Note: Descriptions are shown in the official language in which they were submitted.
n ~ r~ ?
. ? ~
2330~-1213
8MALL ~EPTIDE AND P8BUDOPEPTID~ ANIDE~ IN~IBI~ING
~KB PROLIFERATION OF 8MAL~-CBLL AND EPITHE~IAL-CELL LUNG
CANCER CELL8
The invention relates to novel, therapeutically
active peptide and pseudopeptide amides of the formulae
(1) to (6),
~-HOPA-D-Trp-Phe-D-Trp-Leu ~(CH2NH)Leu-NH2 (1)
10 D-MePhe-D-Trp-Phe-D-Trp-Leu ~(CH2NH)Leu-NH2 (2)
D-MePhe-D-Trp-Phe-D-Trp-Leu-MPA (3)
D-Tyr-D-Trp-Phe-D-Trp-Leu r ~CH2NH)Leu-NH2 (4~
D-Tyr(Et)-D-Trp-Phe-~-Trp-Leu yr(cH2NH)Leu-NH2 (5)
D-MePhe-D-Trp-Tyr-D-Trp-Leu ~(CH2NH)Leu-NH2 (6)
wherein
~-HOPA means ~-hydroxyphenylacetic acid,
D-MePhe stands for D-N-methylphenylalanine,
MPA represents 2-amino-3-methylpentane and
r(CH2NH) is a methyleneamino group bein~ present instead
of a peptide bond,
as well as their pharmaceutically acceptable acid
addition salts and pharmaceutical compositions containing
these compounds.
Furthermore, the invention relates to the
preparation of the above compounds and compositions.
The compounds of formulae (1) - (6) according to
the invention are new and possess valuable
pharmacological activity. More particularly, they inhibit
the proliferation of the cells of the small-cell and
epithelial cell lung cancers.
It is known [Nature 316, 823 ~1985)~ that both
bombesin (BN) occurring in the amphibia as well as the
gastrin-releasing peptide (GRP), which is structurally
closely related to bombesin and occurs in mammals, act as
A4851-67-MRJKmO
autocrine growth factors in the small-cell lung cancer
(SCLC). The formula of bombesin is
Glp-Gln-Arg-Leu-Gly-A3~-Glr~-Trp-Ala-Val-Gly-llis-Leu-Met-NH2,
whereas the fragment 14 to 27 of GRP is represented by
the formula
Met-Tyr-Pro-Arg-Gly-AQn-Hi~-Trp-Ala-Val-Gly-Hi~-Leu-Met-NH2.
It is also known [Am. J. Physiol. 252, 6439 (1987);
Regulatory Peptides 19, 10~ (1987); as well as Eur. J.
Pharmacol. 190, 31 (1990)] that structural analogues of
BN or GRP (BN antagonists) inhibit the binding of [125I-
Tyr4]-bombesin to BN receptors being present on Swiss 3T3
cells and prevent the proliferation of the small-cell
lung cancer cell line; said proliferation can be measured
by the incorporation of 3H-thymidine.
It has been observed [Proc. Natl. Acad. Sci. USA
82, 7616 (1985); Br. J. Cancer 57, 579 (1988)] that the
peptide of the formula D-Arg-D-Pro-Lys-Pro-Gln-Gln-D-Trp-
-phe-n-Trp-Leu-Leu-NH2 (Spantide), i.e. an antagonist
against "Substance P" (SP), acts as a BN antagonist on
Swiss 3T3 mouse embryonal fibroblasts and inhibits the
proliferation of the cells of the small-cell lung cancer.
Furthermore, it has been described [Biochem.
Biophys. Res. Commun. 156, 323 (1988)] that the N-
-acylated pentapeptide of the formula
HOPA-D-Trp-Phe-D-Trp-Leu-Leu-NH2 (I)
is a relatively strong SP antagonist (with a PA2 value of
5.73 on the guinea pig ileum) inhibiting the smooth
muscle-contracting effect of SP.
Our investigations directed to the C-terminal
pentapepti~e analogues of Spantide have shown the low
molecular SP antagonist of the formula (I) to inhibit the
binding of the labelled ~l25I-Tyr4]-bombesin to Swiss 3T3
cells and prevent the proliferation of NCI-H69 small-cell
lung cancer cell line possessing no BN receptors.
Thus, the aim of the present invention is to
2 ~ t ! ~ ~ 7
prepare analogues not inhibiting the binding of [125I-
Tyr4]-BN to Swiss 3T3 cells, i.e. analogues which are
therefore not BN antagonists but exert a strong
inhibitory action on the proliferation of the cells of
the small-cell and epithelial-cell lung cancers. Such
compounds might be very useful in the therapy of lung
cancer.
It has been found that the analogues of formulae
(1) to (6), containing a modified, mainly reduced peptide
bond, show such an effect.
Abbreviations used in the description are as
follows:
FCS foetal calf serum
D-MEM Dulbecco's minimal essential medium
15 BSA bovine serum albumin
HEPES N-(2-hydroxyethyl3piperazine-N'-(2-ethanesul-
fonic acid)
PBS phosphate-buffered saline
SDS sodium dodecyl sulfate
20 TCA trichloroacetic acid
Other abbreviations are in agreement with those
described in "Nomenclature and Symbolism for Amino Acids
and Peptides, Recommendations 1983" of the IUPAC-IUB
Joint Commission on Biochemical Nomenclature (JCBN)
tBiochem. J. 219, 345 (1984)].
The novel compounds of formulae (1) to (6) and their
acid addition salts can be prepared in such a way that,
by using Leu r(CH2NH)Leu-NH2 or Leu-MPA as starting
substances, the peptide of formula (1), or the derivatives
of peptides of formulae (2) to (6) containing N-terminal
benzyloxycarbonyl or tert-butoxycarbonyl protective group
are built up, preferably by the successive use of the
steps of reactive ester coupling and deprotection of the
~-NH2 group, then the protective group is removed by
catalytic hydrogenation or acidolysis and, if desired,
r ~ ~?, ~
the product obtained is tra~sformed into a pharma-
ceutically acceptable acid addition salt or the free base
is liberated from such a salt.
In the case of the compound of formula (1), the N-
-terminal ~-HOPA is incorporated without any protective
group into the molecule, to obtain the final product by
building up the peptide chain.
The methyleneamino (reduced peptide) bond of the
pseudopeptide Leu- ~(CH2NH)Leu-NH2, used as starting
substance, can be formed by reductive alkylation
performed in a known manner [see e.g. Nature 299, 555
(1982)]. Thus, Leu-NH2 hydrochloride is alkylated with Z-
-Leu-H (Z-Leu-aldehyde) and the Schiff's base obtained
is reduced in situ by sodium cyanoborohydride. The N-
-terminal benzyloxycarbonyl protective group of the
protected pseudodipeptide Z-LeuYr~CH2NH~Leu-NH2 is
removed by catalytical hydrogenation.
Leu-MPA may be prepared as follows. In the first
step, 2-amino-3-methylpentane is acylated by a mixed
anhydride formed from Z-Leu-OH and pivaloyl chloride,
then the benzyloxycarbonyl protective group is removed by
catalytical hydrogenation and the mixture of Leu-MPA
isomers is separated by chromatography on a silica gel
column. Thereafter, the Leu-MPA isomer described in the
experimental part is used.
After accomplishment of the synthesis, the obtained
crude pentapeptide amides according to the invention are
purified by chromatography on a silica gel column.
The biological investigation of the new ccmpounds
of the invention was c~rried out in four tests. The BN-
-antagonizing effect of the compounds in question was
determined in Test 1, based on measurin~ the displacement
of the labelled t12~I-Tyr4]-BN from the BN receptors
being present on the Swiss 3T3 mouse fibroblast cells.
Test ~ and Test 3 were used to study the inhibitory
- 5
effect of the compounds of the invention on the
proliferation and propagation of NCI-H69 small-cell lung
cancer cells. The inhibitory effect of the compounds of
the invention on non-small-cell lung cancer cells and
epithelial-cell lung cancer cells was investigated in
Test 4.
Test 1
Study of the binding of target compounds to BN
receptors on the Sviss 3T3 mouse fibroblast cell
line
Swiss 3T3 mouse fibroblast cells were grown on
tissue-cultivating plates containing 24 holes in a D-MEM
culture medium containing FCS of 10 % until a confluent
and restinq state (3x105 cell/hole). The cells were
washed 3 times with 0.5 ml of D-MEM of pH 7.4 (containing
0.2 ~ of BSA and 24 mmol of HEPES, and cooled to 4 ~C),
i.e. incubation mixture, each. Subsequently, the cells
were incubated in a final volumP of 250 ~l at 4 C for 3
hours with an incubation mixture containing [125I-Tyr4~-
-BN in a constant concentration (1 nmol) and the compound
to be tested in an amount of 1.13 to 75 ~mol. At the end
of the reaction the cells were washed twice with the
incubation mixture and then ~ times with PBS (pH = 7.4).
The washings were carried out with 750 ~1 volumes
of the ice-cold liquids each. The cells were solubilized
by adding 750 ~l of 0.5 M sodium hydroxide solution,
carried over into test-tubes by pipet and the radio-
activity bound to the cells was measured. The grade of
aspecific binding was determined in the presence of a
high concentration (1o~6 molJl) of cold ~Tyr4~-BN. The
amount of the specifically bound, labelled hormone was
expressed as the percentage of the maximum binding, i.e.
the specific binding in the absence of the compounds to
be tested, as shown in Table l.
2 ? ~? ~ 7
-- 6 --
T~bl~ 1
Displacement of tl25I-Tyr4]-BN on the Rwis~ 3T3 mou~e
fibrobl~t c~ll line
Displacement as percentage of control
net binding
Concentration Compound
r~mol/l~ 1)(2~ (3) (4) (5)
0 100 100100 100 100 100
1.18 108 lOS - 96 85 113
2.36 98 96 92 108 88 111
4.71 83 99109 92 79 106
9.42 55 10393 103 72 117
18.8 33 10699 108 61 116
37.6 18 10794 113 52 95
23 10591 97 45 81
The data of Table 1 show that the BN-antagonizing
effect being characteristic of SP antagonists is
abolished by incorporating the methyleneamino kond into
SP antagonists [compounds of formulae (1), (2), (4) and
(5)] or by substituting an aliphatic amine for the C-ter-
minal amino acid of the SP antagonists [compound (3)].
Test 2
ID~estigation of the inhibitory effect of target
compoun~s on the prolifer~tion of NCI-~69 3mall-
cell lunq oanc~r cell line po~essing no BN
3 0 recep~ors by measuring the incorporation of 3~-
thymidine
The cells gr~wn in suspension, i.e. in RPMI-1640
culture medium ~DIFC0, U.S.A.3 supplemented with FCS of
10 %, were collected by centrifuging at 4 C with 1200
rpm. After decanting the supernatant, the cells were
2'?`~
-- 7
suspended in RPMI-1640 HITES medium (containing lx10-8
mol/l of hydrocortisone, 5 mg/l of insulin, 10 m~/l of
transferrin, lxl0-8 mol/l of estradiol and 3x10-8 mol/l
of sodium selenite). The cell number was adjusted to
5 5x105 cell/ml; 90 ,ul of this cell suspension were added
into each hole of a tissue-cultivating plate containing
96 holes. 10 1~1 of solutions, each containing the target
~ompounds in various concentrations, were added to the
cells. The cells were grown at 37 C under air containing
5 % by volume of carbon dioxide for 72 hours, then 37 kBq
(1 ,uCi) of 3H-thymidine, dissolved in 10 ~l of culture
medium, were added into the holes and the incubation was
continued for additional 24 hours. Subsequently, the
cells were solubilized in 10 ~l of 20 % SDS solution. 25
,ul of solution from each hole were dropped onto Whatman 3
filter paper. After drying, the filter paper was washed 3
times with 5 % a~ueous cold trichloroacetic acid
solution, and then with 96 % ethanol in order to de-
hydrate it and to remove the traces of trichloroacetic
acid.
After drying, the filter paper pieces were placed
in scintillation tubes and 5 ml of scintillation cocktail
[a mixture containing lO00 ml of toluene, 400 ml of abs.
ethanol, 10 ml of dioxane, 6 g of 2,5-diphenyloxazole and
0.15 g of 1,4-bis(5-phenyl-2-oxazolyl)benzene] were added
to each. The activity of the samples was measured by
using on I,KB Wallac 1211 beta-counter. The data were
expressed as the percentage of control, where the 3H-
-thymidine uptake of NCI-H69 cells not treated with the
compounds was considered to be 100 ~6 (control~ (see Table
2).
Table 2
Effect of the target compounds on the 3~-thymi~ine upta~e
of NCI-~69 small-cell lu~g cancer cell line
Uptake as percentage of control
Concentration Compound
[umol/l~ (I) (1) f2) (3) (4) (5) ~6)
0 100 100 100 10~ 100 100 100
0.75 92 - 75 115 101 105
1.56 77 - 48 104 76 155 82
3.125 104 77 51 49 88 137 65
6.25 66 34 2~ 34 59 67 63
12.5 63 44 8 18 51 17 58
6 39 4 31 9 9 49
4 30 4 7 5 7 44
100 3 2 4 - - -
200 1.7 1.3 1.7 - - -
-
Te~t 3
Investigation of the i~hibitory effec~ of target
compoun~s on the prop~gation of NCI-~69 small-cell
lung c~ncer callq
NCI-H69 cells grown in suspension, i.e. in RPMI-
1640 culture medium supplemented with FCS of 10 ~, were
collected by centrifuging at 4 C with 1500 rpm. After
decanting the supernatant, the cells were suspended in a
fresh medium and the cell number was adjusted to 1 x 105
cell/ml. 5 ml of cell suspension each were applied into
tissue-culti~ating vessels of 25 cm2 size. The compounds
to be tested w re used in a final concentration of lQ and
50 ~mol/l, respectively. The cell number was determined
from 2x200 ~1 of cell suspension in each defined time
3~ interval. The volume of sample taken QUt was replaced with
23305-1213
g
a medium containing the target compound in a con-
centration of 10 or 50 ~mol/l, respectively. The cell
dilution resulted was corrected by calculation. The data
were expressed as percentage of the starting cell number
(lx105 cell/ml was considered to be 100 ~). The change in
the number of cells not treated with the compounds was
also indicated as control (see Table 3).
T~ble 3
Effect of the target compounds on the propagation of
NCI-~69 qm.ll-cell lung ca~cer cell line
Cell number as percentage of the starting
cell number
Compound
Concent- (C) (I) (2) (3) (4) (5)
ration
t~mol/l] o 10 50 10 50 10 50 50 50
t days
0 lV0100 100 100 100 100 100 lO0 100
3 157213 90 130 25 110 65 57 60
164- - 110 47 15~ 93 - 73
7 17~233 120 140 20 190 107 80 122
204182 75 100 10 150 97 87 105
12 257- - 110 20 170 120 74 120
Note: C = control
It can be seen from the data of Tables 2 and 3 that
the target compounds retained the inhibitory effect on
the maturation and propagation of NCI-H69 small-cell lung
cancer cells, which is characteristic of SP antagonists;
the inhibitin~ effect of the compound of formula (2) is
even stronger than that of the reference substance of the
~5 formula ~I).
2 ~ ~ t r
-- 10 --
TQ~t 4
~xAmination of the effect of t~rget co~poun~s on
the proliferation of 8R-M~B-1 epithelial-cell lung
cancer cell line by determining the incorporation
of 3H-thymidine
The cells cultivated in a monolayer culture in
D-MEM medium supplemented with FCS of 10 % were suspended
by treatment with trypsin of 0.25 % and the cell
concentration was adjusted to 5 x 105 cell/ml by adding
fresh culture medium.
100 ~l of cell suspension were applied into each of
96 holes of a tissue-cultivating plate. After 48-hour
incubation at 37 C under air containing 5 % by volume of
carbon dioxide, the culture medium on the adhered cells
was exchanged for 90 ~l of D-MEM-HITES-0.1 % BSA. 10 ~l
of solutions each containing the target compounds in
various concentrations were added to the cells and after
24 hours, 37 kBq (1 ~Ci) of 3H-thymidine were added into
each hole, than the incubation was continued for additio-
nal 24 hours.
The samples were taken and measured as describedabove for Test 2.
The data were expressed as percentage of the
control. The 3H-thymidine uptake o SK-MES-l cells not
treated with the compounds was considered to be 100 %
(control).
Table ~
~ffect on the 3H-t~ymidine uptake of ~K-M~S-l
epithalial-cell lung cancer cell line
Thymidi~e ~ptake n~ perce~tage of control
Concentration Compound
~mollll (I) (2) (3) (4) (5) (6)
~ 100 lO0 lO0 lOQ 100 100
6.2~ 1~8 33 10~ 81 44 63
s
Table ~ (cont~.)
Thymi~ine upt~e as perce~tage of control
Concentration Compound
~umolll] (I) (2) 13) (4) (5) (6~
12.~ 179 25 92 98 6 58
187 17 65 64 lO 49
67 1 1 6 1 44
The data of Table 4 indicate that the spectrum of
inhibitory effects of the compounds according to the
invention on the proliferation of lung cancer cells is
broader than those of the known SP antagonists [see the
reference substance of formula (I)~. The compounds of the
invention prevent also the proliferation of SK-MES-l
epithelial-cell lung cancer cell line.
According to a further aspect of the present
invention there are provided pharmaceutical compositions
comprising as aotive ingredient at least one peptide or
pseudopeptide amide of the formulae (1) - (6) of the
present invention or an acid addition salt thereof in
admixture with suitable inert pharmaceutical carriers.
The pharmaceutical compositions of the present invention
can be used in therapy for inhibiting the proliferation
of cells of the small-cell and epithelial-cell lung
cancer.
The peptide and pseudopeptide amides of the
formulae (1) - (6) and salts thereof are formulated in
forms generally used in therapy by methods of
pharmaceutical industry known per se. The pharmaceutical
compositions of the present invention may be formulated
in solid, liquid or semiliquid forms and may contain one
or more of generally used conventional carriers,
diluents, fillers, auxiliary agents (e.g. stabilizing
agents, salts for modifying the osmotic pressure), agents
for adjusting the pH value and further additives.
The solid pharmaceutical compositions may be e.g.
tablets, dragées, capsules, wafers or powder ampouls
useful in the preparation of injections. The liquid
compositions may be e.g. injections, infusions,
spoonfuls, wet packs and drops. The semiliquid
compositions may be e.g. creams, ointments, balms,
shaking mixtures or suppositories.
The pharmaceutical compositions of the present
invention are administered in an amount which contains
sufficient active ingredient to exhibit the desired
effect.Thesaid dosedependsonthetypeand severenessofthe
disease, the body weight of the patient and his (or her~
sensitivity against the active ingredient, the mode of
application, the daily number of treatments, etc. The
dose to be applied can be safely determined by the
physician based on all circumstances of the given case.
In order to enable simple administration, the
active ingredient is preferably finished im the form of
dosage units which contain the active ingredient in the
amount to be administered or a small multiple or part
(e.g. half, one-third, one-fourth part) thereof. Such
dosage units are e.g. the tablets which may be provided
by groove(s) in order to simplify the division of a
tablet into two or four parts.
To ensure the hypogastric absorption of the active
ingredient Ihe tablets may be provided with a coating
not soluble i~ acidic medium; i.e the tablets can be
rendered enterosolvent. Similar effect can be achieved by
encapsulating the active inqredient.
The pharmaceutical compositions of the present
invention ~ay generally contain from about 1 mg to about
100 mg of the active ingredient per dosage unit. The
above values are naturally of a mere illustrative
character and the actual active ingredient content can be
below or above the said limits as well.
The invention is illustrated in detail by the aid
of the following non-lim-ting Examples.
Melting points were determined on a Tottoli's
device (Buchi, Switzerland).
Thin layer chromatography (TLC) examinations were
carried out on prefabricated silica gel adsorbent layers
(DC-Fertigplatten, Merck) by using the following solvent
systems:
1. Ethyl acetate:stock solution = l9:1 (sign1)
2. Ethyl acetate:stock solution = 4:1 (sign2)
3. Ethyl acetate:stock solution = 39:1 (sign3)
4. Ethyl acetate:methanol:n-hexane = 6:1:3 (sign4).
The stock solution was a 20:6:11 mixture of
pyridine/acetic acid/water. The above quotients are
volume ratios.
The chromatograms were detected by ninhydrin as
well as by using KI/o-tolidine reagent after chlorina-
tion.
High pressure liquid chromatography (HPLC) examina-
tions were carried out on a BST Nucleosil 300 C18 5 ~m
column. A Gilson 305 detector connected with a Gilson
pump was used in these examinations. As eluent a 2:3 mix-
ture of acetonitrile/water containing 0.1 ~ of trifluoro-
acetic acid was used with a flow rate of 1.2 ml/min.
When investigated in this system, the purity of the
compounds of the invention was found to be at least 95 %.
For amino acid analyses, the samples were parallel
hydrolyzed in 6 mol/l hydrochloric acid an~ mercapto-
sulfonic acid at 110 C under nitrogen for 2~ hours. The
hydrolysates were examined in a Biotro~i~ LC 5000 amino
acid analyzerO In addition to the common amino acids,
MePhe and Leu ~ CH2NH)Leu pseudodipeptide were also
detected in the hydrolysate.
NMR spectra of the new compounds ~ere taken on a
Varian VXR-300 type NMR spectrometer at room temperature
in deuterated dimethylsulfoxide (DMSO-d6) solution, by
using tetramethylsilane (TMS~ as internal standard. The
signal of the CH2 moiety of the methyleneamino group
appeared in the lH-NMR spectrum as a multiplet close to
2.4 ppm, whereas the signal of the CH2 moiety showing a
triplet multiplicity was found close to 52 ppm in the
13C-NMR spectrum and simultaneously, the signal of the
carbon atom in the C=O moiety of the peptide bond was
absent.
kxample 1
Preparation of Z-NePhe-D-Trp-Phe-D-Trp-Leu ~(C~2N~)Leu-
NH2
(1) (a) Z-~eu r(CH2N~)Leu-N~2
After suspending 3.6 g (100 mmol) of lithium alumi-
num hydride in 30 ml of tetrahydrofuran (THF) cooled to
O C, the solution of 14.6 g (200 mmol) of diethylamine
in 50 ml of a-hexane is dropwise added thereto at O C.
After stirring the suspension at O C for 10 minutes,
21.3 g (76 mmol~ of Z-Leu-OCH3 dissolved in 120 ml of n-
-hexane are portionwise added. After stirring at O C for
90 minutes, the reaction mixture is diluted with 70 ml of
ethyl acetate and acidified to pH 2 by adding 6 mol/l
hydrochloric acid solution under strong cooling. After
separation of the two phases, the aqueous layer is
extracted with ethyl acetate, then the combined organio
phase is washed with cold water. The ethyl acet~te
solution is dried over anhydrous sodium sulfate and
evaporated.
The Z-Leu-H aldehyde obtained (18 g, 69 mmol) is
dissolved in 300 ml of a 99:1 mixture of methanol/acetic
acid cooled to O C and 8 g (50 mmol) of Leu-NH2.HCl are
added. To the solution obtained 4.8 g (80 mmol) of sodium
cyanoborohydride dissolved in 100 ml of anhydrous
~ ~ . t
23305-1213
- 15 -
tetrahydrofuran are dropwise added at O C. The reaction
mixture is stirred at o C for 30 minutes and then at
room temperature for 2 hours.
After evaporating the solvent, the residue is dis-
solved in 300 ml of ethyl acetate, washed twice with 100ml of 5 % sodium hydrogen carbonate solution each and
twice with water. After drying, the solution is evapo-
rated and the residue is solidified by adding 50 ml of n-
-hexane. The crude product is recrystallized from 20 ml
of ethyl acetate to obtain 7 g (38 % yield calculated for
Leu-NH2 HCl) of Z-Leu ~(CH2NH)Leu-NH2, m.p.: 108-110 C.
(1)(b) H-Leu ~(CH2N~)Leu-NH2~2HCl
To a solution containing 5.0 9 (13.7 mmol) of
Z-Leu r(CH2NH)Leu-NH2 in 100 ml of methanol, 5 ml of 5.3
mol/l hydrogen chloride solution in dioxane and then 0.5
g of 10 % palladium-on-carbon catalyst are added, then
hydrogen is bubbled through the suspension.
The reaction becomes complete within 2 hours. After
filtering off the catalyst, the filtrate is evaporated
under reduced pressure, the residue is solidified by
adding 50 ml of ether and the crude product obtained is
recrystallized from an 1:5 mixture of ethanol and ether
to give the aimed product in a yield of 3.4 g (82 S);
t~]D = +7-3 (c = 2, methanol), m.p.: 110 C ( decomposi-
tion).
c) 5~-MeP~e-D-Trp-Phe-D-'rrp-Leu ~( CH2N~{)Leu-N~2
1.12 ml (8 mmol) of triethylamine and 4.0 g (8.5
mmol) of Boc-D-Trp-OPfp (OPfp means pentafluorophenoxy
group) are added to a solution of 2.4 g (8 mmol) of
Leu (C~2NH)Leu-NH22HCl in 30 ml of dimethylformamide at
O C. After stirring the reaction mixture at room tem-
perature ~or 90 minutes, meanwhile portionwise adding 1.5
ml of triethylamine, the reaction mixture is evaporated
4 ~
- 16 -
and the residue is dissolved in 100 ml of ethyl acetate.
The solution is twi~e extracted with 30 ml of 1 mol/l
hydrochloric acid each and then with 30 ml of water. The
combined aqueous phase is neutralized by adding sodium
carbonate and extracted twice with 30 ml of ethyl acetate
each. After drying over anhydrous sodium sulfate, the
ethyl acetate solution is evaporated. The protected
tripeptide is solidified by adding ether to obtain a
yield of 3.2 g (77 %), m.p.: 180 - 182 C.
Rfl = 0 3 (1 means solvent system 1, 2 means solvent
system 2, etc.)
After dissolving 3.0 g (5.8 mmol) of Boc-D-Trp-Leu-
~(CH2NH)Leu-NH2 in 15 ml of an 5.3 mol~l hydrogen
chloride solution in dioxane, the solution is diluted
with 100 ml of ether after 20 minutes. The precipitate is
filtered off and washed with ether to give 2.8 g (98 %)
of product, m.p.: 170 C (decomposition); Rf2 = 0.1.
After adding 1.4 ml (10 mmol) of triethylamine and
2.3 g (5.3 mmol) of Boc-Phe-OPfp to t~e solution of 2.45
g (5 mmol) of H-D-Trp-Leu r(CH2NH)Leu-NH2-2HCl in 30 ml
of dimethylformamide, the reaction mixture is stirred at
room temperature for 1 hour, then the dimethylformamide
is distilled off and the residue is solidified by adding
50 ml of water. The precipitate obtained is filtered,
washed with 50 ml of water and 30 ml of ethanol to obtain
the pseudotetrapeptide Boc-Phe-D-Trp-Leu ~(CH2NH)Leu-NH2
in a yield of 2.73 g (82 ~), m.p.: 212-214 C (decompo-
sition); Rf2 - 0 7
2.5 g (3.7 mmol) of the pseudotetrapeptide
Boc-Phe-D-Trp-Leu ~ CH2NH3Leu-NH2 are dissolved in 30 ml
of 5.3 molJl hydrogen chloride solution in dioxane and
after 30 minutes, the mixture is diluted with 100 ml of
eth~r. After filtering and washing with 25 ml of ether,
the precipitate is dissolved in 25 ml of dimethyl-
formamide. To this solution, 1.3 ml of triethylamine and
- 17 - 23305-1213
1.9 ~ (4 mmol) of Boc-D-Trp-OPfp are added, the reaction
mixture is stirred at room temperature overnight, then
dimethylformamide is distilled off. The residue is dis-
solved in 50 ml of ethyl acetate and the solution is
twice shaken with 20 ml of 1 moltl hydrochloric acid
solution each and then with 20 ml of water. The aqueous
phases are extracted with 20 ml of ethyl acetate each.
The combined organic solution is dried over anhydrous
sodium sulfate and evaporated. The residue is solidified
by addin~ ether to obtain the pseudopentapeptide in a
yield of 2.83 g (90 %~, m.p.: 133 C (decomposition);
Rf2 = 0 7
2.6 g (3 mmol) of Boc-D-Trp-Phe-D -Trp-Leu ~-
-(CH2NH)Leu-NH2 are treated with 20 ml of 5.3 mol/l
hydrogen chloride solution in dioxane, and after 30
minutes, the solution is diluted with 100 ml of ether.
After filtering the precipitate and washing with ether,
the free pseudopentapeptide dihydrochloride is obtained
in a yield of 2.4 g (92 %), m.p.: 167 C (with
decomposition); Rf2 = 0.15.
After dissolving 1.23 g (1.48 mmol) of H-D-Trp-Phe-
-D-Trp-Leu ~(CH2NH)Leu-NH2 2HCl in 20 ml of dimethylform-
amide, 0.96 g (2 mmol) of Z-D-MePhe-OPfp and 0.7 ml (5
mmol) of triethylamine are added to the above solution.
After stirring the reaction mixture at room temperature
for 1 hour and evaporating, the residue is dissolved in
30 ml of chloroform. The chloroform solution is twice
washed with 10 ml of water each. The organic solution is
dried over anhydrous sodium sulfate and then evaporated.
The final product is solidified by adding 50 ml of ether
to obtain Z-NePhe-D-Trp-Phe-D-Trp-Leu~r-(CH2NH)Leu-NH2 in
a yield of 1.32 g (~5 %), m.p.: 88 C (decomposition);
t~D20 = +5.7~ (c = 1, methanol).
?
- 18 -
Exampl~ 2
Preparation of H-Leu-MPA
After suspending 26.7 g (60 mmol) of Z-Leu-OH di-
cyclohexylamine salt in 300 ml of ethyl acetate, the salt
is decomposed by 150 ml of 1 mol/l sulfuric acid
solution. The ethyl acetate solution is dried over
anhydrous sodium sulfate and evaporated~ Z-Leu-OH ob-
tained as a residue is dissolved in 160 ml of anhydrous
tetrahydrofuran. After cooling the solution to -10 C,
lo 9.4 ml (67 mmol) of triethylamine are added, and then 9.O
ml (73 mmol) of pivaloyl chloride are dropwise added
while maintaining the temperature at -10 C. After
stirring the reaction mixture at -lQ C for 15 minutes, a
solution containing 6.7 g (74 mmol) of 2-amino-3-
-methylpentane in 20 ml of tetrahydrofuran are portion-
wise added. The reaction mixture is stirred at 0 C for
30 minutes and at room temperature for 3 hours and then
evaporated. The residue is dissolved in 300 ml of ethyl
acetate and the solution is successively washed twice
with 120 ml of 1 mol/l hydrochloric acid solution each,
twice with 5 % sodium hydrogen carbonate solution and,
finally, once with 120 m' of water. After drying and
evaporation, Z-Leu-methylpentylamide is obtained as an
oil in a yield of 20.5 g (98 ~); Rf3 -- 0.8.
Th~ obtained oily product is dissolved in 200 ml of
methanol and hydrogenated in the presence of 2.5 g of 10
~ palladium-on-carbon catalyst. The reaction becomes
complete within 90 minutes. Subsequently, after filtering
off the catalyst and evaporating methanol, the oily
residue is separated to two components on a silica gel
column (80x2.5 cm size; 250 g of silica gel; eluent:
6:1:3 mixtuxe of ethyl acetate/methanol/n-hexane; flow
rate: 40 ml/hour) and the fractions collected are
evaporated. The yield of the thus obtained isomer of H-
Leu-MPA is 4r2 g ~19.5 mmol); Rf4 = 0-3, r~D24 = +11.6
-- 19 --
(c = 1, methanol); [~]D24 = -20.4 (c = 1, ethyl
acetate).
In the following, this isomer is used as described
in Example 1. The characteristics of the protected
peptide amides and pseudopeptide amides obtained in this
synthesis are indicated in Table 5.
~xnmpl~ 3
Preparation of D-MePhe-~-Trp-Phe-D-Trp-~eu ~CH_NH)Leu-Na
A solution containing 1.0 g (0.96 mmol) of Z-D-
-MePhe-D-Trp-Phe-D-Trp-Leu r(CH2NH)Leu-NH2 in 25 ml of
methanol is hydrogenated in the presence of 0.3 g of
palladium-on-carbon catalyst for 3.5 hours. After filter-
ing off the catalyst and evaporating the solution, the
residue is solidified by adding ether to obtain the crude
title product ln a yield of 0.73 g.
The crude final product is purified on a silica gel
column by using a 4:1 mixture of ethyl acetate with a
20:6:11 mixture of pyridine/acetic acid/water as eluent
with a flow rate of 20 ml/hour. Fractions of 10 ml each
are collected; the purity of the fractions is observed by
thin layer chromatography. After evaporating the frac-
tions containing the pure final product, repeatedly twice
10 ml of water each and 10 ml of ethanol each are added
to the evaporation residue in such a way that before
adding an additional amount of liquid, the amount of
liquid previously added is distilled off. The final evap-
oration residue is solidified by adding ether and after
filtering, the precipitate is washed with ether to obtain
the amorphous title product in a yield of 0.32 g;
~]D20 = -22.8 (c = 0.5, 10 % acetic acid).
Amino acid analysis: Phe 1.0 (1), Trp 1.7 (2),
MePhe, Leu ~(CH2NH)Leu.
The characteristics of the new compounds are
summarized in Table 6.
? ~`t
I V . '
- 20 - 23305-1213
Table 5
Compound Yield M.p. [~]D2
( c=l, methanol )
1%) (C) ( )
Z-D-Me-Phe-D-Trp-Phe-D-Trp-
-Leu ~(CH2NH)Leu-NH2 75 120 (d~ +1.7
Z-D-Me-Phe-D-Trp-Phe-D-Trp-
-Leu-MPA 56 109 (d) -22.9
Boc-D-Tyr-D-Trp-Phe-D-Trp-
-Leu r(cH2NH)Leu-NH2 71 136 ~d) -19.2
Boc-D-Tyr(Et)-D-Trp-Phe-D-
-Trp-Leu ~(CH2NH)Leu-NH2 58 114 (d) -23.4
Z-D-Me-Phe-D-Trp-D-Tyr(Bzl)-
15 -D-Trp-Leu r(CH2NH)Leu-NH2 74 88 (d) +5.7
Note: (d) means: decomposition.
Table C
20 Compound M.p.[~]D2
( C)( ~
p-HOPA-D-Trp-Phe-D-Trp-
-Leu ~r(CH2NH)Leu-NH2 132-137-29.1 (c=l, 50% AcOH)
D-Me-Phe-D-Trp-Phe-D-Trp-
25 -Leu r(CH2NH)LeU-NH2 90 (d) -22.8 (c=0.5, 10~ AcOH)
D-Me-Phe-D-Trp-Phe-D-Trp-
-Leu MPA 153-155 -41.0 (c=l, EtOH)
D-Tyr-D-Trp-Phe-D-Trp-
-Leu ~(CH2NH)Leu-NH2 las (d~ -17.1 (c=l, MeOH)
D-Tyr~Et)-D-Trp-Phe-D-Trp-
-Leu ~ (cH2NH)Leu-NH2 166 (d) -35.2 ~c=1, MeOH)
D-Me-Phe-D-Trp-Tyr -D -Trp-
-Leu ~(CH2NH)Leu-NH2 110 (d) -18.5 (c=l, 10~ AcOH)
Note: (d) means: decomposition.
- 21 -
Ex~mple ~
Powder ampoule for injection purposes
500 mg. of active ingredient and 9.5 g. of lactose
are dissolved in 80 ml. of distilled water suitable for
injection purposes. 0.1 g. of methyl ~-hydroxybenzoate
are added to the solution, then the volume of this
solution is adjusted to 100 ml. by using distilled water
suitable for injection purposes. The homogenic solution
is filtered to sterile, filled in a volume of 1 ml. each
into vials fitted with gum cap, subjected to freeze-
drying and finally the vials are provided with a gum
plug. Powder ampoules containing 5 mg. of active
ingredient each are obtained.
If one intends to obtain ampoules with a different
active ingredient content, the amount of lactose is
selected in such a manner that, based on 100 ml. of the
solution, the common weight of the active ingredient and
lactose be about 10 g. One can use mannitol in the same
amount instead of lactose.
When administering an injection the powder of these
ampoules is dissolved in an aqueous sodium chloride
solution of such concentration which allows to obtain an
isotonic solution.
