Note: Descriptions are shown in the official language in which they were submitted.
CA 02427594 2003-05-02
S
S-aryl cysteines are useful intermediates in the synthesis of a variety of
pharmaceutically active compounds. Chiral S-aryl-L-cysteines have been used
successfully to target the human immunodeficient.virus (HIV) and are used in
the
treatment of AIDS (Kaldor et al., J. Med. Chem. 1997, 40 (24), 3979-3985).
Representative potent and tight binding inhibitors of the human ''~
immunodeficient virus protease which contain an arylthio group are shown in
Figure 1.
Many currently available synthetic methods for S-aryl cysteines involve the
preparation of racemic mixtures. There are, however, a number of disadvantages
associated with racernic mixtures of such compound. A racemic mixture of an S-
aryl
cysteine results in production of racemic drugs. It is well known that certain
physiological properties of chiral drugs are dependent upon stereochemistry of
the
drug and the undesired side-effects are often attributed to the presence of
the
undesired stereoisomer of the chiral drug. Accordingly, a high
enantioselective
synthesis of a chiral drug will result in a drug having a desired therapeutic
activity
with a reduced amount of undesired side-effects. Of course, the synthesis of a
chiral
drug can include a step of separating a racemic mixture; however, this is
often time
consuming and costly. In addition, racemic synthesis requires discarding one
half of
the compound unless the undesired isomer can be converted to a desired isomer.
Moreover, not all racemic compounds can be resolved to provide a satisfactory
yield
of a desired enantiomer.
CA 02427594 2003-05-02
Current methods for enantioselective synthesis of S-aryl cysteines involve
enzyrriatic methods (see, e.g., USP 5.756.319 or European Patent Application
No.
754>759, which are assigned to Mitsui Toatsu Chemicals, Inc.), applicable,
however,
to the preparation of only a limited number of S-aryl cysteines.
Most of the current chemical synthetic methods for enantioselective
preparation of S-aryl cysteines result in a racemic mixture, use elaborate
reagents
dramatically increasing the overall cost, or result in too low levels of
enantioselectivity to be useful in a pharmaceutical process.
Recently, D.W. Knight and A.W. Sibley (J. Chem. Soc., Perkin Trans. 1, 1997,
2179-2187) reported that reaction of methyl (S)-2-benzyloxycarbonylamino-3-
methylsulfonyloxy-propanoate with freshly prepared sodium thiophenylate in DMF
at about 0°C afforded the desired N-carbobenzyloxy-S-phenyl-L-cysteine
methyl
ester (or methyl (R)-2-benzyloxycarbonylamino-3-phenylthiopropanoate) in 98%
yield providing a reported optical rotation of (a]20D - 17.2 (c, 1.8; MeOH).
No
enantiomeric ratio of the product was reported. Furthermore, the use of sodium
phenolate, prepared from sodium hydride, thiophenol and DMF is not amenable to
large scale manufacture.
Therefore, there is a need for an efficient, concise and enantioselective
method
suitable far the large scale manufacture of S-aryl cysteines using relatively
inexpensive reagents.
2
CA 02427594 2003-05-02 'y
$ Definitions
"Alkyl" comprises straight or branched chain groups having 1 to about 10
carbon atoms. Alkyl groups optionally can be substituted with one or more
substituents, such as halogen, aryl, hydroxy, alkoxy, carboxy, oxo and
rycloalkyl.
There may be optionally inserted along the alkyl group one or more oxygen,
sulfur or
substituted or unsubstituted nitrogen atoms. Exemplary alkyl groups include
methyl, ethyl, i-propyl, n-butyl, t-butyl, n-pentyl, heptyl, benzyl, and
octyl.
"Aryl" means an aromatic group which has at least one ring which has a
conjugated pi electron system and includes, without limitation, carbocydic
aryl,
heterocyclic aryl, biaryl groups and heterocydic biaryl, all of which can be
optionally
substituted.
"Heterocyclic aryl groups" refer to groups having at least one heterocydic
aromatic ring containing from I .to 3 heteroatoms in the ring with the
remainder
being carbon atoms. Suitable heteroatoms include, without limitation, oxygen,
sulfur, and nitrogen. Exemplary heterocyclic aryl groups include furanyl,
thienyl,
pyridyl, pyrrolyl, N-alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl,
benzofuranyl,
quinolinyl, and indolyl.
Preferably, the.aryl group is substituted or unsubstituted aryl selected from
the
group consisting of phenyl, naphthyl, pyridyl, furyl, thiophenyl, pyrazyl and
pyrrolyl.
More preferably, the aryl group is substituted or unsubstituted aryl group
selected
from the group consisting of phenyl, naphthyl and pyridyl, still more
preferably the
aryl group is selected from the group consisting of substituted and
unsubstituted
phenyl, and most preferably the aryl group is phenyl.
3
~l
CA 02427594 2003-05-02
The term "S-aryl" refers to a substituent wherein an aromatic group is bonded
to a sulfur atom. "S-aryl" group may also be referred to as an "aryl
thioether" group.
The term "S-arylation" refers to the process of substituting a compound or
groups
with an S-aryl group.
The term "metal" comprises alkali metals, alkaline-earth metals, transition
metals, noble metals, platinum metals, rare metals, rare-earth metals,
actinide metals,
light metals, and heavy metals. Examples of such metals are aluminum, iron,
copper,
cobalt, potassium, sodium, tin and zinc.
The term "catalyst" refers to any substance of which a fractional percentage
notably affects the rate of a chemical reaction without itself being consumed
or
undergoing a chemical change, as defined in "Hawley's Condensed Chemical
Dictionary", 11th ed., revised by N. Irving Sax and Richard J. Lewis, Sr., Van
Nostrand Reirihald Company, New York; p. 748 (1987).
The term "stoichiometric" refers to the use or addition of an equivalent mole
ratio or amount of a reagent relative to a selected substrate, molecule, or
compound
in a reaction.
The term "chiral" has the usual meaning known to a person skilled in the art.
The term "enantiomeric excess" refers to a difference between the amount of
one enantiomer and the amount of the other enantiomer that is present in the
product mixture. Thus for example, enantiomeric excess of 96% refers to a
product
mixture having 98% of one enantiomer and 2% of the other enantiomer.
The following abbreviations and terms are used herein:
"CBZ" means benzyloxycarbonyl or carbobenzyloxy.
"DMF" means dimethylformamide.
"Tosylate" or "Tos" means p-toluenesulfonate ester.
4
CA 02427594 2003-05-02
"Mesylate" or "Ms" means methanesulfonate ester.
"TBPB" means tetrabutylphosphonium bromide.
"TBAB" means tetrabutylammonium bromide.
"TBAC" means tetrabutylammonium chloride.
"PTC" means phase transfer catalysis or catalyst.
"Aliquat 336~" is tricaprylylmethylammonium chloride {TCMC).
The present invention is directed to a method for preparing S-aryl cysteines
(which are useful intermediates in a variety of pharmaceutically active
compounds)
in high enantiomexic excess. Specifically, the process of the present
invention
provides enantiomerically enriched S-aryl L- or D- cysteine. Preferably, the
process
of the present invention provides S-aryl cysteine in enantiomeric excess of
greater
than about 96%, more preferably greater than about 98%, and most preferably
greater than about 99.5%. Unless the context requires otherwise, reference to
any
compound is to be considered as a reference to an individual enantiomer of the
compound, and to racemic or non-racemic mixtures thereof.
5
CA 02427594 2003-05-02
BRIEF DESCRIPTION OF FIGURES 1-4:
Figure 1 shows.representative compounds of HIV protease inhibitors which
contain a moiety derived from S-aryl cysteine.
Figure 2 illustrates one embodiment of the present invention for preparing an
S-aryl cysteine compound from cystine.
Figure 3 illustrates another embodiment of the present invention for
preparing an S-aryl cysteine compound which utilizes cysteine.
Figure 4 illustrates yet another embodiment of the present invention for
preparing an S-aryl cysteine compound which utilizes serine.
6
CA 02427594 2003-05-02
$ As shown in Figure 2, one embodiment of the present invention provides a
method for producing S-aryl cysteine by contacting cystine with a metal and
contacting the resulting compound with an aryl hal-ide for a time and . under
conditions effective to produce S-aryl cysteine. When cystine is used as a
starting
material the amount of aryl halide used is preferably from about 1 equiv. to
about 6
equiv., more preferably from about 2 equiv. to about 6 equiv., still more
preferably
from about 2 equiv. to about 4 equiv., aiid most preferably about 3 equiv.
Preferably
halide is selected from the group consisting of iodide, bromide and chloride,
more
preferably halide is selected from the group consisting of bromide and iodide,
and
most preferably halide is bromide. Contacting cystine with a metal results in
cleavage
of the disulfide bond generating a metal thiolate of cysteine which undergoes
a
coupling reaction to form the desired product and a metal halide. Thus, any
metal
which can cleave the disulfide bond can be used in the process of the present
invention. Preferably the metal is selected from the group consisting of
aluminum,
iron, copper, cobalt, potassium, sodium, tin, zinc and a mixture thereof, more
preferably the metal is selected from the group consisting of copper, sodium,
tin, zinc
and a mixture thereof, and most preferably the metal is copper. The
temperature of
the reaction is preferably from about 80° to 150°C, more
preferably from about 100
to 130°C, and most preferably from about 115°C to about
125°C. The reaction time
can vary depending upon the identity of the metal and/or the aryl halide; but
generally it has been found that the reaction time of at least about 1 hour
produces
the desired product in a relatively high yield, preferably at least about 15
hours, and
more preferably at least about 18 hours.
As shown in Figure 3, another embodiment of the present invention provides
a method for producing S-aryl cysteine by contacting cysteine with an aryl
halide in
7
CA 02427594 2003-05-02
the presence of a metal oxide. When cysteine is used as a starting material in
a
coupling reaction, the amount of aryl halide used is preferably from about 1
equiv. to
about 5 equiv., more preferably from about 1.1 equiv. to about 4 equiv., still
more
preferably from about 1.2 equiv. to about 3 equiv., and most preferably about
1:5
equiv. Preferably, the metal oxide is selected from the group consisting of
copper
oxide, zinc oxide, stannous oxide and a mixture thereof, more preferably the
metal
oxide is copper oxide. It has been found that the production of S-aryl
cysteine by
this method is facilitated by heating the mixture. Preferably, the reaction
temperature is from about 80° to 150°C, more preferably from
about 100 to 130°C,
and most preferably from about 115°C to about 125°C. A variety
of aryl halides can
be reacted with the thiolate to produce the S-aryl cysteine compound.
Preferably, the
aryl halide is phenyl bromide.
A method of the present invention for producing S-aryl cysteine includes the
presence of a coupling agent which generates from cystine or cysteine the
reactive
thiolate compound of the formula
S-M
OP2
HN
P1 O
wherein M is a metal,
P~ is hydrogen or an amino protecting group and
PZ is hydrogen or carboxylic protecting group.
It will be appreciated that the above strucure merely represents an idealized
representation of thiolate. The exact structure of the thiolate can be a
dimes, trimer
or other polymeric form of the reactive intermediate, e.g., with a metal
binding more
than one thiolate group. Moreover, the metal can also bind other ligands such
as
8
CA 02427594 2003-05-02
solvent molecules or other species which may be present in the reaction
mixture. Any
metal or its derivative which produces a coupled product from the thiolate and
an
aryl halide can be used in the present invention. Generally, a coupling agent
is
selected from the group consisting of a metal, metal oxide, metal salt and
mixtures
thereof. As used in this invention, a "metal salt" refers to any organic or
inorganic
metal salt in which the oXidation state of the metal is not zero. Exemplary
metal salts
include, ferrocene, ferric chloride, ferric acetate, ferrous acetate, ferrous
acetylacetonate, feric acetylacetonate, ferrous chloride, cupric iodide,
cuprous iodide, '''
cupric bromide, cuprous bromide, cupric chloride, cuprous chloride, cupric
fluoride,
cupric acetate, cupric acetylacetonate, cupric hydroxide, copper sulfate,
cupric
cyanide, cupric oxide, and cuprous oxide. Preferably the coupling agent is
selected
from the group consisting of copper, copper halide, copper oxide, zinc, zinc
halide,
zinc oxide, aluminum, aluminum halide; aluminum oxide, iron, iron oxide, iron
halide, cobalt, cobalt oxide, cobalt halide, tin, tin oxide, tin halide,
potassium,
potassium oxide, potassium halide, sodium, sodium oxide, sodium halide and
mixtures thereof. More preferably, the coupling agent is selected from the
group
consisting of copper, copper halide, copper oxide and mixtures thereof. And
most
preferably, the coupling agent is selected from the group consisting of
copper, cupric
bromide, cupric oxide, cupric chloride, cupric iodide, cuprous bromide cuprous
chloride, cuprous iodide, cuprous cyanide, cuprous oxide, cupric fluoride,
cupric
acetate, cupric acetylacetonate, cupric sulfate, cupric hydroxide and mixtures
thereof.
While the method of the present invention can proceed with a metal it can
also include the presence of a metal salt MaXb, with a and b representing
corresponding amounts. of M or X depending on the oxidation state of M and X.
The
metal salt can be an inorganic salt. such as a metal halide including copper
(I) or (II)
9
CA 02427594 2003-05-02
bromide, chloride, iodide, and halide of other above mentioned metals, or an
organic
salt such as copper (I) or (II) acetylacetonate; acetate and organic salt of
other above
mentioned metals. It is believed that when copper metal and copper (II) salt
are
present together, they undergo disproportionation to form copper (I), which
may be
the active species for the process. Suprisingly and unexpectedly, it has been
found
that while Cu(0) or other metal can be an effective coupling agent, the
presence of
Cu(I) or Cu(II) salt, such as copper (I) or (II) bromide, in addition to Cu(0)
increases the rate of reaction. Preferably, from about 0 mol% to about 100
mol% of '
copper (I) or (II) bromide, relative to aryl halide, is added, more preferably
from
about 0.2 mol% to about 5 rriol%, and most preferably about 1 mol% to about 3
mol%. Typically about 6 mol% of copper (I) or copper (II) bromide is added. It
should be recognized that other functional groups present in cystine or
cysteine can
be protected or unprotected.
Typically, the amount of coupling agent, relative to aryl halide, used is from
about 0.3 equiv. to about 1 equiv., preferably from about 0.5 equiv. to about
0.75
equiv., and more, preferably from about 0.6 equiv. to about 0.7 equiv. It has
been
found that after the reaction, the coupling agent or the resulting product of
the
coupling agent, e.g., copper (I) salt such as copper bromide, may be isolated
and
recycled to be used in another coupling reaction. In this manner, the cost of
coupling
agent and the disposal cost of the resulting coupling agent product, e.g.,
copper (I)
bromide, can be substantially reduced.
A method of the present invention for producinb S-aryl cysteine can further
include an oxidizing agent. An oxidizing agent is any compound which can
generate
the reactive species of the coupling agent. Preferably oxidizing agent is
selected from
the gioup consisting of bromine, iodine; chlorine and mixtures thereof.
CA 02427594 2003-05-02
Although the method of the present invention can be conducted in the
absence of a solvent, it has been found that the presence of a relatively high
boiling
point solvent provides a reaction medium which can be heated to a desired
temperature. Thus, the solvent has boiling point higher than the desired
reaction
temperature. Preferably the solvent is selected from the group consisting of
acetonitrile, glymes, dimethylacetamide, dimethylformamide, dimethylsulfoxide,
diethylacetamide, dimethylbutyramide and N-methyl-2-pyrrolidone, and more
preferably the solvent is dimethylformamide. The reaction time can vary
depending
upon the identity of the metal oxide andlor the aryl halide, but generally it
has been
found that the reaction time of at least about 1 hour produces a relatively
high yield
of the desired product with high enantioselectivity, preferably at least about
15 hours,
and more preferably at least about 18 hours
Typically, the thiolate is generated in situ and is used without further
purification.
Another embodiment of the present invention for the preparation of
enantiomerically enriched S-aryl cysteine by contacting a serine derivative
with an
aryl thiol in the presence of a base, i.e., a substitution reaction, as shown
in Figure 4.
"Serine derivative" refers to a compound wherein the hydroxy group of serine
is
replaced by a leaving group. The term "leaving group" has the meaning well
known
to a person skilled in the art. Preferably, the leaving group is selected from
the group
consisting of a halogen atom (i.e. chlorine, bromine or iodine), tosyloxy and
mesyloxy and most preferably is tosyloxy or mesyloxy.
11
CA 02427594 2003-05-02 ')
In accordance with this embodiment of the present invention,-a_compound of
the formula:
X
OP2
HN
P1 0
wherein X is a halogen atom, a mesyloxy or tosyloxy group,
is contacted with an aryl thiol in the presence of a base.
Useful bases include carbonates such as sodium carbonate, potassium
carbonate and lithium carbonate; bicarbonates such as sodium bicarbonate,
potassium bicarbonate and lithium bicarbonate; hydroxides such as sodium
hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide and
potassium hydroxide; sterically hindered amines such as triethyl amine and
diisopropyl ethyl amine; hydrides such as sodium hydride, potassium hydride
and
lithium hydride; amides such as lithium diisopropyl amide and sodium
diisopropyl
amide; and other bases such as sodium hexamethyl dimethyl silazide.
Preferably, the
base is selected from the group consisting of carbonates, bicarbonates and
hydroxides, more preferably the base is selected from the group consisting of
carbonates and bicarbonates, and most preferably the base is a carbonate. It
will be
appreciated that the aryl thiol can be contacted with the base prior to adding
any
serine derivative with a leaving group, or the base can be added to a mixture
of the
aryl thiol and the serine derivative, or the aryl thiol can be added to the
mixture of
the base and the serine derivative. In a particular aspect the serine
derivative may be
prepazed from serine by protection. of the amino group and the carboxy group
followed by conversion of the hydroxy group to a leaving group.
,.
12
CA 02427594 2003-05-02 ')
The temperature of the reaction can affect the enantiomeric excess of the
product. To minimize loss of stereochemical configuration of the product
and/or
the starting material, the temperature of the reaction between the serine
derivative
and the aryl thiol is maintained from about -5°C to about 35°C,
preferably from
about 15°C to about 30°C. Preferably the reaction time is from
about 1 h to about 48
hs, more preferably from about 10 hs to about 30 hs, and most preferably from
about
hs to about 25 hs.
The method of this embodiment may further include the addition of a phase
transfer catalyst. The term "phase transfer catalyst" means a catalyst or
agent
which is added to a reaction mixture of components, and operates to transfer
one or
15 more of the reacting components to a location where it can conveniently and
rapidly
react with another reacting component. Examples of phase transfer catalysts or
agents that may be employed are reviewed in C.M. Starks, C.L. Liotta, and M.
Halpern, "Phase-Transfer Catalysis", Chapman & Hall, New York, 1994.
Especially
preferred phase transfer catalysts include TBAB, TBAC, TBPB and Aliquat 336~.
20 Any appropriate solvent can be used in this embodiment of the present
invention. However, when using a phase transfer catalyst, it is preferred that
a
relatively non-polar solvent be used. Exemplary relatively non-polar solvents
which
are useful include toluene, ethyl acetate and hexane. Preferably, the
relatively non
polar solvent is selected from the, group consisting of toluene, ethyl acetate
and a
mixture thereof.
The above methods of the present invention can include protecting the amino
group of the amino' acid (cystine, cysteine or serine). Any of the known amino
protecting groups can be used. Examples of some protecting groups are
described in
"Solid Phase Peptide Synthesis" by G. Barany and R. B. Merrifield in Peptides,
Vol. 2,
13
CA 02427594 2003-05-02
S edited by E. Gross and J. Meienhoffer, Academic Press, New York, N.Y., pp.
100-118
( 1980), and "Protective Groups in Organic Synthesis" by Green, T., John Wiley
&
Sons, Inc., New York, NY., 1981, pp. 218-287. Exemplary N-amino protecting
groups include acetyl, formyl, benzoyl, substituted benzoyls, FMOC, Bspoc,
Bsmoc,
t-butyloxycarbonyl (BOC), t-amyloxycarbonyl (Mcb), 2-(p-biphenylyl)-propyl-2-
oxycarbonyl (Bpoc), benzyloxycarbonyl (or carbobenzyloxy, CBZ), phthaloyl,
piperidino-oxycarbonyl, trifluoroacetyl and the like. Other N-amino protecting
groups include optionally protected a-amino acids which are linked with the
carboxyl moiety of the oe-amino acids. Preferably the amino protecting group
is
metyl carbamate or CBZ. Amino protecting group can be removed under various
conditions, including mild acidic or basic conditions. The preferred
protecting
groups are those which can be cleaved by an acid or a base, or reductive
conditions.
For example, the amino group can be protected by contacting the amino acid
with
benzyl chloroformate in the presence of a base. Any base that can neutralize
the
acidic proton that is formed by the reaction of benzyl chloroformate and the
amino
group can be used. Exemplary bases useful in protection of the amino group
include
carbonates such as sodium carbonate, potassium carbonate and lithium
carbonate;
bicarbonates such as sodium bicarbonate, potassium bicarbonate and lithium
bicarbonate; hydroxides such as sodium hydroxide, lithium hydroxide, calcium
hydroxide, magnesium hydroxide and potassium hydroxide; and sterically
hindered
. amines such as triethyl amine and diisopropyl ethyl amine. Preferably, the
base is
selected from the group consisting of carbonates, bicarbonates and hydroxides,
more
preferably the base is selected from the group consisting of carbonates and
bicarbonates, and most preferably the base is a bicarbonate. in one particular
embodiment of the present invention, the amino group is protected by
contacting
14
CA 02427594 2003-05-02
the amino acid with benzyl chloroformate in the presence of sodium bicarbonate
to
provide N-benzyloxycarbonyl protected amino acid.
For the protection of the carboxy group of the amino acid any of the known
carboxy protecting groups can be used. Protection of the carboxy moiety of
amino
acids are described in "The Peptides," E. Gross and J. Meienhofer, Eds.,
Vol.3,
Academic Press, NY (1981), pp. 101-135, and "Protective Groups in Organic
Synthesis" by Green, T., John Wiley & Sons, Inc., New York, NY., 1981, pp. 152-
192.
Exemplary carboxy protecting groups include esters such as alkyl esters
including
methyl, ethyl, tert-butyl, methoxymethyl, 2,2,2-trichloroethyl and 2-
haloethyl; benzyl
esters such as triphenylmethyl, diphenylmethyl, p-bromobenzyl, o-nitrobenzyl
and
the like; silyl esters such as trimethylsilyl, triethylsilyl, t-
butyldimethylsilyl and the
like; amides and hydrazides. Other carboxy protecting groups can include
optionally
protected a-amino acids which are linked with the amino moiety of the a-amino
acids. Preferably the carboxylic acid protecting group is an ester, more
preferably the
carboxylic acid protecting group is an alkyl ester, and most preferably the
carboxylic
acid protecting group is selected from the.group consisting of methyl ester
and ethyl
ester. In a particular embodiment of the present invention, the carboxylic
acid of
the amino acid is protected as methyl ester by contacting the amino acid with
thionyl
chloride in the presence of methanol. Alternatively, the carboxylic acid is
protected
as methyl ester by contacting the amino acid with gaseous hydrochloric acid in
the
presence of methanol.
The above method can also include protecting both the amino group and the
carboxy group of the amino acid. ' It will be appreciated that the amino and
carboxy
groups in the amino acid can be protected in any sequence.
CA 02427594 2003-05-02
The S-aryl cysteine reaction product of the present invention which is
recovered from the reaction mixture can be further purified by distillation or
crystallization. For instance, a reaction product obtained in liquid form, is
dissolved
in toluene and crystallized from a relatively non-polar recrystallization
solvent to
afford a product of higher purity. Preferably, the non-polar recrystallization
solvent
is selected from the group consisting of hexane, ethyl acetate, toluene,
xylene,
benzene, pentane, ethers and mixtures thereof. The S-aryl cysteine can be
recovered
as a salt. .For example, adding an acid such as hydrochloric acid; or an
organic acid
including tartaric acid, acetic acid, and/or citric acid to S-aryl cysteine
can result in
the formation of the corresponding S-aryl cysteine salt which can be easily
isolated.
Alternatively, the free carboxy group can react with a base to generate a
carboxylate
salt which can form a solid. In yet another alternative, the presence of both
free
amino and carboxy groups results in the formation of a zwitter ion' which can
precipitate as a solid.
The present invention is further illustrated by the following examples
EXAMPLE 1
Preparation of N,N'-bis-benzyloxycarbonyl cystine dimethyl ester using
thionyl chloride and cystine.
In a 500 ml jacketed round bottomed flask is placed cystine (20 g, 83.2 mmol)
and methanol (250 ml). The reaction mixture is cooled to 0-5°C and
thionyl
chloride (12.7 ml, 0.175 mol) is added while maintaining the temperature at
less than
10°C. At the end of the addition, the reaction is heated to rellux for
4 hours and
excess methanol is removed by distillation. As the product begins to
precipitate,
16
CA 02427594 2003-05-02
water was added followed by a slow. addition of sodium bicarbonate (29.4 g,
0.35
mole) to the solution at less than 5°C.
After the bicarbonate is added, benzyl chloroformate (25 ml, 0.175 mole) is
slowly added. The reaction mixture is maintained at <5°C for 1 hour and
slowly
warmed to room temperature. The reaction mixture is heated to 30 to
40°C and the
organic phase is separated and the aqueous phase is washed with toluene (3 x
20 ml).
The combined organic solution is washed once with sodium bicarbonate (25 ml),
followed by 5% HCI, and saturated NaCI, dried over MgS04, and concentrated in
'
vacuo by rotoevaporation to afford 44.25 g of an oil (0.0825 mole, 99.1%
yield).
A portion of this oil (31.55 g) was recrystallized from EtOAc-hexane to give a
colorless solid (24.95 g, 79.1 % yield). mp 70-72°C ; assayed as 97.5 %
by AIN
HPLC.
EXAMPLE 2
Preparation of N,N'-bis-benzyloxycarbonyl cystine dimethyl ester using
MeOH/HCl and cystine.
To a cold (-5 to -10°C) solution of methanol (100 ml) was bubbled
HCl gas.
About 11.0 g of HCl gas was absorbed into the solution. To a slurry of cystine
in 100
ml methanol was added the above prepared HCl/methanol solution. The mixture
was heated to reflux. After about one hour, a clear solution was obtained. The
mixture was heated to reflux for a total of 4 hours, cooled to room
temperature and
transferred to a one neck round bottomed flask. Concentration of the mixture
by
rotoevaporation afforded white solids.
The white solids were suspended in 400 ml of toluene. A solution of NaHC03
(31.5 g) in 300 ml of water was added to give a clear two phase solution.
Benzyl
17
CA 02427594 2003-05-02
chloroformate (25.0 mI, 0.175 mole) was added dropwise at 16-18°C. The
reaction
mixture was then stirred at 16-18°C for 4 hours.
The aqueous phase was separated and extracted with toluene (3 x 50 ml). The
combined toluene extracts were washed with water, dilute sodium carbonate
solution, 5% HCl solution, and saturated sodium chloride solution. The organic
phase was dried over MgS04 and concentrated in vacuo to give an oil. The oil
was
dissolved in ethyl acetate ( 100 ml). Hexane ( 175 ml) was added and the
mixture was
seeded. The solution was stirred at room temperature overnight. The product
was '
filtered, washed with a hexane:EtOAc (9:1, 100 ml) mixture and dried under
vacuum
at 45°C to afford the product in 87.4%.yield, 39.0 g, assayed as 98.5%
by A/N HPLC.
EXAMPLE 3
Preparation of N-CBZ S-phenyl-L-cysteine methyl ester using crude N,N'-
bis-benzyloxycarbonyl cystine dimethyl ester.
Into a 250 ml round bottomed flask was added N,N'-bis-benzyloxycarbonyl
cystine dimethyl ester (12.7 g, 237 mmol), copper powder (3.08 g) and
dimethylformamide ( 130 ml). The stirred mixture was heated to ?0°C.
Bromobenzene, i.e., phenyl bromide, (10 ml, 95 mmol) was charged to an
addition
funnel and added dropwise to the reaction mixture at about 70-80°C over
30
minutes. The reaction mixture was kept at 75-80°C for 35 minutes,
warmed to 90°C
for 25 minutes, heated to 100°C for 4 hours, and then at 110°C
for another 48 hours.
The reaction was monitored by thin layer chromatography. The reaction
mixture was cooled to 50~C arid DMF was distilled off under reduced pressure
at 50-
60°C where 80 ml of distillate was recovered. The reaction mixture was
diluted with
toluene (150 ml) and water (50 ml). The resulting mixture was heated to reflux
for
minutes and the solution was quickly filtered over a pad of celatom. The
celatom
18
CA 02427594 2003-05-02
pad was washed with excess toluene and the mixture was diluted with water. The
phases were separated and the toluene phase was washed with water, 10%
HCl/water
(100 ml, vol/vol), and saturated sodium chloride.
The resulting solution was concentrated by rotoevaporation to afford a light
yellow oil. The oil was dissolved in toluene ( 15 ml) and seeded to induce
crystallization. Hexane ( 15 ml) was added followed by another 60 rnl of
hexane, and
the slurry was stirred overnight at room temperature. The product was
filtered,
washed with excess hexane, and air dried to afford a colorless solid (80.7%
yield; mp
62-64 °C).
EXAMPLE 4
Preparation of N-CBZ S-phenyl-L-cysteine methyl ester using purified N,N'-
bis-benzyloxycarbonyl cystine dimethyl ester.
Copper powder (4.62 g), bromobenzene (15.0 ml, 142 mmol), and DMF (55
ml) was charged to a 250 ml 3-neck round bottomed flask. The resulting mixture
was heated to 110°C using an external oil bath. To the stirred mixture
was added a
solution of purified N-CBZ cystine methyl ester (19.05 g, 35.5 mmol, 97.5% by
assay)
in DMF {40 ml) over 2 hours and 30 minutes. The resulting mixture was stirred
at
110°C for 18 hours and then at 130 t 2°C for about 24 hours.
The resulting mixture was cooled to 65°C and DMF was distilled off
under
reduced pressure (50-60°C) until a thick slurry was formed. The mixture
was diluted
with toluene ( 150 ml) and heated to 70 to 75°C for 15-20 minutes. The
solution was
quickly filtered over a pad of celatom and the celatom pad was washed with
warm
(70°C) toluene. The toluene phase was washed with water (2 x 100 ml),
10%
aqueous HCl ( 1 x 100 ml), water ( 1 x 100 ml) and saturated sodium chloride (
1 x 100
ml).
19
t
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CA 02427594 2003-05-02
The reddish brown solution was dried over anhydrous magnesium sulfate.
The solution was filtered through a pad of Filtrol-13 (an acid activated clay
from the
Filtrol Corp.) to afford yellow solids. The filtrol pad was washed with
toluene (100
ml). Toluene was distilled under reduced pressure at 40-45°C until an
oil was
obtained. The oil was dissolved in toluene (25 ml), hexane (25 ml) was added
and
the mixture was seeded to precipitate out the desired product. Hexane (200 ml)
was
added and the resulting mixture was stirred at room -temperature for 2.5 days.
The
product was filtered, washed with excess hexane and dried-under vacuum at
45°C to
afford the desired product in 66.9% yield (16.4 g, assayed as 98.3% by A/N
HPLC
(area normalized high performance liquid chromatography)).
Concentration of the mother liquors afforded 6.2 grams of an oil (41%
product by A/N assay).
EXAMPLE 5
Preparation of N-CBZ S-phenyl-L-cysteine methyl ester from N-CBZ cysteine
methyl ester.
A round bottomed flask was charged with CBZ-cysteine methyl ester (5.4 g,
20.0 mmol), copper oxide (2.8 g), and bromobenzene (4.2 ml, 39.9 mmol) in
dimethylformamide (25.0 ml). The mixture was heated to reflux (145 ~
2°C) for 19
hours. Analysis by HPLC revealed 25.9% N-CBZ S-phenyl-L-cysteine methyl ester
and 6.0% N-CBZ S-benzyl cysteine methyl ester.
EXAMPLE 6
Preparation of N-CBZ S-phenyl-L-cysteine from N,N'-bis-benzyloxycarbonyl
cystine.
A mixture of N,N'-bis-benzyloxycarbonyl cystine (3.81 g, 7.5 mmol), copper
powder (0.95 g, 15.0 mmol), and bromobenzene (3.32 ml, 4.958, 31.5 mmol) in
CA 02427594 2003-05-02
dimethylformamide (20 ml) was heated to 120°C for 19 hours. The
resulting mixture
was cooled to 80°C and dimethylformamide was removed by vacuum
distillation at
80-95°C, where 15 ml of distillate was collected.
The resulting residue was diluted with toluene (70 ml) and stirred for 1 hour
at 70-75°C. The product was filtered and washed with excess toluene.
The combined
organic phases were washed with 10% aqueous HCl (1 x 70 ml), water (2 x 70
ml),
saturated sodium chloride ( 1 x 70 ml), and the solution was dried over
anhydrous
magnesium sulfate. ,
The solution was filtered, the cake was washed with excess toluene, and the
organic solution was concentrated via rotoevaporation to afford 4.8 g of a
light
brown oil which solidified upon standing. 96.6% yield, 4.8 g> assayed as
96.33% by
A/N HPLC; containing 3.67% of the corresponding S-benzyl acid.
Phase Transfer Catalyzed Nucleophilic Displacement Reactions
Phase-transfer catalyzed nudeophilic displacement of N-protected, serine ester
mesylate (Ms) or tosylate (Tos) using aryl thiols to prepare the corresponding
N-
protected S-aryl-L-cysteine derivatives is ,provided in the following
representative
experimental descriptions. Tables I-4 provide results from various alkylation
reactions employing different reactions conditions, including variations in
the type
of phase-transfer catalyst, reaction stoichiometry, base, and substrates.
21
CA 02427594 2003-05-02
Table 1. Phase Transfer Alkylation with Thiophenol
1 2 3 4
Substrate mesylate mesylate mesylate mesylate
PhSH (equiv) 1.0 1.0 1.0 1.0
PTC TBAB TBAB TBAB TBAB
PTC (mol%) 5.0 10 10 . WO '
Base K2C03 Nal-i~C03 18% NaOH K2C03
Base (equiv) 1.20 1.10 1.10 2.00
Solvent (ml) toluene toluene toluene toluene
(10) (10) (10) (10)
Temp (C) 25 25 25 25
Time (h) 22 23 23.5 22
Crude Yield (g) --- --- 0.800 --
Chrom Yield (g) 0.255 0.411 0.713 0.301
Chrom Yield (%) 24.5 39.4 68.4 28.9
R:S 99.5:0.5 98.4:1.6 80.3:19.7 99.1:0.9
ee (%) 99.0 96.8 60.6 98.2
22
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Table 2. Phase Transfer Alkylation with Thiophenol
5 6 7 8
Substrate mesylate mesylate mesylate mesylate
PhSH (equiv) 1.1* 1.0 1.0 1.0
PTC TBAB TBAB TBAB TBRB
PTC (mol%) 10 10 5.0 5.0
Base 18% K2C03 K2C03 K2C03
NaOH
Base (equiv) 1.04 2.00 1.50 1.50
Solvent (ml) toluene EtOAc EtOAc EtOAc
(10) (10) ~ (10) (10)
Temp (C) 25 25 25 25
Time (h) 22.5 19 23 19
Crude Yield (g) 0.940 0.959 0.954 0.967
Chrom Yield (g) 0.848 0.787 0.830 0.878
Chrorri Yield (%) 81.4 75.5 79.6 84.3
R:S 90.3:9.7 90.2:9.8 89.4:10.6 89.8:10.2
ee (%) 80.6 80.4 78.8 79.6
* Preformed NaSPh
23
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Table 3. Phase Transfer Alkylation with Thiophenol: Tosylate as Substrate
9 . 10 11 12
Substrate tosylate tosylate tosylate tosylate
PhSH (equiv) 1.0 1.0 1.0 1.5
PTC --- TBAB TBPB TBAB
PTC (mol9io) --- 5.0 5.0 5.0
Base K2C03 K2C~3 --K2CO3
Base (equiv) 1.50 1.50 1.50 1.50
Solvent (ml) Toluene toluene Toluene toluene
(10) (10) (10) (10)
Temp (C) 25 25 25 25
Time (h) 22.5 17 24 22
Crude Yield (g) 1.159 0.959 0.986 1.042
Chrom Yield (g) 0.171 0.877 0.88 0.918
Chrom Yield (%) 16.4 84.2 82.3 88.0
R:S 98.4:1.6 99.2:0.8 99.8:0.2 97.3:2.7
ee (%) 96.7 98.4 99.6 94.6
24
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Table 4. Phase Transfer Alkylation with Thiophenol
13 14 15 16
Substrate mesylate mesylate mesylate mesylate
PhSH (equiv) 1.0 1.0 1.0 1.5
PTC TBPB TBAB Aliquat Aliquat
336 336
PTC (mol%) 5.0 5.0 4.0 4.0
Base K2C~3 K2C03 K2CO3 K2C03
Base (equiv) 1.50 1.50 1.50 1.50
Solvent (m) toluene tol:EtOAc toluene toluene
(10) ($:2) (10) (10)
Temp (C) 25 25 25 25
Time (h) 20.5 18.5 41 3.0
Crude Yield (g) 0.94$ 0.959 0.992 0.974
Chrom Yield (g) 0.409 0.841 0.795 0.817
Chrom Yield (%) 39.2 80.7 76.3 78.4
R:S 98.2:1.8 93.9:6.1 90.9:9:1 92.9:7.1
ee (%) 96.4 87.8 81.8 85.8
25
CA 02427594 2003-05-02
EXAMPLE 7
Preparation of N-CBZ S-phenyl-L-cysteine methyl ester by the displacement
of N-carbobenzyloxy-O-p-toluenesulfonyl-L-serine methyl ester.
A mixture of 1.231 g (3.02 mmol) of N-carbobenzyloxy-O-p-toluenesulfonyl-
L-serine methyl ester, anhydrous potassium carbonate powder (0.626 g, 4.53
mmol,
1.5 equiv.), tetrabutylphosphonium bromide (TBPB, 51 mg, 0.151 mmol, 5 mole
%),
thiophenol (0.31 ml, 3.02 mmol), and toluene was stirred at 25~C for 24 hours.
Water (20 ml) was added and the phases were separated. The organic phase
was washed with water (20 ml), dried over magnesium sulfate, and filtered.
Concentration on a rotoevaporator at 30-35~C and 30 mmHg afforded an oil.
Drying of the oil under vacuum at 25~C for 18 hours (ca. 0.5 mmHg) afforded a
colorless solid (0.986 g, 94:6% yield).
EXAMPLE 8
Preparation of N-CBZ S-phenyl-L-cysteine methyl ester by the displacement
of N-carbobenzyloxy-O-methanesulfonyl-L-serine methyl ester.
Thiophenol (0.31 ml, 0.333 g, 3.02 mmol) was added via syringe to a
suspension of N-carbobenzyloxy-O-methanesulfonyl-L-serine methyl ester (1.00
g,
3.02 mmol), powdered anhydrous K2C03, tetrabutylammonium bromide (TBAB,
49 mg, 0.151 mmol, 5 mole%), and toluene (20 ml). The suspension was stirred
at
25~C for 22 hours. ,Water (20 ml) was added and the phases were separated. The
organic phase was washed with 20 ml water, 10 ml of ethyl acetate was added,
dried
over magnesium sulfate, and filtered. Concentration on a rotoevaporator in
vacuo at
35°C and 35 mmHg afforded a wet colorless solid.
The solid was dissolved in methylene chloride and separated on a 4 mm silica
gel chromatotron plate. The product was separated using hexane (250 ml), 10%
26
CA 02427594 2003-05-02
ethyl acetate in hexane (800 ml), ethyl acetate (200 ml), and methanol (200
ml). The
combined fractions were concentrated on a rotoevaporator at 75 mmHg and 30-
35°C. The residual oil was triturated with hexane and the solids were
dried.under
vacuum (<0.5 mmHg) for 7.5 hours at 25°C to afford 0.255 g of a
colorless solid
(24.5% chromatographed yield, 98.2% R : 0.50% S by assay).
EXAMPLE 9
Preparation of N-CBZ S-phenyl-L-cysteine methyl ester from N-
carbobenzyloxy-O-p-toluenesulfonyl-L-serine methyl ester.
A mixture of 12.31 g (30.2 mmol) of N-carbobenzyloxy-O-p-toluenesulfonyl
L-serine methyl ester 6.26 g (45.3 mmol, 1.5 equivalents) of anhydrous
powdered
potassium carbonate, 512 mg ( 1.51 mrnol, 5.0 mol%) tetrabutylphosphonium
bromide, 100 ml toluene, and 3.1 ml (3.33 g, 30.2 mmol) thiophenol was stirred
at
25°C for 31 hours.
Reaction progress was followed by liquid chromatography (LC): After 1 h,
85% tosylate remaining, after S hs 55%, after 25 hs 4.6%, after 30 hs tosylate
not
detected any more. Water (40 ml) was added and the layers separated. The
organic
layer was washed with 20 ml of water then distilled at atmospheric pressure to
reduce the volume of toluene (bath 125°C, under dry N2). The weight of
solution
remaining after the distillation was 28.75 g.
The solution after cooling contained a small amount of solid residue (salts
from residual water in the toluene after the phase split). These solids were
removed
by gravity filtration. The mother liquor was slowly diluted with 80 ml of
hexane.
The precipitate was suction filtered, washed on the funnel with 20 ml hexane,
then
dried under vacuum at 25°C for 22 h to afford 8.309 g of colorless
solid, mp. 64.7-
65.2°C; yield 79.9%. LC assay for optical purity: 0.1% S.
27
CA 02427594 2003-05-02
EXAMPLE 10
Preparation of N-CBZ S-phenyl-L-cysteine methyl ester from N-
carbobenzyloxy-O-p-toluenesulfonyl-L-serine methyl ester.
A mixture of 30.00 g (73.63 mmol) of N-carbobenzyloxy-O-p-
toluenesulfonyl-L-serine methyl ester, 15.27 g ( 110 mmol, 1.5 equivalents) of
anhydrous powdered potassium carbonate 1.249 g (3.68 mmol, 5.0 mol%) of
tetrabutylphosphonium bromide, 150 ml toluene, and 7.6 ml (8.11 g, 73.6 mmol)
thiophenol was stirred at 25°C for 8 days.
Water (75 ml) was added and the layers separated. The organic layer was
washed with 75 ml water and then distilled at reduced pressure to reduce the
volume
of toluene (heating bath at 125°C, under dry N2): The weight of
solution remaining
after the distillation was 41.6 g.
The residual oil was diluted with 100 ml of hexane. The precipitate was
suction filtered, washed on the funnel with 75 ml hexane, then dried under
vacuum
at 25 °C for 19 hours to afford 22.75 g of colorless solid. yield
89.5%. LC assay for
optical purity: 0.5% S. LC assay for chemical purity: 97.9%.
EXAMPLE 11
preparation of N-CBZ S-(3-methoxyphenyl)-L-cysteine methyl ester from
N;N'-bis-benzyloxycarbonyl cystine dimethyl ester and 3-bromoanisole.
Starting material, N,N'-bis-benzyloxycarbonyl cystine dimethyl ester (3.0 g,
5.6 mmol) was dissolved in anhydrous DMF at a solvent ratio of 10:1. To the
solution were added 2.0 equivalents of copper powder (0.71g, 11.2 mmol) and
4.0
equivalents of 3-bromoanisole (4.1 g, 22.3 rnmol), and the mixture was heated
to
120-130 °C. After stirring overnight the reaction mixture was checked
by HPLC and
was determined to have <5% starting material. The reaction mixture was cooled
and
28
CA 02427594 2003-05-02
the DMF as well as most of the excess 3-bromoanisole was removed under reduced
pressure. The crude mixture was dissolved with 10 volumes of toluene and the
copper and copper salts were removed by filtration.
The filtrate was washed once with 30 ml of a 50:50 mixture (10% NH40H,
15% w/w NH4C1) followed by one wash with saturated brine. The organic layer
was
concentrated under reduced pressure and the crude product was purified by
silica gel
chromatography resulting in a light yellow oil; yield 2.73g ( 65%).
EXAMPLE 12 '
Preparation of N-CBZ S-(2-(6-methoxynaphthalenyl))-L-cysteine . methyl
ester from N,N'-bis-benzyloxycarbonyl cystine dimethyl ester and .2-bromo-6-
methoxynaphthalene.
N,N'-Bis-benzyloxycarbonyl cystine dimethyl ester (2.75 g, 5.12 mmol) was
dissolved in anhydrous DMF at a solvent ratio of 10:1. To the solution were
added
2.0 equivalents of copper powder (0.65 g, 10.2 mmol) and 4.37 equivalents of 2-
bromo-6-methoxynaphthalene (5.3 g, 22.4 mmol), and the mixture was heated to
120-130°C. After stirring overnight the reaction mixture was checked
for completion
by HPLC and was determined to have <5% starting material. The reaction mixture
was cooled and the DMF was removed under reduced pressure. The crude mixture
was dissolved with 10 volumes of toluene and the copper and copper salts were
removed by filtration.
The filtrate was washed once with 30 ml of a 50:50 mixture ( 10% NH40H,
15% w/w NH4C1) followed >~y one wash with saturated brine. The organic layer
was
concentrated under reduced pressure and the crude product was purified by
silica gel
chromatography resulting in a light brown oil 1.96 g (45%).
29
CA 02427594 2003-05-02
EXAMPLE 13
Preparation of N-CBZ S-(3-pyridyl)-L-cysteine methyl ester from N,N'-bis-
benzyloxycarbonyl cystine dimethyl ester and 3-bromopyridine.
N,N'-bis-benzyloxycarbonyl cystine dimethyl ester (3.0 g, 5.6 mmol) was
dissolved in anhydrous DMF at a solvent ratio of 10:1. To the solution were
added
2.0 equivalents of copper powder (0.718, 11.2 mmol) and 4.0 equivalents of 3-
bromopyridine (3.5 g, 22.3 mmol), and the mixture was heated to 120-130
°C. After
stirring overnight the reaction mixture was checked with HPLC and was
determined
to have <5% starting material. The reaction mixture was cooled and the DMF was
removed under reduced pressure. The crude mixture was dissolved with 10
volumes
of toluene and the copper and copper salts were removed by filtration.
The filtrate was washed once with 30 ml of a 50:50 mixture ( 10% NH40H,
15% w/w NH4C1) followed by one wash with saturated brine. The organic layer
was
concentrated under reduced pressure and the crude product was purified by
silica gel
chromatography resulting in a reddish-brown oil 2.05 g (53%).
EXAMPLE 14
Preparation of N,N'-bis-(3-acetoxy-2-methylbenzoyl)-cystine dimethyl ester
from cystine dimethyl ester hydrochloride and 3-acetoxy-2-methylbenzoyl
chloride.
Cystine dimethyl ester hydrochloride (5.36 g, 0.0157 mole), 3-acetoxy-2-
methylbenzoyl chloride (6.67 g, 0.0314 mole), and triethyl amine (6.35 g,
0.0628
mole) were dissolved in dichloromethane (50 ml) and the resulting mixture was
stirred at ambient temperature overnight. The organic phase was washed with 2
N
hydrochloric acid (2 x 20 ml) and then with water (2 x 20 ml).
The organic layer was dried over MgS04 and concentrated on a rotary
evaporator to give a light yellow solid. Trituration of the solid with 100 ml
of toluene
CA 02427594 2003-05-02
gave a light yellow crystalline solid, which was isolated by filtration and
dried at 50°C
(under vacuum) to give N,N'-bis-(3-acetoxy-2-methylbenzoyl)-cystine dimethyl
ester (4.30 g, yield 44.1%). A second crop was obtained by silica gel column
chromatography of the mother liquors, eluting with 50% methylene chloride in
ethyl
acetate.
EXAMPLE 15
Preparation of N-(3-acetoxy-2-methylbenzoyl)-S-phenyl=L-cysteine methyl
ester from N,N'-bis-(3-acetoxy-2-methylbenzoyl)-cystine dimethyl ester and
bromobenzene.
Into a 3-necked 50 ml round bottomed flask equipped with an overhead
stirrer under nitrogen atmosphere was added N,N'-bis-(3-acetoxy-2-
methylbenzoyl)-cystine dimethyl ester (4.3 g, 0.00693 g), bromobenzene (6.5 g,
4.4
ml, 0.0416 mole), copper (0.92 g, 0.0145 mole), and dimethyl formamide (25
ml).
The resulting mixture was heated to 120 ~ 3°C for 19 hours.
Volatiles were removed by distillation at 80-85°C on a rotary
evaporator
under reduced pressure to afford a brown residue. The residue was dissolved in
30
ml of dichloromethane and washed with 3 N hydrochloric acid (20 ml), followed
by
washing with water (2 x 20 ml). The organic phase was dried over MgS04,
filtered,
and concentrated on a rotary evaporator to afford a light yellow solid (3.6 g,
yield
53%).
EXAMPLE 16
Preparation of N-CBZ S-phenyl-L-cysteine methyl ester from N,N'-Bis-
benzyloxycarbonyl cystine dimethyl ester using copper powder and catalytic
CuBr2.
N,N'-Bis-benzyloxycarbonyl cystine dimethyl ester (84.00 g, 7.45 mmol),
copper powder (4.72 g, 74 mmol), cupric bromide {CuBr2, 1.84 g, 8.23 mmol),
31
CA 02427594 2003-05-02
phenyl bromide (2.4 ml, 22.7 mmol), and dimethylformamide (24 ml) were
combined in a 50 ml round bottomed flask. The resulting stirred mixture was
immersed in a preheated oil bath at 140°C for 3 hours.
HPLC analysis showed 1.5% starting material remaining. Dimethylformamide
was removed by distillation at 120°C and 50 mmHg. The residue was
diluted with 15
ml of toluene and filtered through a pad of celatom (diatomaceous earth) to
remove
the solids. The filtrate was washed twice with 30 ml of 1 M HCI and once with
30 ml
of water. Toluene was removed on a rotary evaporator under reduced pressure.
The residue was crystallized by dissolving in approximately 5 ml of toluene
and adding hexane (SO ml) slowly at 40°C to initiate precipitation. The
mixture was
cooled to ambient temperature, filtered and the product was washed with 20 ml
of
hexane. The solid was dried under vacuum at 50°C to give 3.05 g of an
off white
solid (yield 59%).
m.p. 61.9-63.5°C.
EXAMPLE 17
Preparation of N-CBZ S-phenyl-L-cysteine methyl ester from N,N'-bis-
benzyloxycarbonyl cystine dimethyl ester using copper powder and catalytic
CuBr2.
To a 25 ml 2 neck round bottomed flask purged with nitrogen and equipped
with condenser were added N,N'-bis-benzyloxycarbonyl cystine dimethyl ester
(2.0
g, 3.73 mmol), copper powder (0.5g, 7.87 mmol), dimethylformamide ( 10 ml),
and
phenyl bromide (2.0 ml, 1.34 g, 8.54 mmol).
The resulting stirred mixture was heated to 115 - 125°C for 22
hours. Two
drops of bromine were added to the reaction and the resulting mixture was
heated
32
CA 02427594 2003-05-02
for another 22 hours where no starting material remained. HPLC analysis
revealed
88.4% of the desired product, N-CBZ S-phenyl-L-cysteine methyl ester.
EXAMPLE 18
Preparation of N-CBZ S-phenyl-L-cysteine methyl ester from N,N'-bis-
benzyloxycarbonyl cystine dimethyl ester.
Into a 250 ml round bottomed flask equipped with a thermometer,
mechanical stirrer and nitrogen inlet were added N,N'-Bis-benzyloxycarbonyl
cystine dimethyl ester (13.25 g, 0.025 mole), copper powder (4.0 g, 0.0625
mole), and '
copper (II) bromide (0.34 g, 0.00152 mole). To the mixture was added
bromobenzene (31.6 ml, 0.3 mole) and dimethyl sulfoxide (67 ml, dried over 4A
sieve).
The stirred content was heated to 130°C for 1 hour and 15 minutes
where no
starting material remained. Analysis by HPLC showed 94.3% N-CBZ S-phenyl-L-
cysteine methyl ester and no starting material. The resulting mixture was
cooled to
120°C for another 18 hours and analyzed for reaction completion.
HPLC analysis showed 81.7% N-CBZ S-phenyl-L-cysteine methyl ester, 3.9%
benzyl alcohol, 4.2% diphenyl sulfide, and 3.4% N-CBZ S-phenyl-L-cysteine
benzyl
ester.
EXAMPLE 19
Preparation of N-CBZ S-phenyl-L-cysteine methyl ester from N,N'-bis-
benzyloxycarbonyl cysti.ne dimethyl ester.
Into a 250 ml round bottomed flask equipped with a thermometer,
mechanical stirrer and nitrogen inlet were added N,N'-Bis-benzyloxycarbonyl
cystine dimethyl ester (6.7 g, 0.013 mole), copper powder (2.0 g, 0.0315
mole), and
33
CA 02427594 2003-05-02
copper (II) bromide (0.2g, 0.895 mmole). To the mixture was added bromobenzene
( 15.8 ml, 23.56 g, 0.15 mole) and dimethyl sulfoxide (33 ml, dried over 4A
sieve).
The stirred content was heated to 100°C for 18 hours and sampled for
reaction completion. Analysis by HPLC showed 88.8% N-CBZ S-phenyl-L-cysteine
methy~1 ester and no starting material. The resulting mixture was cooled to
room
temperature and analyzed for reaction completion.
HPLC analysis showed 88.8% N-CBZ S-phenyl-L-cysteine methyl ester, 2.5%
benzyl alcohol, 0.5% methyl carbamate, 2.1% diphenyl sulfide, and 1.2%
diphenyl
disulfide.
34
