ring junction
products
as a mixture of diastereomers, see: Ornstein, P. L.; Arnold, M. B.,
Augenstein, N. K.;
Paschal, J. W. ,I. Org. Chem. 1991, .Sb., 4388.
A 2~-mL, round-bottomed flask was charged with methyl 3S .WBoc-3-
decahydro-isoquinolinecarboxylate (0.733 g, 2.46 mmol) and 7 mL ofmethanol. An
I O aqueous 1 N NaOH solution (5 mL) was then added and the resulting mixture
was
stirred at room temperature for 22 h. The mixture was then concentrated and
the
resulting residue was dissolved in water and cooled at 0 ° C in an ice-
water bath. The
pH was then adjusted using a 1 N HCi to pH 4, and the cloudy mixture was
diluted
with ethyl acetate. The aqueous layer was separated and extracted with two
portions
I ~ of ethyl acetate, and the combined organic phases were washed with
saturated sodium
chloride solution, dried over sodium sulfate, filtered, and concentrated to
give 3S-N
Boc-3-decahydro-isoquinolinecarboxylic acid (0.667 g) as a very thick yellow
oil.
The 1H NMR spectrum shows the presence of two diastereomers; cis-ring junction
isomers from the previous :hydrogenation reaction.
Step B: Preparation of title; compound
A 25-mL, round-bottomed flask was charged with Intermediate 1
(0.102 g, 0.182 mmol) and then a solution of the acid of Step A (0.07 g, 0.201
mmol) in I.2 mL of methylene chloride was added. The mixture was cooled at 0
° C
2~ in an ice-water bath and then NMM (0.10 mL, 0.910 mmol), HOBt ~ H20 (0.027
g,
0.201 mmol}, and EDC ~ HCl (0.039 g, 0.201 mmol) were added. The resulting
mixture was stirred at room temperature for 22 h, and was then diluted with
methylene chloride and wa~,shed with two portions of 1 N HCl solution,
saturated
sodium bicarbonate solution, water, and saturated sodium chloride solution,
dried
over sodium sulfate, filtered, and concentrated. The crude product was
purified by
flash chromatography {9:1. methylene chloride:acetone) to give the N-Boc
protected
title compound (0.096 g) as a white solid. ESI-MS calcd for C37H49N4SO6CI:
712:
Found: 713 (m + 1 ). The ' H NMR spectrum shows the presence of two cis-ring
junction diastereomers from the hydrogenation reaction.
SUBS'~TITUTE SHEET ( rule 2f )
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A 25-mL, ro~znd-bottomed flask was charged with the N-Boc protected
title compound (0.080 g, 0.112 mmol) and 0.3 mL of methylene chloride.
Trifluoroacetic acid (0.3 mI,) was then added and the mixture was stirred at
room
temperature for 65 min. The mixture was diluted with toluene and concentrated,
and
the resulting oii was diluted with toluene again and concentrated. The residue
was
dissolved in ethyl acetate arid washed with 1 N NaOH solution, and the
combined
organic phases were dried aver potassium carbonate, fiitered, and concentrated
to give
a clear oil. The free amine 'was then dissolved in 0.5 mL of ethyl acetate and
0.13 mL
of a 1 N HCl solution in ether was added dropwise via syringe. The mixture was
1~0 diluted with ether and the precipitate was then filtered under nitrogen to
give the title
compound (0.056 g) as a white powder. ESI-MS calcd for C3~H41N~S44C1: 612:
Found: 613 (m + 1 ).
F.XAMPT.F ?_
C
H
H HN
H
O
HCI
302CH3
Std A: Preparation of [3SI;3a,4a[i,8a~3)]-N Boc-decahydro-3-
isoquinolinecarboxylic
acid
Bc~cN
HO
H
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A 2~-mL, round-bottomed flask equipped with a reflux condenser was
charged with commercially available ~3S(3a,4a~3,8a(3)]-N tert-butyldecahydro-3-
isoquinolinecarboxamide (1.0 g, 4.19 mmol) and 10 mL of aqueous 6 N HCl
solution.
The solution was heated at 80 °C fox 23 h and then cooled to 0
°C and diluted with 12
mL of 5 N NaOH solution (pH = 13). The aqueous solution was extracted with
ethyl
acetate and then transferred to a 100-mL, round-bottomed flask and diluted
with 25
mL of dioxane. Di-tert-butyl dicarbonate (1.0 g; 4.61 mmol) was then added and
the
mixture was stirred at roonn temperature for 23.5 h while occasionally
adjusting the
pH using 5 N NaOH solution (pH =10). The resulting mixture was diluted with
ethyl
acetate and water and the layers were separated. The aqueous layer was cooled
at 0
°C in an ice-water bath anti then 1 N HCl solution was added
portionwise until pH =
2. The aqueous layer was extracted with three portions of ethyl acetate, and
the
combined organic phases were washed with saturated sodium chloride solution,
dried
over sodium sulfate, filterf;d, .and concentrated to give [3S(3a,4a~i,8aj3)]-N
Boc-
1 S decahydro-3-isoquinolinec;arboxylic acid (0.427 g) as a white solid.
St_ ep B: Preparation of Title Compound
A 2~-mL, round-bottomed flask was charged with Intermediate 1 TFA
salt (prepared as Intermediate 1 except TFA is used in place of HCI, 0.123 g,
0.219
mmol) and 1.3 mL of rnetJhylene chloride and then the mixture was cooled at 0
° C in
an ice-water bath. Acid from Step A (0.068 g, 0.241 mmol), NMM (0.10 mL, 0.9I0
mmol), HOBt ~ HBO (0.033 g, 0.241 mmoi), and EDC ~ HCl {0.046 g, 0.241 mmol)
were added. The resulting mixture was stirred at room temperature for 22 h,
and was
then diluted with methylene chloride and washed with two portions of 1 N HCl
solution, saturated sodium bicarbonate solution, water, and saturated sodium
chloride
solution, dried over sodiwm sulfate, filtered, and concentrated. The crude
product was
purified by flash chromatography (9:1 methylene chloride:acetone) to give N-
Boc
protected title compound(0.112 g) as a white solid. ESI-MS calcd for
C3~HagNaSOsCI: 712: Found: 713 (m + 1 ).
A 2~~-mL, ;round-bottomed flask was charged with the N-Boc protected
title compound (0.110 g, 0.154 mmol) and 0.4 mL of methylene chloride.
Trifluoroacetic acid (0.4 rnL) was then added and the mixture was stirred at
room
temperature for 45 rnin. ~Che mixture was diluted with toluene and
concentrated, and
the resulting oiI was diluted with toluene again and concentrated. The residue
was
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dissolved in ethyl acetate and washed with 1 N NaOH solution {back-extracted
with
two portions of ethyl acetat:e), and the combined organic phases were dried
over
potassium carbonate, filtered; and concentrated to give a yellow oil. The free
amine
was then dissolved in 0.5 n~L of ethyl acetate and 0.18 mL of a 1 N HCl
solution in
ether was added dropwise via syringe. The mixture was diluted with ether and
the
precipitate was then filtered under nitrogen to give the title compound (0.072
g) as a
white powder. ESI- .MS calcd for C32HaiNaSOaCI: 612: Found: 613 (m + 1).
EXAMPLE 3
1~
TFA H
H HN
N
O O H
CI~
,N
C
N
~S02CH 3
STEP A: Preparation of [?~S(3a,4aa,8aa)]-N Boc-decahydro-3-
isoquinolinecarboxylic acrid, (R)- a-methylbenzylamine salt
H
BacN
F'h NHz HO _
H
O
A 2~-mL, round-bottomed flask was charged with acid 3S-N-Boc-3-
decahydroisoquinolinecarboxylic acid from Example 1 (0.453 g, 1.60 mmol) and
14
mL of ethyl acetate. (R)-~x-methylbenzyl amine (0.21 mL, 1.60 mmol) was then
added and the resulting mixture sat at room temperature under nitrogen for 24
h. The
. ~8 .
SUBSTITUTE SHEET ( ruie 26 )
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precipitate was then filtered to give 0.393 g of a white powder. The white
powder
was then recrystallized {etlhyl acetate-ethanol) to give [3S(3a,4aoc,8aa)]-l~'-
Boc-
decahydro-3-isoquinolinec:arboxylic acid, (R)- a-methylbenzylamine salt (O.I30
g) as
a white solid.
STEP B: Preparation of Title Compound
A 15-mL, round-bottomed flask was charged with the free acid of the
product of Step A (obtaine;d by washing intermediate from STEP A with 10%
aqueous citric acid) (0.020 g 0.049 mmol) and 0.3 mL of rnethylene chloride.
Intermediate 1 {0.022 g, 0.045 mmol), NMM (0.020 mL; 0.180 mrnol), HOBt ~ H20
(0.007 g, 0.049 mmol), and EDC ~ HCl (0.009 g, 0.047 mmol) were added. The
resulting mixture was stiwed at room temperature for 17 h, and was then
diluted with
methylene chloride and washed with two portions of 1 N HCl solution, saturated
sodium bicarbonate solution, water, and saturated sodium chloride solution,
dried
over sodium sulfate, filtered, and concentrated. The crude product was
purified by
flash chromatography (1:1 ethyl acetate-hexane) to give N-Boc protected title
compound (0.023 g) as a white solid.
A 10-mL, round-bottomed flask was charged with the N-Boc protected
title compound (0.022 g, 0.031 mmol) and 0.2 mL of methylene chloride.
Trifluoroacetic acid (0.2 rnL) was then added and the mixture was stirred at
room
temperature for 1 h. The :mixture was diluted with toluene and concentrated
twice,
and then the oil was dilutE;d with ether and concentrated to afford the title
compound
(0.021 g) as a white solid. ESI-MS calcd for C32HaiNaSOaCI: 612: Found: 613 (m
+
I ).
2~
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E:KAMPLE 4
HN ~ OH
N
O HCI
CI
N
N
~SO2CHg
To a solution of InterrrAediate I (450.1 mg, 1.0I mmol} in methylene
chloride (10 mL) was added N-Boc-7-hydroxy-L-1,2,3,4-tetrahydroquinoline-3-
carboxylic acid (7-OH-N-Bac-L-Tic, 355.5 mg, 1.21 mmol), HOBt (164.1 mg, 1.21
mmol), EDC (232.3 mg, I .2 i mmol), and NMM (0.5 mL, 4.55 mmol). The mixture
was stirred at room temperature overnight and then quenched with EtOAc {50
mL}.
The organic solution was washed with 5 % aq HCl solution (50 mL), saturated
aqueous NaHC03 {50 mL), and brine (50 mL), and dried over anhydrous Na2S04,
filtered, and concentrated. The crude product was purified by MPLC (4:1
DCM:aceton) to give the td-Boc protected title compound as a white solid
{558.8 mg,
76.6%). ESI-MS calc. for C3~H43CIN40~S 722; Found 723 (M+1}.
To a solution of N-Boc protected title compound (79.5 mg, 109.9
wmol) and anisole (0.1 mL) in DCM ( 1.0 mL) was added TFA (0.5 mL). The
mixture
was stirred at room temperature until no starting material left (TLC). The
solvents
were removed under reduced pressure and diethyl ether was added to give a
white
solid (TFA salt). The salt was added to an aquoues solution of I N NaOH (15
mL)
and extracted with ethyl acetate (2x1 ~ mL). The combined organics were dried
over
anhydrous Na,SOa, filtered, and concentrated. The residue was dissolved in
ethyl
acetate (0.5 mL) to which was added 1N HCl in diethyl ether to yield the title
compound as a white solid (53.6 mg, 73.9%, HCl salt). ESI-MS calc. for
C32H35CIN405S 622; Found 623 (M+1).
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E:KAMPLE 5
OCH3
HN
.w N ( /
=O O
CI N TFA
.N
~S02CH3
To a mixture of the N=Boc protected compound of Example 4 ( 153.4
mg, 212.1 ~mol) and KZCt)3 (58.8 mg, 425.5 wmol) in DMF {5.0 mL) was added
methyl iodide (20.0 ~L, 321.4 wmol). The mixture was stirred at room
temperature
overnight and then quenched with 1N HCl (aquoues, 50 mL), and extracted with
ethyl
acetate {3x50 mL). The combined organic layers were washed with saturated
aqueous
NaHC03 (50 mL) and brime (50 mL), dried over anhydrous Na2S04, filtered, and
concentrated to afford the N-Boc protected title compound as a colorless oil
{147.0
mg, 94.0%). ESI-MS calc. for C38H4,C1N407S 736; Found 737 (M+1).
To a solution of the N-Boc protected title compound {179.9 mg, 244.0
~mol) and anisole {0.1 mL) in DCM {2.0 mL) was added TFA ( 1.0 mL). The
mixture
was stirred at room temperature until no starting material left (TLC). The
solvents
were removed under reduced pressure and diethyl ether was added to provids the
title
compound as a white solid {143.2 mg, 78.1%). ESI-MS calc. for C33H3~C1N4OSS
636; Found 637 {M+1).
EXAMPLES 6-15
The general procedure described in Example 4 was followed to provide
compounds of Examples fi-15 using the Intermediates and acids listed below:
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SUBSTITUTE SHEET ( rule 25 )
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~!!I
HN
M
RII ~~ O O
N~
x -~~ ~ N
S02CH3
Stereoconfiguration same as the Tic starting materiai
Ex.Interm.Acid Salt Rii 8112Y EI-MS
6 3 N-Boc-I)-Tic TFA Br H H 651 {M+1)
653 (M+3)
7 3 N-Boc-I,-Tic TFA Br H H 651 {M+1)
653 (M+3)
8 4 7-OH-N-Boc-L-Tic:TFA Cl OH F 641 (M+1}
9 3 7-OH-N-Boc-D-TicTFA Br OH H 667 (M+1}
669 (M+3)
3 7-OH-N-Boc-L-Tic;TFA Br OH H 667 (M+1)
669 {M+3)
11 1 7-OH-N-Boc-D-TicTFA Cl OH H 623 (M+1)
12 S N-Boc-l~-Tic TFA CH3 H H 587 (M+1)
13 1 N-Boc-l~-Tic HCl Cl H H 6S3 (M+H)
14 2 N-Boc-D-Tic HCl CH30 H H
2 N-Boc-L-Tic HCl CH30 H H
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EXAMPLE 16
HN f \
\ N /
~~ O - HCI
CI
N
O
N
H
To a stirred solution of Intermediate 25 (150 mg, 0.35 mmol), N-Boc-
D-Tic (162 mg, 0.3~ mmol), PyBrop (164 mg, 0.35 mmol) and DMAP (172 mg, 1.4
mmol) in dichloromethane, 6 mL was added DIEA (26 mg, 0.21 mmol). The solution
was stirred 16 hr. concentrated and chromatographed directly (Si02,19:1 EtOAc/
methanol) to provide 200 mg of the N-Bac protected title compound as a white
solid.
ESI-MS calc. for C39H45C1N4O5: 684; Found 685 (M+H), 585 (M+H-Boc).
To a stirred. solution oiE the N-Boc protected title compound from the
previous step ( 160m g, 0.2 3 mmol) in methanol (0.25 mL), HCl-EtOAc was added
(5
mL). The reaction was stirred for 20 minutes and the solvent was removed in
vacuo
to afford I38mg of the title compound as a white solid. ESI-MS calc. for C34
H37 N4
03 Cll: 584; Found 585 (M+H), 607 (M+H+NH3).
EXAMPLES 17-29
The general procedure described in Example 13 was followed using
Intermedates 26-38 to synthesize the following compounds:
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SUBSTITUTE SHEET' ( ruie 26 )
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HN
H
C p ~ HCI
NHR'
Example Intermediate E~t EI-MS
17 26 CONH-cyclopropyl
18 27 S02CH3
19 28 CO-3-pyridyi
20 29 CO-4-pyridyl
21 30 CO-2-pyrazinyl 649 (M+H)
22 31 CONH-2-pyrimidinyl 664 (M+H)
23 32 CO-N(CH2)5 654 (M+H)
24 33 CO-N(CH2)20(CH2)2 656 (M+H)
25 34 CONH-2-thiazolyl 669 (M+H)
26 3~ CO-2-pyridyl 648 (M+H)
27 36 CO-Ph 647 (M+H)
28 37 S02Ph 683 (M+H)
29 38 CO-2-thienyl 653 (M+H)
EXAMPLE 30-34
Following l:he general procedures described for Intermediate 25 and
Example 16 and starting with Intermediates 40-44, the following compounds were
prepared:
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SUBSTITUTE SHEET ( rule 26 )
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HN
NH
/ ~O O
CI N HCI
R
ExampleIntermediateR ESI-MS
30 40 C02CH3 592 (M+H)
31 42 C(O)N(CH3) 2 603 (M+H)
32 43 CH2-1.,2,4-triazol-1-yl6I5 (M+H)
33 41 CH20H 564 (M+H)
34 44 CH20CH3 578 (M+H)
-s~-
SUBSTITUTE SHEET ( ruin 26 )
