Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
POLYMORPHIC FORMS OF A GROWTH HORMONE
SECRETAGOGUE
5 BACKGROUND OF THE INVENTION
Growth hormone, which is secreted from the pituitary,
stimulates growth of all tissues of the body that are capable of growing.
In addition, growth hormone is known to have the following basic
effects on the metabolic processes of the body: (1) Increased rate of
10 protein synthesis in all cells of the body; (2) Decreased rate of
carbohydrate utilization in cells of the body; (3) Increased mobilization
of free fatty acids and use of fatty acids for energy. A deficiency in
growth hormone secretion can result in various medical disorders, such
as dwarfism.
Various ways are known to release growth hormone.
For example, chemicals such as arginine, L-3,4-dihydroxyphenyl-
alanine (L-DOPA), glucagon, vasopressin, and insulin induced
hypoglycemia, as well as activities such as sleep and exercise,
indirectly cause growth hormone to be released from the pituitary by
'20 acting in some fashion on the hypoth~l~mus perhaps either to
decrease somatostatin secretion or to increase the secretion of the
known secretagogue growth hormone releasing factor (GRF) or an
unknown endogenous growth hormone-releasing hormone or all of
these.
'25 In cases where increased levels of growth hormone were
desired, the problem was generally solved by providing exogenous
growth hormone or by ~lmini~tering GRF or a peptidal compound
which stimulated growth hormone production and/or release. In
either case the peptidyl nature of the compound necessitated that it be
~lmini~tered by injection. Initially the source of growth hormone
was the extraction of the pituitary glands of cadavers. This resulted
in a very expensive product and carried with it the risk that a disease
associated with the source of the pituitary gland could be transmitted
to the recipient of the growth hormone. Recombinant growth
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hormone has become available which, while no longer carrying any
risk off disease tr~n~mi~sion, is still a very expensive product which
must be given by injection or by a nasal spray. Other compounds
have been developed whichl stimulate the release of endogenous
5 growth hormone.
In particular, c ertain spiro compounds are disclosed in
U.S. F'atent No. 5,536,716, PCT Patent Publication WO 94/13696
and Proc. Natl. Acad. Sci. USA~ 92, 7001-7005 (July 1995) as being
non-peptidal growth hormone secretagogues. These compounds have
10 the ability to stimulate the release of natural or endogenous growth
hormone and thus may be used to treat conditions which require the
stimulation of growth hormone production or secretion such as in
humans with a deficiency of natural growth hormone or in ~nim~l.c
used for food or wool production where the stimulation of growth
lS hormc,ne will result in a larger, more productive ~nim~l.
Among the preferred compounds disclosed therein is
spiro[3H-indole-3,4'-piperclin]-1'-yl)carbonyl]-2-(phenylmethyl-
oxy)ethyl]-2-amino-2-methylprop~n~mide methanesulfonate which has
the structure:
H H \ CH3
~--O'--C~N~NH2 CH3S02H
SO2CH3
U.S. Patent No. 5,536,716 and PCT Patent Publication WO
94/13696 disclose methods for preparing this compound (see Examples
18, 19 and 55). In particuLar, Example 55 states that the compound
prepared by recryst~lli7~tion from ethyl acetate-ethanol-water had a
25 melting point of "166-168~('". This compound was subsequently
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identified as being of the polymorphic form designated "Form II"
herein. Proc. Natl. Acad. Sci. USA. 92, 7001-7005 (July 1995) notes
that the compound isolated as a monohydrate had a melting point of
168-170~C, but only discloses very general methods for preparing the
5 compound and does not disclose how the compound was crystallized.
Morphological forms of pharmaceutical compounds may be
of interest to those involved in the development of a suitable dosage
form because if the morphological form is not held constant during
clinical and stability studies, the exact dosage used or measured may not
:L0 be comparable from one lot to the next. Once a pharmaceutical
compound is produces for use, it is important to recognize the
morphological form delivered in each dosage form to assure that the
production process use the same form and that the same amount of drug
is included in each dosage. Therefore, it is imperative to assure that
15 either a single morphological form or some known combination of
morphological forms is present. In addition, certain morphological
forms may exhibit enhanced thermodynamic or hydroscopic stability
and may be more suitable than other morphological forms for inclusion
in pharmaceutical formulations. As used herein, a "polymorphic form"
,'0 of a chemical compound is the same chemical entity, but in a different
crystalline arrangement.
SUMMARY OF THE INVENTION
The present invention is concerned with polymorphic
,!5 forms of the compound: N-[l(R)-[(1,2-dihydro-1-methanesulfonyl-
spiro['3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyl-
oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate, as well as
processes for the preparation of such polymorphic forms.
The present invention is also concerned with
30 pharmaceutical formulations comprising these polymorphic forms as ~m
active ingredient and the use of these polymorphic forms and their
formulations in the treatment of certain disorders.
The polymorphic forms of this invention are growth
hormone secretagogue that are useful in food ~nim~ to promote their
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growth thereby rendering the production of edible meat products more
efficient, and in humans, to treat physiological or medical conditions
characterized by a deficiency in growth hormone secretion, and to treat
medical conditions which are improved by the anabolic effects of
S growth hormone.
These polymorphic forms have advantages over the other
known forms of N-[l(R)-c(l~2-dihydro-l-methanesulfonylspiro[3H
indole-3 ,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-
amino-2-methylpropanamide methanesulfonate in terms of
10 thermodynamic stability and suitability for inclusion in pharmaceutical
formulations.
DETA.ILED DESCRIPTION OF THE INVENTION
The present invention is directed to novel polymorphic
15 forms of the compound N-[l(R)-[(1,2-dihydro-1-methanesulfonyl-
spiro[3H-indole-3,4'-piperdin]- ] '-yl)carbonyl]-2-(phenylmethyl-
oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate and the
processes for the preparation of these polymorphic forms.
The compound N- [ 1 (R)- [( 1 ,2-dihydro- 1 -methanesulfonyl-
20 spirol[3H-indole-3,4'-piperdin]- ] '-yl)carbonyl]-2-(phenylmethyl-
oxy)e:thyl]-2-amino-2-methylpropanamide methanesulfonate has the
strucl~ure:
CH3
~C' ~ IH, CH3SO3H
CH3SO~,~ ~J
and is a growth hormone secretagogue which induces the release of
25 growth hormone in humans and ~r~im~l~. This property can be utilized
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to promote the growth of food ~nim~l.s to render the production of
edible meat products more efficient, and in humans, to treat
physiological or medical conditions characterized by a deficiency in
growth hormone secretion, and to treat medical conditions which are
improved by the anabolic effects of growth hormone.
These particular polymorphic forms (herein designated
"Form I", "Form II", "Form III", "Form IV", "Form V", "Forrn VI",
"Form VII","Form VIII","Form IX","Form X",) have superior
properties over other crystalline forms of the compound in that they are
more suitable for inclusion in pharmaceutical formulations. A
preferred crystalline form for pharmaceutical development is Form I
based on its thermodynamic stability and non-hygroscopic properties.
Another preferred crystalline form for pharmaceutical development is
Form IV based on its formulation properties, particularly with respect
to compression for tablet preparation. Form IV has been found to have
a higher buL~ density than other forms.
The present invention is also concerned with a process for
the preparation of Form I of N-[l(R)-[(1,2-dihydro-1-methanesulfonyl-
spiro[ 3H-indole-3 ,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyl-
oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which
comprises:
treating a solution of the free base of N-[l(R)-[(1,2-
dihydro- 1 -methanesulfony]-spiro[3H-indole-3,4'-piperdin]- 1'-
yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide
in ethyl acetate containing ethanol (about 8 volume %) with
methanesulfonic acid (about 1.1 equivalents) at approximately 50~C,
heating to approximately 55~C, and cooling to approximately 45~C.
Optionally, the temperature subsequently may be raised to
approximately 51~C where it is m~int~ined for 2-24 hours.
'30 The present invention is further concerned with an
alternate process for the preparation of Form I of N-[l(R)-[(1,2-
dihydro- 1 -methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1'-
yl)carbonyl] -2-(phenylmethyl-oxy)ethyl] -2-amino-2-methylpropanamide
methanesulfonate which comprises:
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alternatively adding to a solution of the free base of N-
[ l (R) [( 1 ,2-dihydro- 1 -methanesulfonyl-spiro[3H-indole-3 ,4'-piperdin] -
1 '-yl)carbonyl] -2-(phenylmethyl-oxy)ethyl] -2-amino-2-methylpropan-
amide in ethyl ~cet~te cont~inin~ ethanol (about 8 volume %) at
5 approximately 50-55~C,
methanesulfonic acid (about 1.1 equivalents), and
Form I of N-[l(R)-[(1,2-dihydro-1-methanesulfonyl-
spiro[3H-indole-3 ,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyl-
oxy)ethyl]-2-amino-2-methyl-propanamide methanesulfonate (wherein
10 the relative order of addition is not critical),
followed by heating at approximately 55~C for
approximately 2-15 hours, cooling to approximately 25-30~C, and aging
for ap,proximately 2-3 hours.
The present invention is further concerned with an
15 alternate process for the prepa]ration of Form I of N-[l(R)-[(1,2-
dihyd:ro- 1 -methanesulfony:l-spiro[3H-indole-3,4'-piperdin]- 1'-
yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide
methanesulfonate which comprises:
stirring a solution of Form II of N-[l(R)-[(1,2-dihydro-1-
20 methanesulfonyl-spiro[3H- indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
(phen ylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide
metha.nesulfonate in isopropanol at approximately 25~C for about 2-24
hours .
The present invention is also concerned with a process for
25 the preparation of Form II of N-[l(R)-[(1,2-dihydro-1-methane-
sulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenyl-
methyl-oxy)ethyl]-2-amino-2-methylprop~n~mide methanesulfonate
which comprises:
treating a solution of the free base of N-[l(R)-[(1,2-
30 dihyd]ro- 1 -methanesulfonyil-spiro[3H-indole-3,4'-piperdin]- 1'-
yl)carbonyl]-2-(phenylmethyl-oxy)ethy:1]-2-amino-2-methylpropanamide
in eth'yl ~cet~te containing ethanol (about 8 volume %) with
methanesulfonic acid (about 1.1 equivalents) at approximately 50~C,
heating to approximately 55~C, and cooling to ambient temperature.
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The present invention is further concerned with a process
for the preparation of Form IV of N-[l(R)-[(1,2-dihydro-1-
methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
5 (phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide
methanesulfonate which comprises:
dissolving N-[ 1 (R)-[( 1 ,2-dihydro- 1 -methanesulfonyl-
spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyl-
oxy)ethyl]-2-amino-2-methylprop~n~mide methanesulfonate of optional
10 morphological composition in a solution of ethanol/water (preferably
25:75 v/v);
evaporating the solvent from the solution, preferably at a
temperature of 40~C;
grinding the resultant solid to a fine powder; and
exposing the fine powder to a relative humidity of
approximately 75%.
The present invention is further concerned with an
alternate process for the preparation of Form IV of N-[l(R)-[(1,2-
dihydro- 1-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1'-
20 yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide
methanesulfonate which comprises:
recryst~lli7~tion of N-[l(R)-[(1,2-dihydro-1-
methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
(phenylmethyl-oxy)ethyl] -2-amino-2-methylpropanamide
25 methanesulfonate of optional morphological composition from a
solution of ethylacetate/ethanol/water (preferably 24.8/1.6/1.95 vlvlv).
The present invention is further concerned with an
alternate process for the preparation of Form IV of N-[l(R)-[(1,2-
dihydro- 1-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1'-
30 yl)car~onyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide
methanesulfonate which comprises:
exposing Form I of N-[l(R)-[(1,2-dihydro-1-
methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
(phenylmethyl-oxy)ethyl]-2-amino-2-methylprop~n~mide
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methanesulfonate to a relative humidity of greater than about 75%,
more preferably a relative humidity of about 88%, at ambient
temperature for a sufficient time.
The present invention is further concerned with an
5 alternate process for the p:reparation of Form IV of N-[l(R)-[(1,2-
dihyd.ro- 1 -methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1'-
yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide
methcmesulfonate which comprises:
isolation from~ a slurry of Form I of N-[l(R)-[(1,2-
10 dihydro- 1-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1'-
yl)carbonyl]-2-(phenylmet:hyl-oxy)ethyl]-2-amino-2-methylprop~namide
meth~mesulfonate in isopropyl acetate/ethanol (90:10 v/v) containing
approximately 2.8 wt% water at approximately 25~C.
The present invention is further concerned with a process
15 for the preparation of Form V of N-[l(R)-[(1,2-dihydro-1-
methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1'-yl)carbonyl]-2-
(phenylmethyl-oxy)ethyl]- 2-amino-2-methylpropanamide
metha,nesulfonate which comprises:
exposing Forr~ IV of N-[l(R)-[(1,2-dihydro-1-
20 metha~nesulfonyl-spiro[3H- indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
(phenylmethyl-oxy)ethyl]-'2-amino-2-methylpropan~mide
metha.nesulfonate to below 30% relative humidity at ambient
tempe~rature.
The present invention is further concerned with a process
25 for thl~ preparation of Form VI of N-[l(R)-[(1,2-dihydro-1-
metha.nesulfonyl-spiro[3H- indole-3 ,4'-piperdin]- 1 '-yl)carbonyl]-2-
(phen ylmethyl-oxy)ethyl]-~-amino-2-methylpropanamide
methanesulfonate which comprises:
drying Form ~l of N-[l(R)-[(1,2-dihydro-1-methane-
:30 sulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenyl-
methyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate in
the absence of moisture at approximately room temperature, such as
under an atmosphere of sieve dried nitrogen at approximately 25~C.
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The present invention is further concerned with a process
for the preparation of Form VII of N-[l(R)-[(1,2-dihydro-1-
methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
(phenylmethyl-oxy)ethyl] -2-amino-2-methylpropanamide
S methanesulfonate which comprises:
isolation from a slurry of Form I or Form IV of N-[l(R)-
[( 1 ,2-dihydro- 1 -methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1'-
yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylprop~mide
methanesulfonate in isopropyl acetate/ethanol (90:10 v/v) cont~ining
lO approximately 1.5 wt% water.
The present invention is further concerned with a process
for the preparation of Form VIII of N-[l(R)-[(1,2-dihydro-1-
methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide
:LS methanesulfonate which comprises:
dehydrating Form VII of N-[l(R)-[(1,2-dihydro-1-
methane-sulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
(phenyl-methyl-oxy)ethyl] -2-amino-2-methylpropanamide
methanesulfonate under a dry inert gas, such as dry nitrogen, for a
20 sufficient time.
The present invention is further concerned with a process
for the preparation of Form IX of N-[l(R)-[(1,2-dihydro-1-
methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
(phenylmethyl-oxy)ethyl] -2-amino-2-methylpropanamide
25 methanesulfonate which comprises:
preparing a solution of N-[l(R)-[(1,2-dihydro-1-methane-
sulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenyl-
methyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate of
optional morphological composition in water, followed by isolating the
30 solid formed by controlled evaporation at 20% relative humidity at
approximately room temperature.
The present invention is further concerned with a process
for the preparation of Form X of N-[l(R)-[(1,2-dihydro-1-
methamesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
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- 10 -
(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide
methcmesulfonate which comprises:
drying Form ][X of N-[l(R)-[(1,2-dihydro-1-methane-
sulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenyl-
S methyl-oxy)ethyl]-2-amino-2-methylprop~n~mide methanesulfonate at
ambient room temperature and humidity for a sufficient time.
The present invention is further concerned with an
alternate process for the preparation of Form X of N-[l(R)-[(1,2-
dihydro- 1 -methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1'-
10 yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide
methcmesulfonate which comprises:
exposing Forrn I of N-[l(R)-[(1,2-dihydro-1-methane-
sulfonyl-spiro[3H-indole-3,4'-piperdin:l- 1 '-yl)carbonyl]-2-(phenyl-
methyl-oxy)ethyl]-2-amino-2-methylprop~n~n~ide methanesulfonate to
15 lOO~o relative humidity for approximately 1 to 4 days.
Similarly, the present invention is also directed to a process
for the preparation of morphologically homogeneous N-[l(R)-[(1,2-
dihydro- l-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1'-
yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylprop~n~mide
20 methanesulfonate comprising any of the processes mentioned herein.
The compounds of this invention, the novel polymorphic
forms of N- [ 1 (R)- [( 1 ,2-dih ydro- 1 -methanesulfonyl-spiro[3H-indole-
3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-
methylpropanamide methanesulfonate, are growth hormone
25 secretagogues that are usef'ul in food animals to promote their growth
thereby rendering the production of edible meat products more
efficient, and in humans, to treat physiological or medical conditions
characterized by a deficiency in growth hormone secretion, and to treat
medical conditions which are improved by the anabolic effects of
30 growth hormone. Accordingly, the present invention is further
concerned with pharmaceutical formulations comprising a polymorphic
form as an active ingredient, and the use of this polymorphic form and
its foImulations in the treatment of certain disorders.
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Differential Sc~nnin~ Calorimeteric Cell rDSCl
The DSC curve for Form I of N-[l(R)-[(1,2-dihydro-1-
methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide
methanesulfonate at 10 ~C/min in an open cup under nitrogen flow
exhibits a single endotherm, due to me]ting, with a peak temperature of
about 180~C and an extrapolated onset temperature (melting point) of
about 170~C with an asociated heat of approximately 53 J/g.
The DSC curve for Form II of N-[l(R)-[(1,2-dihydro-1-
methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide
methanesulfonate at 10 ~C/min in an open cup under under nitrogen
flow e xhibits a single endotherm, due to melting, with a peak
temperature of about 174~C and an extrapolated onset temperature
(melting point) of about 165~C with an asociated heat of
approximately 37 J/g.
The DSC curve for Form IV of N-[l(R)-[(1,2-dihydro-1-
methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
(phenylmethyl-oxy)ethyl] -2-amino-2-methylpropanamide
methanesulfonate at 10 ~C/min in an open cup under under nitrogen
flow e:xhibits a water loss endotherm at about 45~C followed by an
endotherm with a peak temperature of about 134~C and an extrapolated
onset temperature (melting point) of about 129~C, due to melting of
Form VI, with an asociated heat of approximately 23 J/g.
'25
DSC Data [samples are heated at a rate of 10 ~C/min under a nitrogen
atmosphere (extrapolated onset temperature)]:
Form I: 170~C (melting endotherm)
Form II: 165~C (melting endotherm)
Form VI: 129~C (melting endotherm)
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Form I of N-[l(R)-[(1,2-dihydro-1-methanesulfonyl-
spirol 3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyl-
oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate is a
relati~ely anhydrous polymorph characterized by the following
S properties: a melting point of 169~C and solubility in isopropanol of
4.6 m,g/mL.
Form II of N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-
spirol3H-indole-3,4'-piperldin]- 1 '-yl)carbonyl]-2-(phenylmethyl-
oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate is an
anhyclrous polymorph characterized by the following properties: a
melting point of 158~C and solubility in isopropanol of 12.3 mg/mL.
Form III of N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-
spiro[3H-indole-3,4'-piper~lin]- 1 '-yl)carbonyl]-2-(phenylmethyl-
oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate is a hydrate
characterized by the following properties: a water loss endotherm at a
peak l:emperature of 46~C, followed by a minor melting/decomposition
endot]herm with an extrapolated onset temperature of 123~C.
Form IV of N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-
spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyl-
oxy)ethyl]-2-amino-2-methylprop~n~mide methanesulfonate is a hydrate
characterized by the following properties:
a water loss endotherm at a peak temperature of 45~C,
followed by a melting/decomposition endotherm with an extrapolated
onset temperature of 129~C (presumably due to the melting/
decomposition of Form VI).
Form IV N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-
spiro[3H-indole-3,4'-piperdin]- 1'-yl)carbonyl]-2-(phenylmethyl-
oxy)el:hyl]-2-amino-2-meth,ylprop~n~mide methanesulfonate appears to
be a hygroscopic hydrate containing 3..~ moles of water per mole of
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N-[l(~R)-[(l ,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4'-
piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-
methylprop~n:~mide methanesulfonate.
Form V N-[l(R)-[(1,2-dihydro-1-methanesulfonyl-
spirol~3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyl-
oxy)ethyl]-2-amino-2-methylprop~n:lmide methanesulfonate appears to
be a hygroscopic hydrate containing 1 moles of water per mole of
N-[l(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4'-
piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-
methylprop~n~mide methanesulfonate.
Form VI N-[ 1 (R)- [( 1 ,2-dihydro- 1 -methanesulfonyl-
spirol 3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyl-
oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate appears to
be an anhydrous polymorph and is characterized by a melting point oi'
129 C~.
Form VII of N-[l(R)-[(1,2-dihydro-1-methanesulfonyl-
spirol 3H-indole-3 ,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyl-
oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate is a hydrate
characterized by the following properties: a broad water loss
endotherm at a peak temperature of 60~C, followed by a
melting/decomposition endotherm with an extrapolated onset
temperature of 144~C (presumably due to the melting/decomposition of
Form VIII).
Form VIII N-[l(R)-[(1,2-dihydro-1-methanesulfonyl-
spirol 3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyl-
oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate is an
anhyclrous polymorph characterized by a melting point of 144~C.
Form X of N-[l(R)-[(1,2-dihydro-1-methanesulfonyl-
spirol 3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyl-
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- 14-
oxy)ethyl]-2-amino-2-methylprop~n~mide methanesulfonate is
characterized by a broad water loss endotherm at a peak temperature of
49~C.
X-Ray Powder Diffraction (XRPD)
X-Ray powder diffraction studies are widely used to
eluci~date molecular structures, crystallinity and polymorphism. X-ray
powcler diffraction (XRPD) patterns were collected using a Philips
APD3720 Automated Powder Diffraction instrument with copper Ka
radiation. Measurements were made from 2~ to 40~ (2 theta) with the
sample maintained at amb~ient room temperature.
Form I was characterized by an X-ray powder diffraction
pattern with principal reflections at approximately: 6.5, 14.7, 16.9,
17.1, 17.9, 19.5, 21.1, 21.7, and 22.0~ (2 theta).
Form II was characterize(l by an X-ray powder diffraction
pattern with principal reflections at approximately: 4.8, 11.8, 17.5,
19.4, 21.6, 21.9, 22.5, ancl 22.7~ (2 theta).
Form III was characterized by an X-ray powder diffraction
pattern with principal reflections at approximately: 13.8, 14.1, 18.0,
18.8, 19.5, 20.1, 20.6, 21.8, and 25.7~ (2 theta).
Form IV was characterized by an X-ray powder diffraction
patte]n with principal reflections at approximately: 16.0, 16.2, 18.3,
20.1, 21.0, and 24.2~ (2 th~eta).
Form V was characterized by an X-ray powder diffraction
patteln with principal reflections at approximately: 14.8, 17.1, 17.6,
19.0, 19.1, 19.4, 20.6, 21.5, and 21.8~ (2 theta).
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Forrn VI was characterized by an X-ray powder diffraction
pattern with principal reflections at approximately: 9.8, 14.0, 14.8,
17.1, 17.6, 19.0, 19.5, 20.6, and 21.6~ (2 theta).
Form VII was characterized by an X-ray powder
diffraction pattern with principal reflections at approximately: 9.1,
11.3, 17.1, 17.4, 20.0, 22.1, and 24.5~ (2 theta).
Form VIII was characterized by an X-ray powder
10 diffra~tion pattern with principal reflections at approximately: 11.5,
11.6, 18.1, 19.6, 22.5, 24.7, and 24.8~ ~2 theta).
Form IX was characterized by an X-ray powder diffraction
pattenn with principal reflections at approximately: 8.0, 12.1, 15.3,
15.8, 19.6, 19.7, 21.1, 22.3, and 23.7~ (2 theta).
Form X was characterized by an X-ray powder diffraction
pattern with principal reflections at approximately: 15.5, 15.8, 18.0,
18.4, 18.6, 19.4, 20.7, 20,8, 23.9, and 24.8~ (2 theta).
These XRPD patterns confirm that all of Forms I - X are
'~0 distinct crystalline forms.
Microscopy
Ex~min~tion of the polymorphic forms was conducted at
lOOX m~gnification under plain and polarized light. Form I and Form
25 II were needle shaped particles. Both Form I and Form II appeared
birefringent under polarized light.
Hy~roscopicity
The total volatiles content (as established by TGA analysis)
30 of solid samples of Forrns I, II, III and rv upon exposure to various
controlled humidities is tabulated below. It was found that Form I
posses,sed 0.79 wt % water; Form II possessed 0.56 wt % water; Form
III possessed 4.5 - 5.0 wt % water; and Form IV possessed 9.5 - 10.0 wt
% water.
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Hygroscopicity was assessed by storing the solid compound
in constant relative humidity chambers. A comparison of anhydrous
Forms I and II at room ternperature indicates Form II is hygroscopic
and e:~hibits a large increase in moisture starting at 65% RH. No
5 subst~mtial moisture increase is exhibited by Form I except when stored
above 76% RH. The results are tabulated below in Tables 1 and 2.
TABLE 1
Room Temperature (48 Hrs)
Form I Form II
%RH % GainJLoss % Gain/Loss
O -0.02 +0.08
1 1 -0.05 +0.02
33 - +0.33
47 +0.21 +0.39
+0.37 >lO.Oa
76 +0.12 >lO.Oa
100 >12.0 >lO.Oa,b
( a = Sample becomes a gummy semi-solid)
( b = Converts to Form IV upon exposure to ambient
humidity)
TABLE 2
Roorn Temperature (96 Hrs)
Form I
% RH % Gain/Loss
7 +0.4
22 +0.3
47 +0.5
:30 68 +0.6
88 +12.9*
100 +23.9*
(* = deliquescent material)
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Hydrated forms III and IV of N-[l(R)-[(1,2-dihydro-1-
methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
(phen~ylmethyloxy)ethyl]-2-amino-2-methylpropanamide
methanesulfonate were also evaluated by storage in relative humidity
5 chambers for 48 hrs at room temperature. Form III shows an increase
in moisture after 48-hr storage at 33% RH. Form IV, although not
gaining an appreciable amount of moisture until storage at 100% RH,
loses i.ts water of hydration when stored at 1 1% RH or below. The
results are tabulated below in Table 3.
'10
TABLE 3
Room Temperature (48 Hrs)
Form III Form IV
% RH % Gain/Loss % Gain/Loss
0 - -7.8
1 1 -0. 1 5 -4.9
33 +2.23 +0.3
47 +3.99 +0.7
66 - +1.4
'~0 76 +3.76 +1.5
100 - +5 3
This data indicates that Form I is relatively anhydrous.
Solubility
The solubility of Form I in distilled water at room
temperature is >100 mg/mL. The aqueous solubility (RT) of Form II in
buffer~d solutions (pH 4-9) is >100 mg/mL. The solubility of Form I
in ethcmol/water mixtures is shown below:
Solubility
% Ethanol/H~O (m~/mL)
25l75 >100
50/50 >100
75/25 >90
100% ethanol >90
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Therrnal Stability - Neat Compound
The solid state stability of neat compound was assessed by
storagre of the drug in glass screw-cap vials in the dark. Samples were
assayed by HPLC and the parent compound was quantitatively analyzed.
5 The isocratic method that ~as employed is outlined below:
Column: Beckman Ultrasphere ODS (250x4.6 mm, 5,u)
Mobile Phase: 0.'1% TEA, pH 4.0 with H3PO4:Acetonitrile (65:35)
Flow Rate: 1.0 mL/min
Detection Wavelength: 228 nm
Run Time: 14 minutes
Colurnn Temperature: Ambient
Injection Volume: 20 ,uL of N-[l(R)-[(1,2-dihydro-1-
meth~mesulfonyl-spiro[3H -indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
(phenylmethyl-oxy)ethyl] -'2-amino-2-methylpropanamide
15 metha,nesulfonate (50 ,ug/r~
The results shown below f'or Forms I and II were
calculated as weight percent.
Form l
'~o Inital
~C 6Wks 17Wks
100.1 98.7
100.7 101.3
100.7 99.4
Form lI
% Initial
~C 1 Wk 2 Wks 4 Wks 8 Wks 12 Wks24 VVks
99.7 100.7 99.5 -- 100.1 100.1
99.4 99.6 100.3 100.0 101.0 100.8
99.2 100.3 99.~ 99.8 100.4 --
These results indicate that neat solid Form I and Form II
had good thermal stability.
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The processes to prepare the subject compound are outlined
as fol]lows:
SCHEME I:
s
Oq,OBn Oq,OBn
N N N PdlC H2
BnOCOCI ,~ ~ 1 04 eq. (COC1)2 ~ ~
K2CO3, H20 '~ cat. DMF ~ DIEA
lh:-- ,:6cle
COOH COOH COCI
2 3 4
Oq,OBn Oq,OBn ~OBn
PhNHNH2 (~ TFA ~ NaBH4/MeOH
CHO CH=NNHPh N
O 7 8
OBn ~ OBn H
MsCI ~ N 1 ) Activated ~ N
DIEA ~ ) 2)100to Pd/C
g SO2M e SO2M e
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SICHEME I (CONT'D)
H Ph~o~N H BOC
Ph~O ~ 2 N ~
10 NHBOC,~".~ > 1) MsOH, EtOH
~~ N DCC W~ N 2) basic workup
SO2M e iPrOAc:H~,O S OzM e
1b 11
Ph'~o~\5 NH2 Ph O ~ ~NHBOC
~ H3C~CH3 O O
HO2C NHBOC r N
3 ~> 1) MsOH, EtOH
~:~ N DCC ~~ N 2) basic workup
iPrOAc.H20
S O2M e SO2M e
12 14
PhO~5~ ~NH2 ~<NH2- MsOH
00 0 0
~N ~N
3~ MsOH ~)
SO2M e S O2M e
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As depicted in Scheme I, the CBZ-Spiroindoline 1 is treated
with Darco (20% by weight) prior to hydrogenation. The
hydro~genation is carried out in ethanol at 65~C over 10% Pd/C with
vigorous stirring.
S A solution of lb in isopropyl acetate and water is coupled
with commercially available N-BOC-O-benzyl-D-serine in the presence
of dicyclohexylcarbodiimide (DCC) and l-hydroxybenzotriazole
(HOBt). After filtration of the dicyclohexylurea (DCU) side product,
the 2-phase filtrate is separated and the organic layer is washed
successively with lM aqueous sodium hydroxide solution, 0.5M aqueous
hydrochloric acid and finally saturated aqueous sodium hydrogen
carbonate. Improved results in this coupling are achieved when a
solution of the free amino in iPrOAc/H2O is treated with DCC, HOBT
followed by addition of the amino acid at ambient temperature and
:L5 followed by reaction for 3-5 hrs The batch is then concentrated in
vacuo and the solvent is switched from isopropyl acetate to ethanol.
This solvent switch generally proceeds swiftly by "feeding and
bleeding" 3x batch volumes to remove isopropyl acetate
The BOC-group of 11 is removed by treatment with
methanesulfonic acid (MsOH) (3 eq) in ethanol at 35-40~C. Partitioning
between isopropyl acetate and aqueous lM sodium hydroxide solution
affords 12.
The coupling of 12 with N-BOC-a-aminoisobutyric acid is
best conducted in a two-phase solvent system, isopropyl acetate/water
(1:1) in the presence of DCC and HOBt (1.1 eq. each). Removal of the
DCU by filtration, separation of the layers and washing the organic
layer s,uccessively with lM aqueous sodium hydroxide, 0.5M aqueous
hydrochloric acid and saturated aqueous sodium hydrogen carbonate
affords 14.
The mixture is solvent switched to ethanol for the
subsequent methanesulfonic acid cleavage of the Boc group.
Deprotection of 14 is more difficult than that of 11 and requires a
concentrated solution of ethanollmethanesulfonic acid and heating to 35-
40~C. After extractive workup (EtOAc-NaOH), the free amine 15 is
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isolated. The organic layer is washed well with lN NaOH to ensure
complete removal of meth~mesulfonic acid.
The ethyl acetate solution of the free base 15 is
concentrated to low bulk i~l vacuo and is azeotroped dry (KF <500
5 mgml~l) by "feeding and bleeding" 2x batch volumes of 90/10, ethyl
acetate/ethanol followed bv 2x batch volumes of ethyl acetate. The
resulting dry, slightly hazy solution of the free base 15 in ethyl acetate
is treated with Darco
G-60 (25 weight %) at room temperature for about 10 hours. Removal
10 of the Darco by filtration with a filtration agent gives the free base 15.
Formation of Ihe methanesulfonic acid salt 16 from 15 is
carrield out in EtOAc with 1.1 eq of MsOH at about 50~C. The free
base ] S is treated with 8 volume % of E~tOH and 1 eq of H20 and heated
to 55''C until complete dissolution. Cooling to ambient temperature and
15 stirring the resulting slurry for 4 hours gives crystalline material of 16
designated as crystal Form II [solubility in IPA = 12 mg/rnL].
The conversion of Form II to Form I is accomplished
where the salt is formed in EtOAc-EtOH as above, but instead of
cooling the initial solution of the salt (at 55~C) to ambient temperature,
.~0 it is cooled to 45~C. Crystals should start appearing at that temperature
and the slurry should becorne thicker with time. The temperature is
then raised to 51~C and the slurry is aged overnight. Complete
conversion to Form I of 16 should be expected.
Preferably, the conversion of Form II to Form I is
25 achieved by adding seed crystals of Form I to a solution of the free base
in EtOAc-EtOH at 50-55~C' followed by aging. Accordingly, the free
base 15 may be treated with 1.1 equivs. of methanesulfonic acid in 8%
ethanol in ethyl acetate at 50-55~C. The batch is then seeded with
appro ~imately 2% by weight of Form I of the methanesulfonate salt 16,
30 and then aged at 55~C overnight. The batch is cooled to room
temperature and aged for approximately 2-3 hours. The product is
isolated by filtration at room temperature under a nitrogen atmosphere,
dried at 35~C in vac~o and sieved to give the methanesulfonate salt 16.
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The methanesulfonic acid salt 16 may also be formed by
alternating the stepwise addition of MsOH (1.1 eq) and seed crystals of
Form I to a solution of the free base in EtOAc-EtOH at about 50~C,
wherein the order of addition of the MsOH and the seed is not critical.
The utility of the polymorphic compounds of the present
invention as growth hormone secretagogues may be demonstrated by
methodology known in the art, such as an assay described by Smith, et
al., Science~ 260, 1640-1643 (1993) (see text of Figure 2 therein). In
partic-ular, all of the polymorphic forms of the present invention had
activil:y as growth hormone secretagogues in the aforementioned assay.
Such ;a result is indicative of the intrinsic activity of the polymorphic
forms of the present invention as growth hormone secretagogues.
The growth hormone releasing compounds of the present
invenl:ion are useful in vitro as unique tools for understanding how
growth hormone secretion is regulated at the pituitary level. This
includLes use in the evaluation of many factors thought or known to
influence growth hormone secretion such as age, sex, nutritional
factors, glucose, amino acids, fatty acids, as well as fasting and non-
fasting states. In addition, the compounds of this invention can be used
'20 in the evaluation of how other hormones modify growth hormone
releasing activity. For example, it has already been established that
somatostatin inhibits growth hormone release. Other hormones that are
important and in need of study as to their effect on growth hormone
release include the gonadal hormones, e.g., testosterone, estradiol, and
'25 progesterone; the adrenal hormones, e.g., cortisol and other corticoids,
epinephrine and norepinephrine; the pancreatic and gastrointestinal
hormones, e.g., insulin, glucagon, gastrin, secretin; the vasoactive
peptides, e.g., bombesin, the neurokinins; and the thyroid hormones,
e.g., thyroxine and triiodothyronine. The compounds of the present
invention may also be employed to investigate the possible negative or
positive feedback effects of some of the pituitary hormones, e.g.,
growth hormone and endorphin peptides, on the pituitary to modify
growth hormone release. C)f particular scientific importance is the use
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- 24 -
of these compounds to elucidate the subcellular mechanisms mediating
the release of growth hormone.
The compounds of the present invention may be
~mini~tered to ~nim~l~, including man, to release growth hormone in
5 vivo. For example, the compounds can be ~(lmini~tered to
commercially important ~nim~l~ such as swine, cattle, sheep and the like
to accelerate and increase their rate and extent of growth, to improve
feed e fficiency and to increase miL~ production in such ~nim~l.s. In
addition, these compounds can be ~-lmini~tered to humans in vivo as a
10 diagnostic tool to directly determine whether the pituitary is capable of
releasing growth hormone. For example, the compounds of the present
invemtion can be ~lmini~tered in vivo to children. Serum samples taken
before and after such ~c~mini~tration can be assayed for growth
hormone. Comparison of the amounts of growth hormone in each of
15 these samples would be a rneans for directly determining the ability of
the pa~tient's pituitary to release growth hormone.
Accordingly, the present invention includes within its scope
pharrrlaceutical compositions comprising, as an active ingredient, at least
one oi- the compounds of the present invention in association with a
20 pharlrlaceutical carrier or dliluent. Optionally, the active ingredient of
the pharmaceutical compositions can comprise an anabolic agent in
addition to at least one of the compounds of the present invention or
another composition which exhibits a different activity, e.g., an
antibiotic growth permittant or an agent to treat osteoporosis or in
'25 combination with a corticosteroid to minimi7e the catabolic side effects
or with other pharmaceutically active materials wherein the
combination enhances efficacy and minimi~es side effects.
Growth promoting and anabolic agents include, but are not
limited to TRH, diethylstilbesterol, estrogens"B-agonists, theophylline,
30 anabo:lic steroids, enkephalins, E series prostaglandins, compounds
disclo,sed in U.S. Patent No. 3,239,345, e.g., zeranol, and compounds
disclo,sed in U.S. Patent No. 4,036,979, e.g., sulbenox or peptides
disclo~ed in U.S. Patent Na,. 4,411,890.
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A still further use of the compounds of this invention is in
combination with other growth hormone secretagogues such as the
growl:h hormone releasing peptides GHRP-6, GHRP-l as described in
U.S. ]?atent Nos. 4,411,890 and publications WO 89/07110, WO
5 89/07111 and B-HT920 as well as hexarelin and GHRP-2 as described in
WO "3/04081 or growth hormone releasing hormone (GHRH, also
designated GRF) and its analogs or growth hormone and its analogs or
somal:omedins including IGF-1 and IGF-2 or a-adrenergic agonists such
as clonidine or serotonin SHTID agonists such as sumitriptan or agents
10 which inhibit somatostatin or its release such as physostigmine and
pyridostigmine. In particular, the compounds of this invention may be
used in combination with growth hormone releasing factor, an analog of
growth hormone releasing factor, IGF-1, or IGF-2. For example, a
compound of the present invention may be used in combination with
lS IGF-1 for the treatment or prevention of obesity. In addition, a
compound of this invention may be employed in conjunction with
retinoic acid to improve the condition of musculature and skin that
results from intrinsic aging.
The present invention is further directed to a method for
20 the manufacture of a medicament for stimulating the release of growth
hormone in humans and ~nim~l~ comprising combining a polymorphic
form of the present invention with a pharmaceutical carrier or diluent.
As is well known to those skilled in the art, the known and
potemtial uses of growth hormone are varied and multitudinous. The
25 ~lmirli~tration of the compounds of this invention for purposes of
stimnl~ting the release of endogenous growth hormone can have the
same effects or uses as growth hormone itself. These varied uses of the
present compounds thus may be sllmm~rized as follows: stimulating
growth hormone release in elderly humans; treating growth hormone
30 deficient adults; prevention of catabolic side effects of glucocorticoids;
treatment of osteoporosis; stimulation of the immune system,
acceleration of wound hea]ing; accelerating bone fracture repair;
treatment of growth retardation; treating acute or chronic renal failure
or insufficiency; treatment of physiological short stature, including
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growth hormone deficient children; treating short stature associated
with chronic illness; treatrnent of obesity and growth retardation
associated with obesity; treating growth retardation associated with
Prade:r-Willi syndrome and Tumer's syndrome; accelerating the
5 recovery and reducing hospit~li7~tion of burn patients or following
major surgery such as gastrointestinal surgery; treatment of intrauterine
growth retardation, and skeletal dysplasia, treatment of peripheral
neuropathies; replacement of growth hormone in stressed patients;
treatm1ent of osteochondrody-splasias, Noonans syndrome,
10 schizophrenia, depression, Alzheimer's disease, delayed wound healing,
and psychosocial deprivation; treatment of pulmonary dysfunction and
ventilator dependency; attenuation of protein catabolic response after a
major operation; treating malabsorption syndromes; reducing cachexia
and protein loss due to chronic illness such as cancer or AIDS;
:15 accelerating weight gain and protein accretion in patients on TPN (total
parenteral nutrition); treatment of hyperinsulinemia including
nesidioblastosis; adjuvant treatment for ovulation induction and to
prevent and treat gastric and duodenal ulcers; to stimulate thymic
development and prevent the age-related decline of thymic function;
20 adjunctive therapy for patients on chronic hemodialysis; treatment of
immunosuppressed patients and to enhance antibody response following
vacciniation; increasing the total lymphocyte count of a human, in
particular, increasing the T4/T8-cell ratio in a human with a depressed
T4/Tg-cell ratio resulting, for example, from physical trauma, such as
75 closed head injury, or fromL infection, such as bacterial or viral
infection, especially infection with the human immunodeficiency virus;
improvement in muscle strength, mobility, maintenance of skin
thickn~ess, metabolic homeostasis, renal hemeostasis in the frail elderly;
stimulation of osteoblasts, bone remodelling, and cartilage growth;
30 stimulation of the immune system in companion :~nim~ls and treatment
of disorders of aging in cornpanion ~nim~l~; growth promotant in
livestock; and stimulation of wool growth in sheep. Further, the instant
compounds are useful for increasing feed efficiency, promoting growth,
increasing milk production and improving the carcass quality of
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- 27 -
livestock. Likewise, the instant compounds are useful in a method of
treatm~ent of diseases or conditions which are benefited by the anabolic
effects of enhanced growth hormone levels that comprises the
~rlmini.~tration of an instanl compound.
In particular, the instant compounds are useful in the
prevention or treatment of a condition selected from the group
consisting of: osteoporosis; catabolic illness; immune deficiency,
including that in individuals with a depressed T4/T8 cell ratio; hip
fracture; musculoskeletal impairment in the elderly; growth hormone
~LO deficiency in adults or in children; obesity; cachexia and protein loss
due to chronic illness such as AIDS or cancer; and treating patients
recovering from major surgery, wounds or burns, in a patient in need
thereof.
In addition, the instant compounds may be useful in the
] 5 treatment of illnesses induced or facilitated by corticotropin releasing
factor or stress- and anxiety-related disorders, including stress-induced
depression and headache, abdominal bowel syndrome, immune
suppression, HIV infections, Alzheimer's disease, gastrointestinal
disease, anorexia nervosa, hemorrhagic stress, drug and alcohol
O withdrawal symptoms, drug addiction, and fertility problems.
It will be known to those skilled in the art that there are
numer,ous compounds now being used in an effort to treat the diseases
or therapeutic indications enumerated above. Combinations of these
therapeutic agents some of which have also been mentioned above with
2'5 the growth hormone secretaLgogues of this invention will bring
additional, complementary, and often synergistic properties to enhance
the growth promotant, anabolic and desirable properties of these
various therapeutic agents. In these combinations, the therapeutic agents
and the growth hormone secretagogues of this invention may be
independently present in dose ranges from one one-hundredth to one
times the dose levels which are effective when these compounds and
secretagogues are used singly.
Combined therapy to inhibit bone resorption, prevent
osteoporosis and enhance the healing of bone fractures can be illustrated
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by combinations of bisphosphonates and the growth horrnone
secreltagogues of this invention. The use of bisphosphonates for these
utilities has been reviewed., for example, by Hamdy, N.A.T., Role of
Bisphosphonates in Metabolic Bone Diseases, Trends in Endocrinol.
5 Metab., 4, 19-25 (1993). Bisphosphonates with these utilities include
alendronate, tiludronate, dimethyl-API), risedronate, etidronate, YM-
175"~lodronate, pamidronate, and BM-210995. According to their
potency, oral daily dosage levels of the bisphosphonate of between 0.1
mg and 5 g and daily dosage levels of the growth hormone
10 secrel:agogues of this invention of between 0.01 mg/kg to 20 mg/kg of
body weight are ~lmini~tered to patients to obtain effective treatment of
osteo]porosis.
In the case of alendronate daily oral dosage levels of 0.1
mg tc~ 50 mg are combined for effective osteoporosis therapy with 0.01
15 mg/~g to 20 mg/kg of the growth hormone secretagogues of this
invention. Osteoporosis and other bone disorders may also be treated
with compounds of this invention in combination with calcitonin,
estrogens, raloxifene and calcium supplements such as calcium citrate.
Anabolic effects especially in the treatment of geriatric
20 male patients are obtained with compounds of this invention in
combination with anabolic steroids such as oxymetholone,
methyltesterone, fluoxymesterone and stanozolol.
The compounds of this invention can be ~clmini~tered by
oral, ]parenteral (e.g., intramuscular, intraperitoneal, intravenous or
25 subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual, or
topical routes of ~(lmini~tration and can be formulated in dosage forms
appropriate for each route of ~-lministration.
Solid dosage iorms for oral ~(lmini~tration include
capsules, tablets, pills, powders and granules. In such solid dosage
30 forms, the active compound is admixed with at least one inert
pharmaceutically acceptab:le carrier such as sucrose, lactose, or starch.
Such dosage forms can also comprise, as is normal practice, additional
substcmces other than inert diluents, e.g., lubricating agents such as
magnesium stearate. In the case of capsules, tablets and pills, the dosage
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- 29 -
forms may also comprise bllffering agents. Tablets and pills can
additionally be prepared with enteric coatings.
Liquid dosage forms for oral ~-lmini~tration include
pharmaceutically acceptab]e emulsions, solutions, suspensions, syrups,
the elixirs cont~ining inert diluents commonly used in the art, such as
water. Besides such inert diluents, compositions can also include
adjuvants, such as wetting agents, emulsifying and suspending agents,
and sweetening, flavoring, and perfuming agents.
Preparations a~ccording to this invention for parenteral
~lmini~tration include sterile aqueous or non-aqueous solutions,
suspensions, or emulsions. Examples of non-aqueous solvents or
vehicles are propylene glycol, polyethylene glycol, vegetable oils, such
as olive oil and corn oil, gelatin, and injectable organic esters such as
ethyl oleate. Such dosage forms may also contain adjuvants such as
preselving, wetting, emulsifying, and dispersing agents. They may be
sterilized by, for example, filtration through a bacteria-ret~ining filter,
by incorporating sterilizing agents into the compositions, by irradiating
the ca,mpositions, or by heating the compositions. They can also be
manu:~actured in the form of sterile solid compositions which can be
dissolved in sterile water, or some other sterile injectable medium
imme,diately before use.
Compositions for rectal or vaginal ~-lmini.stration are
preferably suppositories which may contain, in addition to the active
substamce, excipients such as cocoa butter or a suppository wax.
Compositions for nasal or sublingual ~clmini~stration are
also prepared with standard excipients well known in the art.
The compound of this invention may be ~lmini.~tered to
patients (~nim~l~ and humans) in need of such treatment in dosages that
will provide optimal pharmaceutical efficacy. It will be appreciated that
the dose required for use in any particwlar application will vary from
patient to patient, not only with the palrticular compound or composition
selected, but also with the route of ~rlmini~tration, the nature of the
condition being treated, the age and condition of the patient, concurrent
medication or special diets then being followed by the patient, and other
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factors which those skilled in the art will recognize, with the
a~prol~liate dosage ultimately being at the discretion of the attendant
physi, ian.
The dosage of active ingredient in the compositions of this
S invention may be varied; however, it is necessary that the amount of the
active ingredient be such that a suitable dosage forrn is obtained. The
selected dosage depends upon the desired therapeutic effect, on the route
Of ~lmini~tration, and on the duration of the treatment. Generally,
dosage levels of between 0.0001 to 10 mg/kg. of body weight daily are
10 ~lmini~tered to patients and ~nim~l~, e~g., m~mm~l.c, to obtain effective
release of growth hormone. Preferably, the dosage level will be about
0.001 to about 25 mg/kg per day; more preferably about 0.01 to about
10 mg/kg per day.
Methods for preparing the polymorphic forms of the
lS present invention are illustrated in the following Examples. The
following examples are given for the purpose of illustrating the present
invention and shall not be c onstrued as being limitations on the scope or
spirit of the instant invention.
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EXAMPLE 1
N-[l('R)-[(1,2-Dihydro-l-methanesulfonylspiro[3H-indole-3,4'-
piperclin]- 1 '-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-
meth~rlpropanamide
Step A: 1,2-Dihydro-l-methanesulfonylspiro[3H-indole-3,4'-
piperdinelhydrochloride
To a solution of 1.20 g (5.8mmol) of 1'-methyl-1,2-
dihydro-spiro[3H-indole-3,4'-piperdinel (prepared as described by H.
Ong, et al., J. Med. Chem., 23, 981-986 (1983)) in 20 mL of dry
dichloromethane at 0~C was added triethylamine (0.90 mL; 6.4 mmol)
and mlethanesulfonyl chloride (0.49 mL; 6.35 mmol) and stirred for 30
min. The reaction mixture was poured into 15 mL of saturated aqueous
sodium bicarbonate solution and extracted with dichloromethane (2X10
mL). The combined organics were washed with brine (20 mL), dried
over a.nhydrous potassium carbonate, filtered and the solvent removed
under reduced pressure to yield 1.44 g of the methanesulfonamide
derivative as pale yellow oil which was used without purification.
To a solution of above crude product in 20 mL of dry 1,2-
dichloroethane at 0~C was added 1.0 mL (9.30 mmol) of l-chloroethyl
chloroformate, and then stirred at RT for 30 min and finally at reflux
for lh. The reaction mixture was concentrated to approximately one
third of the volume and then diluted with 20 mL of dry methanol and
'25 refluxed for l.5h. The reaction was cooled to RT and concentrated to
approximately one half of lhe volume. The precipitate was filtered and
washed with a small volume of cold methanol. This yielded 1.0 g of the
piperidine HCl salt as a white solid. The filtrate was concentrated and a
small volume of methanol was added followed by ether. The
precipitated material was once again filtered, washed with cold
methanol, and dried. This gave an additional 0.49 g of the desired
product. Total yield 1.49 g (70%).
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lH NMR (CDCl3, 200MHz) o 7.43-7.20 (m, 3H), 7.10 (dd, lH), 3.98
(bs, 2]H), 3.55-3.40 (bd, 2H), 3.35-3.10 (m, 2H), 2.99 (s, 3H), 2.15 (t,
2H), 2.00 (t, 2H).
S Step E,: N-[l(R)-[(1,2-Dihydro-1-methanesulfonylspiro[3H-indole-
3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-
2-[(1,1 -dimethylethoxy)carbonyl]amino-2-methyl-
prop~n~mlde
To 0.35g (1.1'; mmol) of (2R)-2-[(1,1-dimethylethoxy)-
lO carbonyl]amino-3-[2-(phenylmethyloxy)ethyl]- 1 -propanoic acid in 13
mL of dichloromethane was added 1,2-dihydro-1-methanesulfonylspiro-
[3H-indole-3,4'-piperdine] hydrochloride (0.325 g; 1.07 mmol), 0.18
mL (1.63 mmol) of N-methylmorpholine, 0.159 g (1.18 mmol) of 1-
hydroxybenztriazole(HOB'r) and stirred for 15 min. EDC (0.31 g; 1.62
15 mol) was added and stirring was continued for lh. An additional 60 ,uL
of N-methylmorpholine was added and stirred for 45 min. The reaction
mixture was poured into 5 mL of water and the organic layer was
separated. The organic layer was washed with 5 mL of O.5N aqueous
hydrochloric acid and 5 mI, of saturated aqueous sodium bicarbonate
20 solution. The combined organics were dried over anhydrous
magnesium sulfate, and concentrated to yield 0.627 g of the product as a
yellov~ foam which was used without purification.
To a 0.627 g (1.07 mmol) of the above product in 5 mL of
dichloromethane was added 1.0 mL of trifluoroacetic acid and stirred at
25 RT for 75 min. An additional 1.00 mL of trifluoroacetic acid was
added and stirred for 10 min. The reaction mixture was concentrated,
diluted with 5.0 mL of dichloromethane and carefully basified by
pourimg into 10 mL of 10~ aqueous sodium carbonate solution. The
organic layer was separated and the aqueous layer was further extracted
30 with 2X15 mL of dichloromethane. The combined organics were
washed with 5 mL of water, dried over potassium carbonate, filtered
and concentrated to give th~e 0.486 g of the amine as a light yellow foam
which was used without purification.
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- 33 -
To 0.486 g (1.01 mmol) of the amine and 10 mL of
dichloromethane was added 0.26g (1.28 mmol) of 2-[(1,1-dimethyl-
ethoxy)carbonyl]amino-2-methyl-propanoic acid, 0.173 g (1.28 mmol)
of l-hydroxybenztriazole l HOBT) and EDC (0.245 g; 1.28 mol) and
S stirried at RT overnight. The reaction mixture was poured into 5.0 mL
of water and the organic Llyer was separated. The aqueous layer was
back extracted with S mL of dichloromethane. The combined organics
were washed with 5.0 mL of 0.5N aqueous hydrochloric acid, S mL of
saturated aqueous sodium bicarbonate solution dried over anhydrous
magnesium sulfate, and concentrated to yield 0.751 g of the crude
prodwct as a yellow foam. A solution of this crude product in
dichloromethane was chromatographed on 25 g of silica gel and eluted
first with hexanes/acetone/'dichloromethane (70/25/S) and then with
hexanes/acetone/dichloromethane (65/30/S). This gave 0.63 g of the
lS title compound as a white solid.
lH NMR (CDC13, 400MHz) Compound exists as a 3:2 mixture of
rotamers o 7.40-7.10 (m, GH), 7.06 (d, 1/3H), 7.02 (t, 1/3H), 6.90 (t,
1/3H'I, 6.55 (d, 1/3H), S.l'i (m, lH), 4.95 (bs, lH), 4.63 (bd, 1/3H),
4.57-4.40 (m, 2 2/3 H), 4.10 (bd, 1/3H), 4.00 (bd, 1/3H), 3.82 (t, lH),
3.78-3.62 (m, 2H), 3.60-3.50 (m, lH), 3.04 (q, lH), 2.87 (s, lH), 2.86
(s, 2~I), 2.80-2.60 (m, lH), 1.90 (bs, lH), 2.85-2.75 (m, lH), 1.82-1.60
(m, 3H), 1.55-1.45 (m, lH), 1.45 (s, 4H), 1.42 (s, 2H), 1.39 (s, 9H).
Step (': N-[ l (R)- [(1,2 Dihydro- 1 -methanesulfonylspiro[3H-indole-
3,4'-piperidin l- 1 '-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-
2-amino-2-methylpropanamide hydrochloride
To 0.637 g (0.101 mmol) of the intermediate from Step B
in S rnL of dichloromethane was added 2.5 mL of trifluoroacetic acid
and sltirred at RT for 30 min. The reaction mixture was concentrated to
an oil, taken up in 10 mL of ethyl acetate and washed with 8 mL of 10%
aqueous sodium carbonate solution. The aqueous layer was further
extracted with S mL of ethyl acetate. The combined organics were
washed with 10 mL of water, dried over magnesium sulfate, filtered
and concentrated to give the 0.512 g of the free base as a white foam.
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- 34 --
To 0.512 g of Ihe free base in 5 mL of ethyl acetate at 0~C
was a.dded 0.2 mL of saturated hydrochloric acid in ethyl acetate and
stirred for 1.5 h. The white precipitate was filtered under nitrogen,
washed with ether, and dried to give 0~50 g of the title compound as a
white solid
lH NMR (400MHz, CD3C)D) Compound exists as 3:2 mixture of
rotamers. a 7.40-7.28 (m, 4H), 7.25-7.17 (m, 2H), 7.08 (t, 1/3H), 7.00
(t, 1/3H), 6.80 (d, 1/3H), 5.16 (ddd, lH), 4.60-4.42 (m, 3H), 4.05 (t,
lH), 3.90 (bs, 2H), 3.83-3.70 (m, 2H), 3.30-3.15 (m, lH0, 2.97 (s, lH),
2.95 I~s, 2H), 2.90-2.78 (m, lH), 1.96 (t, 1/3H), 1.85-1.65 (m, 4H), 1.63
(s, 2~1), 1.60 (s, 4H).
EXAMPLE 2
lS N-[l(R)-[(1,2-Dihydro-1-rnethanesulfonylspiro[3H-indole-3,4'-
piperdin]-l'-yl) carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-
meth!~lpropanamide
Step A: (2R)-[[[-2-(1, :l -dimethylethoxy)carbonyl]amino]-2,2-
dimethyl-1-oxoethyl]amino-2-(phenylmethoxy)ethyl]-1-
propanoic acid allyl ester
Prepared from (2R)-2-[(1,1-dimethylethoxy)carbonyl]-
amino-3-(phenylmethyloxy)ethyl-propanoic acid and allyl alcohol by
carrying out the coupling reaction in CH2cl2 in the presence of EDC
and DMAP.
lH NMR (400MHz, CDC13) o 7.25 (s, SH), 5.8 (m, lH), 5.2 (dd, 2H),
S.0 (bs, lH), 4.7 (m, lH), 4.6 (m, 2H), 4.4 (dd, 2H), 3.9 (dd, lH), 3.6
(dd, lH), 1.45 (d, 6H), 1.39 (s, 9H).
Step ~3: (2R)-[[[-2-(1,] -dimethylethoxy)carbonyl]amino]-2,2-
dimethyl- 1 -oxoethyl]amino-2-(phenylmethyloxy)ethyl)- 1 -
propanolc acld
To a stirred solution of the crude intermediate obtained in
Step A (6.7 g, lS.9 mmol), tetrakis (triphenylphosphine)-p~ lium (1.8
g, 0.1 eq) and, triphenyl phosphine (1.25 g, 0.3 eq) was added a solution
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- 35 -
of potassium-2-ethyl hexanoate (35 m~" 0.5M solution in EtOAc). The
reaction mixture was stirred at room temperature under nitrogen
atmosphere for lh and then diluted with ether (100 mL) and poured
into ice-water. The organic layer was seperated and the aqueous
5 fraction was acidified with~ citric acid (20%), then extracted with
EtOAc. The EtOAc extracts were washed with brine, dried over
magnesium sulfate, filtered and evaporated to give the title compound as
a soli~d.
lH NMR (400Hz, CD30D) ~ 7.3 (s, 5H), 4.7 (m, lH), 4.5 (s, 2H), 4.0
10 (m, lH), 3.6 (m, lH), 1.4 I'd, 6H), 1.3 (s, 9H).
Step C: N-[ l (R)-[(1,2- Dihydro- 1-methanesulfonylspiro[3H-indole-
3,4'-piperdin] - 1 '-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-
2-[(1,1 -dimethyl-ethoxy)carbonyl]amino-2-methyl-
l S propanamide
To a solution of 1.0 g (3.44 mmol) of l-methanesulfonyl-
spirolindoline-3,4'-piperidine] hydrochloride, 1.44 g (3.78 mmol) of
(2R)-1[[-2-(1,1-dimethylethoxy)carbonyl)amino]-2,2-dimethyl-1-oxo-
ethyl]-amino-2-(phenylmethyloxy)ethyl)-1-propanoic acid, N-methyl
20 morpholine (0.58 mL; 5.2() mmol), and l-hydroxybenztriazole (HOBT)
(0.58 g; 3.78 mmol), in 50 mL of dichloromethane was added EDC
(1.03 g; 5.20 mmol) and stirred at RT for 16h. The reaction mixture
was diluted with an additional 50 mL of dichloromethane and washed
with aqueous sodium bicarbonate solution (50 mL), dried over
25 anhyd~rous magnesium sulfate, filtered, and concentrated. Flash
chromatography (50 g silica gel) of the crude oily residue gave 2.148 g
(90%'~ of the desired material as a colorless foam.
1H NMR (CDCl3, 400MHz) Compound exists as a 3:2 mixture of
rotamers ~ 7.40-7.10 (m, 6H), 7.06 (d, 1/3H), 7.02 (t, 1/3H), 6.90 (t,
30 1/3H), 6.55 (d, 1/3H), 5.15 (m, lH), 4~95 (bs, lH), 4.63 (bd, 1/3H),
4.57-4.40 (m, 2 2/3 H), 4.]0 (bd, 1/3H), 4.00 (bd, 1/3H), 3.82 (t, lH),
3.78-3.62 (m, 2H), 3.60-3.50 (m, lH), 3.04 (q, lH), 2.87 (s, lH), 2.86
(s, 2H[), 2.80-2.60 (m, lH)~ 1.90 (bs, lH), 2.85-2.75 (m, lH), 1.82-1.60
(m, 3H), 1.55-1.45 (m, lH), 1.45 (s, 4H), 1.42 (s, 2H), 1.39 (s, 9H).
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Step O: N-[ l (R)- [(1,2-Dihyd~ro- 1 -methanesulfonylspiro[3H-indole-
3,4'-piperdin]- 1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-
2-amino-2-methylpropanamide hydrochloride
S To a solution of 2.148 g (3.41 mmol) of the intermediate
from Step C in 10 mL of dichloromethane was added 5 mL of
trifluoroacetic acid and stirred for lh. The reaction mixture was
concentrated and basified with 100 mL of 5% aqueous sodium carbonate
solution and extracted with dichloromethane (3X50 mL). The
10 combined organics were washed with brine (50 mL), dried over
anhydrous potassium carbonate, filtered, and concentrated to yield a
colorlless foam. To a solution of the foam in 25 mL of ethyl acetate at
0~C was added 4 mL of l~I solution of hydrochloric acid in ethyl
acetate. The precipitate was filtered and washed first with ethyl acetate
15 and then with ethyl acetate-ether (1:1), dried to yield 1.79 g (93%) of
the tit:le compound as a colorless solid.
lH N~R (400MHz, CD30D) Compound exists as 3:2 mixture of
rotam,-rs. ~ 7.40-7.28 (m, 4H), 7.25-7.17 (m, 2H), 7.08 (t, 1/3H), 7.00
(t, 1/3H), 6.80 (d, 1/3H), 5.16 (ddd, lH), 4.60-4.42 (m, 3H), 4.05 (t,
20 lH), 3.90 (bs, 2H), 3.83-3.70 (m, 2H), 3.30-3.15 (m, lH0, 2.97 (s, lH),
2.95 (s, 2H), 2.90-2.78 (m, lH), 1.96 (t, 1/3H), 1.85-1.65 (m, 4H), 1.63
(s, 2H), 1.60 (s, 4H).
EXAMPLE 3
XS
N-[l(R)-[(1,2-Dihydro- l-methanesulfonylspiro[3H-indole-3,4'-
piperdin]- 1 '-yl)carbonyl]-2 -(phenylmethyloxy)ethyl]-2-amino-2-methyl-
prop~n~mide mesylate
This compound was prepared by the treating the free base
30 obtained in Example 5, Step D, with methane sulfonic acid. The title
compound was obtained by recryst~lli7.ing it from ethyl acetate-ethanol-
water. m.p. = 166~-168~C. This sample was subsequently identified as
being polymorphic Form I]:. It was characterized by an X-ray powder
diffraction pattern with principal reflections at approximately: 4.7,
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11.6,17.4,19.2, and 21.6''(2 theta). It was further characterized by a
DSC at 10 ~C/min in an olpen cup under under nitrogen flow and
exhibited a single endotherm, due to melting, with a peak temperature
of about 174~C and an extrapolated onset temperature (melting point) of
S about 165~C with an asociated heat of approximately 37 J/g.
E X A M P L E 4
O;~,OBn
~3
CO2H
Isonipecotic acid-N-benzy] carbamate (3)
M atelials:
Isonipecotic acid (2) T.C.I. 4.02 kg (31.1 mol)
Benzyl chloroformate (Schweitzerhall) 6.91 kg (40.5 mol)
K 2 CC)3 10.1 kg (72.9 mol)
15 W ater 40.2 L
Isonipecotic acid (2) and K2co3 were dissolved in 40.2 L
of water in a 100 L 4 neck flask with mechanical stirring under N2 and
the solution was cooled to 10~C. Benzyl chloroformate was added,
20 maintaining the temperature between 9 and 14~C, and the mixture was
warmed up to 22~C after the addition was complete and aged for 58 h.
The addition was completed in 4 h at which point the pH was 9Ø After
aging for 58 h there was no change in the pH.
The reaction rnixture was transferred to a 200 L extractor
25 and washed with 3 x 13 kg (15 L) of IPAC and 1 x 12 L of EtOAc.
The a,queous layer was exb~acted with 8 L of toluene. After the washes
the benzyl alcohol content was reduced from 3.8 % to 1.4% by HPLC
analysis. HPLC analytical: Dupont Zorbax 25 cm RXC8 column with
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- 38 -
1.5 mL/min flow and detection at 254 nm; isocratic mixture with 35~o
MeC~, 65% of 0.1% aqueous H3PO4; retention times: 3 = 6.9 min,
benzyl alcohol = 3.3 min, l:oluene = 17.3 min.
The aqueous phase was acidified with 37% aqueous HCl to
5 pH 1. ~. Carbon dioxide was evolved during the addition of HCl, but gas
evolultion was easily controlled. The addition of HCl took <1 h and
required 10 L of conc. HC]. The aqueous phase was extracted with 3 x
6.6 L of toluene. The toluene extracts were dried with 2 kg of sodium
sulfate and ~lltered through a pad of Solka-flocTM. The combined
10 filtrates weighed 17.8 kg. The crude yield of carbamate 3 was 7.89 kg
(97%,1 (as obtained by evaporation of weighed aliquots of the filtrates to
dryness). The filtrates were transferred through a 10 ,u inline ~llter to a
100 L flask. The extracts ~ere concentrated at 10 mbar at ~25~C to a
volume of 18 L. The ~Inal concentration of carbamate 3 was 440 g/L.
15 The concentration of the toluene filtrate served to azeotropically
remo~e final traces of water (final KF = 170mg/L). The product was
99.1 a~rea % pure with 0.9 area % benzyl alcohol as the only impurity.
EXAMPLE 5
O~,OBn
~4
COCI
Isonipecotic acid chloride-,?V-benzyl carbamate (4)
Materials:
Isonipecotic acid N-benzyl carbamate (3) 7.89 kg (30.0 mol) in
in toluene. (MW = 263.30) 17.9 L
Oxalyl chloride (MW = 126.93) 3.94 kg (31.0 mol)
DMF (MW = 73.10) 10 mL
Toluene 12 L
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To the toluene solution of benzyl carbamate 3 from the
preceding step was added 5 mL of DMF and 10 L of toluene. The
oxaly]l chloride was added over a period of 20 min. The reaction
mixture was aged for 16 h at 18~C under a slow stream of nitrogen.
5 HPLC' analysis of the reaction mixture showed that 1.3 % of the
carboxylic acid 3 still remained unreacted. The reaction mixture was
warmed to 26~C, and 5 mL, of DMF were added. The mixture was aged
for 2.5 h. A 1.0 mL aliquot of the reaction mixture was quenched with
5.0 mL of tert-butylamine ,and analyzed after evaporation by HPLC: 25
10 cm Dupont Zorbax RXC8 column at 50~C with 1 mL/min flow and
detection at 220 nm; isocratic 42% MeCN, 58% of 0.1% aqueous
H3PO4. This method showed that <0.05% of the acid 3 remained (as
judged by A) and showed ~3 area % B (>1 mol% (COCl)2).
Oq~,Oe,n
N~ Oq~ N H-t-Bu
0~ N H-t-Bu
CONH-t-Bu
A B
The mixture was concentrated at 10 mbar and a
temperature of 20-25~C until 5 L of solvent had been removed.
The typical HPLC profile of concentrated toluene solution
after t-BuNH2 quench described above is as follows:
Retention time (min) Area % Identity
'~0 2.1 <0.5% carboxylic acid 3
7 . 8 <0.5% benzyl chloride
11.0 >99% Cbz-t-butylcarboxamide A
12.1 NA toluene
12.7 <0.5% ditert-butyloxamide B
,~5
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- 40 -
EXAMPLE 6
O~,OBn
CHO
Piperidine-4-carboxaldehyde-1-benzyl carbamate (5)
5 Materials:
Isonipecotic acid chloride N-benzyl carbamate (4) 3.38 kg (12.0
mol)
in toluene (MW = 281.74) in 5.54 kg
DIE~ (KF = 18 mg/L) 1.55 kg (15.0 mol)
10% Pd/C (KF < 20 mg/g) 101 g
thioanisole (MW = 124.21, d = 1.058) 0.56 g
The DIEA anld thioanisole were added to the solution of (4)
in toluene from the previous step and the catalyst was suspended in this
15 mixture. The mixture was immediately placed into the 5 gal autoclave
and hydrogenated at 20~C and 40 psi of H2. After 18 h the reaction had
taken up 70% the theoretical amount of hydrogen and HPLC analysis of
an aliquot that was quenched with tert-butylamine indicated that 14.2
area 'Yo of acid chloride 2 remained. HPLC conditions same as above.
20 Retention time: 5 = 8.1 mm.
A second charge of catalyst (101 g) and thioanisole (0.54 g)
were added as a slurry in 1375 mL toluene to the hydrogenator. After
23 h ]HPLC analysis of an aliquot that was quenched with tert-
butylamine indicated that 1.8 area % of acid chloride 2 remained. The
25 mixture was purged with nitrogen and the catalyst and precipitated
DIEA-HCl were removed by filtration through Solka-flocTM. The filter
cake was washed with 10 L of toluene. The filtrates were transferred
through a 10 ,u inline filtel to a 50 L extractor and washed with 2 x 7.2
L of I M aqueous HCl ancl 2 x 7.2 L of water. The mixture was
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- 41 -
concentrated at 10 mbar and a temperature of 25-30~C until 5 L of
residue remained.
Retention time (min) Area % Identity
2.1 <2 carboxylic acid 3
6.6 <1 dimer 21
8.1 >95 aldehyde 5
The assay yield of aldehyde 3 was 94 % by HPLC analysis.
EXAMPLE 7
O~,OBn
~9
~CN~H
CBZ-Spiroindoline (9)
L 5 Materi als:
Piperidine-4-carboxaldehycle-1-benzyl 1.71 kg (6.89 mol)
carbamate (5) in toluene solution in 21.4 kg
Phenylhydrazine 900 mL, 981 g (9.15 mol)
Trifluoroacetic acid (TFA) 2.20 L, 3.26 kg (28.6 mol,)
NaBH,4 300 g, (7.93 mol)
Toluene 34.4 kg
MeCN 7.0 L
MeOH 7.0 L
The crude aldehyde 5 solution from the previous step was
transferred through a 10 ,u inline filter to a 100 L reactor equipped with
Teflon coated copper coils for cooling or heating and a mechanical
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- 42 --
stirrer. Toluene (34.4 kg) and MeCN (7 L) were added, and the
resulting solution was coo]ed to 0~C. Phenylhydrazine was added in
portions and the temperature was maintained at -1 to 3~C while nitrogen
was c:ontinuously bubbled through the reaction mixture.
The phenylhydrazine was added until TLC and HPLC
analysis indicated complete consumption of the aldehyde 5 and the
appearance of a slight excess (<5%) of phenylhydrazine. TLC
conditions: Silica, E. Merck Kieselgel G60 F254 0.25 mm; diethyl
etherlpentane (4/1); and developing agent 0.5% ceric sulfate, 14%
ammonium molybdate in 10% aqueous sulfuric acid then heat; Rf:
aldehyde 5 = 0.52, phenylhydrazone 7 = 0.61, phenylhydrazine 6 =
0.21.
HPLC conditions: 25 cm Dupont Zorbax RXC8 column at 30~C with 1.0
mL/min flow and detection at 254 nm; gradient schedule:
Time (min) acetonitrile:water
0 57:43
65:35
75:25
18 75:25
retention times: phenylhydrazine 6 = 4.5 min, toluene = 7.2 min,
phenylhydrazone 7 = 11.4 min.
The reaction rnixture was aged for 30 min at 0-2~C, and
TFA was added maint~ini~g the temperature between 2 and 7~C. The
25 reaction mixture was warmed to 50~C over 30 min, and maintained for
17 h. The nitrogen sparge through the reaction mixture was stopped
and a slow stream of nitrogen was maintained over the reaction
mixture. During the first hour at 5~C the color gradually darkened to a
deep green, and a relativel'y small amount of a white crystalline
30 precipitate (ammonium trifluoroacetate) formed. After 17 h HPLC
analysis (same conditions as above) indicated that the reaction mixture
contaiined 91.6 area % indolenine 8 and 1.5% of unreacted
phenylhydrazone remainecl. Aging the mixture for longer periods of
time did not increase the assay yield of indolenine 8.
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The reaction rnixture was cooled to 12~C, and 7.0 L of
MeO]~I was added. NaBH~ was added in small (<20 g) portions
maintaining the temperature below 15~C. The addition took 30 min.
Moderate hydrogen evolution was obserlved during the addition, but it
5 was easily controlled and there was virtually no frothing. Near the end
of the addition the color rapidly changed from green to brown and then
brighl: orange. A small amlount (<200 mL) of a heavier phase had
separated (presumably aqueous salts). HPLC analysis (conditions as
before) indicated that all of the indolenine 8 had been consumed (90.4
10 area '~o CBZ-indoline 9); retention times: indolenine 8 = 7.5 rnin,
indoline 9 = 8.2 min. TLC': ethyl ether as solvent, ceric sulfate-
ammonium molybdate stain or 1% anisaldehyde stain; retention factors:
indolenine 8 = 0.18, CBZ-indoline 9 = 0.33.
The color change from green to orange corresponds very
15 closely to reaction end point. The quantity of NaBH4 required to
complete the reaction is heavily dependent on the temperature and rate
of addition of NaBH4, but the yield and quality of the product is
virtually unaffected provided that the reaction is complete. The
reaction mixture was cooled to 5~C over a period of 30 rnin. Then 8 L
20 of 3~c~ aqueous NH40H (8 L) were added to bring the pH of the aqueous
phase to 7.4, the mixture w as agitated, and allowed to settle. The
temperature rose to 15~C. The cloudy yellow lower aqueous phase was
separated. The organic phase was washed with 4 L of 3% aqueous
NH4OH, 2 x 4 L of water, and 2 x 4 L of brine. The weight of the
25 organic phase after the washings was 53.5 kg, and the assay yield was
94%.
The washed toluene solution was combined with the washed
organic phases of two other similarly processed reactions. The total
aldeh'yde used in the three reactions was 5.06 kg, (20.5 mol). The total
30 weigh~t of CBZ-indoline 9 assayed in the combined organic phases was
5.91 kg, (18.3 mol, 90% assay yield). The combined organic phases
were ,~ried with 5 kg of sodium sulfate, treated with 250 g of Darco
G60 carbon for 30 min, and filtered through Solka-flocTM. The filtrates
were vacuum concentrated at 10 mbar at <25~C until the residue was
CA 0223~371 1998-04-20
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- 44 -
near dLryness. The solvent switch was completed by slowly bleeding in
30 L of IPAC and reconcentrating to 14 L at 200 mbar at 50-60~C. The
mixture was heated to reflux in order to obtain a clear homogeneous
deep orange solution. lH NMR analysis indicated that the solution
5 contained ca. 6 mol% of residual toluene after solvent switch.
The solution u~as cooled to 68~C and seeded with 4 g of
crystalline CBZ-indoline 9. The solution was allowed to gradually cool
to 26~C over 6 h and aged for 9 h at 20-26~C. The slurry was cooled to
2~C over 1 h and aged at 2~C for lh. The product was isolated by
lO filtration, and the filter cake was washed with 2 x 2 L of 5~C IPAC and
2 x 2 L of 5~C MTBE. The product was dried in the vacuum oven at
30~C under a nitrogen bleed to give 4.37 kg (74%) of the title
compound 9 as a light tan crystalline powder. HPLC analysis of the
product indicated 99.5 area % purity. The mother liquor (11 L) and
15 the washes contained 1.15 kg (19%) of additional product 9 and ca 3%
of Cb;~-isonipecotic acid phenylhydrazide (retention time = 4.8 min).
EXAMPLE 8
O~,OBn
~N~
[~
SO2Me
20 CBZ-'ipiroindoline-methanesulfonamide (1)
Materiials:
CBZ-'ipiroindoline (9) 1.69 kg (5.23 mol)
Methanesulfonyl chloride 599 g (5.23 mol)
Et3N l(KF = 151) 635 g (6.27 mol )
THF (KF = 41) 12 L
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A 22 L flask was charged with the solid CBZ-spiroindoline
9 and then 11.5 L of THF and the Et3N were transferred into the flask
throu'gh a 10 ,u inline filter. The resulting homogenous solution was
cooleld to 0~C. A 1 L dropping funnel was charged with the
5 meth~mesulfonyl chloride cmd 500 mL of THF. The solution of the
MsCl in THF was added to the reaction mixture m~int~ining the
temperature between 0 and 4~C. The addition took 5 h and was
exothermic. A white precipitate, presumably triethylammonium
hydrochloride formed during the addition. HPLC analysis indicated
10 that the reaction was complete at the end of the addition (9 was
undetectable).
HPLC conditions: 25 cm Dupont Zorbax RXC8 column
with ]~.5 mL/min flow and detection at 254 nm. Gradient Schedule:
Time (min) 0.1% aq. H~P04:MeCN
0 71~:30
3 71~:30
12 21~:80
25 21~:80
Retention times: 9 = 7.6 min, 1 = 13.6 min.
After the addition was complete the reaction mixture was
warmed to 18~C and aged for 16 h. There was no change in the
appearance of the reaction mixture? and HPLC profile between the end
25 of the addition and after the 16 h age. The reaction mixture was slowly
transferred over lh into a vigorously stirred solution of 30 L of water
and 200 mL of 37% aqueous HCl in a 50 L flask. The temperature in
the 50 L flask rose from 22 to 28~C. The product separated as a pale
tan gummy solid which changed to a granular solid. The aqueous
30 suspension was cooled to 22~C and aged for 1 h. The suspension was
filtered, and the filter cake was washed with 2 x 4 L of MeOH/water
(50/5()). HPLC analysis indicated that <0.1% of the CBZ-Spiroindoline-
methanesulfonamidel was in the mother liquors.
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The filter cake was washed with 4 L of MeOHlwater
(50/5()) to which 50 mL of 28% aqueous NH40H had been added. The
filter ,cake was washed with 2 x 4 L of MeOH/water (50/50), and the
solid was dried in the vacuum oven at 50~C under a nitrogen bleed to
5 give 2.03 kg (97%) of the title product 1 as an off-white powder.
HPLC' analysis of the solids indicated 93.7 area % 1.
EXAMPLE 9
O~,OBn
~N~
N
10 Optional Procedure for Isolation of Intermediate
CBZ-Spiroindolenine (8)
Materials:
Piperidine-4-carboxaldehy~de-1-benzyl 12.37 g (0.050 mol)
carbamate (5)
Phenylhydrazine 5.41 g (0.050 mol)
Trifluoroacetic acid (TFAj 11.56 mL,17.10 g
(0.150 mol)
Methylene chloride 500 mL
The CBZ-aldehyde 5 was dissolved in dichloromethane in a
1 L flask equipped with Teflon coated magnetic stirring bar. The
resulting solution was cooled to 0~C. Phenylhydrazine was added via a
weighled syringe over 5 min and the temperature was m~int~ined at -1 to
25 3~C while nitrogen was continuously bubbled through the reaction
mixture. TLC and HPLC cmalysis indicated complete consumption of
the C]BZ-aldehyde 5 and the appearance of a slight excess (<2%) of
phenylhydrazine. TLC conditions: Silica, E. Merck Kieselgel G60 F254
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- 47 -
0.25 nnm; diethyl ether/pentane (4/1); and developing agent 0.5% ceric
sulfate, 14% ammonium molybdate in ] 0% aqueous sulfuric acid then
heat; Rf: aldehyde ~ = 0.52, phenylhydrazone 7 = 0.61,
phenylhydrazine 6 = 0.21. HPLC conditions: 25 cm Dupont Zorbax
S RXC8 column at 30~C with 1.0 mL/min flow and detection at 254 nm;
gradient schedule:
Time (min) acetonitrile:water
0 5,7:43
:10 10 6'j:35
7'j:25
18 7'j:25
retention times: phenylhydrazine 6 = 4.5 min, toluene = 7.2 min,
phenylhydrazone 7 = 11.4 min.
The reaction rnixture was aged for 10 min at 0-2~C, and
TFA was added by syringe maint~ining the temperature between 2 and
7~C. The reaction mixture was warmed to 35~C over 30 min, and
maintained for 17 h. The nitrogen sparge through the reaction mixture
was stopped and a slow stream of nitrogen was maintained over the
20 reaction mixture. During the first hour at 35~C the color gradually
darkened to a rosy pink then to a deep green, and a relatively small
amount of a white crystalline precipitate (ammonium trifluoroacetate)
formed. After aging for 17 h HPLC analysis (same conditions as above)
indicated that the reaction mixture contained 93 area % indolenine 8
25 and <().5% of unreacted phenylhydrazone remained. Aging the mixture
for longer periods of time clid not increase the assay yield of indolenine
8. The reaction mixture was cooled to 10~C, and a mixture containing
60 mL, 28-30% ammonium hydroxide, 90 mL water and 150 g crushed
ice was added with good stirring. The color of the mixture changed to
30 a salmon color. The organic phase was separated and washed twice with
400 mL water then 100 mL saturated aqueous NaCl. The organic phase
was dried over magnesium sulfate and filtered through a plug of 5 g of
silica. The filtrate was evaporated to give 15.84 g (99%) of indolenine
8 as a pale orange oil.
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EXAMPLE 10
O~,OBn
~1
~ ' SO2Me
Procedure for the Preparat:ion of CBZ-Spiroindoline-
5 methanesulfonamide (1) without Isolation of Interrnediate CBZ-
Spiroindoline (9)
Step 1: CBZ-Spiroindoline (9)
:l0 Materials:
Piperildine-4-carboxaldehyde-1-benzyl 49.5 g (0.20 mol)
carbarnate (5)
Phenylhydrazine (Aldrich) 23.7 g (0.22 mol)
Trifluoroacetic acid (TFA) 75.4 g (0.66 mol)
:l5 Toluene (KF < 250 mg/L) 654 mL
MeCN (KF < 250 mg/L) 13.3 mL
NaBH4 11.3 g, (0.30 mol)
Toluene 20 mL
MeOH: 50 mL
,~0
A 2% (by volume) solution of MeCN in toluene was made
up usi:ng 654 mL of toluene and 13.3 mL of MeCN. In a 2 L 3 neck
flask equipped with a mech.anical stirrer 617 ml of the above solution
were clegassed by passing a. fine stream of nitrogen through the solution
25 for 5 min. Phenylhydrazine and TFA were added to the mixture while
still degassing.
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The CBZ-aldehyde 5 was dissolved in the rest of the
solution prepared above (50 mL) and degassed by bubbling nitrogen
through the solution while in the addition funnel. The solution in the
flask was heated to 35~C, and the aldehyde solution was slowly added to
S the phenylhydrazine-TFA over 2 h. The mixture was aged at 35~C for
16h.
HPLC conditions: 25 cm Dupont Zorbax RXC8 column at
50~C with 1 mL/min flow and detection at 220 nm; isocratic 55%
MeCN, 45% of 0.1% aqueous H3PO4. Typical HPLC profile after 16 h
10 age:
Retention time (min) Area ~c, Identity
1.6 0.1-0.5 phenylhydrazine 6
4.1 <0.1 dimer 21
4.7 <0.1 aldehyde S
lS 5.0 NA spiroindoline 9
6.3 NA toluene
6.9 97 spiroindolenine 8
10.3 <0.2 phenylhydrazone 7
2-3 tot. other impurities <0.2% ea.
The mixture was cooled to -10~C and MeOH was added. A
suspension of sodium borohydride in 20 mL toluene was added in small
portions (1 mL) over 30 min taking care that the temperature did not
exceed -2~C.
Area % Identity
0.1-1 phenylhydrazine 6
85-90 C'BZ-spiroindoline 9
<0.1 C'BZ-spiroindolenine 8
10-lS tot. other impurities (<3% ea.)
The temperature was raised to 10~C over lh, and 6%
aqueous ammonia (200 mI,) was added. The mixture was agitated for
10 min, allowed to settle for another 10 min, and the lower aqueous
phase was drawn off. Acetonitrile (20 mL) and MeOH (20 mL) were
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- 50 -
addecl to the organic phase and it was washed with 150 mL of 15%
brine. The organic phase was found to contain a 92% assay yield of
CBZ-spiroindoline 9.
5 Step 2: CBZ-Spiroindoline-methanesulfonamide (1)
M atelials:
CBZ-Spiroindoline (9) (MW = 322.51) (0.184 mol)
Methanesulfonyl chloride 21.1 g (0.184 mol)
DIEA~ (KF = 150 mg/L) 29.7 g, 40.1 mL (0.230
mol)
THF (KF = 41 mg/L) 150 mL
The crude solution of CBZ-spiroindoline 9 solution from
15 Step l above was concentrated in a lL 3 neck flask (60-70~C, 150-200
Torr) until 250 g of residue remained. The THF and DIEA were
lefl, and the resulting homogenous solution was cooled to 0~C. A 125
mL dropping funnel was charged with the methanesulfonyl chloride and
50 m]L of THF. The solution of MsCl in THF was added over 2 h to the
20 reaction mixture maintaining the temperature between 0 and 4~C and
the mixture was aged for 2 h at 5-8~C. The addition was slightly
exothermic. A white precipitate, presumably DIEA-hydrochloride,
formed during the addition. HPLC conditions were the same as above.
HPLC analysis indicated that the reaction was complete 1 h after the end
25 of the addition (9 was undetectable) and the assay yield was 94% from
9. Retention time: 1 = 7.8 min. Typical HPLC profile of reaction
mixture after 2 h age:
Area % Identity
<0.1 C'BZ-spiroindoline 9
90-92 C'BZ-sulfonamide 1
8-10 tot. other impurities (<2% ea.)
The mixture was warmed to 20~C, and 200 mL of lM
aqueous HCl was added. The mixture was warmed to 50~C, and the
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WO 97/15574 PCT~US96/16955
aquec,us phase was separated. The organic phase was washed
sequentialy with 100 mL water, 100 mL 5% aqueous sodium
bicarbonate, and 100 mL water. The organic phase was transferred to a
1 L 3 neck flask equipped for mechanical stirring and distillation. The
5 mixture (ca 400 mL) was distilled at atmospheric pressure until 150 mL
of distillate had been collected. The head temperature reached 107~C;
the pot temperature was 110~C. The distillation was continued with
continuous addition of n-propanol at such a rate as to m~int~in a
constant volume (ca 350 mL) in the pot. The distillation was stopped
10 when a total of 525 mL of n-PrOH had been added and a total of 800
mL oi' distillate had been collected.
The temperature of both the head and pot rose from 94~C
to 98''C during the solvent switch. Toluene and n-PrOH form an
azeotrope boiling at 97.2~C' composed of 47.5% toluene and 52.5% n-
15 PrOH. The mixture was allowed to cool gradually to 20~C over 3h andaged i'or 12 h. The mother liquor was found to contain 2% toluene and
4 mg/mL of sulfonamide. The solubility of the sulfonamide in various
mixtures of toluene and n-}'rOH has been determined by HPLC assay:
%toluene in n-PrOH solubility of 1 in m~/mL
o 2.36
3.02
4.23
7.51
10.3
The crystalline slurry was ~lltered and washed with 3 x 100
mL of n-PrOH. The product was dried in a vacuum oven at 50~C with
a nitrogen bleed for 16 h to furnish 65.5g (82 % from aldehyde 5) of 6
30 as a tan solid with 93.5 wt'~o purity.
Typical HPLC profile of solid:
Area % Identity
<0.1 CBZ-spiroindoline 9
>99 CBZ-sulfonamide 1
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- 52 -
<1 tot. other impurities (<0.2% ea.)
For additional puri~ïcation, a 40.0 g sample of the n-PrOH
crystallized sulfonamide was dissolved in 134 mL of EtOAc at 60~C and
5 treated with 8.0 g of Darco G-60 carbon for 1 h at 60~C. After the
addition of 2.0 g SolkaflocTM, the slurry was filtered through a pad of
4.0 g SolkaflocTM, and the pad was washed with 90 mLof EtOAc at
60~C. Prior to the addition of the carbon the solution was a brown
color. The filtration proceeded well without plugging to give a golden
10 yellow filtrate. The filtrate was distilled at atmospheric pressure in a
500 mL flask (pot temperature 80-85~C) until 100 g (100 mL) of
residue remained. This solution was allowed to cool to 35~C over 3 h.
Over a lh period, 116 mL of cyclohexane was added with good
agital:ion at 35~C. The mixture was cooled to 20~C over 1 h and aged at
15 20~C for 12 h. At 35~C much of the sulfonamide has crystallized out
and t]he mixture was thick. Addition of cyclohexane at 20~C makes
agitation difficult. After the aging period, the supernatant was found to
contain 2.5 mg l/g. The crystalline slurry was filtered and the cake was
washed with 77 mL of 2:1 cyclohexane-EtOAc and 2 x 77 mL of
20 cyclohexane. The product was dried in a vacuum oven at 50~C with a
nitrogen bleed for 16 h to furnish 34.2 g of 1 (MW = 400.3) as a white
crystalline solid (85 % recovery from crude 1, 70 % from 5 with >99.9
wt % purity).
EXAMPLE 11
H
~N~ HCI
I~J la
1~ N~
SO2Me
HCl Salt of Spiroindoline-methanesulfonamide (la)
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- 53 -
Matelials:
CBZ-spiroindoline-methanesulfonamide (1) 941 g (2.35 mol)
Pearlman's catalyst 20% Pd(OH)2/C 188 g
THF 8 L
MeO~I 7 L
The catalyst was suspended in 7 L of MeOH and transferred
into the 5 gal autoclave followed by the solution of 1 in 8 L of THF.
The mixture was hydrogenolyzed at 25~C at 80 psi of H2. After 2.5 h
the temperature was raised to 35~C over 30 min.
HPLC analysis indicated complete consumption of Cbz-
spiroindoline-methanesulfonamide. HPLC conditions: 25 cm Dupont
Zorbax RXC8 column with 1.5 rnL/min flow and detection at 254 nm.
Gradient Schedule:
Time (min) 0.1% aq. H~PO4:MeCN
0 71~:30
3 71~:30
12 21~:80
25 21~:80
retention times: Spiroindoline = 7.6 min,
Cbz-spiroindoline-methanesulfonamide = 13.6 min.
The mixture was purged with nitrogen and the catalyst was
removed by filtration through Solka-flocTM while still warm. The
catalyst was washed with 4 L of THF and 2 L of MeOH. The pale
yellow filtrates were concentrated to a thick oil at 10 mbar and <25~C.
The solvent switch was cornpleted by slowly bleeding in 15 L of EtOAc
and reconcentrating to dryness. The residue solidified to a hard off-
white mass. MeOH (1.5 L'~ was added and the mixture was heated to
70~C to give a homogenous solution. While the solution was at 70~C,
10.5 L of EtOAc at 20~C was added. The temperature fell to 40~C, and
the mixture remained homogenous.
Subse~uent experiments suggested that it is more
convenient to solvent switch the MeOH-THF filtrates to MeOH,
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concentrate to the desired volume, and then add the EtOAc. This avoids
the solidification of the residue upon concentration of the EtOAc
solution.
Hydrogen chloride diluted with about an equal volume of
5 nitrogen was passed into the solution. The temperature rose to 60~C
over the course of 15 min, and a white precipitate of the hydrochloride
salt formed. Diluting the HCl with nitrogen only avoids the reaction
mixture sucking back and may not be necessary.
The mixture was cooled in an ice bath, and the hydrogen
l0 chloride addition was continued for lh. The temperature gradually fell
to 20~C. The suspension was aged for 2 h while the temperature was
lowered to 10~C. The crystalline product was isolated by filtration, and
the filter cake was washed with 3 L of EtOAc. It was dried in the
vacuum oven at 35~C to give 1.18 kg (86%) of the title product la as an
l5 off-white crystalline solid of >99.5 area % purity by HPLC analysis.
HPLC conditions: 25 cm Dupont Zorbax RXC8 column with 1.5
mL/min flow and detection at 230 nm; isocratic 35% MeCN, 65% of
0.1% aqueous ammonium acetate. Retention time: la = 5.4 min.
EXAMPLE 12
H
I
[~ 1b
[~
SO2Me
Spiroindoline-methanesulfonamide (Free base form) (lb)
A 250 mL aliquot of the filtrate from the Cbz-
hydrogenolysis containing 4.67 g of lb (free base) was concentrated to
25 ca 10 mL. The residue was dissolved in 20 mL of EtOAc and the
solution was reconcentratecl to ca 10 mL. This was repeated once more,
and 10 mL of EtOAc was added to the residue. A crystalline precipitate
began to form. MTBE (20 mL) was added in one portion. Additiona]
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crystalline solid precipitated, but the supernatent still contained a
substantial quantity of dissolved product which did not precipitate on
standiing. Hexanes (70 mL,) were added dropwise over 2 h to the
mixture with vigorous stirling. The slow addition of the hexanes is
5 neccessary to avoid the oiling out of the amine.
The agitated rnixture was aged for lh and filtered. The
filter cake was washed with 20 mL of 1: 1 MTBE-hexanes and then with
20 m]_ of hexanes. The product was dried under a stream of nitrogen
to give 3.86 g (82%) of the free amine of lb as an off white crystalline
10 solid of >99.5 area % puril:y. HPLC conditions: 25 cm Dupont Zorbax
RXC8 column with 1.5 mI,/min flow and detection at 230 nm; isocratic
35% ]\~eCN, 65% of 0.1% aqueous ammonium acetate. Retention time:
lb = 5.4 min.
EXAMPLE 13A
H
I
[~ ~ 1b
l~ ~N
SO2Me
Spiroindoline-methanesulfonamide (Free base form) (lb)
Materials:
CBZ-,Spiroindoline-sulfonamide (1) 833.5 gr (2.08 mol)
Pd(OH)2/C (20% weight of Pd(OH)2) 124.5 (l5~o)
THF 6.5 L
MeOH 19.5 L
NH4OH(conc) 60 mL
The hydrogenation was run three (3) times due to
equipment limitations; this procedure refers to a single run. The CBZ
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- 56 -
spiroindoline sulfonamidel was dissolved in THF (6.5 L, KF = 53
,ug/~uL) and then MeOH (KF=18 ,ug/mL, 4L) was added followed by
addition of the catalyst and the slurry was transferred to a 5 gal
autoclave. The remainder of the MeOH (2.5 L) was used for rinsing.
S The mixture was heated to 40~C at 50 psi for 24 hours. The catalyst
loading and reaction time are a function of the purity of starting
material 1. This material was unique requiring > 15% catalyst and long
reaction time. Purer batches of spiroindoline required only 5% of
catalyst and 4-6 hrs reaction time.
Upon complelion (<0.1 A% 1 by LC) the mixture was
filtered thru Solka FlocTM and the carbon cake washed with MeOH (13
L) containing NH40H (0.5%, 60 mL). The combined filtrates (assay
shows 1587 g of spiroindoline amine lb) were concentrated in vacuo
and the resulting solids were partitioned between 40 L (of toluene:THF
15 (3:1) ,and O.5N NaOH (18 L). Although the layers separated easily a
heavy precipitate could be seen in the aqueous layer. The aqueous
suspension was thus extracted with CH2cl2 (15 L). The aqueous and
organic layer separated slowly. Prior to CH2Cl2 addition THF was
added to the aqueous layer along with enough NaCl to saturate the layer.
20 However dissolution of the product was not achieved which necessitated
the use of CH2C12.
The combined toluene, THF and CH2cl2 layers were
combiined and concentrated in the batch concentrator. The residue was
flushed with 7 L of CH3CN. Finally 1() L of CH3CN were added and
25 the solution stood overnight under N2 atmosphere.
EXAMPLE 13B
H
I
~N~ lb
3 ~ sO2Me
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- 57 -
Spiroindoline-methanesulfonamide (Free base form) (lb)
M ateIials:
CBZ-Spiroindoline-sulfonamide (1) 3 kg (7.49 mol)
Darco G-60 600 g
Ethyl Acetate 36 L
Absolute Ethanol 189 L
10% ]?d/C 450 g
Ammonia Solution 500 ml
SoL~a F~LocTM 2.5 kg
Isopropyl Acetate 65 L
A mixture of ICBZ-spiroindoline (1) (1 kg) and Darco G-
60 (2()0 g) in ethyl acetate (9 L) was stirred and heated at 60-65~C
15 under a nitrogen atmosphere for 8 hours. The Darco was removed by
filtration at 60-65~C, the solid washed with hot ethyl acetate (3 L) and
the fi]trate and washings combined. LC wt/wt assay confirmed
negli~rible loss to the Darco. The ethyl acetate solution was evaporated
to dryness in vacuo using a 20 L Buchi apparatus and then flushed with
20 absohlte ethanol (2 x 5 L). This material was then slurried in absolute
ethanol (8 L) warmed to 65-70~C and placed in the 20 L autoclave. The
batch was rinsed in with absolute ethanol (1 L). A slurry of 10%
Pallaclium on charcoal (75 g, 7.5% by weight) in absolute ethanol (750
ml) was then added to the ,autoclave and rinsed in with a further portion
25 of absolute ethanol (250 ml).
The batch was hydrogenated at 65~C with vigorous stirring
under 40 psi hydrogen pressure for 3 hours, a second portion of 10%
palladLium on charcoal (75 g) was added, the batch was hydrogenated for
a further 2 hours and then sealed overnight. The batch was transferred
30 (still hot, 60-65~C) to a 20 L Buchi apparatus and degassed in vacuo to
remove formic acid by "feeding and bleeding" absolute ethanol (18 L
total).
This procedure was repeated twice more and the three
batches were combined in a 10 gallon glass-lined vessel and the
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- 58 -
combiined batch was degassed again by the addition and distillation (in
vacuo) of absolute ethanol (2 x 10 L). Solka flocTM (0.5 kg) was added
to the batch and rinsed in with ethanol (10 L). An Estrella filter was
loaded with SolkaflocTM (2 kg) as a slurry in ethanol (20 L). The
5 resultiing mixture was warmed to 60-65 C and then transferred at this
temperature via heated filter using pump to two tared stainless-steel
bins. The initial vessel, the filter, the pump and the lines were rinsed
with aL hot (60-65~C) mixture of aqueous ammonia (500 ml) in absolute
ethanol (25 L). The filtrate and washings were combined in the two
10 stainless-steel bins.
The batch was then transferred to a vessel using an in-line
filter containing a 10 micron cartridge, and then concentrated in vacuo
to lo~ bulk (~15 L). The ethanol was replaced by isopropyl acetate by
the "fi eding and bleeding" of 3x batch volumes of isopropyl acetate (45
15 L total), while maintaining a batch volume of ~15 L. The solvent
switch, when complete, contained <1% residual ethanol by GC. The
batch was then diluted to ~33 L by the addition of isopropyl acetate (20
L), and this solution of spiroindoline-amine lb (1.855 kg by LC
analysis) in isopropyl acetate was used for the next stage of the process.
'~O
EXAMPLE 14A
H ,H
, N - BOC
~J =O
~N~ 1 1
SO2Me
Boc-O-Benzylserine Spiroindoline (11)
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_ 59 _
Materials:
Spiroiindoline-amine (lb) 1587 g (5.966 moles)
Amino acid (10) 1938 g (6.563 moles)
Ph~'O~co2H
NHBOC
S DCC 1334.5 g (6.563 moles)
HOBT 884 g (6.563 moles)
CH3CN 25 I
0.5N NaOH 18 I
0.5N ]HCl 18 I
NaHC03 sat. 18 I
iPrOAc 28 I
The spiroindo]line-aminelb in CH3CN or iPrOAc:H20 (25
L) at ;Imbient temperature under N2 was treated in sequence with
15 HOBT (884 g; 1.1 eq) as a solid, DCC (1334.5 g, 1.1 eq) as the melt
(heating in hot water at 60''C for ca. 1 hr) and finally the amino acid 10
(1938 g) as the solid. The mixture was stirred for 3 hr upon which
time heavy precipitation of DCU occurred and LC analysis showed ca.
0.5 A~% of amine lb rem~ining. IPAc (9 L) was added, the slurry was
20 filtered through Solka FlocrM and the cake was washed with IPAc (19
L). The combined organic solution was washed in sequence with 0.5N
NaO~ (18 L), 0.5N HCl (18 L) and saturated NaHC03 (18 L). A final
water wash at this point resulted in an emulsion and was thus elimin~ted.
The organic layer was concentrated in vacuo and the
25 residue was dissolved in MeOH or EtOH (10 L final volume). Assay
yield 3026 gr (89%).
The use of alternative peptide coupling agents such as
carbonyldiimidazole or forxnation of mixed anhydrides, such as sec-
butyl carbonate, gave inferior yields of 11 and/or 14 with a high
30 degree of epimerization in the case of the former compound. Other
peptide coupling reagents were prohibitively expensive.
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EXAMPLE 14B
H, N- BOC
=O
[~N~ 1 1
~ ' SO2Me
Boc-O-Benzylserine Spiroindoline (11)
S Materials:
Spiroindoline-amine (lb) 1.855 kg (6.96 mol)
Isopropyl acetate 29 L
Dicyclohexylcarbodiimide (DCC) 1.58 kg (7.65 mol)
l-Hydroxybenzotriazole (HOBt) 1.03 kg (7.62 mol)
N-Boc-O-benzyl-D-Serine 2.26 kg (7.65 mol)
lM A.queous sodium hydroxide 26 L
0.5M Aqueous hydrochloric acid 26 L
Satd. Aqueous sodium hydrogen carbonate 26 L
Absolute Ethanol 50 L
Water (20 L) was added to a stirred solution of the
spiroindoline-aminelb (1.855 kg) in isopropyl acetate (33 L) in a
reaction vessel. The following chemicals were then added sequentially
at roc,m temperature under a nitrogen atmosphere: DCC (1.58 kg, 1.1
20 equivs.), HOBt (1.03 kg, 1.1. equivs.) and finally N-Boc-O-benzyl-D-
Serine (2.26 kg, 1.1 equivs.). The reagents were rinsed in with
isopropyl acetate (7 L). The batch was stirred at room temperature
under nitrogen atmosphere for 5 hours when LC showed the ratio of
product/starting material to be 99.4/0.6. The mixture was then filtered
25 through an Estrella filter using cloth and cardboard only and utilizing a
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- 61 -
pump into another vessel. The sending vessel was rinsed with isopropyl
acetate (22 L) and this was used to rinse the filter, the pump and the
lines into the receiving vessel. The
2-phase mixture in the vessel was stirred for 10 minutes and then
5 allowed to settle for 15 minutes. The lower aqueous layer was
separated off and the organ,ic solution was left to stand at room
temperature overnight.
The next day, the organic solution was washed with lM
aqueous sodium hydroxide solution (26 L) then O.5M aqueous
lO hydro,chloric acid (26 L) and finally saturated aqueous sodium hydrogen
carbonate (26 L). LC analysis gave an assay yield of 3.787 kg, 93%
overa]l yield from 7.49 moles (3 kg) of starting CBZ-spiroindoline (1).
The batch was concentrated in vacuo (internal temperature = 13-15~C.
jacket temperature = 40~C, Vacuum = 29") to low bulk (~15 L) and
l5 solvent switched to ethanol by "feeding and bleeding" ethanol (50 L)
whilst maintaining the volume at ~15 L. GC showed c1% isopropyl
acetate rem~ining. This solution was used for the next stage of the
process.
EXAMPLE l5A
~H,NH2
=O
~'
~~ ' SO2Me
O-Benzylserine Spiroindoline (free base form) (12)
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Materials:
Boc-O-Benzylserine Spiroindoline (11) 3026 g (5.57 moles)
Methane sulfonic acid (MsOH) 1.16 L (17.9 moles)
MeOH 10 L
iPrOAc 24 L
0.5NNaOH 35L
The Boc-O-benzylserine spiroindoline 11 in 10 L of
MeO]H (or EtOH) was treated with neat MsOH (1.16 L) added over ca.
10 30-40 min, (initial temperature 16~C, final temperature 28~C). The
dark red solution was aged overnight under N2. The mixture was then
pumped into a 100 L extractor containing 24 L iPrOAc and 35 L 0.5 N
NaOH. The pH of the aqueous layer was 7. NaOH (6M) was added
until ]pH 2 10.5. As the pH increased the color changed from red to
15 yellow. The layers were separated and the organic layer (24 L) was
shown by NMR to contain 13 mole % of MeOH in iPrOAc [5 volume
%]. I,C assay 2.48 kg.
EXAMPLE l5B
~ ~ 0 C ~ 2
~J =o
~N~ 12
SO2Me
O-Benzylserine Spiroindoline (free base form) (12)
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Materials:
Boc-O-Benzylseriine Spiroindoline (11) 3.787 kg (6.96 mol)
Methanesulphonic acid 2.006 kg (20.87mol)
Isopropyl acetate 38 L
lM A,queous sodium hydroxide 16 L
50% ~queous sodium hydroxide 1.6 L
Methanesulphonic acid (2.006 kg, 1.355 L, ~3 equivs.) was
addecl to the stirred solution of Boc-O-benzylseriine spiroindoline (11)
10 (3.787 kg) in ethanol (total volume ~15 L) in a reaction vessel. The
batch was warmed to 35-40~C. After 7 hours, LC showed the absence
of starting materiial and the reaction was allowed to cool to room
temperature overnight. Thle next day, water (44 L) was added to the
batch with stirring. The batch was cooled to ~5~, stirred for 30 minutes
15 and then filtered through an in-line filter (loaded with a 10~1 cartridge)
into a bin. The batch was lthen sucked back into the vessel. A water
rinse (10 L) was used to rinse the vessel and lines into the bin and this
was used to then rinse bacl~ into the vessel. Isopropyl acetate (38 L) was
added~ followed by a lM aqueous sodium hydroxide (16 L). The batch
20 was cooled to 10-15~C, the pH of the lower aqueous layer was
confiImed as ~7 and 50% aqueous sodium hydroxide solution was added
(1.6 L,) (pH >10). The batch was stirred at 10-15~C for 25 minutes and
then allowed to settle for 10-15 minutes. The lower aqueous layer was
separated (78.1 kg). LC assay indicated 28.4 g of 12 (0.85% of theory)
25 contaiined in the aqueous liquors. Volume of the organic solution = 51
L. LC assay indicated 3.057 kg, 92% overall yield from 3 kg,
7.49 rnoles of CBZ-spiroindoline sulfonamide (1). This solution was
used i'or the next stage.
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EXAMPLE 16A
¢~~~~C~ N ~N~ BOc
=O O H
N
14
~N~
SO2Me
Boc-Aminoisobutyryl O-Benzylserine Spiroindoline (14)
Materials:
Spiroindoline amine (12) 2481 g (5.57 moles)
amino acid peptide (13) 1247.1 g (6.16 moles)
CH~,<CH3
HO2C NH-BOC
DCC 1266.7 g (6.16 moles)
HOBT 827 g (6.16 moles)
IPAc 52 I,
H20 37 ~,
0.5N NaOH 36 L
0.5N HCl 36 L,
Sat.NaHCO3 36L,
The solution of the amine 12 in IPAc was diluted to a total
volume of 39 L with IPAc and 37 L of H20 was added. The biphasic
mixture was then treated in sequence with HOBT (827 g) as a solid,
20 DCC (1266.7 g) as a melt, and amino acid 13 at ambient temperature
under nitrogen. The reaction mixture was stirred for 2 h upon which
time L,C analysis indicated dissappearance of the starting material 12
(<0.3 A~o). The mixture was filtered through Solka FlocTM and the
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solids were washed with 13 L of IPAc. The material may be stored at
this point as a biphasic mixture overnight.
The mixture was transferred to a 100 L extractor, the
a~ueous layer was separated and the organic layer was washed
5 successively with 36 L of Cl.5N NaOH, O.5N HCl and saturated
NaHC'03. Assay yield 3160 g (81% from spiroindoline + 5% for
volurrle measurement error). The solution was concentrated to a small
volume and was flushed wiith ethanol (2 x 4 L). If desired, the
inermediate compound 14 may be isolated by adding water to crystalize
lO it out.
The use of alternative peptide coupling agents such as
carbonyldiimidazole or forlmation of mixed anhydrides, such as sec-
butyl carbonate, gave inferior yields of 14 with a high degree of
epimerization. Other peptide coupling reagents were prohibitively
1 5 expensive.
EXAMPLE 16B
H H CH3 CH3
~ C ~ N ~N' BOC
=O O H
14
~C
SO2Me
Boc-Aminoisobutyryl O-Benzylserine Spiroindoline (14)
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Materials:
Spiroindoline amine (12) 3.057 kg (6.89 mol)
Dicyclohexylcarbodiimide (DCC) 1.56 kg (7.56 mol)
1-Hydroxybenzotriazole (HOBt) 1.02 kg (7.55 mol)
Boc-2-Aminoisobutyric acid (13) 1.54 kg (7.58 mol)
Isopropyl acetate 32 L
lM Aqueous sodium hydroxide 38 L
0.5M Aqueous hydrochloric acid 38 L
Satd. aqueous sodium hydrogen carbonate 38 L
l0 Absolute ethanol 45 L
Water (49 L) was added to the stirred solution of the
spiroindoline amine 12 (3.057 kg) in isopropyl acetate (total volume
~51 L) in a reaction vessel at room temperature under a nitrogen
l5 atmosphere. The following chemicals were then added sequentially:
DCC l'1.56 kg, ~1.1 equivs.), HOBt (1.02 kg, ~1.1 equivs.) and finally,
N-Boc-2-aminoisobutyric acid 13 (1.54 kg, ~1.1 equivs.). The mixture
was stirred vigorously at room temperature for 2 hours when LC
showed the reaction to be complete. The mixture was filtered to to
,~0 another vessel via an Estrella filter using a pump. Isopropyl acetate (22
L) was used to rinse vessel, the filter, the pump and the lines into the
receiving vessel. The 2-phase mixture was then stirred for 5 minutes
and the layers were allowed to separate. The lower aqueous layer was
separated without incident (weight of aqueous liquors = 51.1 kg). The
25 organic solution was then washed sequentially with lM aqueous sodium
hydro:~ide (38 L), 0.5M aqueous hydrochloric acid (38 L) and finally,
satural~ed aqueous sodium hydrogen carbonate (38 L) without incident.
The organic solution was then transferred using a pump via
an in-line filter (containing a 10,u cartridge) to another vessel for the
30 solven,t switch to ethanol. The vessel was rinsed with isopropyl acetate
(10 L) and this was used to rinse the pump, the filter and the lines into
the receiving vessel. The filtrate and washings were combined. Total
volume = 75 L (by dipstick). LC assay gave 4.395 kg of Boc-
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aminc~isobutyryl O-benzylserine spiroindoline (14), i.e. 93% overall
from 7.49 moles of starting CBZ-spiroindoline sulfonamide (1).
The batch was concentrated in vacuo to low bulk (~15 L)
and thle isopropyl acetate switched to ethanol by "feeding and bleeding"
5 absolute ethanol (45 L total). At the end of the solvent switch? GC
showed <1% isopropyl acetate rem~ining. This solution (25 L)
containing 4.395 kg of 14 was used for the next stage. If desired, the
inerrnediate compound 14 may be isolated by adding water to crystalize
it out.
EXAMPLE 17A
H H CH3,CH3
~C~ N ~If NH2
=O O
N~
~ SO2Me
Aminoisobutyryl O-Benzylserine Spiroindoline (15)
lS Materials:
Boc Spiroindoline (14) 316() g (5.03 moles)
Methanesulfonic acid (MsOH) 979 mL (lS.l moles)
EtOH 6.2 L
H2O 3() L
lNNa,OH 11 L
EtOAc 26 L
Darco 60 activated carbon l Kg
The Boc spiroindoline 14 was dissolved in 6.2 L of EtOH
25 and treated with MsOH (979 mL). The temperature rose from 20 to
30~C and the reaction was ,allowed to proceed overnight. After 12
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hours at 20~C there was still 15 A% of starting material left so the
mixture was heated to 35~C for 6 hours. Upon completion (<0.1 A%
14) the reaction was cooled to 20~C and 30 L of H2O were added and
the solution was filtered through a glass funnel with a polypropylene
5 filter to filter off residual DCU. The mixture was transferred to a 100
L extractor and 26 L of EtOAc were added. The aqueous layer was
basified via addition of chilled lN NaOH (11 L) and 1 L of 50% NaOH.
Addition of ice was required to keep the temperature below 14~C.
Higher temperatures resulted in significant emulsion problems.
The organic layer was distilled at 50~C at ca. 21" of Hg
until :KF c1000 ~g/mL. Lower KF's result in more efficient carbon
treatments and better recovery at the salt formation step. KF's of 160
llg/m]L were achieved at the 700 g scale. The solution was diluted with
ethyl acetate to a total volume of 31 L (LC assay 2.40 kg). Activated
15 carbon (Darco G-60) was added and the mixture was stirred for 24 h.
The mixture was filtered through Solka FlocTM and the filter cake was
washed with ethyl acetate ( 16 L), assay 2.34 Kg.
EXAMPLE 17B
H H CH3,CH3
¢~0 C~ ~ NH2
=O O
~) 15
SO2Me
Aminoisobutyryl O-Benzylserine Spiroindoline (15)
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Materials:
Boc Spiroindoline (14) 4.395 kg (6.99 mol)
Methanesulfonic acid 2.017 kg (20.99 mol)
Ethyl acetate 185 L
lM Aqueous sodium hydroxide 16 L
50% ~queous sodium hydroxide 2.6 L
Darco G-60 900 g
Solka FlocTM 2.5 kg
Methanesulfonic acid (2.017 kg, 1.36 L, ~3 equivs.) was
added to the stirred solution of the Boc spiroindoline 14 (4.395 kg) in
ethanol (total volume ~25 ]_) in a reaction vessel at room temperature.
The b;~tch was warmed to 35-40~C, and stirred overnight. On the next
day, the batch contained ~1.1 A% of starting material and so the
15 reaction was continued for a further 4 hours, then LC showed ratio of
product/ starting material to be 99.6/0.4. The batch was concentrated in
vacuo to ~15 L volume ancl then diluted with water (44 L). The batch
was cooled to 5~C, stirred ior 30 minutes and then filtered through a
Sparkler in-line filter (cont~ining a 10~ cartridge) using a pump to
20 another vessel to remove a small amount of residual DCU.
The vessel, the pump, the filter and the lines were rinsed
with water (10 L), and this was added to the vessel. Ethyl acetate (36
L) was added to the vessel and the stirred mixture was cooled to 10~C.
A solution of cold (5-10~C) lM aqueous sodium hydroxide solution (16
25 L) ancl cold (5-10~C) 50% ;Iqueous sodium hydroxide solution (2.6 L)
were added at 10~C and the temperature rose to 14~C. The resulting
mixture was stirred for 15 ]ninutes at <14~C and then the lower aqueous
layer separated off.
The batch was concentrated in vacuo to ~20 L volume and
30 then a mixture of ethyl acetate (35 L) and ethanol (5 L) was fed in while
maintaining the volume at -20 L. At the end of this distillation the KF
was 9:l60 mgml~ 1. The batch was solvent switched to ethyl acetate by
"feeding and bleeding" ethvl acetate (40 L total). At the end of this
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distillation, KF was 446 mgml~ 1. The batch was diluted with ethyl
acetate (10 L).
Darco G-60 (900 g) was added to the hazy mixture. This
was rinsed in with ethyl acetate (6 L). This mixture was stirred at room
S temperature overnight. Next day, Solka FlocTM (0.5 kg) was added to
the stirred batch in the vessel and then Solka FlocTM (2.0 kg) was stirred
in a little ethyl acetate and loaded into an Estrella filter . The excess
solvent was pumped away through a Sparkler in-line filter containing a
10~ cartridge. The slurry was transferred from the vessel through a
10 filter using a pump and then through another filter to 2 x 40 L stainless
steel bins. Visual inspection showed the liquors to be clear and clean.
The vessel was rinsed with ethyl acetate (22 L) and this was used to
rinse through the route outlined above to the stainless steel cans. The
contents of both cans was transferred into a reaction vessel and the
15 solution was mixed thoroughly.
The batch (58 L) had a KF of 2950 mgml~l and so was
redried by concentrating in vacuo to 20-25 L volume. The batch was
diluted to 46 L volume (dipstick) by the addition of ethyl acetate (25 L).
The ~F was 363 mgml~l. The batch was diluted to 62 L volume by the
20 addition of ethyl acetate (] 7 L) and was used for the final stage of the
process.
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EXAMPLE 18A
H H CH ~CH3
~0 C~ ~ NH2- CH3SO3H
=O O
[~
SO2Me
Spirol 3H-indole-3,4'-piperldin]- 1 '-yl)carbonyl]-2-(phenylmethyl-
oxy)ethyll-2-amino-2-methylpropanamide Methanesulfonate (16)
s
Materials:
Amine (15) 2 340 g (4.43 moles)
Metha,ne sulfonic acid (MsOH) 316 mL (4.88 moles)
EtOAc 60 L
EtOH 4~8 L
8% EtOH in EtOAc 20 L
The volume oi the solution of 15 from the previous step
was adjusted to 60 L with ethyl acetate and EtOH (4.8 L) was added.
lS The ~IsOH (316 mL) was added in 3 L of EtOAc at 45~C. To the deep
red homogeneous solution was added 496 g of the title compound Form
I seed (10% seed based on the weight of the free amine was employed).
The temperature rose to ca 48~C and the reaction was aged at 52~C for
l.S hours. Analysis indicaled complete conversion to the title
20 compound (Form I). (At less than 10% seed longer age (> 3 hours) was
requirled). The slurry was allowed to cool to 20~C overnight and was
filtered in a centrifuge under N2. The cake was washed with 20 L of
8% EtOH in EtOAc. N2 is essential during filtration because the wet
crystals are very hygroscopic. The batch was dried at 35~C under
25 vacuum to afford 2.7Kg (56% overall yield) of the title compound
(Form I) (99.9 A% purity; < 0.1% enantiomer).
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The conversion of Form II to Form I is also accomplished
where the salt is formed in EtOAc-EtOH by addition of MsOH as above
and the initial solution of the salt (at 55~C) is cooled to 45~C. Crystals
start appearing at that temperature and the slurry becomes thicker with
5 time. The temperature is then raised to 51~C and the slurry is aged
overnight. Complete conversion to Form I of 16 should be expected.
This procedure may also be employed to prepare seed crystals of Form
I of 16.
EXAMPLE 18B
H H CH ~,CH3
~0 C~ ~ NH2- CH3SO3H
=O O
SO2Me
Spirol[3H-indole-3,4'-piperdin]- ] '-yl)carbonyl]-2-(phenylmethyl-
oxy)ethyll-2-amino-2-methylpropanamide Methanesulfonate (16)
MateIials:
Amine (15) 3.1 kg (5.86 mol)
Methcmesulfonic acid 620 g (6.45 mol)
Ethyl acetate 37 L
Absolute ethanol 8.7 L
20 Spiro[3H-indole-3,4'-piperdin]- 1 '-yl)-
carbonyl] -2-(phenylmethyl-oxy)ethyl]-
2-amino-2-methylprop~n~rnide
methanesulfonate (Form I) 70 g (0.1 1 mol)
Absolute ethanol (6.4 L) was added to the solution of the
amine (15) (3.1 kg) in ethyl acetate (total volume ~62 L) in a reacttion
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vessel. The batch was warmed to 50~C and a solution of
meth~mesulfonic acid (620 g, 412 ml, 1.1 equivs.) in ethyl acetate (11
L) was added over ~5 mim1tes at 50-54~C. The batch was seeded with
spirol 3H-indole-3,4'-piperdin]- 1 '-yl)-carbonyl]-2-(phenylmethyl-
5 oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate (Form I)
(70 g) and the resulting slurry was stirred and heated at 55~C under
nitrogen atmosphere overnight.
The next day, the slurry was cooled to 15-20~C, held for 2
hours and then dropped to the 50 cm polypropylene filter under
10 nitro,~,en atmosphere. The solid product was washed with a mixture of
absoll1te ethanol (2.3 L) in ethyl acetate (26 L). The white, solid
product was dug off and d]ied in an Apex oven in vac~o at 35~C for an
appropriate time (approx. two days). The dried spiro[3H-indole-3,4'-
piperdin]- 1 '-yl)-carbonyl]- 2-(phenylmethyl-oxy)ethyl]-2-amino-2-
15 methylpropanamide metharlesulfonate (3.352 kg) was sieved using aJackson-Crockatt sieve to give 3.347 kg (including seed, 70 g) } yield =
3.277 kg.
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HPLC Conditions:
LC Retention times on Zorbax RX-C8 (4.6 mm x 25 cm),
~ = 210 nm, flow rate = 1.5 ml/min.
Compound 1: 60:40 CH3CN-H2O (1% H3PO4) RT = 5.0 min
Compound lb: 35:65 CH3CN-H2O (0.1 w % NH40Ac) RT = 6.2 min.
Compound 10: 60:40 CH3cN-H2o (0.1 H3PO4) RT = 2.9 min.
Compound 11: 60:40 CH3CN-H2O (0.1% H3PO4) RT = 5.4 min.
Compound 12: 40:60 CH3CN-H20 [pH 5.25 NaH2PO4 (6-9 g/L of H2O)
(adjust pH with NaOH)] RT = 5.6 min
Compound 14: 60:40% CH3CN-H2O (0.1% H3PO4) RT = 4.65 min
Compound 15: 40:60% CH3cN-H2o [pH = 5.25 NaH2PO4 (6.9 g/L
of H2O)] adjust pH with NaOH)RT = 4.9 min
LC Retention times on Zorbax RX-C8 (4.6 mm x 25 cm),
= 210 nm, flow rate = 1.2 ml/min, column temperature = 48~C
Solvent A = 0.05% Phosphoric acid + 0.01% Triethylamine in water
Solvent B = Acetonitrile
Gradient system:
Time % A % B
0 min 95 5
35 min 10 90
38 min 95 5
40 min 95 5
Retention time (mins)
Compound 1 25.2
Compound lb 8.5
Compound 10 20.5
Compound 11 26.3
Compound 12 14.8
Compound 14 25.6
Compound 15 15.7
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EXAMPLE 19
Preparation of Form I of N-[l(R)-[(1,2-Dihydro-1-methanesulfonyl-
spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyloxy)-
5 ethyll-2-amino-2-methylprop~ mide methanesulfonate
The conversion of Form II to Form I may be accomplished
by the: procedure of Example 18A where the salt is formed in EtOAc-
EtOH by addition of MsOH and the initial solution of the salt (at 55~C)
is cooled to 45~C. Crystals should start appearing at that temperature
10 and th~e slurry should become thicker with time. The temperature is
then r;aised to 51~C and the slurry is aged overnight. Complete
conversion to Form I should be expected.
EXAMPLE 20
Preparation of Form I of N-[l(R)-[(1,2-Dihydro-1-methanesulfonyl-
spiro[ 3H-indole-3,4'-piperclin]- 1 '-yl)carbonyl]-2-(phenylmethyloxy)-
ethyll-2-amino-2-methylpropanamide methanesulfonate
The conversion of Form II to Form I is accomplished by
20 stirring a solution of Form II of N-[l(R)-[(1,2-dihydro-1-
methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide
methanesulfonate in isopropanol at approximately 25~C for about 2-24
hours.
~5
EXAMPLE 21
Preparation of Form IV of N-[l(R)-[(1~2-Dihydro-1-methanesulfonyl-
spiro[:3H-indole-3,4'-piperdlin]- 1 '-yl)carbonyl]-2-(phenylmethyloxy)-
30 ethyll-2-amino-2-methylpropanamide methanesulfonate
A sample of 8.4 g N-[l(R)-[(1,2-dihydro-1-methane-
sulfonyl-spiro[3H-indole-3 ,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenyl-
methyloxy)ethyl]-2-amino-'2-methylpropanamide methanesulfonate of
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- 76 -
optional morphological form is dissolved in a mixture of 24.8 ml ethyl
acetate, 1.6 ml ethanol and l.9S ml water with stirring at 42~C.
The solvent is evaporated from the solution at a temperature of
40~C, the resultant solid is ground in a morter to a fine powder and
the fine powder is exposed to a relative humidity of approximately
75% to give the title Form IV.
EXAMPLE 22
10 Preparation of Form IV of N-[l(R)-[(1~2-Dihydro-l-methanesulfonyl-
spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyloxy)-
ethyll-2-amino-2-methylprop~n~mide methanesulfonate
A sample of N-[l(R)-[(1,2-dihydro-1-methane-sulfonyl-
spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenyl-methyl-
IS oxy)el:hyl]-2-amino-2-methylpropanamide methanesulfonate of optional
morphological form is recry.ct~lli7e~1 from a solution of ethylacetate/
ethanol/water (24.8/1.6/1.95 vlvlv) to give the title Form IV.
EXAMPLE 23
,70
Preparation of Form IV of N-[l(R)-[(1,2-Dihydro-1-methanesulfonyl-
spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-(phenylmethyloxy)-
ethyll-2-amino-2-methylprop~n~mide methanesulfonate
A slurry of Form I of N-[l(R)-[(1,2-dihydro-1-
25 methanesulfonyl-spiro[3H-indole-3,4'-piperdin]- 1 '-yl)carbonyl]-2-
(phenylmethyl-oxy)ethyl] -2-amino-2-methylpropanamide
methanesulfonate in isopropyl acetate/ethanol (90:10 v/v) containing
approximately 2.8 wt% water is stirred at approximately 25~C
overnight and the resultant solid is isolated.
'30
CA 0223~371 1998-04-20
W O 97/155741 PCTAUS96/16955
While the invention has been described and illustrated with
reference to certain particular embodiments thereof, those skilled in the
art wiill appreciate that various adaptations, changes, modifications,
substiitutions, deletions, or additions of procedures and protocols may be
S made without departing from the spirit and scope of the invention. For
example, effective dosages other than the particular dosages as set forth
herein above may be applicable as a consequence of variations in the
responsiveness of the m~mm~l being treated for any of the indications
with lhe compound of the iinvention indicated above. Likewise, the
10 specii.~lc pharmacological responses observed may vary according to and
depending upon the particular active compound selected or whether
there are present pharmaceutical carriers, as well as the type of
formulation and mode of admini~tration employed, and such expected
variations or differences in the results are contemplated in accordance
15 with lhe objects and practices of the present invention. It is intended,
therefore, that the invention be defined by the scope of the claims which
follow and that such claims be interpreted as broadly as is reasonable.
