Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
0050/44849
2189690
Novel active peptide and its preparation
The invention relates to a novel active peptide and to its
preparation and suitable starting materials therefor.
The PCT Application WO 93/23424 describes peptide-based active
substances which have interesting antineoplastic activities. A
particularly good effect is shown by the pentapeptide of Example
234 in the said application, which has the following formula:
4O7jO9
NH
Me2Va1-Val-MeVal-Pro-Pro-NHBz1
Me2Val is N,N-dimethyl-L-valine, MeVal is N-methyl-L-valine and
Bzl is benzyl.
The peptide can, according to the said PCT application, be pre-
pared by a solid-phase method starting from proline. This gives a
poor yield of impure active substance. Elaborate chromatographic
purification is necessary. The solid-phase method is, moreover,
suitable only for preparing small amounts of substance. It has
not to date proven possible to prepare the substance of Example
234 of WO 93/23424 in crystalline form. The active substance is
in the form of a resin. This makes it difficult to remove
residual solvent completely. Costly purification steps (spray
drying, freeze drying) are necessary. Pharmaceutical processing
of the substance is impeded. Larger amounts of substance are
necessary for testing and launching it. A process which can be
implemented industrially is needed to prepare the active sub-
stance in crystalline form if possible.
We have now found a process which affords the active substance
without racemization in high purity so that the substance can be
converted without difficulty into a crystalline salt, the hydro-
chloride.
0050/44849
2189690
i 2
The invention relates to a process for preparing Me2Val-Val-MeVal-
Pro-Pro-NHBzl=HC1, which comprises in a compound of the formula
II
Z-Val-Val-MeVal-Pro-OR1 II
where
Ri is C1_5-alkyl and
z is a benzyloxycarbonyl protective group which may be
substituted on the phenyl ring,
A a)eliminating the protective group Z on the N terminus, and in
the resulting compound
1) dimethylating the free amino group on the N terminus,
2) hydrolyzing the alkoxy group -OR1 on the C terminus and
b) coupling the resulting compound of the formula
Me2Val-Val-MeVal-Pro-OH V
with proline benzylamide XII
or
B a)removing the alkoxy group -OR1 on the C terminus and
b) coupling the resulting compound of the formula
Z-Val-Val-MeVal-Pro-OH IX
with proline benzylamide XII, and in the resulting compound
of the formula
Z-Val-Val-MeVal-Pro-Pro-NHBzl XI
1) removing the protective group Z on the N terminus and
2) dimethylating the free amino group on the N terminus
and converting the resulting compound into its hydrochloride.
0050/44849 2189690
U
3
Process A takes place in accordance with the following reaction
scheme:
Z- Val - Val - MeVal - Pro - OR1 II
~
III
Val - Val - MeVal - Pro - OR1
1
Me2Va1 - Val.- MeVal - Pro - ORI IV
I
Me2Val - Vatll - MeVal - Pro - OH V
~
Me2Val - Val - MeVal - Pro - O9 Me VI
~
Me2Val - Val - MeVal - Pro - OCOR2 VII
~ J~ ,NH ~ ' XI I
N 17
K 0
Me2Val - Val - MeVal - Pro - Pro - NHBzl x HC1 I
The meanings of the substituents in the above scheme as elsewhere
in the description are as follows:
R'i: C1_5-alkyl such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, tert-butyl, isobutyl, pentyl and preferably methyl
and ethyl,
R2: tert-butyl, 2-ethylhexyl, C1_4-alkoxy such as methoxy, ethoxy,
isobutoxy,
0050/44849
i 2J8969D
Z: benzyloxycarbonyl, which is unsubstituted or substituted on
the phenyl ring by halogen, C1_4-alkyl, C1_4-alkoxy, C1_4-acyl-
oxy or nitro and in particular by 2-Cl, 3-Cl, 4-Cl, 4-Br,
4-CH30-, 4-CH3COO-, 2-NO2 and 4-NO2
Mfl: R0, NaP, Li or an ammonium ion such as 9NH(C2H5)3
Bzl:benzyl
Me: methyl.
The tetrapeptide ester II is dissolved in a suitable solvent, eg.
an alcohol such as methanol, ethanol, isopropanol, butanol, an
ether such as THF, dioxane, MTBE, an ester such as ethyl acetate,
or glacial acetic acid. After addition of a suitable catalyst,
eg. Pd/C or Pt/C, hydrogen is passed in at from 0 to 50*C, prefer-
ably from 10 to 30*C. Introduction of hydrogen can take place
under atmospheric pressure or up to 10 bar. The reaction rate can
be increased by allowing a certain amount of gas to escape. After
hydrogen uptake is complete, 2-5 equivalents of formaldehyde are
added in the form of an aqueous solution, of the gas or of para-
formaldehyde. Subsequently hydrogen is passed in further under
the conditions described above. The catalyst is then filtered
off. IV can be purified by crystallization as hydrochloride from
a suitable solvent or mixture of solvents, isopropanol/methyl
tert-butyl ether having proven suitable. Traces of the Z-tetra-
peptide ester II in IV can be removed by extraction methods.
The ester IV is hydrolyzed in a suitable solvent, eg. an alcohol
such as methanol, ethanol, isopropanol, an ether such as MTBE,
THF, dioxane, a hydrocarbon such as toluene, xylene, or a chlori-
nated hydrocarbon such as 1,2-dichloroethane, methylene chloride,
chloroform, with and without addition of water and with a suit-
able base such as NaOH, ICOH, LiOH. The ester cleavage can also be
carried out by acids. Particularly suitable when Ri - tert-butyl
are CF3CO2H and a solution of HC1 in dioxane.
The resulting tetrapeptide acid V must subsequently be coupled
with proline benzylamide XII to give the pentapeptide I. Race-
mization readily takes place in such coupling reactions.
G. Pettit et al. (J. Am. Chem. Soc. 1,11, 6692-3 (1991)) therefore
use DEPC [(OEt)ZPOCN] as coupling reagent for an analogous
coupling with the tetrapeptide acid V. DEPC cannot be bought in
large amounts. The method therefore requires additional process
steps with poisonous phosphorus and cyanide reagents.
Cyanide-containing wastes cause disposal problems. The process is
therefore unsuitable for industrial implementation. A peptide
coupling method which can be carried out particularly
0050/44849
~ 2189690
straightforwardly on the industrial scale is the mixed anhydride
method (see, for example, J. Meienhofer in The Peptides, Analy-
sis, Synthesis, Biology, Volume 1, Academic Press, Orlando, 1979,
pages 264-314). This entails the acid V being deprotonated with a
suitable base, eg. a tertiary amine such as triethylamine,
N-methylmorpholine, dicyclohexylethylamine, diisopropyl-
ethylamine, to VI. The esters of the formula IV can also be
converted directly into the salts VI with bases such as NaOH,
KOH, LiOH. The compounds VI are reacted with an acid chloride
C1COR2 to give the mixed anhydride of the formula VII. Besides
pivaloyl chloride it is also possible to use other acid chlorides
such as 2-ethylhexanoyl chloride, ethyl chloroformate, methyl
chloroformate and isobutyl chloroformate. The mixed anhydrides
are very prone to racemization (see, for example, J. Meienhofer
in The Peptides, Volume 1, Academic Press, Orlando, 1979,
pages 276 et seq.).
surprisingly, it was then possible to react the tripeptide acid V
by the mixed anhydride method completely without racemization.
Particularly good results were obtained with the mixed anhydride
prepared from V and pivaloyl chloride. In contrast to recently
published results (N.L. Benoiton et al., Can. J. Chem. 65,
619-625 (1987)), reaction with pivaloyl chloride gives better
results, in terms of selectivity and yield, than reaction with
chloroformic esters. Preparation of the mixed anhydride VII and
the subsequent coupling of proline benzylamide are carried out at
from -20 to +S*C in a suitable solvent such as dioxane, NMP, THF,
toluene, methylene chloride, dimethylformamide. In place of pro-
line benzylamide XII it is also possible to use a suitable salt
of this compound, eg. the bisulfate, the methylsulfonate, the
hydrochloride or the hydrobromide. in this case it is necessary
to add another equivalent of base, eg. triethylamine. After the
peptide coupling and conventional workup by extraction, the crude
product is dissolved in a suitable solvent, eg. a hydrocarbon
such as toluene, xylene, or an ether such as diethyl ether, THF,
dioxane, methyl tert-butyl ether, a ketone such as acetone,
methyl ethyl ketone, diethyl ketone, cyclohexanone, or chlori-
nated solvents such as methylene chloride, chloroform, 1,2-dich-
loroethane. The hydrochloride I is precipitated by introducing
gaseous HC1 or metering in a solution of HC1 in a suitable sol-
vent, eg. THF, methanol, isopropanol, n-pentanol, diisopropyl
ether. A particularly suitable process has proven to be one in
which the free base of the pentapeptide is initially dissolved in
methyl ethyl ketone, and subsequently a solution of HC1 in iso-
propanol is added.
0050/44849
2189690
6
Process B takes place in accordance with the following scheme:
Z- Val - Val - MeVal - Pro - ORi II
~
Z- Val - Val - MeVal - Pro - OH. IX
~
Z -Val - Val - MeVal - Pro - 0 - CO - RZ + XII X
4
Z - Val - Val - MeVal - Pro - Pro - NHBzl XI
4
Me2Val - Val - MeVal - Pro - Pro - NHBzl x HC1 I
The hydrolysis of the ester II, the preparation of the mixed
anhydrides X and the peptide coupling to give XI take place as
for the sequence IV ~ V-> VI -~ VII ~ I. Elimination of the
Z protective group and dimethylation to give I take place as for
the conversion II -> III -> IV.
The mixed anhydride method also, surprisingly, takes place with-
out racemization in variant B. In contrast to the results pub-
lished by Benoiton, the best yields are obtained with the mixed
anhydride prepared from the acid IX and pivaloyl chloride.
The starting material II needed to prepare the peptide I can be
prepared from Z-Val-O-CO-R2 (XIII) and Val-MeVal-Pro-OR1 (XIV).
The novel process provides active substance I in crystalline
form. The peptide can be further purified in a simple way by
recrystallization. Elaborate chromatographic purification steps
are unnecessary.
The invention also relates to the following precursors for pre-
paring I:
Z-Val-Val-MeVal-Pro-OR' II
Val-Val-MeVal-Pro-OR1 III
Me2Val-Val-MeVal-Pro-O-CO-R2 VII
Z-Val-Val-MeVal-Pro-OH IX
0050/44849
M9690
7
Z-Val-Val-MeVal-Pro-O-CO-R2 x
Z-Val-Val-MeVal-Pro-Pro-NHBzl XI
where Ri and Z have the meanings stated in claim 3, and R2 is
tert-butyl, 2-ethylhexyl, C1_4-alkoxy, methoxy, ethoxy and iso-
butoxy.
The compound I has activity on solid tumors (tumors of the lungs,
the breast, the intestine, the bladder, the rectum, the uterus,
the prostate) on leukemia, lymphomas and other neoplastic
disorders.
The following examples illustrate the invention.
Example 1 (process A)
A. Preparation of the starting materials
a. Z-Val-Val-MeVal-Pro-OMe (II, R1 = Me)
39.6 kg (83.3 mol) of Z-Val-MeVal-Pro-OMe (VIII) in 320 1 of
methanol were introduced together with 4 kg of 5 8
palladium/carbon into a 400 1 hydrogenation vessel. Then, while
cooling at 20-30*C, hydrogen was passed in until precursor was no
longer detectable in the reaction solution. The catalyst was fil-
tered off as the contents of the vessel were discharged. The fil-
trate was worked up by concentration to 50 1 in a 400 1 enameled
vessel under waterpump vacuum. Subsequently 50 1 of toluene were
run in and the mixture was extracted with 40 1 of 2N HC1. The
toluene phase was extracted once more with 40 1 of iN hydro-
chloric acid and then discharged. The collected acidic aqueous
phase was returned to the vessel and, after addition of 40 1 of
methylene chloride, adjusted to pH 9 by running in 50 % strength
sodium hydroxide solution while stirring vigorously and cooling.
After phase separation, the methylene chloride phase was dis-
charged and the aqueous phase was extracted twice more with 40 1
of methylene chloride each time. The collected methylene chloride
solution of the product is washed with water until neutral. The
methylene chloride phase is subsequently concentrated to 90 1 to
afford Val-MeVal-Pro-OMe (XIV, R1=CH3).
Yield: 24.2 kg = 85.2 B.
17.84 kg (70.88 mol) of Z-valine and 4.59 kg (74.42 mol) of tri-
ethylamine are dissolved in 170 1 of methylene chloride in a
400 1 vessel. 8.58 kg (70.88 mol) of pivaloyl chloride are
metered into this solution at -5 to -10*C. After a reaction time
0050/44849
+ 8 2189690
of 2 h at -S*C, a solution of 24.2 kg of Val-MeVal-Pro-OMe in 86 1
of methylene chloride was run in at -5*C. After a further 2 h at
-50C, the mixture was heated to 20*C and stirred at this tempera-
ture for 12 h. For workup, 50 1 of water were added and, after
removal of the aqueous phase, the organic phase was extracted
once with 40 1 of 2N hydrochloric acid and twice with 40 1 of 2N
sodium hydroxide solution each time. After the organic phase has
been washed with water to neutrality, the methylene chloride sol-
vent was removed by distillation and replaced by 300 1 of diiso-
propyl ether. The product was crystallized by heating the emul-
sion of the oily product to 60'C, adding seed crystals and keeping
at 600C for 7 h. The crystallization was completed by stirring
successively at 50*C for 5 h and at 400C for 5 h and then cooling
to 20*C. The crystals were filtered off through a 120 1 pressure
filter and dried in a stream of nitrogen.
Yield: 32.2 kg = 79 %
Melting point: 134 - 135*C
b. Proline benzylamide hydrochloride (XII x HC1)
48.2 g of pivaloyl chloride were added dropwise to a solution of
99.7 g of Z-proline and 58 ml of triethylamine in 1 1 of CH2C12 at
-10 to -150C. The mixture was stirred at -10*C for 45 min and
then, over the course of 0.5 h, 42.8 g of benzylamine in 500 ml
of CH2C12 were added at -10*C. The mixture was then stirred at
room temperature for 1 h. The CH2C12 solution was subsequently
washed twice with 500 ml of water, twice with 500 ml of 10 %
strength aqueous NaHCO3 solution, twice with 500 ml of water,
twice with 500 ml of 5 % strength aqueous citric acid solution
and twice with 500 ml of water, dried over Na2SO4 and evaporated.
The 120 g of residue were taken up in 200 ml of ethyl acetate.
1.2 1 of n-heptane were added to the solution, the mixture was
stirred for one hour, and the product was filtered off with suc-
tion and dried at 50*C under reduced pressure.
Yield: 110 g, 81.3 $
Melting point: 93 - 94*C
110 g of the Z-proline benzylamide obtained in this way were dis-
solved in 1.5 l of methanol. After addition of 0.5 g of Pd/C
(10 %) hydrogen was passed in. The solution took up 0.5 1 of H2
over the course of 1.5 h at room temperature. After removal of
the catalyst by filtration and evaporation, 4.6 g of a yellow oil
remained.
AMENDED SHEET
0050/44849
218 9 6 9 0
9
413 g of the proline benzylamide obtained in this way were dis-
solved in 400 ml of isopropanol. 630 ml of a saturated solution
of HC1 in isopropanol were added, the resulting suspension was
stirred at 0-5*C for 2 h, and the solid was filtered off with suc-
tion and washed twice with 250 ml of isopropanol. The residue was
dried at 50*C under reduced pressure to obtain 401 g of proline
benzylamide hydrochloride; aD20:- 450
B. Preparation of the final product
a.1
Me2Val-Val-MeVal-Pro-0Me x HC1 (IV x HC1, R1= Me)
kg (34.8 mol) of Z-Val-Val-MeVal-Pro-OMe (II, Ri=Me) were
15 introduced together with 2 kg of 5 % palladium/carbon into 200 1
of methanol in a 400 1 hydrogenation vessel. Then, while cooling,
hydrogen was passed in at 20*C until precursor was no longer
detectable in the reaction solution. Subsequently 8.46 kg of 37 B
strength (104 mol) aqueous formaldehyde solution were added, and
20 hydrogenation was continued at 20*C until hydrogen uptake ceased.
The catalyst was filtered off as the contents of the vessel were
discharged. The filtrate was worked up by concentrating to 50 1
in a 400 ml enameled vessel under waterpump vacuum. Then 200 1 of
isopropanol were added, and the mixture was again concentrated to
50 1. The residue was then dissolved in 135 1 of methyl tert-
butyl ether, and one equivalent of isopropanolic HC1 was added
while cooling at 20*C. The resulting suspension was stirred fur-
ther at 20*C for 3-4 h and at 0-5*C for 2 h and then filtered
through a 120 1 pressure filter. The filter cake was washed once
with 50 1 of fresh methyl tert-butyl ether.
Yield: 16.2 kg = 92.3 t
Melting point: 2240C (decomposition)
a.2
It was also possible to isolate the intermediates Val-Val-MeVal-
Pro-OMe (III, Ri=CH3) when workup was carried out as follows after
the first hydrogenation stage:
The reaction solution was separated from the catalyst and concen-
trated. The residue was taken up in ethyl acetate. The ethyl ace-
tate solution was extracted twice with 2N hydrochloric acid. The
acidic aqueous phase was adjusted to pH 9 with sodium hydroxide
solution and extracted twice with methylene chloride. The methy-
lone chloride phase was then washed until neutral and evaporated.
AMENDED SHEET
0050/44849
2118 96 90
HPLC 96,8 S
iH-NMR (400 MHz, CDC13 / TMSiat.):
5 S(ppm): 0.84 - 1.08 (m, 18H); 1.45 - 1.6 (S, broad, M);
1.85 - 2.15 (m, 4H); 2.18 - 2.38 (m, 3H);
3.15 (s, N-M); 3.25 (d, 1H); 3.65 - 3.75 (m, 1H);
3.73 (s, O-M ); 3.9 - 4.05 (m, 1H); 4.38 - 4.45 (m, 1H);
4.73 - 4.83 (m, 1H); 5.12 (d, 1H); 7.9 (d, NFj)
a.3
Me2Val-Val-MeVal-Pro-OMe x HC1 (IV x HC1, R1=Me) can also be
prepared by the following method which dispenses with isolation
and purification of the intermediate Z-Val-Val-MeVal-Pro-OMe (II,
R1=Me):
128 g (0.51 mol) of Z-valine and 55.1 g (0.54 mol) of triethy-
lamine were dissolved in 1.2 1 of methylene chloride in a 4 1
flask. 62.1 g (0.51 mol) of pivaloyl chloride were added to this
solution at -50C to -106C. After a reaction time of 2 h at -50C, a
solution of 174.6 g (0.51 mol) of Val-MeVal-Pro-OMe in 0,8 1 of
methylene chloride was run in, and the mixture was stirred at -5*C
for a further 2 h and then, after warming to 200C, for a further
12 h. 370 ml of water were then added to the mixture. After phase
separation, the methylene chloride phase was washed once with
290 ml of 2N hydrochloric acid, twice with 290 ml of 2N sodium
hydroxide solution each time and three times with 370 ml of
water. The methylene chloride solvent was subsequently evaporated
off and replaced by 3 1 of methanol. To this solution was added a
suspension of 30 g of 5 8 palladium/carbon in 110 ml of water and
hydrogenation was carried out at 25*C using a gas-introduction
stirrer and hydrogen burette until one equivalent of hydrogen had
been taken up. Then 123 g (1.53 mol) of 37 % strength aqueous
formaldehyde solution were added and hydrogenation was continued
until a further 2 equivalents of hydrogen had been taken up. The
catalyst was then removed and the solution was evaporated in a
rotary evaporator. The remaining oil was dissolved in 670 ml of
isopropanol and 2.6 1 of methyl tert-butyl ether. One equivalent
of isopropanolic HC1 was added to this solution. The resulting
suspension was stirred at 20*C for a further 12 h and then fil-
tered with suction. The filter cake was washed with a little
methyl tert-butyl ether and subsequently dried at 40*C in a vacuum
oven.
Yield: 182.8 g - 71 ~
Melting point: 224*C (decomposition)
0050/44849
~ 2189690
11
b. Me=Val-Val-MeVal-Pro-Pro-NHBz1 x HC1 (I)
15.9 kg (31.5 mol) of MeZVal-Val-MeVal-Pro-OMe x HC1 (IV x HC1,
RI=CH3) were introduced together with 140 1 of toluene and 15 1 of
methanol into a 400 1 vessel. To this were added 3.15 kg
(76.38 mol) of sodium hydroxide pellets. After hydrolysis was
complete, ie. after 3 h at 200C, the mixture was neutralized by
adding isopropanolic HC1. It was subsequently azeotropically dis-
tilled with toluene under 100 mbar until free of alcohol and
water. The solvent which was distilled off was successively
replaced by toluene. Subsequently, 80 1 of methylene chloride and
6.44 kg (63.0 mol) of triethylamine (99 % pure) were added, the
mixture was cooled to -5*C and, at this temperature, 3.84 kg
(31.5 mol) of pivaloyl chloride were metered in. After reaction
for 2 hours, 7.6 kg (31.5 mol) of Pro-NHBzl x HC1 were added a
little at a time at -5*C to O*C. After the mixture had stood at
-5*C for 2 h it was warmed to 20*C and left to react for a further
6 h. Subsequently the added methylene chloride was removed by
distillation under 500 mbar, and 80 1 of toluene were added. Then
50 1 of water were added and the pH of the aqueous phase was
adjusted to pH 9. After vigorous stirring, the aqueous phase was
separated off, and the organic phase was washed once with 25 1 of
water. The organic phase was subsequently extracted twice with
50 1 of 2N hydrochloric acid each time. The product was back-
extracted from the acidic aqueous phase after adjustment of the
pH to 9 by extraction 3 times with 50 1 of inethylene chloride
each time. After the methylene chloride phase had been washed
with water to neutrality, the methylene chloride was removed by
distillation and replaced by 180 1 of methyl ethyl ketone. The
solution was warmed to 408C and one equivalent (31.5 mol) of iso-
propanolic HCl was added. The resulting suspension was warmed to
60*C and subsequently stirred for 12 h without further input of
heat. It was then cooled to 20*C and stirred for a further 5 h. It
was subsequently cooled to 5*C and filtered through a 120 1
pressure filter. The filter cake was washed with 60 1 of fresh
methyl ethyl ketone at 5*C. After initial drying on the filter,
the product is dried to constant weight in a vacuum oven at 40*C.
Yield: 14.36 kg = 67 %
Melting point: 214*C (decomposition)
0050/44849
2189b90
12
Example 2 (process B)
a. Z-Val-Val-MeVal-Pro-0H (IX)
117 g (0.2 mol) of Z-Val-Val-MeVal-Pro-OMe (II, R1:Me,
Example lAa) are dissolved in 900 ml of methanol and 47.5 ml of
water in a 2 1 flask. Subsequently 18 g (0.45 mol) of sodium
hydroxide pellets were added, and the mixture was stirred at 200C
for 12 h. For workup, 250 ml of water were added and the methanol
was distilled off. Then sufficient ethyl acetate was added to
produce clear phase separation (about 500 ml). The ethyl acetate
phase was separated off. The aqueous phase was acidified to pH 1
with hydrochloric acid and extracted twice with 500 ml of methy-
lene chloride. The organic phase was then evaporated to dryneas.
Yield:. 10-5 g = 96.4
8
'H-NMR (200 MHz, CDC13/TMSiat.)
8(ppm): 0.6 - 1.2 (m, 18H); 1.7 - 2.45 (m, 7H); 3.2 (s, N-M);
3.55 - 3.95 (m, 2H); 4.05 - 4.2 (m, 1H); 4.35 - 4.5
(m, 1H); 4.68 - 4.85 (m, 1H); 4.98 - 5.2 (m, 3H); 5.93
(d, Val-1N$); 7.2 - 7.4 (m, 5H); 7.53 - 7.68 (Val-2Nx);
9.6 - 10.2 (s, broad, COO$)
b. Z-Val-Val-MeVal-Pro-Pro-NHBzl (XI)
5 g of Z-Val-Val-MeVal-Pro-OH (8.75 mmol) (IX) were dissolved in
50 ml of CH2C12, 1.79 g (17.5 mmol) of triethylamine were added
dropwise, the mixture was cooled to 10*C and, at this temperature,
1.08 g (8.75 mmol) of pivaloyl chloride were added dropwise.
After stirring at 10*C for 2 h, a solution of 2.11 g (8.75 mmol)
of Pro-NHBzl x HC1 in 10 ml of methanol was added dropwise at
this temperature, and the mixture was stirred at 10*C for 2 h and
at room temperature overnight.
The reaction mixture was washed 3 times with 50 ml of water each
time, once with 50 ml of water at pH 9 and twice more with 50 ml
of water each time. The methylene chloride solution was evapo-
rated in a rotary evaporator to leave 5.3 g (81.4 %) of white
crystalline product (purity: 88.7 %).
Melting point: 118 - 122*C
c. Me=Val-Val-MeVal-Pro-Pro-NHBz1 x HC1 (I)
0050/44849
13 2189690
~
12 g of Z-Val-Val-MeVal-Pro-Pro-NHHzl (XI) were dissolved in
200 ml of methanol. To this were added 2 g of 5 % palladium/
carbon (suspended in 20 ml of water) and hydrogenation was
carried out at 20*C until hydrogen uptake ceased. Subsequently,
6.5 g of 37 t strength aqueous formaldehyde solution were added,
and hydrogenation was continued until hydrogen uptake ceased. The
catalyst was then separated off and,the reaction solution was
evaporated. The residue was taken up in toluene and concentrated
again, and a further 200 ml of toluene were added and the mixture
was filtered. The toluene solution was subsequently extracted 2x
with 50 ml of 2N hydrochloric acid. The acidic aqueous phase was
adjusted to pH 9 with sodium hydroxide solution and extracted
three times with 50 ml of methylene chloride. The methylene chlo-
ride phase was washed with water until neutral and concentrated.
The crude base was taken up in a solution of 150 ml of methyl
ethyl ketone and 7.5 ml of isopropanol. The product salt was pre-
cipitated from this solution by adding 4 g of 25 % strength iso-
propanolic HC1 at 400C. The suspension is stirred at 200C for 3 h
and at 0-5*C for one hour and then filtered with suction.
Yield: 7.1 g
Purity: 99.1 B(HPLC percent area)
Melting point: 2140C (decomposition)
30
40
* .
