Note: Descriptions are shown in the official language in which they were submitted.
~ \
o~
This in~ention relates to the tetxadecapeptide D-Val-
Gly-L-C~s-L~Lys-L-Asn~L-P~e-L-Phe-D~T~p~L~Lys~L~Thr~L -
Phe-L-Thr-L-Ser~L-Cys-OH, formula I-, as well as to its pharma-
ceutically acceptable acid addition salts and to intermediates
produced during the synthes~s of this tetradecapeptide.
Somatostatin (also known as somatotropin release in-
hibiting factor) ~s a tetradecapeptide of the formula
L-Ala-Gly-L-Cys-L~Lys-L-Asn-L-Phe-L-Phe-L-Trp-L-Lys-L -
Thr-L-Phe-L-Thr-L-Ser-L-Cys-OH. This tetradecapeptide was
isolated from ovine hypothalamic extracts and was found to be
active in inhibiting the secretion of growth hormone (GH), also
known as somatotropin. In this regard, see P. Brazeau, W. Vale,
R. Burgus, N. Ling, M. Butcher, J. Rivier, and R. Guillemin,
Science, 179, 77 (1973).
In addition, the compound conveniently designated as
D-Trp -somatostatin was previously reported by Brown et al.,
Endocri _logy, 98, No. 2, 336-343 (1976).
. -- '
The biologically active tetradecapeptides of formula I
have the formula defined above and include the non-toxic acid
addition salts thereof. Their structures differ from that of
somatostatin by the presence of a D-tryptophan resi~ue in posi-
tion 8 in place of an L-tryptophan residue and a D-valine
-- residue in position 1 in place of an L-alanine residue. For
convenience sake, the tetradecapeptides of formula I can be
referred to as D-Val , D-Trp -somatostatin.
Thus, this invention relates to a compound selected
from those of the formula H-D-Val-Gly L-Cys-L-Lys-L-Asn-L-Phe -
L-Phe-D-Trp-L-Lys-L-Thr-L-Phe-L Thr-L-Ser-L-Cys-OH and their
; pharmaceutically-acceptable non-toxic acid addition salts, and,
as intermediates, ~ Gly-L-Cys(Rl)-L-Lys(R2)-L-Ans-L Phe-L-
Phe-D Trp(R5)-L-LysrR2)-L-Thr(R3)-L-phe-L-Thr(R3)-L-ser(R4)-L
Cys(Rl)-X, formula II, in which
2 -
: : i ,
~Z~3~
R is hydrogen or an a-amino protecting group;
Rl is hydrogen or a thio protecting group;
R2 is hydrogen or an ~-amino protecting group;
R3 and R4 each are hydrogen or a hydroxy protecting
group;
R5 is hydrogen or formyl; and
X is hydroxy or
0= ~ ~esin
-0-~ --o~
in which the resin is polystyrene; with the proviso that, when X
is hydroxy, each of R, Rl, R2, R3, R4, and R5 is hydrogen, and,
when X is
o = ~ ~ esin
~o~
each of R, Rl, R2~ R3, and R~ is other than hydrogen.
The no~el tetradecapeptide of formula I above is pre-
pared by xeacting the corresponding straight-chain tetradecapep-
2Q tide of ormula III, D~Val-Gly-L-Cys-L-Lys-L-Asn-L-Phe-L-Phe- ~ -
D-Val-Gly-L~Cys-L-Lys-L-Asn-L-Phe-L~-Phe-D-Trp-L-Lys-L-Thr-L-
Phe-L-Thr-L-Ser-L-Cys-OH, with an oxidizing agent. This reaction
converts the two sulfhydryl groups to a disulfide bridge.
Pharmaceutically acceptable non-toxic acid addition
salts include the organic and inorganic acid addition salts, for
example, those prepared from acids such as hydrochloric, sulfuric,
sulfonic, tartaric, fumaric, hydrobromic, glycolic, ci~ric, maleic
phosphoric, succinic, acetic, nitric, benzoic, ascorbic, p-tolue-
nesulfonic, benzenesulfonic, naphthalenesulfonic, and propionic.
Preferably the acid addition salts are those prepared from acetic
acid. Any of the above salts are prepared by conventional methods
Also contemplated as ~ein~ within the scope of this
invention are intermediates of the formula II, D-Val~Gly-L-Cys
(Rl)~L-Lys(R2)~L-Asn-L-Phe-L~Phe-D~Trp(R5)-L~Lys(R2)-L-Thr(R3)-
L-Phe-L-Thr(R3~-I,-Ser(R4)-L~Cys(Rl)-X wherein the various symbols
are defined as before.
H-D-Val-Gly-L-Cys-L-Lys-L-Asn-L~Phe-L Phe-D-Trp-L-Lys-
L-Thr-L-Phe-L-Thr-L-Ser~L-Cys-OH;
N-(BOC)-D-Val-Gly-L-(PMB)Cys-L-(CBzOC)-Lys-L-Asn-L-
Phe-L-Phe-D-Trp-L-(CBzOC)-Lys-~-(Bzl)Thr-L~Phe-L(Bzl)Thr-L-(Bzl)
~==~esin
Ser-L-(PMB)Cys-O-CH2 ~ ~o ;and
The a~o~e formulas defining the intermediates include
protect~ng groups for amino, hydroxy, and thio (sulfhydryl)
functions. The properties of a protecting group as defined herein
are two-fold. First, the protecting group prevents a reactive
; moiety present on a particular molecule from undergoing reaction
; during subjection of the molecule to conditions which could cause
disruption of the otherwise active moiety. Secondly, the pro- -
tecting group is such as can be readily removed with restoration
2Q of the original actiVe moiety and under conditions which would
not undesirably affect other portions of the molecule. Groups
which are useful for these purposes, that is, for protecting
amino, hydroxy, and thio groups, are well recognized by those
skilled in the art. Indeed, entire volumes have ~een directed
specifically to a description and discussion of methods for using
such groups. One such volume is the treatise Protective Groups
in Organ~c he-mis-try, J. F. W. McOmie, Editor, Plenum Press,
~ New York, 1973.
; In the abo~e formulas defining the intermediates, R
represents either an a-amino hydrogen or an a-amino protecting
group. The ~-amino protecting groups contemplated for R are well
recognized by those o~ ordinary skill in the peptide art. Many
''', ~-
: . :
~LZ003C~
of these are detailed in McOmie~ supra, Chapter 2, authored b~
J. W. Barton. Illustrati~e of such protecting groups are benzyl-
loxycarbonyl, _~chlorobenzyloxycarbonyl, ~-bromobenzyloxycarbonyl,
o-chlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, 2,4-
dichlorobenzyloxycarbonyl, o bromobenzyloxycarbonyl, p-methoxy-
benzyloxycarbonyl, ~nitrobenzyloxycarbonyl, t-butyloxycarbonyl
(BOC), t-amyloxycarbonyl, 2~ biphenylyl)isopropyloxycarbonyl
(BpOC), adamantyloxycarbonyl, isopropyloxycarbonyl, cyclopentyl-
oxycarbonyl, cyclohexyloxycarbonyl, cycloheptyloxycarbonyl, tri-
phenylmethyl (trityl), and p-toluenesulfonyl. Preferably, the
~-amino protecting group defined by R is t-butyloxycarbonyl.
Rl represents either the hydrogen of the sulfhydryl
group of the cysteine or a protecting group for the sulfhydryl
substituent. Many such protecting groups are described in
McOmie, supra, Chapter 7, authored by R. G. Hickey, V. R. Rao,
and W. G. Rhodes. Illustrative suitable such protecting groups
are _-methoxybenzyl, benzyl, p-tolyl, benzyhydryl, acetamido-
methyl, trityly _-nitrobenzyl, t-butyl, isobutyloxymethyl, as
well as any of a number of trityl deri~atives. For additional
groups, see, for example, Houben-Weyl, Methodes der Organischen
Chemie, "Synthese von Peptiden", Vols. 15/1 and 15/2, (1974),
Stuttgart, Germany. Preferably, the sulfhydryl protecting group
defined by Rl is p-methoxybenzyl.
R2 represents either hydrogen on the -amino function
of the lysine residu or a suitable ~-amino protecting group.
Illustrative of such groups are the bulk of those mentioned
hereinabove as being suitable for use as an ~-amino protecting
group. Included as typical such groups are benzyloxycarbonyl,
t-butyloxycarbonyl, t-amyloxycarbonyl, cyclopentyloxycarbonyl,
: 3a adamantyloxycarbonyl, ~-methoxybenzyloxycarbonyl, p-chlorobenzyl-
oxycarbonl, p-bromobenzyloxycarbonyl, o-chlorobenzyloxycarbonyl,
2,6-dichlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,
- 5 -
~ z~1~30
o-bromobenzylQxyc~xbonyl) ~nitxo~enzylox~caxhonyl, isopxop~loxy-
carbon~l, cyclohexyloxycarbonyl, cycloheptyloxycarbon~l, and ~-
toluenesul~onyl.
As will become apparent hereinafter, the process for
the preparation of the tetradecapeptides of formula I involves
periodic cleavage of the ~amino protecting group from the ter-
minal amino acid present on the peptide chain. Thus, the only
limitation with respect to the identity of the -amino protecting
group on the lysine residue is that it be such that it will not
be cleaved under the conditions employed to selectively cleave
the ~-amino protecting group. Appropriate selection of the ~-
amino and the - amino protecting groups is a matter well within
the knowledge of a peptide chemist of ordinary skill in the art
and depends upon the relative ease with which a particular pro-
tecting group can be cleaved. Thus, groups such as 2-(_~iphenylyl) -
~isopropyloxycarbonyl (BpOC) and tityl are very labile and can
be cleaved even in the presence of mild acid. A moderately
strong acid, such as hydrochloric acid, trifluoroacetic acid, or
boron trifluoride in acetic acid, is required to cleave other ;;
groups such as t~butyloxycarbonyl, t-amyloxycarbonyl, adamantyl-
oxycar~onyl, and p-methoxybenzyloxycarbonyl. Even stronger acid
conditions are re~uired to effect cleavage of other protecting ~-
~roups such as benzyloxycarbonyl, haloben~yloxycarbonyl, ~-
nitrob~nzyloxycarbonyl, cycloalkyloxycarbonyl, and isopropyloxy-
carbonyl. Cleavage of these latter groups requires drastic acid
conditions such as the use of hydrogen bromide, hydrogen fluoride,
or boron trifluoroacetate in trifluoroacetic acid. Of course, any
of the more labile groups will also be cleaved under the stronger
acid conditions. Appropriate selection of the amino protecting
3Q ~roups thus will include the use of a group at the a-amino
function which is-more labile than that employed as the -amino
protectin~ group coupled with cleavaye conditions designed to
6 ~
,
1~2E)030
selectively re~o~e only the ~Amino function In this context~
R2 preferably is o~chlorobenz~loxycarbonyl or cyclopentyloxy-
carbonyl, and, in con~unction therew-i~h, the ~amino protecting
group of choice for use in each of the amino acids which is
added to the peptide chain prefera~ly is t~utylo~ycarbonyl.
The yroups R3 and R4 represent the hydro~yl hydrogen or
a protecting group for the alcoholic hydroxyl of threonine and
serine, respectively. Many such protecting groups are described
in McOmie, supra, Chapter 3, authored by C. B. Reese. Typical
such protecting groups are, for example, Cl-C4 alkyl, such as
methyl, ethyl, and t-butyl; benzyl; substituted benzyl, such as
~-methoxybenzyl, ~nitrobenzyl, p-chlorobenzyl, and o-chloroben-
zyl; Cl-C3 alkanoyl, such as formyl, acetyl, and propionyl; tri-
phenylmethyl (trityl~. Preferably, when R3 and R4 are protecting
groups, the protecting group of choice in both instances in benzyl.
The group R5 represents either hydrogen or formyl and
defines th~ moiety ~ NR5 of the tryptophan residue. The formyl
serves as a protecting group. The use of such a protecting ~roup
is optional and, therefore, ~5 properly can be hydrogen (N-unpro-
2Q tected) or formyl (N-protected~.
The group X relates to the carboxyl terminal of the
tetradecapeptide chain; it can be hydroxyl, in which case a free
carboxyl group is defined. In addition, X represents the solid
resin support to which the carboxyl terminal moiety of the pep-
tide is linked during its synthesis. Irhis solid resin is repre-
sented by the formula
~ esin
-O--CH2--~
o--~--
; 3Q In any of the above, when X represents hydroxyl, each
of R, Rl, R2, R3, R4, and R5 is hydrogen. When X represents the
solid resin support, each of R, Rl, R2, R3, and R4 is a protec~
ting group.
ï' - 7 -
3!~
The following ~bhreyiations~ most o$ which are ~ell
known and commonl~ used in the art, are employed herein:
Ala - Alanine
Asn - Asparagine
Cys - Cysteine
Gly ~ Glycine
Lys - Lysine
Phe - Phenylalanine
Ser - Serine
Thr ~ Threonine
Trp ~ Tryptophan
~al ~ ~aline
DCC ~ N,N'-Dicyclohexylcarbodiimide
DMF - N,N-Dimethylformamide
BOC - t-Butyloxycarbonyl
PMB ~ ~-Methoxybenzyl
CBzOC ~ o-Chlorobenzyloxycarbonyl
CPOC - Cyclopentyloxycarbonyl
Bzl - Benzyl
For ~ Formyl
- BpOC ~ 2~ biphenylyl~isopropyloxycarbonyl
Although the selection of the particular protecting
groups to be employed in preparing the compounds of ~ormual I
remains a matter well within the ordinary skill of a synthetic
peptide chemistr it is well to recognize that the sequence of
reactions which must be carried out gives rise to a selection of
particular protecting groups. In other words, the protecting
group of choice must be one which is stable both to the reagents
and under the conditions employed in the succeeding steps of the
3a reaction sequence. For example, as alread~ discussed to some
degree hereinabove~ the particular protecting group which is em-
plo~ed must be one which remains intact under the conditions
- 8 ~
.'~.
'
, ~, .
3~
which are emplo~ed ~or cle~y~n~ the ~min~ pxotecting ~roup o~
the terminal amino acid residue of the peptide fragment in prep-
aration for the couplin~ of the next succeeding amino acid frag-
ment to the peptide ch~in. It is also important to select, as a
protectin~ group, one which will remain intact during the building
of the peptide chain and which will be readily removable upon
completion of the synthesis of the desired tetradecapeptide pro-
duct. All of these matters are well within the knowledge and
understanding of a peptide chemist of ordinary skill in the art.
As is evident from the above discussion, the tetr~de-
capeptides of formula I can be prepared by solid phase synthesis.
This synthesis involves a sequential building of the peptide chain
beginning at the C~terminal end of the peptide. Specifically,
cysteine first is linked at its carboxyl function to the resin
by reaction of an amino-protected, S-protected cysteine with a
chloromethylated resin or a hydroxyme~hyl resin. Preparation of
a hydroxymethyl resin is described by Bodanszky et al., Chem. Ind.
(London)~ 38 1597~98 (1966). The chloromethylated resin is com-
mercially available from Lab Sy~tems, Inc., San Mateo, California.
`, 20 In accomplishing linkage of the C-terminal cysteine to
the resin, the protected cysteine first is converted to its
cesium salt. This salt then is reacted with the resin in accor-
dance with the method described by B. F. Gisin, Helv. Chim. Acta,
56, 1476 tl973). Alternatively, the cysteine can be linked to
the resin by activation of the carboxyl function of the cysteine
molecule by application of readily recognized techniques. For
example, the cysteine can be reacted with the resin in ~he pre-
` sence of a carboxyl ~roup activating compound such as N,N'-di-
-~ cyclohexylcarbodiimide (DCC).
Once the free carboxyl cysteine has been appropriately
linked to the resin support, the remainder of ~he peptide buil-
ding sequence involves the step-wise addition of each amino acid
~'
`
to the N~terminal p~rtion of the peptide chain~ Necessaxily~
therefore, the particular sequence which is Lnvolved comprises
a cleavage of the ~-amino protecting group from the amino acid
~hich represents the N~terminal portion of the peptide fragment
~ollo~ed by couplin~ of the next succeedin~ amino acid residue
to the now-free and reactive N~terminal amino acid. Cleavage of
the ~-amino protectinq group can be effected in the presence of
an acid such as hydrobromic acid, hydrochloric acid, trifluoro-
acetic acid~ ~-toluenesulfonic acid, benzenesulfonic acid, naptha-
lenesulfonic acid, and acetic acid, with formation of the respec-
tive acid addition salt product. Another method which is avail-
able for accomplishing cleavage of the amino protecting group
involves the use of boron trifluoride. For example, boron tri-
fluoride diethyl etherate in glacial acetic acid will convert
the amino-protected peptide fragment to a BF3 complex which then
can be converted to the deblocked peptide fragment by treatment
with a base such as aqueous potassium bicarbonate. Any of these
methods can be employed as long as it is recognized that the
method of choice must be one which accomplishes cleavage of the
N-terminal a-amino protec~ing group without disruption of any
other protecting ~roups present on the peptide chain. In this
regard, it is preferred that the cleavage of the N-terminal pro-
tecting group b~ accomplished using trifluoroacetic acid~
Generally, the cleavage will be carried out at a temperature -
from about 0C. to about room temperature.
Once the N-terminal cleavage has been effected, the
product which results normally will ~e in the form of the acid
addition salt o~ the acid which has been employed to accomplish
the cleavage of the protecting groupO The product then can be
converted to the free terminal amino compound by treatment with
a mild base, typically a tertiary amine such as pyridine, or tri-
ethylamine.
- 10 -
!/ ` .
. ..
,
200~
The peptide chain then is xeady fo~ reaction with the
next succeeding amino acid. This can ~e accomplished by employ-
ing any of several recoqnized techniques. In order to achieve
coupling of the next~succeeding amino acid to the N~terminal
peptide chain, an amino acid which has a free carboxyl but which
is suitably protected at the a~amino function as well as at any
other active moiety is employed. The amino acid then is sub-
jected to conditions ~hich will render the carboxyl function
acti~e to the coupling reaction. One such activation technique
which can ~e employed in the synthesis involves the conversion
o~ the amino acid to a mixed anhydride. Thereby, the free car-
boxyl function of the amino acid is activated by reaction with
another acid, typically a carbonic acid in the form of its acid
chloride. Examples of such acid chlorides which can be used to
form the appropriate mixed anhydrides are ethyl chloroformate,
phenyl chloroformate, sec~butyl chloroformate, isobutyl chloro-
formate, and pivaloyl chloride.
Another method of activating the carboxy function of
the amino acid to achieve coupling is by conversion of the amino
acid to its active ester derivative. Examples of such active
esters are, for example, a 2,4,5-trichlorophenyl ester, a penta-
chlorophenyl ester~ a ~-nitrophenyl ester, an ester formed from
l-h~droxybenzotriazole, and an ester formed from N-hydroxysuccin-
imide. Another method for effecting coupling of the C-terminal
amino acid to the peptide fragment involves carxying out the
couplin~ reaction in the presence of at least an equLmolar quan-
tity of N,N~-dicyclohexylcarbodiimide (DCC). This latter method
is pre~erred for preparing the tetradecapeptide of formula II
where x is
R~, i n
-O--CH ~0~ ~
!
~2~ 3~3t
Once the desired amino acid sequence has been prepared,
the resulting peptide can be removed from the resin support.
This is accomplished by treatment of the protected resin-supported
tetradecapeptide with hydrogen fluoride. Treatment with hydrogen
~luoride cleaves the peptide from the resin; in addition, however,
it cleaves all remaining protecting groups present on the reactive
moieties located on the peptide chain as well as the ~-amino pro-
tecting group present at N-terminal amino acid. When hydrogen
fluoride is employed to effect the cleavage of the peptide from
;~ 10 the resin as well as to remove the protecting groups, it is pre-
j,:
ferred that the reaction be carried out in the presence of anisole.
~; The presence of anisole has been found to inhibit the potential
alkylation of certain amino acid residues present in the peptide
` chain. In addition, it is preferred that the cleavage be carried -~
out in the presence of ethyl mercaptan. The ethyl mercaptan
serves to protect the indole ring of the tryptophan residue, and,
furthermore, it facilitates conversion of the blocked cysteines
to their thiol forms. Also, when R5 is formyl, the presence of
ethyl mercaptan facili~ates hydrogen fluoride cleavage of the
formyl group.
Once the cleavage reaction has been accomplished, the
product which is obtained is a straight-chain peptide containing
14 amino acid residues. In order to obtain the final product of
formula I, it is necessary to treat the straight-chain tetrade-
capeptide under conditions which will effect its oxidation by
con~erting the two sulfhydryl groups present in the molecule, one
at each cysteinyl moiety, to a disulfide bridge. This can be
accomplished by treating a dilute solution of the linear tetra-
decapeptid~ ~-ith any of a variety of oxidizing agents including,
for example, iodine, and potassium ferricyanide. Air also can be
employed as oxidizing agent, the pH o~ the mixture ~enerally
being from about 2.5 to about 9.0, and preferably from about 6.2
~ 12 -
., ,~; ,
, .- ~ . .
. .
,- ~
~i21~3
. `
; to about 7.2. When aiX is used ~as oxidizin~ a~ent~ the concen-
tration of the peptide solution ~enerally is not ~reatex than
about 0.4 mg. of the peptide per milliliter of solution, and
usually is about 50 ~./ml.
The compounds of formula I may be administered to warm-
blooded mammals, including humans, and are particularly useful
for relaxing smooth muscle. Specifically, the gastrointestinal
tract can be relaxed by parenteral administration of small am-
ounts o~ these compounds, and preferably, of D-Vall, D-Trp8-
somatostatin. This action, resulting in reduction of gut motil-
ity, is particularly desirable in hypotonic gastrointestinal
radiography. These compounds, furthermore, are useful in treat-
ment of spastic colon, pylorospasm, and other spastic conditions
of the gastrointestinal tract, as well as for ureteral and bi-
liary colic.
Normally, in order to efect xelaxation of smooth
muscle, these compounds are administered at a dose of about 0.1
~g. to about 3 ~g. per kilogram body weight of the recipient and
preferably from about 0.3 ~g. to about 1.5 ~g. per kilogram body
weight. Administration is parenteral and it can be intra-
:
; muscular, subcutaneous, or intravenous; preferably, the compounds
are administered intravenously or intramuscularly.
For parenteral administration~ fluid unit dosage forms
~ generally are prepared using the compound in association with a
-~ pharmaceutical carrier, such as, for example, isotonic saline,
isotonic glycine, lactose, mannitol, dilute acetic acid, bac-
teriostatic water, fox example, water containing about 1~ benzyl
alcohol, and phosphate buffer solutions, as well as appropriate
combinations of any standard carriers. The carrier, relative to
the active compound, yenerally is present in a weight ratio of
~rom about 25:1 to about 1000:1.
,
- 13 -
,: ~
,' . .. .
. j . . . . .
3~
,
The compound~ depending upon the carrier and the con-
centration used~ can either be suspended or dissol~ed in a
suitable sterile vehicle, water being preferred. In preparing
solutions, the compound and carrier can be dissolved in the
selected vehicle, the solution filtered and added to a suitable
vial or ampoule, and the vial or ampoule sealed. Advantageously,
adjuvants, such as a local anesthetic preservative or a buffering
agent, can be dissolved in the vehicle. To enhance stability, the
; compound in association with the carrier can be dissolved in water,
and the a~ueous solution placed into a vial and then lyophilized.
The dry lyophilized solid then is sealed in the vial and an ac-
! companying vial of the vehicle supplied to reconsitute the com-
position prior to use. Parenteral suspensions can be prepared
in substantiall~ the same manner except that the compound is
suspended in the carrier instead of being dissolved.
;~ The compounds of formula I also are active, although
` not necessarily to an equivalent degree, in inhibiting the re-
lease of growth hormone. This inhibitory effect is beneficial
in those instances in which the host being treated re~uires a
therapeutic treatment for excess secretion of somatotxopin, such
secretion being associated with adverse conditions such as
juvenile diabetes and acromegaly. These compounds also exhibit
other physiological effects~ including the inhibition o~ gastric
~ acid secretion, useful in treatment of ulcer conditions; the in-
- hibition of exocrine pancreas secretion, potentially useful in
treatment of pancreatitis; and the inhibition of secretion of
insulin and glucagon. The compounds may be administered by any
of several methods~ including oral, sublingual, subcutaneous,
i intramuscular~ and intravenous. Preferably~ the dose range for
sublinsual or oral administration is about 1 mg. to about 100 mg.
!~ /kg. of ~ody ~eight. Generally, the intra~enous, subcutaneous,
or intrasmuscular dose range for these latter indications is
~.~.ZC~30
from about 1 ~g. to about 1 mg./kg. of body weight, and, pre-
ferably, is from about 50 ~g. to about 100 ~g./kg. of body weight.
It is evident that the dose range will vary widely depending
upon the particular condition which is being treated as well as
the severity of the condition.
1~
;,
': :
'
:
'.'`'' ' .
2Q
;~
:`
''''
.
, ~;
~, .
.:,
. 30
.. . .
'.~
~i ~ 15 ~o 18
. ~
03~
~ o~ )o~ s ()1- ~or~ ! c~ cl~lmirli~
oral1y or sublin~l~lall~ ass()ci~ltil-n with a ~-harmace-l~icc
carrier, for example, in the forrn o~ tal-lets or capsuLes.
Lnert ciiluents or car~iers, for example, Inaqrlesium (~arbonate
or lactose, can be used together with conventional dis-
integrating agents, for example, maize starch and alginic
acid, and lubricating agents, for example, magnesium stearate.
Typically, the amount of carrier or di]uent will range from
about 5 to about 95 percent of the final composition, and
preferably from about 50 to about 85 percent of the final
composltion. Suitable flavoring agents also can be employed
in the final preparation rendering the composition more
palatable for administration.
When the compounds of ~ormula :r are to be adminis-
tered parenterally, suitable carriers may be employed, such
as, for example, any of those described above with reference
to the use of these compounds for relaxing smoo-th muscle.
The following examples are illustrative of the
preparation of compounds of formula I and intermediates
thereto.
Lxam~Le 1
N-t-L~UTYI,OXYC~R~ONYI.-L-CYSTEINYI.(S-~-METIIOXYi3ENZYI)
METIIYLATIFID POIYSTYRE;NE RESIN
To 1000 rnl. of N,N-dimethylformamide (DME) con-
taining the cesium salt of N-t-butyloxycarbonyl-(S-~-
methoxybenzyl)cysteine (prepared from 17.5 g. of the free
acid) were added 100 g. of chloromethylated polystyrene
resin (Lab Systems, Inc~., 0.75 mmoles Cl/gram). The mixture
was stirred at room temperature for five days. The resin
X-4747A -19-
.. ~ , . . .. ~.. ,, ;,
- ~12~3C~
then was filtered ancl was waslle(l alternately Lhre~ times
each with a mi~ture o~ 85 percent DMF and 15 percent water
and with DMF, and then twice witll DMF. 1'o the resin sus-
pended in 1000 ml. of DMF were added a solution of 16 yrams
(83.4 mmoles) of cesium acetate. The mlxture was stirred
for nine days at room -tempera-ture. The resin then was
filtered and was washed alternately -three times each with a
mixture of 85 percent DMF and 15 percent water and with DMF.
The resin was washed with C~IC13 and then was suspended four
times in CIIC13 in a separatory funnel, drawing off the
liquicl each ti~e to remove fines. The resin was Eiltered,
washed with 95~ ethanol and then alternately three times
each with benzene and 95 percent ethanol. The resin then
was dried in vacuo at 30C. to obtain 115.3 g. of the title
product. An amino acid analysis showed 0.254 mmoles of Cys
per gram resin. The cysteine was determined as cysteic acid
from an acid hydrolysis carried out using a 1:1 mixture of
dioxane and concentrated hydrochloric acid to which a small
amount of dimethyl sulfoxide was added.
~xample_2
N-t-B~TYLOXYCARBONYL-D-VALYL-GLYCYL-L-(S-~-METHOXY-
BENZYL)CYSTEINYL-L-(N~-o-CHLOROBENZYLOXYCARBONYL)-
LYSYL-L-A',PARAGINYL-L-PHENYLALANYL-L-PiIENYLAIIANYL-
D-TRypTopHrL~L-(NE-o-cllLoRoBENzyLoxycARBoNyL)LysyL-L-
(O-BENZYL)TIIREONYL-L-PHENYLALANYL-L-(O-BENZYL)THREONYL-
L (O-BENZYL)SERYL-L-(S-~-METHOXYBENZYL)CYSTEINYL
METHYLATED POLYSTYRENE RESIN
The product from Example 1 (5.0 grams) was placed
in the reaction vessel of a Beckman 990 automatic peptide
synthesizer, ancl twelve of the remaining thirteen amino
acids were adclec! employing the automatic synthesizer. The
. .,
X-4747A -20-
. ~
z~3~
resulting protected tridecapeE)tide resin was dividec! into
two equal portions, and thc final residue was introduced to
one of the portions. The amino acids which were employed as
well as the se~[uence o~ their employment is as follows: (1)
N-t-butyloxycarbonyl-(O-benzyl)-L-serine; (2) N-t-butyl-
oxycarbonyl-(O-benzyl)-L-threonine; (3) N-t-butyloxycarbonyl-
L-phenylalanine; (4) N-t-butyloxycarbonyl-(O-benzyl)-L-
threonine; (5) Na-t-butyloxycarbonyl-N~-o-chlorobenzyl-
oxycarbonyl-L-lysine; (6) N -t-butyloxycarbonyl-D-tryptophan;
(7) N-t-butyloxycarbonyl-L-phenylalanine; (8) N-t-butyl-
oxycarbonyl-L-phenylalanine; (9) N-t-butyloxycarbonyl-L-
asparagine, _-nitrophenyl ester; (10) N -t-butyloxycarbonyl-
N~-_-chlorobenzyloxycarbonyl-L-lysine; (11) N-t-butyloxy-
carbonyl-(S-~-methoxybenzyl)-L-cysteine; (12) N t-butyl-
oxycarbonyl-glycine; and (13) N-t-butyloxycarbonyl-D-
valine. The sequence oE deprotection, neutralization,
coupling, and recoupling for the introduction of each amino
acid into the peptide is as follows: (1) three washes (10
ml./gram resin) of three minutes each with chloroform; (2)
removal of BOC ~roup by treatment twice for twenty minutes
each with 10 ml./gram resin of a mixture of 29 percent
trifluoroacetic acid, 48 percent chloroform, 6 percent
triethylsilane, and 17 percent methylene chloride; (3) two
washes (10 ml./(3ram resin) of three minutes each with
chloroform; (4) one wash (10 ml./gram resin) of three
minutes with met-hylene chloride; (5) three washes (10 -
ml./gram resin) of three minutes each with a mixture of 90
percent t-butyl alcohol and 10 percent t-amyl alcohol; (6)
X-4747A -21-
,
1~2~)~3(~
three washes (10 ml./gram resin) of three minutes each withmethylene chloride; (7) neutralization by three treatments
of three minutes each wi,th 10 m]./gram resin oE 3 percent
triethylamine in methylene chloride; (8) three washes (I0
ml./gram resin) of three minutes each wi-th methylene chloride;
(9) three washes (10 ml./yram resin) of three minutes each
with a mixture of 90 percent t-butyl alcohol and 10 percent
~t-amyl alcohol; (10) three washes (10 ml./gram resin) of
three minutes each with methylene chloride; (11) addition of
1.0 mmole/gram resin of the protected amino acid and 1.0
mmole/gram resin of N,N'-dicyclohexylcarbodiimide (DCC) in
10 ml./gram resin of methylene chloride followed by mixing
for 120 minutes; (12) three washes (10 ml./gram resin) oE
three minutes each with methylene chloride; (13) three
washes (10 ml./gram resin) of three minutes each with a
mixture of 90 percent t-butyl alcohol and 10 percent t-amyl
alcohol; (14) three washes (10 ml./gram resin) of three
minutes each wlth methylene chloride; (15) neutralization by -
three treatments of three minutes each with 10 ml./gram
resin of 3 perc~nt triethylamine in methylene chloride; (16)
three washes (1() ml./gram resin) of three minutes each with
methylene chlor~de; (17) three washes (10 ml./gram resin) of
three minutes each with a rnixture of 90 percent t-butyl
alcohol and 10 percent t-amyl alcohol; (18) three washes (10 `'~''''
ml./ gram resin) of three minutes each with methylene
chloride; (19) three washes (10 ml./gram resin) of three
minutes each with DMF; (20) addition of 1.0 mmole/gram resin
of the protected amino acid and 1.0 mmole/gram resin of
N,N'-dicyclohexylcarbodiimide (DCC) in 10 ml./gram resin of
X-4747A -22-
~,~2~
a l:l mixture of DMF and methylene chloride followed by
mixing for 120 minutes; (21) three washes (10 ml./gram
resin) of three minutes each with DMF; (22) three washes (lO
ml./gram resin) o~ three Ininutes each with methylene chloride;
(23) three washes (lO ml.~granl resin) of three minutes each
with a mixture of 90 percent t-butyl alcohol and lO percent
t-amyl alcohol; (24) -three washes (10 ml./gram resin) of
three minutes each with methylene chloride; (25) neutraliza- -
tion by three treatments of three minutes each with 10
10 ml./gram resin of 3 percent triethylamine in methylene ;.
chloride; (26) three washes (10 ml./gram resin) of three
minutes each with methylene chloride; (27) three washes ~10
ml./gram resin) of three minutes each with a mixture of 90
percent t-butyl alcohol and 10 percent t-amyl alcohol; and
(28) three washes (10 ml./gram resin) of three minutes each
with methylene chloride.
The above treatment sequence was employed for
addition of each of the amino acids with the exception of '~
the glycine and asparagine residues. The glycine addition
was carried out using only steps 1-18. The asparagine
residue was incorporated via its ~-nitrophenyl active ester.
In doing so, Step (]1) above was modified to the following
3-step sequence: (a) three washes (lO ml./gram resin) of
three minutes each with DMF; (b) addition of 1.0 mmole/gram
resin of the p-nitrophenyl ester of N-t-butyloxycarbonyl-
L-asparagine in lO ml./gram resin of a 1:3 mixture of DMF
and methylene chloride followed by mixing for 720 minutes;
and (c) three washes (10 ml./gram resin) of three minutes
X-4747A -23-
each with DMF. Also, Step (20) above was rnodified to the
use of the ~-nitrophenyl ester of N-t-butyloxycarbonyl-
L-asparagine in a 3:1 mixture of DMF and methylene chloride
followed by mixing Eor 720 minutes.
The linished peptide-resin was dried ln vacuo.
The product was hydrolyzed ~y reEluxincJ for 72 hours in a
l:l mixture of concentrated hydrochloric acid and dioxane.
Amino acid ana]ysis of the resulting product gave the
following resu~ts, lysine being employed as standard: Asn,
l.00; 2Thr, 2~18; Ser, 0.95; Gly, l.00; Val, 0.99; 3Phe,
3.45; 2Lys, 2.00.
Example 3
D-VALYL-GLYCYL-L-CYSTEINYL-L-LYSYL-
L-ASPA~AGINYL-L-P~ENYLALANYL-L-PHENYL-
ALANYL-D-TRYPTOPHYL-L-LYSYL-L-THREONYL-
L-PHENYLAlJANYI.-L-THREONYL-L-SERYL-r.-CYSTEINE
To a mixture of 7.2 ml. of anisole and 7.2 ml. of
e-thyl mercaptan were added 3.9l4 grams (at substitution
level of 0.155 mmoles/qram) of the protected tetradeca-
peptide-resin oE Example 2. The mixture was cooled in
liquid nitrogen, and 80 ml. of liquid hydrogen fluoride were
added by distilLation. The resulting mixture was allowed to
warm to 0C. and was stirred for 2 hours. The hydrogen
fluoride then was removed by distillation. E-ther was added
to the remainin~ mixture, and it was cooled to 0~C. The
resulting solid was collected by filtration and washed with
ether. The product was dried, and the deprotected tetra- --
decapeptide was extracted from the resin mixture using l~ -
acetic acid and 50~ acetic acid. The acetic acid solution
then was immediately lyophili~ed to dryness in the dark.
X-4747A -24-
.
ZC~3~
The resulting slightly yellow solid was suspended in a
mixture of 10 ml. of degassed 0.2M acetic acid and 4 ml. of
glacial acetic acid. The resulting suspension was heated
slightly with 6 ml. of 50~ acetic acid until a clean, yellow
solution resulted, and the solution was applied to al~ephadex~*
G-25 F column. The chromatographic conditions were:
solvent, degassed 0.2~ acetic acid; column size, 7.5 x 150
cm.; temperature, 26C.; flow rate, 1670 ml./hour; fraction
volume, 25.05 ~1.
Absorbance at 280 m~l of each fraction plotted
versus fraction number indicated one large broad peak with a
following shoulder. UV spectroscopy revealed that the main
part of the peak was the product. The fractions which were
combined and their effluent volumes are as follows: ~ -
Fractions 207-233 (5160-5837 ml., peak = 5515 ml.)
This ollection of fractions did not include the
back side shoul~er. UV spectroscopy indicated that 470 mg.
of the product were present. (yield = 46.4~). An Ellman
titration of an aliquot indicated a free sulfhydryl content
Of 95% of theor~tical.
Example 4
~XIDATIOI~ TO D-Val , D-Trp -SOMATOSTATIN
The sl)lution of the reduced D-Val , D-Trp -
somatostatin (6'7 ml.) from Example 3 was diluted with
distilled water to achieve a concentration of 50 ~Ig./ml.
Concentrated amllonium hydroxide was added to adjust the p~
of the mixture to 6.7. The solution was stirred at 4C. in
the dark for 64 hours after which an Ellman titration
indicated that c,xidation was complete.
X-4747A -25-
* Trademark for a hydrophilic, insoluble molecular sievechromatographic medium, made by cross-linking dextran.
~ ~ r
3~
The mixture was concentrated ln vacuo to a volume
of 45 ml., and 45 ml. of glacial acetic acid were added.
The mlxture then was desalted on a Sephadex C,-25 F column.
The chromatographic conditions were as ~ollows: solvent,
degassed 50% acetic acid; column size, 5.0 x 215 cm.;
temperature, 26C.; flow rate, 148 ml./hour; fraction
volume, 17.3 m~.
Absorbance at 280 m~ for each Eraction plotted
versus fraction number indicated two large peaks. The first
peak represented the aggregated forms of the product, and
the second peak represented monomeric product. The material
represented by the second peak was collected [fractions
116-155 (2000-2685 ml.)]. UV spectroscopy indicated that
279 mg. of product were present in the sample (yield =
59.4%). The solution was lyophilized to dryness in the
dark.
The resulting white solid was rechromatographed in
two approximately equal portions. The first portion was
dissolved in 25 ml. of degassed 50% acetic acid and was
absorbed on a Sephadex G-25 F column. Chromatographic
conditions were: solvent, degassed 50% acetic acid; column
size, 5.0 x 215 cm.; temperature, 26C.; flow rate, 148
ml./hour; fraction volume, 17.3 ml.
Absorbance at 280 m~ for each fraction plotted
versus fraction number showed two large peaks. A conserva- -
tive cut of the second peak was made. Fractions 119-125
(effluent volumes 2128-2256 ml.) were combined. UV spec-
troscopy indicated that 65.3 mg. of product were present in
this sample. The solution was lyophilized to dryness in the
dark to obtain the desired product.
X-4747A -26-
,,
. . .
The second portion was rechromatographed in the
same manner as the flrst with similar results. The two good
products were combined totalling 126 mg. by UV spectroscopy
(45.2~ recovery of purifiecl product). The combined product
was dlssolved in 15 ml. of degassed 0.2~1 acetic acid and was
"
applied to a Sephadex G-25 F column. Chromatographic
conditions were: solvent, degassed 0.2M acetic acid; column
size, 5.0 x 150 cm.; temperature, 26C.; flow rate, 466 ml./hr.;
fraction volume, 16.3 ml.
Absorbance at 280 mll of each fraction plotted
versus fraction number indicated one large peak. UV spec-
troscopy indicated that the major portion of the peak was
excellent product. Fractions 160-180 (2592-2934 ml., peak =
2685 ml.) were combined and were lyophilized to dryness in
the dark. UV spectroscopy indicated the presence of 90.
mg. of product (71.7~ recovery).
Op-tLcal rotation [a]26 = -56.1 (1 percent acetic
acid).
Amino acid analysis: Val, 1.0; Gly, 0.97; 2Cys,
1.62; 2Lys, 2.Q0; ~sn, 1.01; 3Phe, 2.87; Trp, 1.02; 2Thr,
1.83; Ser, 0.81~
The above results are expressed as ratios of Lys/2
= 1Ø All values are averages from two 21 hour hydrolyses
without scavengers. Tryptophan was determined from UV
spectroscopy (as a ratio to Lys/2); serine was not corrected
for losses durinc~ hydrolysis.
The above product contains minor guantities of
impurities. If desired, the product can be further purified
X-4747A -27-
3~
by subjecting it to preparative high pressure liquid chroma-
tography (HPLC).
An alternative method for oxidizing the reduced
D-Val , D-Trp8-somatostatin to D-Vall, D-Trp -somatostatin
is by treatmenl with potassium ferricyanide. The oxidation
is accomplished in an aqueous solution brought to p~l 6.7 as
described earlier in this example. An aqueous solution of
potassium ferricyanide is added to the mixture to produce a
final concentration representinq approximately 3.3 times
that of the reduced D-Vall, D-Trp8-somatostatin. The
solution is stirred in the dark at room temperature for -
about two hours. Completion of the oxidation is verified by
an Ellman titration.
D-Vall, D-Trp8-somatostatin was tested in dogs
for its in vivo inhibition of gastric acid secretion. In
six dogs with chronic fistula and Heidenhain pouch, gastric
HCl secretion was induced by infusion of the C-terminal tetra-
peptide of gastrin at 0.5 ~g./kg.-hr. Each dog served as
its own control. After one hour of steady state secretion
of HCl, D-Vall, D-Trp8-somatostatin was infused at 0.15
~g./kg.-hr. for one hour. Collection of gastric acid `
samples was continued for an additional 1.5 hours at 15
minute intervals. The samples were titrated to pH 7 with an
automatic titrator. The maximal inhibitory effect of the
D-Vall, D-Trp8-somatostatin was extrapolated against the
dose-response curve of somatostatin, and the relative
potency of the analog to that of somatostatin is express~d
as percent activity. D-Val , D-Trp -somatostatin inhibited
steady state acid secretion induced by the C-terminal
~etrapeptide of gastrin by 48.22 - 6.~5% (standard error of
- 28 -
~2~3(:~
mean). This effect is equivalent to that of 0.175 ~ug./kg.-hr.
of somatostatin. Its activity relative to that of somato-
statin thus is 116%. A more highly purified sample of
D-Vall, D-Trp~-somatostatin administered at doses of 0.200,
0.166, and 0.138 ~g./kg.-hr. inhibited steady state acid
secretion induced by the C-terminal tetrapeptide of gastrin
by 77.63, 71.57, and 67.8~, respectively. This activity
relative to that of somatostatin is 302-325~.
D-Vall, D-Trp8~somatostatin also was tested
for its action on gut motility in conscious dogs. Three
dogs having intralumenal catheters placed in the antrum,
duodenum, and pylorus were used~ Pressure changes in the
gut lumen were recorded on a Visicorder using strain gauges
and miniature light beam galvanometers. After a steady
state control was established, test compound was infused
intravenously over a ten minute period. The test compound
initially increased the intralumenal pressure in the pylorus
and then decre~sed it whereas the pressure in the duodenum
and the antrum remained depressed throughout the test. The
minimum effective dose required to increase the p~loric
pressure and to decrease the duodenum and antrum pressures
is about 0.05 ~y./l-g.-10 minutes for D-Vall, D-Trp - -
somatostatin. ~ compares to a value for somatostatin
itself of 0.125-0.25 ~g./kg.-10 minutes.
D-Vall, D-Trp8-somatostatin also was tested
for its activity wibh respect to the release of growth
hormone. The procedure which was employed is carried out
using normal male Sprague-Dawley rats weighing 100-120 grams
(Laboratory Supply Company, Indianapolis, Indi~na). The
test is a modification of the method of P. Brazeau, W. Vale,
- 29 ~
~9LZ~ 3~
and R. Guilleman, Endocrinology, ~4 184 (1974). In this
assay, a total of five groups of eight rats each weré
employed for the testing of each compound. Sodium
pentobarbital was administered intraperitoneally to
all o the rats to stimulate ~rowth hormone secretion.
One group s~rved as the control and received onl~ saline.
Two of the groups xeceived somatostatin, one at 2 ~g./rat,
subcataneously, and the other at 50 ~g./rat, subcut-
aneously. The other two groups received test compound,
one at 10 ~g./rat, subcutaneously, and the other at
0.4 ~g./rat, subcutaneously. The serum concentration
of growth hormone was measured 20 minutes after simult-
aneous a~ministration of so~ium pentobarbital and test
compound. The degree of inhibition of serum growth hormone
concentration then was determined with respect to the
control group, and the relative activities of test compound
and of somatostatin itself were compared.
At dose levels of 0.4 ~g./rat and 10 ~g./rat,
D-Vall, D-Trp8-s~toStatin inhibited the increase in growth
hormone secretion by 14% and by 42% over control,
respectively. Somatostatir, at a dose level of 2,ug./rat
had no effect on the increase in growth hormone secretion
whereas at 50 ~g./rat it inhibited the increase in growth
hormone secretion ~y 56% over control.
D-Vall, D-Trp3-somatostatin was tested for its
in vivo activity in inhibiting glucagon and insulin
secretion upon stimulation with L-alanine. Normal mongrel
dogs of either sex were ~asted overnight. Control blood
samples were obtained, and then an intravenous infusior
of saline, somatostatin, or test compound was started.
After 30 minutes~ L-alanine additionally was administered
- 30 -
~3
intravenously for a period of 15 minutes. The infusion
of saline, somatostatin, or test compound was continued
for 15 minutes after completion of the L-alanine infusion.
The infusion of L-alanine caused an abrupt increase in
serum concentration of glucagon and insulin which returned
to control concentration upon termination of the L-alanine
infusion. From the above it was determined that the minimal
dose of D-Vall, D-Trp8-somatostatin for the inhibition
of glucagon secretion is 0.04 to 0.11 ~g./kg./min. and
for the inhibition of insulin secretion is less than
0.004 ,ug./kg./min.
- 31 - -
.j
