Note: Descriptions are shown in the official language in which they were submitted.
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PROCESS FOR THE PREPARATION OF A TETRAPEPTIDE
FIELD OF THE INVENTION
The present invention is directed to a new process for the preparation of a
tetrapeptide,
more specifically the tetrapeptide H-Tyr-D-Ala-Phe(pF)-Phe- NIA, or a
pharmaceutically
acceptable salt thereof. In further aspects, the present invention also
relates to new
intermediates used in process.
io BACKGROUND AND PRIOR ART
The present invention relates to a new process for the preparation of the
peptide H-Tyr-D-
Ala-Phe(pF)-Phe-NH2, or a pharmaceutically acceptable salt thereof.
WO 97/07129 discloses a process for producing inter alia the peptide
is H-Tyr-D-Ala-Phe(pF)-Phe-NH2. The peptide H-Tyr-D-Ala-Phe(pF)-Phe-NH2 is
also
disclosed in WO 97/07130. Said peptide exhibits peripheral analgesic activity
and
selectivity for the ~t subtype of opioid receptors, and is suitable in
particularly in pain
therapy. Furthermore, it is prepared using solid phase synthesis according to
procedures
well established in the art. The drawback with solid phase synthesis, which is
a common
zo and well established method for peptide synthesis, is that it is difficult
to use for large scale
production, in addition to being expensive.
The process of the present invention provides the tetrapeptide H-Tyr-D-Ala-
Phe(pF)-Phe-
NH2 with a higher purity, in a more cost effective and environmentally better
way
2s compared to methods known in the art. Furthermore, the process of the
present application
provides the product in a higher yield.
Thus, the object of the present invention is to provide a novel process
suitable for use in
large scale synthesis. A further object of the present invention is to provide
a process
3o containing as few reaction steps as possible.
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2
OUTLINE OF THE INVENTION
The present invention provides a new process for large scale preparation of
the peptide H-
Tyr-D-Ala-Phe(pF)-Phe-NH2, which is a peptide of the formula (I)
s
O
~NH I
NH, 2
or a pharmaceutically acceptable salt thereof.
The process according to the present invention for preparing a compound of the
formula (1)
io above, comprises the following reaction steps;
St_.eo 1
(~) A coupling step wherein an activated p-fluorophenylalanine derivative
(III),
F
A-
~s
(III)
wherein
A is an amino protecting group, and
R_ is an activating agent residue group;
~u
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3
previously prepared by a pre-activation step or generated in situ, is reacted
with the amino
group of phenylalanine, wherein the carboxyl group is protected as an ester or
amide, i.e. a
compound of the formula Phe-Ri, wherein Rl is the ester or amide residue
group, in the
presence of a solvent, providing a protected dipeptide derivative (IV)
s
F
O
R'
A
O (IV)
wherein
A is an amino protecting group, and
io Ri is an ester or an amide residue group;
(ii) A deprotection step wherein a protected dipeptide derivative (I~ prepared
in the
previous step, is deprotected by either catalytic hydrogenation, base or acid
treatment,
depending on the amino protecting group used, providing the dipeptide
derivative (5),
~s
c
(5)
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4
wherein
Rl is an ester or an amide residue group;
Steu 2
s
(i) A coupling step wherein an activated alanine derivative (VII),
HsC O
(VII)
H O
wherein
A is an amino protecting group, and
R is an activating agent residue group;
previously prepared by a pre-activation step or generated in situ, is reacted
with the product
~s of step 1, i.e. the dipeptide derivative (5) in the presence of a solvent,
providing the
protected tripeptide derivative(VIII)
F
O a
O
A ~ ~~ R~
CH3 O (VIII)
wherein
zo A is an amino protecting group, and
Rl is an ester or an anode residue group;
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(ii) A deprotection step wherein a protected tripeptide derivative (VIII)
prepared in the
previous step, is deprotected either by catalytic hydrogenation or acid
treatment, depending
on the amino protecting group used, providing the tripeptide derivative (9)
C
H2N R' (9)
CH3
s
wherein
Ri is an ester or an amide residue group;
Steo 3
~o
(i) A coupling step wherein an activated tyrosine derivative (X),
ORZ
O
R
O
~s wherein
O
A is an amino protecting group,
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6
R is an activating agent residue group, and
RZ is H or a benzyl-like group;
previously prepared by a pre-activation step or generated in situ, is reacted
with the
tripeptide derivative (9) and being the product of step 2, in the presence of
a solvent,
s providing the protected tetrapeptide derivative (XI)
H ~ ,F
O O
a'~ b ~ b~ , (xi)
O O
CH3
i~
wherein
A is an amino protecting group, and
io Rl is an ester or an amide residue group;
(ii) A optional transformation step performed if the protected tetrapeptide
derivative (I~
prepared in the previous step (i) is an ester, wherein the ester compound (XI)
is reacted
with ammonia in an organic alcohol , preferably ammonia in methanol, providing
the
is protected dipeptide derivative {XII),
O O
txu,
O O NH2
CH3
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(iii) A deprotection step wherein a protected tetrapeptide derivative (XII) is
deprotected
either by catalytic hydrogenation, base or acid treatment, depending on the
amino
protecting group used, providing the final tetrapeptide (I), which optionally
may be
converted to a salt of the tetrapeptide (I).
s
The peptide H-Tyr-D-Ala-Phe(pF)-Phe-NHZ (I), may if desired be reacted with a
pharmaceutically acceptable acid, such as AcOH, H3P04, citric acid, lactic
acid and HCI.
HCl is the preferred acid to use in accordance with the present invention.
Possible salts
which may be used are described in S. M. Berge, L. D. Bighley and D. C.
Monkhouse, J.
~o Pharmaceut. Sci., 66(1977) I-19.
The process according to the present invention described above can therefore
schematically
be described as comprising the following steps;
a Step 1
(i) A coupling step,
(ii) A deprotection step,
Step 2
20 (i) A coupling step,
(ii) A deprotection step
Step 3
(i) A coupling step,
2s (ii) An optional transformation step,
(iii) A deprotection step
The optional transformation step described above in step 3(ii) could instead
be performed
after the coupling step (i) in Step 1 or step 2. Preferably the transformation
step is
3o performed after the coupling step (i) in step 1. The preferred way of
performing the process
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of the present invention could therefore schematically be described as
comprising the
following steps;
Step 1
s (i) A coupling step,
(ii) An optional transformation step,
(iii) A deprotection step,
Step 2
~o (i) A coupling step,
(ii) A deprotection step
Step 3
(i) A coupling step,
is (ii) A deprotection step
The Na-amino protecting group may be selected from any protecting group
suitable in
peptide synthesis, such as tert-butoxycarbonyl (Boc) or benzyloxycarbonyl,
often
abbreviated Z-, just to mention two possible amino protecting groups. However,
2o benzyloxycarbonyl is particularly preferred to use for the present
synthesis since it is easily
removed by catalytic hydrogenation, and contrary to the protecting group Boc,
it does not
require neutralization of the liberated amine. Suitable amino and carboxyl
protecting
groups which may be used in accordance with the present invention will be
appreciated by
a person skilled in the art. Reference is made to J. Meienhofer in The
Peptides, Vol.l, Eds.:
zs E. Gross & J. Meienhofer, Academic Press, Inc, London 1979, pp. 264-309;
The peptides,
Vol. I -9, E. Gross & J. Meienhofer, Eds., Academic Press Inc., London, 1979-
1987;
Houben-Weyl, Methoden der organischen Chemie, E. Muller, ed., Vol. 1 S, Part I-
ll,
Thieme, Stuttgart 1974; and M. Bodanszky, Principles of peptide Synthesis,
Springer
Verlag, Berlin 1984.
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9
The pre-activation step preceding Step 1-3, or the in situ generation of the
activated
activated amino acid derivative, is achieved by reacting an amino acid,
wherein the amino
function has been protected by a suitable protecting group, such as tert-
butoxycarbonyl
(Boc) or benzyIoxycarbonyl (Z), which are either commercially available or
available by
techniques known in the art, with an activating agent in the presence of a
tertiary amine and
an organic solvent, providing the activated amino acid derivative. A schematic
representation of a pre-activation step is shown below
F
A N F activating agent
y
R
O
(II) (III)
aminoacid derivative activated aminoacid derivative
~o
wherein
A is an amino protecting group, and
R is an activating agent residue group;
For the coupling step, in Step 1-3 described above, a variety of powerful
solvents may be
~s used, as long as the amino component is essentially soluble and available
for immediate
reaction with the activated peptide derivative. Examples of suitable solvents
for the
coupling step are acetone, acetonitrile, DMF, N-methyl pyrrolidone (NMP) and
EtOAc, or
mixtures thereof.
2o As used herein, the term " benzyl-like group " denotes any substituted or
un-substituted
benzyl group that is hydrogenolyzed under similar reaction conditions as the
benzyloxycarbonyl group.
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The term "pF" denotes a para-fluoro substituent.
Possible as well as preferred reagents and reaction conditions in each step
are the
following.
The pre-activation step
Suitable activating agents may be selected from those that generates any of
the commonly
used activated amino acid derivatives including, but not limited to,
carbodiimides,
~o activated esters, azide, or anhydrides. IsobutyIchloroformiate (iBuOCOCI)
is the preferred
activating agent. When isobutylchloroformiate (iBuOCOCI) is the activating
agent, the
activated peptide derivative will have the following structure, exemplified on
D-alanine,
H3C -iBu
A, \, O
O O
is
The tertiary amine may be selected from any tertiary amine. However, NMM
(N-methylmorpholine), di-isopropylethylamine and triethylamine are preferred.
Furthermore, a secondary amine which is sterically hindered may also be used.
The organic solvent may be any organic solvent known to a person skilled in
the art to be
suitable in peptide chemistry. However, ethyl acetate, acetonitrile, acetone
and
tetrahydrofurane are preferred solvents in the pre-activation step.
2s
The coupline step; Step 1(i), step 2(i) and step 3 (i~
The solvent used for the coupling step may be selected from a variety of
solvents, as long
as the amino component is essentially soluble and available for immediate
reaction with the
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activated amino acid residue. Examples of suitable solvents for the coupling
steps are
acetone, acetonitrile, DMF, N-methyl pyrrolidone (NMP) and EtOAc, or mixtures
thereof,
of which acetone, EtOAc, NMP and DMF are preferred.
Any temperature where the activated amino acid derivative is not degraded or
the reaction
rate is too slow may be used. The preferred range is from 0°C to -
20°C, and particularly
preferred is from -5°C to -15°C. The rate of addition is
adjusted so that the preferred
temperature is maintained.
io
The deprotection stew; Step 1(ii), step 2(iil and step 3 (iii)
The catalyst used for hydrogenation may be selected from a great variety of
catalysts as will
be appreciated by a person skilled in the art. However 5% Pd on carbon is
preferred. Any
solvent that can dissolve at least some of the peptide is possible to use
except ketones, such
is as acetone, or those solvents that poison the catalyst or react with the
components of the
reaction. The choice of solvent will be appreciated by a person skilled in the
art. DMF is
the preferred solvent.
The optional Step 3(ii) is only required if the protected tetrapeptide
derivative (XI)
2o prepared in step 3(i) is an ester. Thus, if an amide of phenylalanine is
used, step 3(ii) will
be excluded from the synthetic procedure.
If an acid is used for removal of the a-protecting group, an equivalent molar
amount of a
base is required to deprotonate the amino group of the peptide derivative.
zs
In a preferred embodiment of the present invention the protected amino acid,
preferably
using Benzyloxycarbonyl- as Na-anuno protecting group, is activated as a mixed
anhydride
with isobutyloxycarbonylchloride, or a similar type of chloroformate. The
method
employed is based on the general method reviewed by J. Meienhofer in The
Peptides,
so Vol.l, Eds.: E. Gross & J. Meienhofer, Academic Press, Inc, London 1979,
pp. 264-309.
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We have surprisingly found that the activation time can be extended to at
least 30 min at a
temperature about 0 - -15°C, contrary to the recommended 1-2 min at -
15°C. We also
found that strictly anhydrous conditions are not necessary as otherwise is
recommended.
This allows the present method to be used for large scale production where the
longer
s reaction times allow a safe and reproducible process to be carried out. The
stereochemical
integrity has been completely maintained and the chemical purity as well as
yields have
been typically over 90%. The generated mixed anhydride is coupled with the
slow addition
of the amino component (amino acid/ peptide amide or ester) at about 0 - -
15°C and the
reaction mixture is then allowed to reach 20-30°C in about 30-60 min.
or longer before
~o crystallization of the product is initiated directly from the reaction
mixture.
We have also surprisingly found that when using the present method, if
appropriately
selected solvent combinations is used, there is no need for a separate washing
step prior to
crystallization. DMF, acetonitrile, EtOAc and water are preferably used. A
controlled
is crystallization not only achieves an excellent purification but also
shortens the filtering or
centrifugation time during work up as well as shortens the drying time, if dry
intermediates
are required. One important factor is to generate sufficiently large crystals
with a relatively
narrow size distribution not to block the filter medium or centrifugation
cloth. It is very
common for peptides in particular to generate gels or amorphous crystals that
are almost
2o impossible to filter.
The tripeptide derivative (9)
F
w/
O O
H2N ~ ~~ R~
O
CH3
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13
wherein
Ri is an ester or an amide residue group;
is a useful intermediate for the preparation of target compound (I).
DETAILED DESCRIPTION OF THE INVENTION
The preperation of the peptide H-Tyr-D-Ala-Phe(pF)-Phe-NHZ or a
pharmaceutically
io acceptable salt thereof, will now be described in more detail by the
following Examples,
which however should not be construed as limiting the invention. Furthermore,
Scheme 1
below provides a detailed overview of the synthetic route followed for the
preparation of
the peptide of the formula (I) according to the present invention using a
phenylalanine
derivative, wherein the carboxyl group is protected as an ester. Scheme 2
below provides a
~s detailed overview of the synthetic route followed for the preparation of
the peptide of the
formula (I) according to the present invention using a phenylalanine
derivative wherein the
carboxyl group is protected as an amide. The compound numbers referred to in
the detailed
synthesis description in the Examples below, correspond to the compound
numbering in
Schemes 1 and 2.
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14
Scheme 1
(i) cH~
0 0
O-'~ ~ . ~ F CH, o
iBuOCOCI C~
OH
2-Phe(pF) O (ii) NHa Acetone (2)
-10°C
Co1 20 min
NMJM Z-Phe
(pF)-OCOOtBu
(i) Phe-OMe x HCI
(ii) NMM
(a)-10 °C 30 min
(b) 25 °C t h
O
(3)
~(pFrPhe-OMe
Nn3
MeOH
2-3 atm
Hz
Pd/C
NH2
DMF
25 °C 1 h
,~7~
H-Phe(pF)-Phe-NHz
(4)
Z-Phe(pF)-Phe-NHZ
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Scheme 1 lcont'dl
H C (i) iBuOCOCI
O 3 ' OH (ii) NMM _
-- ~ ACN O H3C O O CH3
O ~ O 0 °C 20 min ~
// p O O CH3
Z-D-Ala O
(a) -10 °C 30 mi (7) Z-D-Ala-OCOOiBu
(b) 25 °C 1 h
ACN + DMF H-Phe(pF)-Phe-NH2
- (5)
/ I ~ F
H
O~ a O a ~O 5 % Pd/C
O// ~ ~NH2 3 bar '
O DMF
(8) CH' ~ 25°C 1
Z-D-Ala-Phe(pF)-Phe-NH2
ii
(y)
H-D-Ala-Phe(pF)-Phe-NHZ
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16
Schem-con-
OH
(i)iBuOCOC)
(ii) NMM
ACN -O CH3
O OH -1O°C
~--N 20 min CH3
O (a) -10 °C 30 min
(10) Z-Tyr (d) 25 °C 1 h (11) Z-Tyr-OCOOiBu
H-D-Ala-Phe(pF)-Phe-NHZ (9)
(12)
Z-Tyr-D-Ala-Phe(pF)-Phe-Nli~
F
NH; N ~~NH (t)
- ~;
H-Tyr-D Ala-Phe(pF)-phe-NHZ
HCI/NZO
Acetone
MIBK
H-Tyr-D-Ala-Phe(pF)-Phe-NHZ x HCI
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Step 1
(i) Preparation of Z-Phe(pF'~-Phe-OMe (Compound 3 in Scheme 1)
3.5 mole scale
Z-Phe(pF) (compound 1 )( 1 eq.) is first dissolved in acetone (4.7L/mole) and
cooled before
addition of IBK (0.9-1.2 eq.)(leq actual). The reaction is then controlled by
the rate of
addition (about 20 rriinutes) of NMM (N-methylmorpholine) (0.9-1.2 eq.) (leq
actual). A
reaction temperature between 0 and -15°C is recommended (from -
9°C to -14°C actual)
where the reaction occurs immediately upon addition of NMM, yet prevents the
mixed
anhydride from decomposing to rapidly.
~o
H-Phe-OMe x HCI (0.9-1.3 eq.) (1.04 eq actual) is meanwhile mixed with acetone
(2.6L/mole), neutralized with NMM (0.9-1.5 eq.) (1.04eq actual) and cooled to
0 - -20°C
(about -10°C actual). This slurry is upon completion of the activation
added at a rate that
maintains the temperature around -10°C (from -8°C to -
13°C actual) (about 30 minutes).
is EtOAc (4L/mole) is then charged and the organic phase washed with water
(2x2L/mole)
followed by azeotrop distillation from ACN and dissolution in MeOH prior to
the next
step. 92% purity in a methanol slurry.
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18
Shift Multinlicitv Integral
H
8.5 d 1
7.49 d 1
7.31 m 7
s 7.24 m 5
7.08 m 2
4.93 m 2
4.5 m 1
4.26 m 1
~0 3.58 s 3
2.99 m 3
2.67 m 1
is (ii) Preparation of Z-Phe(yF~-Phe-NH2 (comt~ound 4 in Scheme 1)
2.3-3.3 mole scale
PCT/SE99/00414
Ammonia is charged to the solution of compound 3 prepared in the previous step
(about
8L MeOH/mole) at a pressure between 1-5 bar at 15 to 40°C and for more
than 5 hours or
2o until the reaction is close to completion (actual conversion 99%). Upon
completion the
ammonia is evaporated and the reaction cooled before filtration or
centrifugation. The
product is washed with MeOH and dried under vacuum at 20-50 °C.
Yield 74% calculated from compound 1 (Z-Phe(pF) ) and 100% purity.
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19
Shift Multiplicity Integral
H
8.05 d 1
7.49 d 1
s 7.24 m 11
7.07 m 3
4.94 m 2
4.46 m 1
4.21 m 1
io 3.00 m 1
2.88 m 1
2.66 m 1
~s
Preparation of H-Phe(pF)-Phe-NHS (compound 5 in Scheme 1)
4.3 mole scale
PCT/SE99/00414
Compound 4 prepared in the previous step is mixed with DMF (4.2L/mole) and and
a Pd/C
2o catalyst(5% Pd actual content) is added (0.2-10% w/w / LEF-581) (7% actual)
and the
resulting mixture hydrogenated for more than 0.5 hours ( 1.2h actual) at
25°C and 3bar H2.
The reaction mixture is then filtered and cooled to about -15°C before
the next step. 99.6%
purity in solution.
a Step 2
(i) Preparation of Z-D-Ala-Phe(pF)-Phe-NH~(compound 8 in Scheme 1)
4.4 mole scale
Z-D-Ala (compound 6) { 1 eq.) was dissolved in acetonitrile (ACN) (2.3L/mole)
and cooled
so before addition of IBK (0.9-1.2 eq.)(leq used). NNfM (0.9-1.2 eq.) (leq
used) was then
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PCTISE99/00414
added in the same manner as described above for the preparation of compound 3.
The
solution of compound 5 (24.SL) was then charged during about 30 minutes,
maintaining
the temperature around -10°C (from -8°C to -14°C actual).
After completion of the
coupling the product was crystallized from the reaction mixture by slow
addition of water
s (3x3.6Lmole + lx 1.3L/mole) with about 25 min wait between each addition and
at a
starting temperature of about 30°C and an ending temp of about
20°C. The crystals can
then be centrifuged and washed with waterlacetonitrile (4:1 ) before drying
under vacuum at
20-50°C. Yield 90% and 99.5% purity.
io Shift Multinlicity Integral
H
8.14 d 1
8.05 d 1
7.43 d 1
7.29 m 12
~s 7.05 m 2
S.O1 m 2
4.44 m 2
4.01 m 1
2.92 m 3
20 2.73 m 1
0.96 d 3
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21
Preparation of H-D-Ala-Phe(pF)-Phe-NHS (compound 9 in Scheme 1)
Compound 8 prepared in the previous step is mixed with DMF (4.2Lmole) and a
Pd/C
catalyst (5% Pd actual content) is added (0.2-10% w/w / compound 3)(7% actual)
and the
resulting mixture hydrogenated for more than 0.5 hours (1.2h actual) at
25°C and 3bar H2.
The reaction mixture is then filtered and cooled to about -15°C before
the next step. Purity
97%. Conversion of starting material >98%
io Steu 3
(i) Preparation of Z-Tyr-D-Ala-Phe(pF)-Phe-NH (compound 12 in Scheme 1)
4.1 mole scale
This coupling utilized the same method as the previous two couplings. Z-Tyr
~s (compound 10) (1 eq.) was dissolved in ACN (2.3IJmole)and cooled before
addition of
IBK (0.9-1.2 eq.). NMM (0.9-1.2 eq.) was then added in the same manner as
described
above under compound 3. The solution of compound 3 from the previous step was
then
charged during about 30 minutes, maintaining the temperature around -
10°C (from -7°C to
-14°C actual). After completion of the coupling the product was
crystallized from the
2o reaction mixture by slow addition of acetonitrile and water (2Lmole ACN +
0.3LJmole
25% NH3 in H20 ,hold Zh, add 1.SIJmole ACN:H20 (1:1), hold lh, increase temp
to 35°C,
add seeding crystals (about 1 % w/w), hold 1 h, add 1.3L/mole ACN:H20 ( 1:1 ),
hold 1 h, add
1.2IJmole H20 and hold O.Sh at 35°C, add 1.2IJmole H20 and hold 2h at
20°C, add
1.2TJmole H20 and hold lh at 20°C and add 1.2IJmole H20 and hold O.Sh
at 20°C.
2s Centrifuge and wash first with water and then ACN before drying under
vacuum at
20-50°C.
Yield 81 % calculated from compound 8. 98.4% pure.
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22
Shift Multiplicity Intesral
H
9.18 s 1
8.18 d 1
s 8.11 d 1
8.05 d 1
7.43 d 1
7.24 m 12
7.04 m 4
io 6.63 d 2
4.92 m 2
4.45 m 2
4.22 m 2
3.00 m 1
~s 2.83 m 1
2.64 m 1
0.89 d 3
2o ii Preparation of H-Tvr-D-Ala-Phe(pF)-Phe-NHZ (compound I in Scheme 1)
3.1 and 3.2 mole scale
Compound 4 is mixed with DMF (2-2.6IJmole actual runs) and a 5 % PdJC (actual
content) catalyst is added (0.2-10% w/w / compound 3) (6-7% actual) and the
resulting
mixture hydrogenated for more than 0.5 hours( 1-2h actual run) at 20-
40°C (20-25°C actual
2s runs) and 3bar H2. The reaction mixture is then filtered to remove the PdlC
before
crystallizing the product by addition of EtOAc until all substance has
crystallized (typically
10i./mole). The solid is separated by filtration or centrifugation and washed
with EtOAc
prior to drying under vacuum at 20-50°C.
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23
Preparation of H-Tvr-D-Ala-Phe(pFl-Phe-NHS hvdrochloride
2.1 mole scale
The free base compound I is dissolved in a mixture of water and acetone with
one
s equivalent HCI added and clear filtered ( 146g/mole 25% HCl/H20, 2L
Acetone/mole in
actual run}. The salt has a limited solubility in acetone and therefore the
filter is washed
once with an additional amount of the acetone/water(95:5) mixture (O.SL/mole).
The
crystallization is initiated by a slow addition of acetone (3.4L/mole) at high
agitation rate
and then at least 1 % w/w of seeding crystals is added. After 30 minutes the
first amount of
io MIBK (3L/mole) is slowly charged and left with slow stirring until the
batch clearly
thickens. MIBK (3L/mole) is charged three additional times separated by 30-60
minutes
while maintaining the reactor inner temperature at about 20°C. The
solid is then separated
by centrifugation or filtration and washed with MIBK before drying under
vacuum at
20-50°C for more than 16 hours or until the solvent levels are lower
than specified in the
is release specifications.
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Scheme 2
F
CH3
w H3 ~ N
° - ° C
°~~ °H + H3C °~CI
O
O
iBuOCOCI NMM
Z-Phe(pF)
EtOAc
-10°C
20 min.
F
i H3
N
C~
H3 O
NMM
Z-Phe(pF).OCOOiBu Phe-NH2 x HCI
-10°C 25°C
min ~- 1 h
EtOAc + DMF
F
O
Hi ~NHz
NH
2 DMF H2N
25°C O
-1h
Z-Phe(pF)-Phe-NHZ Phe(pF)-Phe-NHZ
(13)
(14)
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Scheme 2 (cont'd)
i) isuococl ~ ~ CH
ii) NMM
M--.. O ~O O 3
C ~~ O ~ CHI
Z-D-Ala Z-D-Ala-OCOOiBu
H-Phe(pF)-Phe-NHZ
i) -10°C, 10 min
ii) 25°C, 1 h
MeCN
H-D-Ala-Phe(pF)-Phe-NH
5% Pd/C
DMF (~ g)
(15)
Z-D-Aia-Phe
(P~-Phe-NHZ
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Scheme 2 (cont'd)
i) iBuOCOCI
ii) NMM
MeCN
-10°C
Z-Tyr
Z-Tyr-OCOOiBu
H-D-Ala-Phe(pF~-Phe-NHZ
(1g) i) -10°C, 10 min
ii) 25°C, 1 h
MeCN
~ OH ~ F
/ ~/
O O O
~~-NHZ
H~
Z-Tyr-D-Ala-Phe(pF)-Phe-NH2
5% Pd/C
DMF
H-Tyr-D-Ala~Phe(F)-Phe-NHZ
(I)
Aceton
HCI 3% HZO
MIBK
Z-Tyr-D-Ala-Phe(pF)-Phe-NH2 x HCI
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z7
step 1
(i) Preparation of Z-Phe(pF)-Phe~NH2 (Comuound 13 in Scheme 2)
6.7 mole scale
Z-Phe(pF) ( 1 eq.) is first dissolved in acetonitrile (EtOAc)( 1.7L/mole) and
cooled before
addition of i-Butylchloroformiate (0.9-1.2 eq.)( 1.OSeq actual). The reaction
is then
controlled by the rate of addition, (about 20 minutes) 15 min actual, of N
Methylmorpholine (0.9-2.0 eq.) ( l.4eq actual). A reaction temperature between
0 and -
~0 15°C is recommended (from -8°C to -11°C actual) where
the reaction occurs immediately
upon addition of N Methylmorpholine, yet prevents the mixed anhydride from
decomposing to rapidly.
H-Phe-NH2 x HCl (0.9-1.3 eq.) (1.04 eq actual) is meanwhile dissolved in DMF
~s (4.OLmole), neutralized with N Methylmorpholine (0.9-1.5 eq.) ( 1.04eq
actual) and cooled
to 0 - -20°C (about -10°C actual). This slurry is upon
completion of the activation added at
a rate that maintains the temperature around -10°C (from -6°C to
-13°C actual) (about 15
minutes) 8 min actual.
After completion of the coupling the product was crystallized from the
reaction mixture by
2o slow addition of 50% Ethanol/water (3.6L/mole). After 30 min wait a total
of 2.85L/mole
water in three portions were charged with about 25 min wait between each
addition and at
temperature of about 20°C. The crystals can after about 17 hours be
filtered or centrifuged
and washed with 50% Ethanol/water followed by several portions of acetonitrile
before
drying under vacuum at 20-60°C. Yield 90% and 99.9% purity.
2s
Preparation of H-Phe(pF)-Phe-NHS (comr~ound 14 in Scheme 2)
6.7 mole scale
Z-Phe(pF)-Phe-NH2 prepared in the previous step is mixed with DMF (3.SL/mole)
and a
3o Pd/C catalyst (5% Pd actual content) is added (0.2-10% w/w / LEF-582) (5%
actual) and
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28
the resulting mixture hydrogenated for more than 0.5 hours ( 1.3h actual) at
25-30°C and
about 3bar H~. The reaction mixture is then filtered and cooled to about -
15°C before the
next step. 99.6% purity in solution and >99% conversion of starting material.
Sten 2
Vii) Preparation of Z-o-Ala-Phe(pF)-Phe-NHS (compound 15 in Scheme 2)
5.9 mole scale
Z-D-Ala-OH (compound x) ( 1.03 eq. used) was dissolved in acetonitrile (
1.9L/mole) and
~o cooled before addition of i-Butylchloroformiate (0.9-1.2 eq.)( 1.07eq
used). N
Methylmorpholine (0.9-2.0 eq.) ( 1.2eq used) was then added in a similar
manner as
described above for the preparation of Z-Phe(pF)-Phe-NH2. The solution of H-
Phe(pF)-
Phe-NH2 (25L) was then charged during about 15 minutes (8min actual),
maintaining the
temperature around -10°C (from -8°C to -11°C actual).
After completion of the coupling
~s the product was crystallized from the reaction mixture by slow addition of
water
(4x 1.9lJmole) with about 15-30 min wait between each addition and at a
temperature of
about 20°C. The crystals can then be filtered or centrifuged and washed
with
water/acetonitrile (4:1 ) followed by acetonitrile before an optional drying
under vacuum at
20-60°C. Yield calculated from Z-Phe(pF)-Phe-NH2 93.8% and 99.6%
purity.
Preparation of H-n-Ata-Phe(pF)-Phe-NHZ (compound 16 in Scheme 2)
5.5 mole scale
Z-D-Ala-Phe(pF)-Phe-NH2 prepared in the previous step is mixed with DMF
(2.9L/mole)
2s and a Pd/C catalyst (5% Pd actual content) is added (0.2-10% w/w / compound
3)(5%
actual) and the resulting mixture hydrogenated for more than 0.5 hours (3h
actual) at 25-
35°C and about 3bar H~. The reaction mixture is then filtered and
cooled to about -15°C
before the next step. Purity 99.4%. Conversion of starting material >99%
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Step 3
(i) Preparation of Z-Tyr-y-Ala-Phe(nF)-Phe-NH (compound 17 in Scheme 2)
5.5 mole scale
s This coupling utilized a similar method as the previous two couplings. Z-Tyr
(compound x) ( 1.05 eq.) was dissolved in MeCN ( 1.9LJmole)and cooled before
addition of
i-Butylchloroformiate (0.9-1.2 eq.)( 1.05 actual). N Methylmorpholine (0.9-2.0
eq.)( 1.3
actual) was then added in a similar manner as described above for the
preparation of Z-
Phe(pF)-Phe-NH2. The solution of H-D-Ala-Phe(pF)-Phe-NH2 from the previous
step was
~o then charged during about 20 minutes (6min actual), maintaining the
temperature around -
10°C (from -8°C to -9°C actual). After completion of the
coupling the product was
crystallized from the reaction mixture at about 20-45°C by slow
addition of acetonitrile and
water (3.4IJmole MeCN + 0.9Lmole 15% NH3 in H20 hold Smin and seed, hold 4-
24h,
then add a total of 13.9LJmole H20 in four portions with about 30min or longer
hold in
is between each. Filter or centrifuge and wash first with water and then MeCN
before
optional drying under vacuum at 20-60°C.
Yield 87.7% calculated from Z-D-Ala-Phe(pF)-Phe-NH2 and 95.1% pure.
The yield and purity were found to be increased by heating the reaction to
about 60°C with
addition of ammonia to a pH of about 9 for two hours. This will convert the
major
2o impurity, Z-Tyr(O-(i-Butyloxycarbonyl))-D-Ala-Phe(pF)-Phe-NH2, to product.
(ii) Preparation of H-Tvr-D-Ala-Phe(pF)-Phe-NHZ (compound I in Scheme 2)
5.4 mole scale
Z-Tyr-n-Ala-Phe(pF)-Phe-NH2 is mixed with DMF (2.6IJmole actual run) and a 5 %
Pd/C
2s (actual content) catalyst is added (0.2-10% w/w / compound 3) (6.4% actual)
and the
resulting mixture hydrogenated for more than 0.5 hours( 1.8h actual run) at 20-
40°C (20-
25°C actual runs) and about 3bar H2. The reaction mixture is then
filtered to remove the
Pd/C before crystallizing the product by addition of EtOAc until all substance
has
crystallized (typically about l4Lmole). The solid is separated by filtration
or centrifugation
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and washed with EtOAc prior to drying under vacuum at 20-50°C. Purity
96.7%.
Conversion of starting material >99%
Preuaration of H-Tvr-y-Ala-Phe(u~-Phe-NH~,hydrochloride
4.6 mole scale
The free base H-Tyr-~-Ala-Phe(pF)-Phe-NH2 is dissolved in a mixture of water
and
acetone with one equivalent HCl added and clear filtered ( 146g/mole 25%
HCl/H20, 2L
~o Acetone/mole in actual run). The salt is almost insoluble in acetone and
therefore the filter
is washed once with an additional amount of the acetonelwater(95:5) mixture
{0.5L,/mole).
The crystallization is initiated by a slow addition of acetone (3.4L/mole) at
high agitation
rate and then about 1 % w/w of seeding crystals is added. After 30 minutes the
first amount
of IVQSBK (3L/mole) is slowly charged and left with slow stirring until the
batch clearly
is thickens. MIBK (3L/mole) is charged three additional times separated by 30-
60 minutes
while maintaining the reactor inner temperature at about 20°C. The
solid is then separated
by centrifugation or filtration and washed with MIBK before drying under
vacuum at 20-
50°C for more than I6 hours or until the solvent levels are lower than
specified in the
release specifications. Yield 95.8% and a purity of 99.8%.
Reorocessine
Product that fail the specifications for the drug substance may be
recrystallized by the same
procedure as described above for the crystallization of the compound I, but
without the HCl
addition.
2s
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Assisnment of NMR spectra for H-Tyr-D-Ala-Phe(F)-Phe-N x HC1, i.e for
comvound I in its hydrochloride form.
NMR spectra were obtained on a solution of 36mg of the compound in approx. 0.7
ml
DMSO-d6 (99.95 atom-% D) at 27.0° C on a Varian UNITY plus 400 MHz
instrument.
Chemical shift reference for proton spectra was the middle peak of the DMSO-d6
multiplet
taken as 2.49 ppm. Reference for carbon spectra was the middle peak of the
DMSO-d6
multiplet taken as 39.5 ppm.
~o
11
OH ~/ 1 F
'' ~ ~ 0
23 ~ 5 Q 2
20 ~ ~~
NH ~ 9 5 N 1 ~~NH
H i8 2 (1)
O CH3 O 1 io ~ 21
14
/ 24
22
Atom numbering used in assignment is arbitrary and refers to the figure above.
PROTON SPECTRA
is The one dimensional proton spectrum allows groupwise assignment of alpha
protons (3.9 -
4.4 ppm), benzyl-CHI (2.6-3.1 ppm), amide-NH and phenol-OH (8.2-8.5 ppm) and
also
specific assignment for Ala-CH3 ( 14-CH3) (0.74 ppm).
The two dimensional DQFCOSY spectrum allows for groupwise assignment of the
spin
2o systems (alpha, beta and NH protons) in each amino acid residue, and
groupwise
assignment of aryl protons in each aromatic ring. All protons in the Ala
residue can also be
specifically assigned.
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CARBON SPECTRA
The one dimensional carbon spectrum allows for groupwise assignment of alpha
carbons,
benzyl-CH2, carbonyls and aryl carbons and of course specific assignment of C-
14. The
APT spectrum allows assignment of CH-multiplicity for each carbon. Line
splittings due to
C-F couplings allows specific assignment of the carbons in the fluoroaromatic
ring.
TWO DIMENSIONAL HETEROCORRELATED SPECTRA
The two dimensional carbon-proton correlated (HMQC) spectrum gives a
correlation
~o between protonated carbons and all directly bound protons. All protonated
carbons in the
Ala residue can be specifically assigned.
The two dimensional carbon-proton multiple-bond correlated (HMBC) spectrum
gives a
correlation between carbons and protons situated two to three bonds apart.
This allows
~s assignment of amino acid sequence via alpha hydrogens and the carbonyl
group of the
neighboring amino acid residue (three-bond correlation), as well as via NH and
the
carbonyl group of the neighboring amino acid residue (two-bond correlation).
Similarly
two- and three-bond correlations between benzyl-CHI and aryl carbons as well
as between
aryl protons and benzyl-C_H2 allows specific assignment of aryl protons and
carbons of the
2o individual aromatic amino acids.
In this way all (non-exchanging) protons and carbons in all four amino acid
residues can be
specifically assigned in an unambiguous way.
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Table 1
Proton assignments
Chemical shift (ppm)Integral MultiplicityAssignment
9.4 1 H s 300H
8.48 1 H d 9NH
8.37 1 H d 1 NH
8.35 1 H d 8NH
8.26 2H s
7.53 1H s
7.28 2H m 18H, 19H
7.26 2H m 21H, 22H
7.22 2H m 6H, 7H
7.I8 1H m 24H
7.12 1H s
7.04 2H m lOH, 11H
6.98 . 2H m 26H, 27H
6.68 2H m 28H, 29H
4.43 1H m 1H
4.39 1H m 8H
4.26 1 H m 9H
3.96 1 H m 20H
3.39 HDO
3.02 1H m l2Hb
2.99 1 H m 2Hb
2.87 2H m 23Ha, 23Hb
2.85 I H m l2Ha
2.67 1 H m 2Ha
A ~/A '1TT ~ t TT
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Table 2
Carbon assignments
Chemical shift (ppm)MultiplicityAssignment JC-F
172.99 s 13
171.08 s 5
170.81 s 3
167.39 s 17
162.11 s 1 S 241 Hz
159.70 s 15 241 Hz
156.50 s 30
137.98 s 16
133.96 s 4 3.1 Hz
133.93 s 4 3.1 Hz
131.16 d 6.7 8.4 Hz
131.08 d 6.7 8.4 Hz
130.46 d 26.27
129.26 d 18.19
128.09 d 21.22
126.30 d 24
124.73 s 25
115.16 d 28.29
114.63 d 10.11 21.3 Hz
114.41 d 10.11 21.3 Hz
54.33 d 8
54.01 d 1
53.37 d 20
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Table 2 (contd.)
Carbon assignments
Chemical shift (ppm) Multiplicity Assignment JC-F
47.96 d 9
37.57 t 12
36.83 t 2
36.17 t 23
18.45 q I4
