Note: Descriptions are shown in the official language in which they were submitted.
3~6
~ACKGROUND OF THE INVENTION
. ~
Most physiological fur.ctions of the mammalian body
are controlled by enzymes or hormones which may be generically
described as physiological catalysts. Many of these involve
metabolic reaction sequences, one or more steps of which involve
hydrolytic cleavage of a protein. For example, in the sequence
of reactions which results in the coagulation of blood, a
critical step is the conversion of prothro~bin to t~rombin.
This conversion is catalyzed by the enzyme ~actor Xa, which
stimulates the hydrolysis of arginylthreonine and arginylleucine
depeptide ~nits in the prothrombin moleculeO
If these proteolytic control cleavage steps go awry,
there may be unfortunate pathological results. For example,
th~ body protects itself from the danger o circulating blood
clots by dissol~ing them. A part o~ the reaction sequence
which ultimately effects such dissolution is the conversion
of plasminogen to plasmin which is triggered by the enzyme
urokinase. Excess urokinase activity can result in the
~ormation of too much plasmin, and this, in turn, can lead
to inability to form clots. Conseque~tly, the body is unable
to protect i~sel~ from hemorrhage. There a~e many o~her
examples o~ such metabolic mal~nctions.
TKE INVENTION
This inventlon provides a procedure ~or alleviating
the harm~ul ef~ects o~ such imperfections by providing thera-
peut.ic agents which masquerade as one o~ the participants in
metabolic reactions involvins p~oteolysis. These disgu~sed
molecules f unction as unnat~ral su~stra~es w~.ich bind the
natural catalysts or c~talysts which bind the natural subst_ 2tes
and mitigate the results of high concentration or hyperactivity of
the natural substance.
More specifically, the therapeutic agents of this invention
masquerade as one partner in a pro-teolytic pair. A specific agent
may masquerade as a catalyst, e.g., an enzyme or a hormone, or it
may masquerade as a substrate for an enzyme. In any event the
pseudopeptide, or unnatural partner, binds the natural partner and
minimizes its participation in the natural metabolic pathway. If,
for example, the pseudopeptide is a segment of a natural substrate
for a specific enzyme; that enzyme, while bound to the unna-tural
substrate, is not available to perform its usual function. For
instance, the production of dangerous amounts of plasmin can be
inhibited by providing a plasminogen surrogate which binds
urokinase.
The invention provides processes of preparing
therapeutically useful pseudopeptides containing peptide bonds
joining amino acid residues and at least one linkage in which a
normal peptide bond between amino acid residues is replaced with a
thiomethylene group so as to provide a pseudopeptide having at leas-t
one por-tion which is represented by any of the fo:Llowing six partiaL
structures:
(1) - N - CHC112SlH~ -
1 ~1
( 2 ) 1 - CH - Cll - S - 1E~
CH3
ICH2
Hl ~11 CH3 - CH
(3) - N - CH - CH2 - S - CH - -
0~6
H~ ~H
U \NH
IH2 1 2
(4~ - N - H - CH2 - S - CH -
/ C
H H
I H2 ~H2
(5) - N - CH - CH2 - S - H -
( 6 ) - N - CH - CH S I ~
In the foregoing partial structures, R and Rl are amino
acid residues. The open valences on the nitrogen atom are joined to
hydrogen or the carbon atom of a peptide bond. The open valence on
the carbon atom is joined to a hydroxyl group or the nitrogen atom
of a peptide bond (or pharmaceutically acceptable acid addition or
metallic salts thereof).
The process accordln~ to -the invention comprises replacin~
a-t lqast one o:E -khe pep-ti.de bonds ln a conventlonal pept:ide
con-taining pept.ide bonds joining amino acid residues with a
thiome~hylene group accordlng to -t~chnique~ well known ln the
peptide art, and when -khe pharmaceu-tically accepta~le aclcl additi.on
or metall.ic salts thereof are required, reacting the resulting
peptldes wlth suitable pharmaceutically acceptable reagents.
..
- 3a -
The invention also provides pseudopep-tides containing
peptide bonds joining amino acid residues and at least one linkage
in which a normal peptide bond between amino acid residues is
replaced with a thiomethylene group so as to provide a pseudopeptide
having at least one portion which is represented by any of -the
foregoing partial structures, when prepared by the methods
described.
The invention will be better understood by reference to the
figures in which Fig. 1 is a drawing illustrating the spatial
similarity of glycylglycine and its counterpart in which the peptide
bond is replaced with a thiomethylene group. Figs. 2 through 10
illustrate various procedures for preparing products of the
invention. Figs. 11 and 12 illustrate the preparation of pseudo
phenylalanylglycine and pseudo leucylglycine utilizing combinations
of the processes shown in certain of the previous figures.
The compounds of this invention are pseudopeptides
containing up to ten or more bonds joining amino acid segments, and
at least one linkage between amino acid segments is replaced with a
thiomethylene group so as to provide a pseudopeptide at least one
portion of which may be represented by the following par-tial
structure:
C~12~
where:i.n R clnd Rl~ whiGh may be the same or cli~erent, are side
- 3b -
-
chains of amino acid residues. The ~alance or ,he ~onds bet~een
amino acid segments are peptide bonds. In t~e structure, t~e
open valences on the nitrogen atom will normally be completed
by a hydrogen atom or the car~on atom OL a peptide bond
through which another amino acid is joinèd to the structure.
Similarly, the open bond on the carbon atom could ~e completed
by an hydroxyl group or by co~nection to the ~itrogen atom of
a peptide bond.
Thus, for example, the a~ove structure might be
completed ~y union of its amino terminus ~ith the car~oxyl
group of glycine and its carboxyl terminus with the group
of alanine. The resulting pseudopeptide would have the
structure:
~2N-C~2C - N - C~CH2SCH - N - C~COOH
The union might also involve omega amino, carboxyl or
guanadino groups as in lysine, glumatic acid or arginine.
It ~ight also involve an imino group of proline or
hydroxyproline. The important point is that the resultin~
structure is one in which one chemically replicates one
member o~ a proteolytic pair in substantially all respects
except the presence o~ at least cne thiomethylene linkage.
I~ addition, to beingclosely related chemically to
~he aa~urally occur~ing materials, the products o~ this
invention are al50 substa~tially isosteric with their natural
counte~p2rts as well as be~ng substantially e~ui~alent in
b~asicity.~
. ~ . _ . . .. .
The prlncipal reason Lcor the close duplic:ation of
these important physical and chemical parameters or the
natural product is the close rela~ionship of the thiomethylene
3V~6
and the peptide sroups. This relations~Ip is clearly
illustrated in Fig. 1 of this application. ~rom a study o~
the figure it will be seen that the caxbon, hydrogen, nitrogen
and oxygen bonds on both sides of the peptide a~d thiomethylene
groups have substantially the same spatial relationship, and
that the thiomethylene group is approximately the same size
as the peptide group. The in~rastructure of the peptide
group and the thiomethylene group are also very similar so
that the substitution o~ a ~hiomethylene group for a peptide
group of a natural product does not materially affect such
factors as charge distribution, dielectric constants, ana
ability to become hydrated. Accordingly a pseudopeptide of
. .
this invPntion is capable of masquerading for its natural
counterpart and affecting in a contr~lable fashion those
r#~*ions in which the natural product is normally involved.
Two of the most important results which arise ~rom
the close similarlty of the products of this invention and
~heir natural products are (1) they can be used as therapcutic
agents ~o control the rate of naturally occurring metabolic
reactions involving proteolysis by their ability to substitute
~or their natural counterpart and (2) the~ are non-toxic and
~on ~utagenic.
The pseudopeptide illust~ated above i~ pseudotetrapeptid~
containing onl~ one thiomethylene linkage. For some purposes,
smaller pseudopeptides may be adva~tageous, and ~or o~hers,
it may be pre~erred to utilize pseudopep~ides containing as
many as eight or more amino aci~ se~ments. The reasons for
these variations will be apparent ~rom a consideration of the
simple lock and key analogy often used in describing protein -
protein interactions such as the action OI a pr3tein enzymeon a protein or peptide substrate. If the protein or peptlde
substrate is considered the key and the enzyme the lock, the
geometrical configuration of the two structures must complement
each other or the key will not fit the loc.~, and no reaction
will take place.
If the pseudopeptide now is to take the place of
the lock, it must have suf~icient geometric structure so that
the key fits. This requirement may be satisfied by a
pseudodipeptide, but often a higher molecular weight
pseudopeptide may be more sastisfactory.
The compounds of this invention, as will be
recognized by those skilled in the art, are amphoteric in
nature, and may be ~ormed into pharmaceutically acceptable
acid addition and metallic salts. Such salts are within the
scope of the invention. As the description proceeds, it will
be apparent to those skilled in the art that a number of
derivatives of the therapeutically active substances can be
prepared. Simple derivatives include esters and amides.
More complex deriva~ives include products ~ormed by reactions
with ~ree functional groups on the amino acid residues, ~or
example, the hydroxyl g~oup o~ tyrosine. ~hese, too, are
withln the ambit Q~ the inv~tion.
Norn~ally, the compounds of this i~vention will be
base~ upon L-amino acids. However, in certain instances,
it may be advantageous to utilized the D-form of the acid,
Accordingly, both forms are within the scope o~ the invention.
The following de~initions o~ some of the words and
terms used in the description o~ this invention may assist
in understanding.
~o~
Pseudopeptide - a peptide containing amino
acid residues in which at least one of the normal
peptide bonds has ~een replaced by a thiomethylene
group.
Amino acid residue - that portion of an a-amlno
acid molecule which remains arter removal of the
hydroxyl portion of the carboxyl group and a hydrogen
atom of the amino group in the fo~mation of a peptide
bond. It will be readily understood from the
description of the reac~ion sequences used to form
the compounds of this invention that it is not
necessary to employ an ~-amino acid as a starting
material. This will present no difficulty in
~isualizing the meanlng of the term "amino acid
residue" to those skilled in the art.
Amino acid side chai~ - that portion of an
~-~mino acid which is joined to the ~-car~on atom
in addition to the amino group, or in the case of
proline or hydroxproline, the imino grou~ The
term includes, for example, the hydrogen of glycine,
the b~nzyl group o~ phenylalanine, or the isobutyl
group o~ leucine.
Th~ ps~udopeptldes wlthin the scope of ~his invention
will, for conve~ie~ce, b~ designa~ed by th~ Greek letter ~ (Psi)
1~ place of a dash to indicate replacement o~ a peptide
linkage by a ~hiomethylene linkage. Additionally, the
standard abbreviations for the common amino acids will be
employed. ~hus,
Gly - Leu, and
Gly ~ Leu
~f~8~
are glycylleucine and pseudoglycylleucine, respectively.
Their molecular formulas would be:
~ ~ ~3
O CH
Il 1 2
CE2C - ~ - CHCOO~, and
NH2 H
~ ~ CX3
~2
fH2CH2SCHCOOH
N~2
A typical pseudotetrapeptide within the scope of
this lnvention containing only one thiomethylene linkage is:
Leu ~ Leu - Val - Tyr
This compound, its pharmaceutically acce~table salts and
derivatives are USB~Ul in th~ trea~men~ o~ hypertensi.on by
inhiblti.~g the conversion o~ angiotensin to angiotensin I
and angioten~in II by the en~ym~ renin. Treatme~t o~' a
patient with a p~a~maceutical compositio~ con~aining the
pseudopep~ide provides a masqueradi~g subs~rate which competes
with the na~ural substrate angiot~ansin ~or the available renin.
To ~he extent that the renin becomes bound ~o t~e pseudope~tide,
is it unavailable ~or th~ natura~ conversion. 'rherefore, the
pseudopeptide serves as a useful therapeutic agent for the
control of hypertension.
. -8-
30r3a~6
Other pathological conditions can also be treated
with the pseudopeptides of this invention; the pseudopeptide
employed and the metabolic pathway which is in~errupted include:
Hypertension is a common medi.cal problem. A key
step in the regulation of blood pressure is the enzymatic
cleavage of ~2-globulin to yield angiot:ension I which i~
turn is cleaved to angiotension II. Angiotension ~ is a
powerful pressor and blockages of its formatlon provides
an effective therapeutic tool. The conversions in~olve
cleaving Phe-His and Leu-Leu bonds. Introduction o..
Phe ~ His and Leu w Leu i~o selected pseudopeptides and
administration of those pseudopeptides provides a block
to the conversion of ~2-globulin to angiotension II. The
pseudopeptides and their derivatives which are useful ror
this type of block include: Ac-Phe ~ His-Leu-Leu-Val-Tyr-
Ser-OH; Ac-Phe-~is-Leu ~ Leu-Val-Tyr-Ser-OH and Ac-Phe ~
His-Leu ~ Leu-Val-Tyr-Ser-OH. Suitable methods of administra-
tion include parenteral, I.V., nasal aerosol and supository
modes.
Corneal Ulceration
__~___
C~rneal ulcexation is a commonly encourltered
ophthalmological problem with a broad spectrurn o etiologies
including ocular burns, rheum2todial arthri~is and infection.
The ulcerakion results fr~m the action o~ excessive production
o~ collagenase during ~he healing process. The cornea is 70%
collagen. Cleavage o~ collagen by collagenase is hi~hly
~peci~ic involving only Gly-Ile and a Gly-Leu linkages. The
pseudopeptides required to control these destructive cleavages
incorporate, there~ore, Gly ~ Ile and Gly Y' Leu. Typical
therapeutic agents of this invention incorporating the desired
structures are:
Ac-Pro-Gln-Gly ~ Ile-Ala-Gly-Gln-Arg-Gly-OEt
Ac-PrG-Cln-Gly ~ 1eu-Ala-Gly-Gln-Arg-Gly-OEt
Ac-Pro-Leu-Gly ~ Ile-Ala-Gly-.Leu-Arg-Gly-OEt
Ac-Pro-Leu-Gly ~ Teu-Ala-Gly-Leu-Arg-Gly-OEt,
the amide analogs thereof, or the parent compounds in which
the carboxyl group of the glycine molecule is free.
Treatment of the ulcerating cornea is done by topical ~ ~-
application i~ isotonic acqueous s~ ~ approxima~ely
PH 7.4, several times daily.
Since it is known that the C-terminal Arg is removed
when the peptide is in contact with plasma, it is useful to
include Gln ~ Arg or Leu Y' Arg in the same peptide or a
D Arg at the C terminal position. The resulting use~ul
pseudopep~ides are:
Ac-Pro-Gln-Gly ~ Ile-Ala-Gly-Gln ~ Arg
Ac-Pro-Gln-Gly ~ Ile-Ala-Gly-Leu ~ Axg,
or the corresponding esters or amides. The Ile in the
pseudo linkage may be replaced with Leu
Rh~umatoid Art~ritis
~ h~umatoid ar~hrltis is a syndrome afflicting
millions ~ individuals. The destructive aspects of this
disease includes the hydroly~ic action o~ ~issue collag~nase
on the collagen matrix of the af~licked join~s. Since ~he
generic enzyme of inkerest in this syndrome is the same as
tha~ ~or corneal ulcera~ion, the therapeutic pseudopeptides
are identical to those described for that af~liction. A
--10--
common clinical procedure wlth rheumatoid patlen~s is the
removal of excess synovial fluid from i~flamed joints.
Treatment of the patient involves injection or a sterile
sali~e solution or suspension of the pseudopeptides in 2
volume o~ I ml or less replacing only a small fraction of
the fluid withdrawn.
nticoagulants
Large numbers of coronary patients receive
znticoagulants as a precau~ionary measure to avoid the
threat of thrombosis. Agents used are frequently vitamin X
antagonists and, therefore, inte~efere with all kno~ and
unknown actions or vitamin K. A more specific interference
with blood clotting is provided by blocking the conversion
of prothrombin to thrombin by the action of proteolytic
e~zyme factor ~ . This enzyme cleaves specifically at
Arg-Thr (Residues 274-275) and Arg-Ile ~Residues 323-324)
of the prothrombin sequence. Interference with these
cleavages by synthesis and administration of appropriate
pseudopeptides provide highly specific blocking agents to
the onset o~ clotting. These pseudopeptides contain
Arg ~ ~hr and Arg ~
These pseudopeptides are:
Ac-Ile-Glu-Gly-Arg ~ Thr-ser-Glu-OEt
and
Ac-Ile Glu-Gly~Arg ~ Ile-Val-Glu-OEt.
Methods for administration include tablet, I.V., nasal
aersol or swab and suppository modes.
Modulation Of Urokinase Actlon
The enzyme urokinase activates plasminogen to
plasmin. This con~ersion is an impor.ant step in the
control of thrombosis. Accordingly urokinase is widely
used in t~erapy. An unfortunate side effect in urokinase
therapy is bleeding. This invention provides for control
of bleeding by attenuating the activity of urokinase through
a specific inhibitor of the action of urokinase. Thus, when
urokinase is unsuited to trea~ment as evidenced by bleeding
the ap~opriate pseudodipeptides may be administered. The
component pseudodipeptides are Arg ~ Met and Lys ~ Ser.
The completed pseudopeptides used in the block axe:
Ac~Ser-Ile-Arg ~ Met-Ary-Asp-Val-OEt
and
~c-Glu-As~-Arg-Lys ~ Ser-Ser-Ile-Ile-OEt.
Methods of administration include parenteral, I,V. nasal
aerosol or swab and supposi~ory modes, These preparations
are also useful in treatment of hemophilia.
Control Of Destruction By Elastin In Emphysema
-
Individuals W}10 are genetically deficient in
a2-antitrypsin present a high risk for pulmonary emphysema.
Th~ a~-antitrypsi~ ln the pulmorlary circulation ~ormally
prot~cts lung tissue against the action o~ elastase. ~i~hou~
this protection, the de~icie~ individual suf~ers extensive
pulmonary dz~age by the action of elastase. The most co~mon
peptide bo~d cleaved by the elas~ase in an Ala-Aïa linkage.
~ccordingly, the pseudodipeptide incoxporated in the inhibitor
is Ala ~ Ala. The resul~ing therapeutic peptides are Ac-~la-
Ala ~ -Ala OEt and Ac-Ala-Ala-Ala ~ Ala~O~t. ~he r~ethod of
administration is by inhalation of an aqueous aerosol.
~1~81~6
L _
Luteinizing hormone releasing hormone (pGlu-Eis-
Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-~H2) is used for trea~ment
of a variety of endocrine disorder~ which include loss or
lack of fertility, proper function of the menstrual cycle
and ovulation as well as male infer~ility. This hormone
has a short biological half-lifa (2 min..) because of rapid
destruction by proteolysis. A long-lived hormone, therefore,
provides an important therapeutic ins~rumen~. The bonds
subject to proteolysis are Gly-Leu, Tyr Gly and Pro-Gly-NH~.
The required pseudodipeptide u~its requlred are Gly w~ Leu,
Tyr ~ Gly and Pro w Gly-NH2. The long-lived hormo~es, therefore,
are:
pGlu-~is-Trp-Ser-Tyx ~ Gly-Leu-Arg-Pro-Gly-NH2
pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro ~ Gly-NH2
pGlu-~is-Trp-Ser-Tyr ~ Gly-Leu-Arg-Pro ~ Gly-N~2
pGlu-His-Trp-Ser-Tyr-Gly ~ Le~-Arg-Pro-Gly-NH2
Modes o' administration include nasal aerosol or swab,
suppository, parenteral and I.V. methods in a vehicle of
normal saline.
Anticoagulant-Block 0~ The Ac ti~n O
~5 descxibed pxeviously, anticoagulants are important
therapcutic agents~ ~lood coagulation processes involve a
compl~x cascade o activa~ions by proteolytic enæymes which
ulminate in the cleavage o~ ibrinogen with thrombin to
produc~ fibrin which spontaneously assembles to a so~ clot.
The poin~ o~ thrombin cleavag~ in the A-~ chain is an
Arg~Gly bond. AccordingLy, the pseudodipeptide unit required
for incorporation into the inhibitor is Arg ~ Gly. The
_l3_
completed peptide inhibitor of thrombi~ action is:
Ac-Gly-Gly-Gly-Val-Arg ~ Gly-Pro~Arg-Val-NH2
Methods of administration include tablet, I.V., nasal
aerosol or swab, and suppository modes.
Protection Against Peptic Ulcers
. . . _ . . . _ _
Pepsin is the dominant proteolytic enzyme found in
gastric ~uice. Individuals who secrete excessive sastric
fluid suffer ulceration due to proteolysis. Inhibition of
proteolysis due to pepsin protects against enzymatic damage.
The Phe-Phe linkage is especially prone to proteolysis by
pepsin. Accordingly, the pseudodipeptide required for
synthesis is Phe ~ Phe.
The therapeutic inhibitor is:
Ac-Ala-Ala-Phe ~ Phe-NH
Administra~ion of this substance is accomplished
orally in tablet or liquid form in combination with
antiacids.
Pseudopeptides Of Tuftsin
. . ~
Medical syndromes exist in which cells of the
patient are essentially incapable o~ phagocytosis or
pin~cy~osis. A class.o~ phago~ytosis-stimulatin~ pep~ides
which are therap~uticall~ use~ul contain basic and hylroxy
amino acid~ in speci~ic patterns. ~hes~ agents fall into
structural pat~erns o~ ~he type H-B~Pro-~', where H indicates
a position occupied by a hydroxy amino acid (Thr or Ser)
and B and 3' represent basic amino acids such as Arg, O~n or
Lys~ Plasma is known to have carboxypeptidase B activity
and kidney is known ~o contain ami~opeptidase activity.
Pseudotuftsins, thereore, containing Pro `g Lys, Pro ~ Arg,
Ser ~ Lys and Ser ~ Arg (for example) have a longer biological
half life than the normal pe~tide. Exemplary pseudotuftsins,
therefore, include Thr-Lys-Pro ~ Arg, Thr ~ Lys-Pro-Arg and
Thr ~ Lys-Pro ~ Arg. The Arg may be replaced with Orn.
These pseudotuftsins have varying degrees of resistance to
pro~eolysis.
Methods of administration include tablet, injection
and a~ueous oral procedures.
Insulin Of Extended Biological ~alf Life
Insulin is widely used in the treatment of diabetics.
Insulin is inactivated by removal of the C-terminal octapeptide
by trypsin-like activity. Accordingly, pseudoinsulins may be
prepared which are resistant to proteolysis by incorporating
Cys ~ Asp at positions 20-21 in the A chain of human lnsulin
and/or by replacing residues 22 and 23 in the B chain of
insulin by Arg ~ Gly. Coupling (oxidation) of the chains
prepared by solid phase synthesis provides an insulin
resistant to proteolysis. The resulting pseudoinsulins may
be administered in precisely the same manner as natural or
synthetic human insulin.
~1~!~
~ ece~tly the struc~ures o~ endogenous opiate
peptides have b~en discovered and explored. These substances
lenkephali~s and ~-endorphins) have pain relieving quali~ies
similar to opium. Loss of biological activity of Leu5-
enkephalin, which has the structure T~r-Gly-Gly-Phe-Leu, is
due to cleavage of t~e Tyr-Gly bond. Replacement of the
Tyr ~ Gly produces and analog of longer biological half-life
-15-
~v~s
~nd, t~ Iore, of enhanced therapeutic Yalue, The final
pseudo enkep~alin would ~e Tyr ~ Gly-Gly-Phe-Leu. The Leu
may be replaced with Met.
A similar approach can be applied to the
stabilizat~on of ~-endorp~ins ~y substituting a Met ~ Thr
linkage i~ the ~-endorphin. ~eu ~ Thr may be equally efective.
Modes of administration would be primarily, I.V.
but intranasal and intrarectal adminis~ration are also
possible.
Inhibition of Acrosin
.
Acrosin is the key enzyme found in spermatozoa.
this
The function of/trypsin-like enzyme is to facilitate
penetration of the ovum duri~g fertilizatio~. Interruption
of the action of acrosin prevents fertilization. The
inhibitory substance here is Benzoyl-Arg ~ Gly OEt. The
pseudodipeptide would be Arg ~ Gly.
This inhibitor of fertilization is used in an
intravaginal foam similar to Del~in.
Long-Lived Oxytocin
_
Oxytocin causes uterine contraction and is used in
obstetrics whe~ i.nduc~ion o~ uterine contrac~io~ is dasired.
~h~ ~truc~ure of oxy~ocin is:
N~
Cys-Ty~-Ile
I
Cy5 -Asn-G.~n
Pro-Leu-Gly-NH2
-1~
The pseuduo-oxytocin of this ln~ention having a
longer biological half life is:
Cys-Try ~ Ile
S
ll ,
Cys-Asn - n
Pro-Leu ~ Gly-NH2
The pseudodipeptides which would be employed are:
Tyr ~ }le and
~eu ~ Gly-N~2
Long-Lived Vaso~ressin
.
Vasopressin is used in surgical shock as an
adjuvant in elevating blood pressure. It is also used in
the management of delayed postpaxtum hemorrhage and at
delivery to o~ercome uterine inertia.
The structure of vasopressin is:
Cys-Tyr-P. le
,
Cys ~Asn-Gl n
Pro--.~g-Gly~NR2
The pseudodipeptides which are use~ul in accordance
with ~his inve~ion are:
Arg ~ G1Y~NH2
Tyr ~ Phe
Pro ~ Arg
'7
A pseudovasopressin incorporating these products is:
Cys-Tyr ~ Phe
S
S
Cys-Asn - Gln
Pro w Arg ~ Gly-NH2
The produc~ can be adminis~ered intravenously or
intranasally.
All o the above physiological conditions are
grouped, ~or purposes of this description, as metabolic
malfunctions -- simply as a matter of convenience. Some
of them, however, are not, in fact, metabolic aberrations,
although they do involve metabolic reactions. Fox example,
the relie~ o~ pain is not a ~uestion of a metabQlic pathway
gone astray.
One common feature of all of the conditions described
above is that at some point in the succession of reactions
which results in the condition, a substrate containing a
natural peptide bond is cleaved. The feature of this
in~en~ion is tha~ the total productio~ of the products of
that cleavage is reduced by br~ngiIlg ~o the reaction si~e
a pseudopeptide which competes or either ~he substrate o~
the ca~alyst in~ol~ed in the reaction by masquerading for
o~e o~ the par~icipan~s.
Since the de~elopment o~ the gen~tic code, it has
~come clear that pol~mers such as ~NA, ~NA and pro~eins,
as well as peptides ar~ information con~aining products. In
the case of nucleic acids, the in~orma~ion resides in the
sequential appearance o~ purine and pyrmidine bases.
Similarly, when this i~ormation is "tra nslated" to the
level of protein or peptide structures, it is the amino
acid side chains which impart the distinctive characteristics
o~ the protein or 2eptide. The function o~ the peptide
backbone is largely relegated to that o~ a connecting
structure. For example, polyglycine is essentiall devoid
of i~formation. It is the appearance oE the unique side
chains brought into play by the genetic code that dic~ate
the manner in which a pep~ide or protein folds, and
therefore the spatial relationship between the various atoms
and molecular segments, particularly the side chains on the
basic structure. The three dimensional structure which
results permit cooperative interaction between various
proteins, for example enzymes and substrates and thus
determinPstheir biological activities.
The problem solved by this invention is the
construction of a protein or peptide like structure with
substantially the same molecular geometry as i~s natural
counterpart. The problem has been solved by replacing a
pep~ide bond with a thiomethylene bond. Produc~s bui.lt
around ~uch thio~e~hyl~e bond~ have essentially the same
molecular se~metry as natural products, ~hey are capable o~
participating in the ~ame type of reaction. Howe~er, since
the khiomethylene bond is re~istan~ to hydrolysls, the
efect of increasing the concentration o~ th~ pseudopep~ide
in the presence o~ the na~ural product and it natural
coreactant is to decrease the concentration of the product
of the nor~al reaction because of the fact that the pseudo-
peptide has the right geometrical configuration to bind the
natural coreactant. The bound materials are not readily
_~9_
cleaved because of the hydrolytic r~sistance of the
pseudopeptide.
The unusual ad~antage of t~is invention is the
su~stantially complete predictability of the res~lts. On~e
the essential feature of the natural reaction is ~nown~ a
pseudopeptide can be constructed ~it~ confidence t~at it will
be ef~ective. It is kno~n, for example that an enzyme and a
substrate react by a process which includes ~ind~ng at
speci~ic peptide bonds on each reactant, the reac~ion can
be controlled by treatment with a pseudopeptide which
replLcates the particular section of the enzyme or-t~e~~~~~~~~~
substrate containing the peptide ~ond except that the
peptide bond is replaced with a thiomet~ylene group, It
may be necessary to conduct some con~entional studIes to
determine the best met~od of administrat~on or other
parameter using ordinary tec~ni~ues known to those skilled
in the art. The ~asic reaction howe~er r is predicta~le
with assurance~
The products of this invention are use~ul m~mmalian
therapeutic agents and ma~ be effect~e to inhi~it ~ydrolytic
cleavage o~ protein or peptide substrates at extremel~ low
level5, The physician ar veter~narian ~ill determine ~he
dosage which wlll be most s~ita~le ~or a particular application.
It may ~ary ~rom patient to pati~nt depending on the size o~
the pakient, the conditio~ u~der treat~nt and ot~er ~actors
which are readily evaluated b~ those skilled in the art. For
continuous administra~ion over extended periods to individuals
with more or less permanent metabolic abnormalities, the products
will normally be provided in various dos~ge forms varyi~s from
-2~-
relatively large, to build up a prompt blood level, to
relatively small to maintain an effective level. ~or
internlitte~t treatments to combat acute or chronic problems,
various dosage forms may be provided. The products may be
administered at very high levels, even up to two or more
grams per day. Nor~ally, they will be pro~id~d in dosage
units containing about 250 mg of the active ingredient and
the number o~ units appropriate to the condition under
trea~ment can be prescribed per day.
The products of this invention may be administered
alone but will generally be administered with phaxmaceutically
acceptable, non-toxic carriers, the proportions of which are
determined by the suitability and chemical nature of the
particular carrier, the chosen route of administration, and
standard pharmaceutical practice. For example, in dealing
with certain abnormalities or in maintaining therapeutically
ef~ective levels i~ the blood or tissues, they may be
administered orally in the form of tablets or capsules
containing such excipients as starch, milk sugar, certain
type~ o~ cl~y, etc. They may be enteric coated so as to
b~ more re~istant to the acid and digestive enzymes cl~ the
stomach. Por intravenou~ and intramuscular administration,
the~ may be used in the ~orm o~ a sterile solution containing
other solutes, ~or example, enough saline or glucose to make
the solution isotonic.
It is a particular advantage o~ the products of
this i~vention that unlike many pep~ide bond-containing
therapeutic products, they can be administered orally because
they ha~e increased resis~ance to enzymatic hydxolysis by the
enzymes of the lower digesti~e tract. ~ecause of their
v~
amphcteric nature, they may be adsorbed for oral administration
on no~-toxic ion exchange xesins which may be either anionic
or cationic to achiev~ slow release either in the stomach or
the int~stines or both. Furthermore, adsorption on these
resins makes them all the ~ore resistant to enzyme destruction.
Another advantage arising fron the amphoteric nature
of the products of this invention is that, as discussed above,
they can be utilized in the form of pharmacologically acceptable
salts which may be either metallic salts or acid addition salts.
These salts have the adYantage of water solubility and are
- ~ rly useful ror parenteral administration. The
metallic salts, especially the alkali metal salts, are
relatively stable and for that reason are preferred over
acid addition salts. The sodium salts are especially
preferred because of their ease of preparation.
The acids which may be used to prepare the
pharmacologically acceptable acid addition salts of this
invent~on are those con~aining non toxic anions and include,
for example, hydrochloric, sulfuric, phosphoric, acetic,
lactic, citric, tartaric, oxalic, succinic, maleic, gluconic,
saccharic, and similar acids.
~ ny o~ a wide vari~ty o~ ~o~-toxic deri~atives of
the pseudopeptides of this in~ention can be usefully employed.
~he pharmacologically acceptable salts have been mentioned
above. Amides, esters, acylated derivati~es and others can
also be utili~ed.
Other useful derivatives may be obtained by modifying
the free unctional groups on the pseu~opeptide backbone, for
example, free hydroxyl groups or free amino groups. One very
conventient class of derivatives is the class in which a free
-22-
hydroxyl group, for example, the free hydroxyl group of
threonine or tyrosine is esterified with an alkanoyl or
alkenoyl group containing up to eighte~n ox more carbon
atoms. Alternatively, an a~ino group, for example, the
amino group of threonine or lysine, can be acylated with
an alkanoyl or alkenoyl group containing up to about eighteen
car~on atoms. In both instances, the preferred derivatives
are those in which the derivatizing groups contain from
eleven to eighteen carbon atoms because the longer hydrocarbon
chains impart increased lipid solubility to the molecules and
enhance their transport across cell barriers.
Both types of de~.itat~ es may be prepared directly
from the pseudopeptide, but are preferably prepared by
incorporation during synthesic, by reaction of an amino acid
with the selected group, for example, the alXanoyl group
already in place.
The compounds o this invention have a number of
special features. One of the most important from the poi~t
of view of their therapeutie utility is the close configurational
and chemical analogy between them and the natura~ compounds
they are designed t~ replace. For this reason they may be
utilized with minimum da~ger o~ antigenic reaxtions. Addition-
alLy, they appear to have little or no toxicity. ~urther~
they can be metabolized and ~xcreted without danger.
1'he ~atural substrates which the pseudopeptides o~
this invention are designed to replace, in the course of
per~onming th~ir usual physiological functions are hydrolyzed
at a speci~i~ natural peptide bond site. The amino acid
segments on either side of this site..will be.kno~. The
pseudopeptiGe will normally be designed to include a partial
structure in which tAe natural site is repl~ced w~th a
thiomethylene group, and this Dartial structu~e w~'ll h~ve
on at least one side still furt~ex partial structure w~ch
is similar to the corresponding structu:re of the natural
substrate.
One example o~ the low toxicity of the compounds
of this invention is the results achieved with mice on
treatment with Gly ~ Leu. In the test Swiss Wistar mice
were i~jected I.P~ with Gly w ~eu at the dose levels
indlcated ~elow. Five mice were tested at each dose level.
In no case did any of the mice show any signs of ~cute
toxicit~. The behavior of the animals was o~served closely
for 7 days after injectio~. In no case were any adverse
effects noted.
Table 1: Weight Gains of Swiss Wistar Mice (N - 5)
Injected with Gly ~ Leu
Dose Level % Wt. Gain after 3 Days
-
44 mg/Kg 17
94 mg/Kg 19
208 mg/Kg 20
432 mg/Kg 23
Sal~ne Control 18
Possible mutagenic effects of pseudodipeptides were
examlned ~wo ways~ One possible route for z metabolic
complication is via the action o~ methionin~ adenosyl
transerase (M~T). Ethionine ~which contains a thiome~hylene
linkage~ is known to be bo~h a carcinogen and a ~ubstrate for
M~T. ~ile the speci~icity requirements for ~AT are stringent,
the possibility existed.that Gly ~ Leu might be either a
-2~-
substrate or acompetitive inhibitor. As evidenced below,
data obtained using the assay of Horfman, Arch. Biochem
and B~ophys 179,136 (1977), reveals that neithex Gly ~ Leu
nor Gly w D-Leu has a measurable effect on MAT acti~ity.
Table 2: Effect of Gly ~ Leu and Gly w D-Leu on MAT
Substance Concentration Counts/~in transferred
Expt I ~one , -- 15,760
Gly ~ Leu 12 mM 16,620
Gly ~ D-~eu 12 mM 16,760
Expt II None -- 7,380
Gly ~ ~eu 80 mM 7,990
Gly ~ ~-Leu 80 mM 7,710
The mutagenicity of Gly ~ Leu and Gly ~ Ile was
examined by a variation of the Ames test using repair-
de~icient strains of Bacillus subtilis.
The results shown in Table ~b indicate that neither
Gly ~ Leu nor Gly ~ Ile behaves as a mutagen.
Table 2b: Mutagenicity Test Results
Growth Inhibition of Bacterial
Strains in mm ~~-
Concentration ~ MC-l ~or-9 PB-13 Pol A
Gly ~ L~u 0.1 M 0 0 0 0 0
Gly ~ D-Ile 0.1 M 0 0 0
Gly ~ Ile 0.1 M 0 0 0 0 0
Gly ~ D-Ile 0.1 M 0 0 0 0 0
Chloraecetal~ehyde 0.6 M 8 16 4 1 5
The specific procedures b~ which these tests were
conducted are described in the following references:
~L~8~6
1. Laum~ach, A.D., Lee, S., Wong, J~, and Streips,
U. N., Studies on the Mutagenicity of Vinyl
Chloride Metabolites and Related Chemicals.
In: The Prevention and Detection of Cancer,
Part 1, Prevention, Vol 1, Etiology, ~dited
by H. E. Nieburgs, Marcel--Dekker, Inc., New
Yor~ (1977).
2. Kada, T., Tu~ikawa, K. and Sadaie, ~., Mu~a~ion
Research 16 (1972), 165.
A number of procedures are available for the
synthesis of the pseudopeptides of this invention. These
are illustrated in Figs.l through 12. In the figures R and
Rl are amino acid side chains as described above, ~L~ is an
alkyl group containing up to five carbon atoms, X is halogen,
Y is an amino blocking group and TOS is the tosyl group.
Those skilled in the art will recognize that several
of the reaction sequences illustrated in the figures are
based on the ormation of a thiomethylene joining group by
reaction of a primary halide with a secondary thiol or a
secondary halide with a primary thiol. The various synthesis
di~e~ one ~rom the other in the methods by which ~he critical
reactants are prepared.
Fig. Z illustxates a sy~thesis in which a p~imary
thiol prapared through an intermediate mercap~othiazollne is
reacted w~h a~ alpha~halocarbo~:~lic acid whlch is a
s~condary halide.
For ~his synthes i5 the starting materials are amino
acids which are reduced to form amino alcohols. A number of
amino aLcohols are known, and are available commercially.
~26-
~ 6
Others can be produced by reduction with bora~e in accordance
with the procedure described by Yoon et al. in the Journal of
Organic Chemistry, Vol. 38, No. 16, page 2786 (1973).
Generally, the procedure is one in which the amino
acid to ~e red~ced is co~tacted with borane, usually a molar
excess of borane in an ether type solvent. The preferred
solvent is tetrahydro~uran. The reaction ~emperature may be
from about lSC. to 40C. over a reaction period which may
vary from one-half hour to as long as twenty-four hours.
~ ny of a variety of isolation procedures can be
utilized to obtai~ the reaction product. A suitable procedure
is to decompose the excess reducing agent by the addition of
cold water which may be diluted with a small amount OL solvent.
Conversion of the amino alcohol to the desired
mercaptothiazoline is effec~ed in a reaction inert organic
solvent in the presence o~ a strong alkaline reagent utilizing
carbon disulfide as the cyclizing reagent. Typically, a molar
excess, e.g. up to about a 30% molar excess of carbon disulfide,
will be employed to ensure 2S complete a reaction as possible.
The most convenient alkaline reagents are alkali metal
hydroxides, su~h as sodium o~ potassium hydroxides. ~ny
o~ a variety of reaction l~ert polar organic solventc; may
b~ emplo~ed~ Th~ pres~ntly pre~rred are lower alipha~ic
alc~hols, particula~ly rne~h~l and ethyl alcohols.
Tha reaction temperature ~ay vary ~rom approxLmately
30C. to 45~. under standard atmospheric conditions. ~eaction
is normally completed at ~he end o~ ~rom about six to twelve
hours.
~ he desired product is readily recovered by simple
e~aporation o the carbon disulfide and of the solvent. The
-27-
residue is washed with cold dilute acid, e.g., 2% aqueous
hydrochloric acid. It may be recrystallized for purification if
desired, utilizing, in most instances, a 1:1 mixture of water and
alcohol.
While borane is the presently preferred reducing agent,
other reducing agents may also be employed. Alkali metal
borotetrahydrides may be employed as shown in Fig. 2. Of these,
sodium borohydride is generally preferred.
The mercaptothiazoline may be hydrolyzed to form a
primary thiol as indicated in the figure. The overall reaction
sequence thus far is one, therefore, in which the carboxyl group
of an initial amino acid is replaced with a mercapto group.
The mercaptothiazoline is readily converted to a primary
-thiol by a ring opening reaction with a halogen acid, suitably
aqueous hydrochloric acid. The reaction temperature may vary
from about 60C. to 160C. during a period of from eight to
twenty hours. One very convenient procedure is to reflux the
reaction mixture containing a molar excess of six normal
hydrochloric acid for a period of from ten to seventy-two hours.
At the end of the reaction period, the desired product
may be recovered as the hydrochloride salt by evaporation at
50C. to 80C. under reduced pressure. It is most convenient to
isolate the product as the salt since the sal-t may be employed
directly in se~uence reactions. ~owevex, if for any reason, the
fxee base i5 desixed, ik can he obtained by titra-tion to the
:isoelec-tric poink with, for example, dilute potassium carbonate
~llowed by extraction wi-th a water :immiscible organic solven-t.
- 2~ -
The ~-halo~arboxylic acids, the secondary halides,
used in this invention are produced by diazotization reactions
in which the amino group of the corresponding amino acid is
replaced with a halide. The presently prererred second~ry
halides are bromides produced by a diazotlzation in which
nitrosyl bromide is produced by the reaction of dilute
sulfuric acid and sodium nitrite in the presence o potassium
bromide at a low temperature, e.g., -10C. to 10C. The
reaction is a typical diazotization reaction, and ~s so well
known that no detailed description would appear to he required. `
A suitable procedure is described by Pfister et alO in the
Journal of the American Chemical Society, 71, 1096 (1949~.
Reaction of a primary thiol with a secondary halide,
or the alternate reaction of a primary halide with a secondary
thiol may be comple .ed utilizing a~ least equimolar quantities
of the reactants in dilute aqueous media under an inert
atmosphere at a temperatuIe of from about 20C. to 60C.
during a period from 2bout ten to thirty hours. To ensu~e as
complete a reaction as possible, and especially to li~it
inter erence by competing reactions, such as the reaction
in which an amine group, rather than a mercapto group,displaces
the hali~e, it i9 best to employ a molar excess or the thiol.
In ~ac~, the use of ~rom two to three rnolar excess of this
~acta~t is not u~usual.
The presently pre~erxed procedure for isolating the
resulting pseudodipeptide is desaltins using an ion exchange
resin as described by Dreze et al. in An21. Chin. Acta 11,
554 ~1954). The presently prefer~ed resin is Dowex* 2-x8.
~ ig. 3 illustrates a reaction seyuence in which an
amino substituted primary halide ~eacts with an -merc~ptoc2rboxylic
*Trade Mark
~ -29-
,.,
iL~ v~i
acid, i.e., a secondary thiol.
The starting amino alcohol may be prepared as
described above and then converted to a primary halide.
In order to prepare primary halides, the amino
alcoh~ls prepared as indicated above are reacted with halogen
substituting agents. These may include any of a variety of
such agents known to those skilled in the axt. However, the
preferred agents are those indicated in the figure, ~hat is
thionyl chloride or bromide or 48% aqueous hydrogen bromide.
The reaction with ~he thionyl halide may be effected
without a solvent, but for better control the reaction normally
takes place in a reaction inert polar solvent, typically
anhydrous dimethyl formamide or dimethyl sulfoxide. Reaction
is usually complete at the end of approximately one to three
hours at reflux utilizing at least a molar quantity of the
thionyl halide, but normally at least a 10% molar excess of
the reagent.
At the end of the reaction period, the excess thionyl
halide is decomposed by the addition of water. The resulting
mixture is neutralized with dilute aqueous sodium bicarbonate
and ex~racted w~th a water i~miscible organic solvent, such
as di~thyl ether. The nonaqueous layer i~ dried, suitably
over anhydr~us magnesium sul~ate, ~iltered, and the desired
product recovered by e~aporatian ~ the solvent under ~acuum.
The reaction with hydrogen bromide may be carried
QUt in an a~ueous medium utilizing a molar excess of H~r under
reflux during a period o~ approxima~ely one to three hours.
The desired reaction product may be recovered as de~cribed in
the immediately proceding paragraphs, except, of course, that
there is no necessity for decomposing excess reagent.
-30-
~ 3~
The secondary thiol is prepared from a secondary
halide obtained through the diazotization procedure
described above.
The secondary halide may be converted to the desired
thiol ~igure by reaction with thiourea in an aqueous-alkanol
medium in the presence of dilute base. Typically, the
reaction is carried out at a temperatur~ of from about 70C.
to 100C. in 95% aqueous ethanol during a period of from
about one to ~our hours. Equimolar quantities of the
reactants may be employed, but it is generally preferred
to use an excess o~ thiour~a so as to ensure as comDlete a
reaction as possible. The preferred alkaline reagent is
sodium hydroxide, and it is used in essentially catalytic
auantities, e.g., up to a~aut five molar percent.
An i~termediate isothiouronium halide is fonned,
but need not be isolate~. Instead, additional aqueous
al~ali is added to the reaction medium, and the resulting
mixture heated at from about 70 to 100C. for an additiona
one to four hours.
The resulting product is conveniently isolated by
~xtractlQn with a water immiscible organic solvent such as
~ther a~ter neut~aliza~ion wi.th dilute acid, e.g, dilu~e
h~drochloric acld. The product is obtained ~rom organic
solutio~ ater drying over a~ anhydrous reagent, ~ ering,
and removal oE the solvent under vacuum. The procedure is
described in somewha~ more detail in Org. Syn., Coll. Vol.
4, 40~ (1963).
The primary halide is then reacted with the
secondary thiol as described above.
Fig. 4 illustrates another procedure for preparing
a primary thiol ~or reaction with a secondary halide. The
preferred procedure is to replace a halogen of a primary
halide with trithiocarbonate, and to thereafter decompose
the resulting thioester with acid. The procedure is described
in detail by Martin et al. in J. Org. Chem. 33, 1275 (1968).
It is a most convenient synthetic tool.
In the usual procedure for carrying out the reaction,
the trithiocarbonate is formed by reaction of carbon disulfide
and sodium sulfide in aqueous alkali, for example 10% sodium
hydroxide. The primary halide is added to the trithiocar~o~ate
reagent at a temperature of from about 20C. to 60C. The
reaction mlxtuse is maintained at from a~out 20C. to 60C.
~or from about five to fifteen hours to form the thioester.
The ~hioester need not be isolated. In fact, it is
usually most convenient not to do so. Instead, the pH of the
reaction medium is reduced to about two to three by the
addition of an acid suitably a mineral a~id, such as hydro-
chloric acid. The primary thiol which forms may ~e isolated
by extraction with a water immiscible organic solvent, such
as e~her, dryl~g over a~hydrous rea~ent, ~iltering, and
removal o~ th~ ~olvent under va~u~m.
~ h~ procedure ~or reacting the primary thiol with
the seca~dary halid~ has been described above.
The key reaction in the sy~thesis ~hown in Fig. 5
i-~ the conversion o a primary halide to a primary thiol with
thiourea~ The reaction conditions or preparing the primary
thiol by this route are essentially the same as de~3cribed
above ~ox the preparation of a secondary thiol.
-32-
3~
~ ig. 6 illustra~es somewhat more specifi~ally the
reaction sequence which is the last step of the synthetic
sequence shown in Fig. 2. It illustrates the preparat~on of
a secondary halide by diazotization, a~d its conversion into
a pseudodipeptide by reaction with a primary thiol.
Fig. 7 illustrates a corollary of the reaction
sequence shown in Fig. 4,7i11ustrates the preparation of a
secondary thiol by ~he same procedure utilized for ~he
preparation of a primary thiol in the synthesis of Fis. 4.
The synthesis sho~n in Fig. 8.is a corollary o~
the synthesis of Fis~ 5~an ~ ates the ~act thai the
thiourea procedure for prepari~g primary thiols is also
applicable to the preparation of secondary thiols.
Fig. 9 illustrates a process of the invention in
which an amino alcohol/ blocked at the amino group is reacted
with tosyl chloride, and the resulti~g product reacted with
th~ dialkali ~letal salt of an a-mercapto-car~o~lic acid.
The blocking group may be any of a large number of ~nown
amino bloc~ing groups such as the t-butoxy group or the
carbobenzo~ group~ These groups are presently preferred,
but any of a n~er o~ o~hers may be employed. The selection
of the blocking group will generally be made on the basis of
subseque~t reactions in which the resulting dipeptide will
be involved, all i~ accorda~ce wi~h principles well knawn
to those skilled in the art.
For the preparation of the tosyl ester, the blocked
amino alcohol is reacted with a tosyl halide, preferably tosyl
chloride i~ a suitable solvent at a temperature of from -20C.
to 10C. ~or.a period of from one to one-hundred hours.
-33-
A molar excess of the tosyl compound is normally
employed to insure as complete a reaction as possible. Thus,
fr~m a molar equivalent to a molar excess of at least two,
based on the number of moles of amino alcoholwill normally be
employed.
The reaction is one which generates a halogen acid,
so it is normally carried out in the presence of a basic
reagent which neutralizes the ac~d as it is produced.
Normally ~he alkaline reagent chosen will be one which is
soluble in the selected solvent. As will be apparent to those
skilled in the art, the selected solvent must be reactio~
inert. Thus, neither water,ethyl alcohol, or any other
solvent with an active hydrogen could be used as a solvent
since they would react with the tosyl chloride.
The most preferred solvent is pyridine, since the
the liquid combines the ability to neutrali~e the hydrogen
haiide with its ability to dissolve the reactants. Additionally,
it IS readily-removed at the end of the reaction period because
it is soluble in water. Thus it is possible to isolate the
reaction easily since the solven~ is soluble in water a~d the
reaction p~oduct is soluble in organic ~olvents.
Con~rslon o the tosyl der~vative to a pseudodipep~ide
is accomplished by reactio~ with, ~or ~xample, the disodium or
dipotassi~m salt o glycine, ala~ine, leucine or isolucine,
in a reac~ion inert solvent under alkaline condition~ at a
temperature o~ ~rom about 20C. to 75C. ~or from two to
~welve hours. Normally a molar excess of the mercapto compound
will be employed to insure comple~e reac~ion~ However, if the
mercapto compound is the more expensive of the two reactants
an excess of the tosyl derivative can be used. Therefore,
-34-
based on the number of moles of the tosyl derivati~e, tne
number o~ moles of tosyl compound per mercapto compound can
be from 0.5 to 3 moles. Typically useful organic solvents
include polar solvents such as dLmethylformamide and lower
alkanols including methanol and ethanol.
Fig. 10 illustrates a procedure in which the tosyl
derivative of the selected amino alcohol is cyclized to a
cyclic imine. This is accomplished in an aqueous media under
s~rongly alkalin conditions, i.e., a pH o~ a~ least 11 to
1~, at a temperature of from 25C. to 75C. for a period of
from one to four hours, Alkali metal hydroxides are suitable
alkaline reagents.
The substituted imine is llnked to the selected
alpha mercapto acid by reaction in aqueous alkali at a
temperature o ~rom 20C. to 50C. ~or a period of from O.S
to 3 hours. ~he mixture is acidified, suitably with a
mineral acid at the end of the reaction period. The
pseudopeptide can be recovered from the aqueous medium ~y
ex~raction with an organic solvent.
Figs. ~1 and 12 illustrate the application of the
reactions described abave to speci~ic pseudopeptides. Fig.
11 illu~tr~tes th~ pxoduction o~ Boc~Phe ~ ~eu, and Fig. 12
~u ~ Gl~.
In the synthe~is o~ Boc-Phe ~ ~eu, phenylal~e is
fi~st reduced to the correspondinq ~lmino alcohol wl~h borane,
and the amino group is blocked with a t-Boc radical. The
blocked amino alcohol is converted to the O-tosyl derivative
a~d reacted with the disodium salt o~ leucine.
For the procedure illustrated in Fig. 1~, leucine
is converted to 4-isobutyl-2-mexcapto-thia~oline ~y reac.ion
~lB~ 1)6
with carbon disulfide in KOH-EtO~ at 40C. for 24 hours. The
thiazoline ring is opened with acid to form the thioleucinol
acid salt, and this compo~nd is reacted with alpha-iodoacetic
acid to form the desired product.
Those s~illed in the art will recognize that several
of the reactions described in connection with the synthesis
illustrated in Figs. 2 through 12 may involve inversion. The
course of the v.arious reactions can be readily foilowed by
conventional techniques. Thus, if a D-pseudopeptide within
the scope of this inve~tion is to be synthesized, it may be
necessary to utili~e a starting compGund with .~n L-configuration.
Similarly, the preparation of an L-pseudopeptide may require
that the starting compound have a D~confisuration.
A large num~er of pseudopeptides ha~e been prepared
by the procedures descri~ed above. Table 1 lists some of ~hem
and their physical constants.
Table 1 - Characterizatiol~ of Gly ~ Leu, Gly ~ Ile,
and their Stereoisomers
22 Analysis ('~) - P'ound
~]D Calcd. for C H NO S*
Dipe~tide Mp o 8 17 2
Analog (decComp.) (c-2, H O) C ~ N S
2 ~ _ _
Gly ~ Leu 214-216 -23.2 *1.250~10 9.08 7.20 1~.60
Gly ~R Leu 2~1-224 ~24.1 ~1.350.56 9~13 7.37 16,4S
Gly ~ Ile 218-220 ~57.2 ~1.050.57 9.04 7.35 16.65
Gly ~RaXle 210-213 -~37.9 ~S0.~0 8.96 7.36 16.81
*C, 50.23; ~, 8.96; N, 7.32; S, 16,76
A particular ~eature of this invention is the fact
that a pseudodipeptide, once p~epared, can be chemi.cally
trea~ed like a normal peptide. trhus, the chain length can be
increased at both the amino and the carboxyl termini by the
-36-
usual methods employed in pe~tide syntheses.
Perhaps the most con~enient procedure is the
Merrifield technique in which an amino acid, peptide or
pseudopeptide is bound to a resin particle through an es~er
linkage. Successive amino acides, peptides or pseudopeptides
are then added to the growing molecule by techni~ues well
known to those skilled in the art. Once the desired molecule
is generated, it is cleaved from the resin. One useful
reagent for cleavage is anhydrous liquid hydrogen fluoride
in a reaction inert solvent at 0C.
The following non-limiting examples are given by
way of illustration only.
~XAMPLE 1
~ly ~ ~eu
2-Bromo-4-Methyl-~entanoic Acid
A total of 50 g. of D-Leu are taken up in 1200 ml.
o 2.5 N sulfuric acid containing 250 g. of potassium bromide.
The mixture is cooled to 0C. and stirred while adding 65.5 g.
o~ sodium nitrite in small portions over a period of 1 1/2
houxs. The solution is stirred for an additional hour at 0C.,
and then heated to 25C. and stirred another hour. 'rhe mixture
is extracted with ether, the ether layer separated, washed
wikh water, and dried o~er anhydrous sodium sul~ate ~o give
the desixed produc~.
2-~ercapto-4-~ethyl-Pentanoic Acid
~ total o~ 1.25 g. (6.S mmoles) of the product
p~epared above is ta~en up in 2 ml. of water. Sufficient 1 N
sodi~m hydroxide is added to dissolve the bromo acid. To
~his mixture, there is added 5 ml. o 40~ sodium thiocarbonate
and the vessel is closed and allowed to stand at room tempera~ure
-37-
for 24 hours. At the end of this period, the solution is
acidified with 18 N. sulfuric acid, and extracted with 2
10 ml~ portions of ethyl ether. The combined extracts are
dried over anhydrous magnesium sulfate and filtered. The
~iltrate is concentrated to approximately half its volume
and chromatographed on a silica gel column (2,2 X 30 cm).
Tha original solution is eluted with 150 ml. o ethyl ether,
a~d the product recovered by evaporation of the solvent in
the eluate. The yield is approximately 34%. The Rf values
~T~C) are 0.88 and 1.0 on silica gel and cellulose plates
(n - butanol~acetic acid/water --- 12/3/5).
Gly ~ Leu
Ethyleneimlne (30 mmoles) is added to 2.2 mmoles of
the preYious product in 10 ml. of 0.5 N sodil~ bicarbonate.
This solution is maintained a~ about 20C. for 2 hours, and
acidi~ied with 12 N hydrochloric acid. It is then extracted
several times with ether, and the extracts discarded. The
aqueous solution is desalted on a Dowex 2 X8 column according
to the procedure of 3reze et al. The acetic acid fractions
are recove~ed, and the product isola~ed by evaporation. The
residue is ~ecrystallized ~rom 50% ethanol to give a product
with an R~ value o~ 0.82 by TLC on cellulose in the same
butanol/acetio acid/wa~er mix~ure previously described. The
speci~ic rotation, ~]~ is -16.3.
Leu
A total of S.3 g. ~27 mmoles) of the bromo acid
previously described is dissolved in 530 ml. o nitrogen-
purged 0.5 M sodium bicarbonate and 9.2 g, (81 mmoles) of
2-mercaptoe~hylamine hydrochloride is added. The reaction
-~8-
~ 6
vessel is ,lushed with nitrogen for one hour and sealed. It
is then allowed to stand at room temperature for 24 hour
while the desired product forms. The solution is acidified
with 6 N ~Cl and extracted twice with ether. The ether
extracts are discarded, the a~ueous portion neutralized with
2 N sodium hydroxide, and diluted to 2 liters with deionized
water. The salu~ion is desalted on a 5.5 X 30 cm. column of
Dowex 2 X8 resin according to the method of Dreze et al. The
acetic acid fractions are pooled and evaporated to dryness
under reduced pressure, the residue triturated with 20 ml. of
acetone, and crystallized from 47.5% ethanol to give white
needles of the desired product (yield 2.34 g., 44.5%, mp
205-210C. with decomposition).
These same procedures are utilized to prepare
Gly ~ Ile substituting D-alloisoleucine for the D-leucin~.
It is observed that in the course of these reactions
the halogen replaces the original amino gxoup without i~ersion
of configuration. Inversion does take place during t:he
replacement of the bromine. Thus, the amino acid residues o~
the pseudopeptides are in the L-~orm. Analoaous pseudopeptides
in which the components are in the D-~orm are prepared utilizing
the corr~sponding L-~orm o~ ~he staxting amino acids~
EXAMPLE 2
__
Phe ~ His
A kotal of 75 mmoles o~ Phe in 225 mmoles of ethanol
co~tai~in~ 300 mmoles o~ carbon disulfide and 75 mmoles of
solid potassium hydroxide is added with stirring while main-
taining the temperature below 40C. The temperatur.e is then
raised to 40C. arld the reaction mixture held in a closed
_~g_
vessel for 12 hours. At the end of this period, the alcohol
and carbon disulfide are removed by rotary evaporation. The
residue is susended in a small amount of cold water, and the
product recrystall~zed from the water-ethanol mixture.
2-Mercapto-l-Benzyl-Ethylamine
A total of 4 g. o~ the mercapt:othiazoline is taken
up in 25 ml. of 12 N HCl and heated und~r xeflux for five
hours at 155C. At the end of this period, the excess acid
is removed by rot~ry evaporation. The desired product is
recovered as the ~dr~chloridé and recrystallized rrom aqueous
ethanol.
Phe ~ His
This product is prepared from a-bromo-~-(5 Lmidazolyl)
propionic acid and 2-mercapto-1-benzyl-ethylamine by rea~tion
in the presence of sodium carbonate utilizing the procedure
described for the analogous reactio~ involving 2-mercapto-
ethylamlne as described in Example 1.
The pseudopeptide Leu ~ Leu is similarly prepared
utilizing as reacta~ts an isobutyl substituted ethanolamine
and an isobutyl substituted 2-mercapto ethylamine.
Ala Iy Ala is similaxly prepared from 4-methyl-2-
mexcapto thiaz~line ~Gabriel and Ohle 3er., 50, 804 ~1917)]
and ~-bromo prppionic acid.
The procedures describ~d i~ these examples are
utilized ~o prepare the ~ollowing pseudodipeptides:
~rp ,w Gly Arg ~ Val Phe 'Y Tyr Met ~ Lys
Gln 'Y Lys Lys w ~is Arg ~ Gly Gly ~ ~y5
Gln ~ Arg Arg ~ Val Lys w Tyr Lys ~ Arg
Arg ~ Thr Arg 'Y Lys Tyr ~ Leu Phe ~ ~rg
Arg 'Y Leu Arg `Y Val Lys ~ Tyr Arg `Y Pro
Tyr w Gly Lys ~ Lys Tyr 'Y Leu Phe ~ Ser
Phe ~ Met Lys 'Y Leu Pro 'Y Arg Tyr ~ Glu
Phe ~ Leu Phe ~ Trp Tyr ~ Phe Tyr ~ Gly
_~o_
~8~
EXAMPLE 3
Solid Phase Synthesis of
Ac-Pro-Gln-Gly ~ Leu-Ala-Gly-Leu ~ 3 Orn-~ly-NH2
Synthesis of this pseudopeptides is accomplished
using the peptide shaking apparatus with a medium reaction
vessel described by Stewart and Young (Solid Phase Peptide
Synthesis, W. ~. Freema~ and Company, 1969).
Boc-Gly-Resin (4 g.) containing 0.24 mmoles Gly
per g. of resin is used as the starting material. The
following schedule is employed in each cycle of Boc-A.A. or
Boc pseudodipeptide additions to the resin.
Step Rea~ent or Solvent No. Times Vol (ml) 'rime(~in)
1 C~2C12 3 ~0
2a 40% T~A/CH2C12/Anisole 1 40 20
2b A/C~2C12/Anisole 1 40
3 CH2C12 5 40
4 (i-Pr)2N~t/CH2C12 2 40 2
C~2C12 4 40
6 Boc-A.A., (3 eq.) 1 15 20
7 Box-A.A. line flush 1 5
8 DCC (2.7 e~.) 1 15 90
9 CHzCl~ 2 40
EtO~ 2 40
11 C~2C1~ 4 40
1~ ~oc-A.A. * 1 15
13 Boc-A.A. line flush 1 15
14 DCC 1 15
lS DCC line ~lush & couplin~ 1 S ga
16 CH2C12 3 40
-41-
iL~8~ 6
Step Reagent or 501vent No. Times Vol (ml) Time(min)
17 EtOH 3 40
18 D~ 3 40 2
19 Ac20 4 ml. 1 50 50
DMF 2 40 2
21 EtO~ 3 40 2
Fro~ the schedule, it can be seen that a double
coupling with acetyla~ion of any residual amino group is
employed in this synthesis. At each step 3 equivalents of
amino acid or pseudodipeptide are employed per egui~alent of
Boc Gly resin present.
Before coupling of Boc-Leu ~ ~ Orn-and Boc-Gly ~
~eu, these substances are released from their correspondins
dicyclohexylamine salts by suspending 2.5 g. of the salt in
10 ml. water. Citric acid ( 1 M) is slowly added until the
powder is liquified. An equal volume of ether is added.
After thorough mixing, the ether layer is separated. The
ether layer is washed once with w~ter, and then dried over
sodium sulate. The ether is removed at reduced pressure!,
and the sample is dried overnight to give the free pseudo-
dipeptld~3. ~oc-pseudodlp~ptides are coupled in the same
man~er as the ~mino acids.
I~ the case of Gln additi,on, the Boc Gln~p-
nitroph~nyl ester is employed at a 4 old molar excess. DM~
is sukstituted ~or methylene chloride in the coupling, washins
and acetylation steps. A cou~ling t.ime of 19 hrs. is used
o~ ea~h cycle o~ nitrophenyl ester. After ~ro is added, the
deprotection steps and acetyla~ion steps are per~ormed, and
the completed resin coupled peptide released from t:he resin
'd2--
with ~eOH and triethylamine to give the methyl ester which
is then ~reated with anhydxous ammonia in dry methanol to
give the amide.
Conversion o~
Ac-Pro-Gln-~lY-~ Leu-Alz-Çly-Leu ~ ~ Orn-GlY~NH
-- -----2
to
Ac-~ro Gln-Gly Y' Leu-Ala-Gly-Leu ~ Arg-Gly-NH
The peptide ~mide containing Leu ~ ~ Orn is dried
rigorously under vacuum, and then subjected to anhydrous
li~uid hydrogen ~luoride in the presence o~ 100 equivalents
o~ methyl e~hyl sulfide, with stirrins, for 30 min. at 0C.
The peptide is chromatographed on a column of Sephadex* G-100
in 10% aqueous acetic acid. The fractions containing the
deprotected peptide are comDined and lyophilized to gi~e
Ac-Pro-Gln-Gly ~ Leu-Ala-Gly-~eu ~ Orn-Gly-N~2.
This peptide is then treated with o-methyl isourea
according to the method of Merrifield (Cosand and ~errifield,
Proc. Natl. Acad. Sci. USA 74, 2771-2775 (1977) to give the
desired
Ac-Pro-Gln-Gly ~ Leu-Ala-Gly-Leu ~ Arg-Gly-NH2
EXA~LE a
A ~ 2
Tho ~ollowin~ table summari~es ~he xeactions for
th~ prepara~ion of the ti~le compound. The values in the
xlght hand col~ show the amino acid compositlon at each
stage in the synthesis. The values for ~he pseudodipe~tide
which elutes in the position o Phe using a 4.5 h.r citrate
bu~er elution pro~ram are determined a.ter ~C1-propionic acid
hydrolysis using cys~eamine as a scavznge~. Av~ropri2te
*Trade Mark
-43-
3 [)~~
control a~alyses are performed in the absence of pseudopeptide.
The section following the table describes the
various steps in the synthesis.
In the table and description Aoc signi~ies a
~-amyloxycarbonyl group.
Collagenase activity is i~hibited by the presence
of this pseudooctapeptiae which masquerades as a segment of
the natural collagen molecule when the enzyme is assayed
usi~g the octapeptide with the natural sequence as a substrate.
Table 3:
Renz. Resin
~ Aoc-Arg(Tos),DCC
Aoc-.~rg(Tos)-Benz. Resin
~ Boc-Leu,DCC
Boc-Leu-Arg(Tos)-Ben~. Resin Leu l.38
Arg l.00
3 steps Ratio of Residues
Gly l.0
Ala 0.99
Box-Gly ~ Leu-Ala-Gly-Leu-Arg(Tos~-Benz. Leu l~06
Resin Gly ~ Leu l.12
Arg O . g2
~ TFA 40~, 20 min
Gly ~ L~u-Ala~Gly-Leu-~rg(~o~)-Benz. Gly 1.O
Resin ~a l.0
Leu l.Ol
~ Leu l.13
1) 3OC Gln-NP~ Arg .94
2) 40~ T~
Gln-Gly ~ ~eu-Ala-Gly-Leu-~g~os)-Ben~ Gln 0.88
R~sin Gly l. a
~0,276 mmole/g.) Ala l.0
Leu .99
Gly ~ Leu l.lO
¦ Arg .88
~ Ac-Pro,DCC
Ac-Pro-Gln-Gly ~ Leu-Ala-Gly-Leu-Arg(Tos)--Benz. Resin
(O.243 mmole/g.)
-44-
tio of ~ idues
Gln .86
- Pro .93
Gly 1.0
Ala 1.0
Leu 1.01
Gly ~ Leu 1.08
~g O.g6
HF/Anisole
Ac-Pro-Gln-Gly ~ Leu-Ala-Gly-Leu-Arg-NH2 Gln 0.86
Pro 0.87
Gly 1.01
Ala 0.97
Leu 1.08
*Based on hydrolysis in absence of scavenger Gly ~ Leu 0.73*
Arg 1.00
Prepar~tion_of Boc-Gly ~ Leu. 2-tTert-butyloxcar~onyloxyimino)-
2-phenylacetonitrile (Aldrich, 5.4 g, 22 ~moles~ is added
to a mixtuxe of Gly Y Leu ~3.81 g,~0 mmoles), triethylamine
(4.1 ml, 30 mmoles), 12 ml dioxane and 12 ml water at room
temperature. The stirred mixture becomes homogeneous within
one hour and stirring is continued for ten hours. Water
(30 ml) and ethyl acetate (30 ml~ are added. The aqueous
layer is separated and washed with ethyl acetate (2 x 40 ml).
The aqueous layer is acidified with 5% aqueous citric acid
solution and extracted with ethyl acetate. The ethyl acetate
extrack is washed with sa~urated aqueous sodium chlo:ride
twlce and dri~d ov~r anhydrous sodium sulate. Re~o~al o~
drying agent and solvent gave 7.7 g o~ an oil. The oil is
diq~olved in 40 ml of ethyl ether and mixed with 7~4 g (40 mmole)
o~ dicycloh~xylamine. The resulting sus~nsion is s~red at
40 C ~or 4 hr prior to collection of the desired salt. The
sal~ is washed with ether and dried in vacuo over phosphorous
penkoxide to give 10 g (80% yield based on Gly ~ Leu) of white
crystals. Re~rystallization o~ the dicyclohexyla~:ine salt of
Boc-Gly w Leu from methanol: ethex (2:1 v/v) giving the
-~5-
purified product, m.p. 143-144~ C. Anal. calcd for
C25H48N2O4S: C, 63.5; ~I, 10.23; N, 5.93; S~ 6.78
Found: C, 63.73; ~, 10.48; N, 5.66; S, 6.43
Attachment of C-term~nal amino acid to the_resin.
Benzhydrylamine resin HCl (2.0 g, 0.4C meq/g) is
neutralized by stirrlng 50 ml of 25% triethylamine in methylene
chloride for ten minu~es. The resin is separated by filtration
and washed with additional methylene chloride ~25 ml per gm
resin). The resin is then suspended in methylene chloride
(40 ml) and shaken for 10 hx in the presence of a 2.5 fold
molar excess of Aoc-Arg(Tos). Af~er washing with methylene
chloride, the remaining unreacted amino groups on the resin
are acetylated with a mixture of a~etic anhydride a~d
N-methylmorpholine in dimethylformamideO Amino acid
analyses of a weighed portion of hydrolyzed Aoc-Arg(Tos) -
resin gives a value of 0.24 mmole of arginine released
per gm resin.
Chain elongation of double cycle addition and acetylation,
After the first amino acid is coupled to the resin,
the remaining amino acids are added sequentially by t.he
~ollowlng procedures: The resin is washed thxice with
meth~lene chloride and ~hen prewashed with trifluoroacetic
acid-anisole-methylene chloride ~40:2~0, v/v). Amino groups
axe relea~ed by s~aking with a ~F~ solukion o~ the same
composition ~or 20 minutes. ~ive washin~s with methylene
chloride are ~ollowed by two washings with 7~ (v/~) N,N
diisopropylethylamine in methylene chloride. Excess amine
is removed by washing five times with methylene chloride.
t-Butyloxycarbonyl amino acid (2.5 molar excess) is added
to the reaction vessel in methylene chloride, Dicyclohexyl-
carbodiimide (2.3 molar excess) is added in methylenechloride (final volume o~ 40 ml). The reaction vessel is
shaken for two hr. Two washinss with methylene chloride
are ~ollowed by two ethanol washings. The five methylene
chlorlde prewashes, as well as the coupling steps, are
repeated. After three washings with dimethylformamide,
residual unacylated amino groups on the resin are
acetylated with a mixture of acetic anhydride (4 ml),
N-methyl~crpholine (2 ml) and dime~hylformamide (34 ml)
during 20 minutes of shaking. The double cycle of coupling
is complete arter three washings with dimethylformaI~de
followed by three washings with ethanol. The double cycle-
acetylation sequence described is followed for addition or
each amlno acid resiaue including acetyl proline, but with
the exception of Gln. Gln is introduced (double cycle) as
t-~oc-p-nitrophenyl ester in dimethylfor~amide (omitting
dicyclohexylcarbodiimide) and a ten hr. reac~io~ time is
used instead of two hr. The progress of ~he sy~thesis is
followed by frequent hydrolysis and analysis of the growing
peptide-resin.
~ v ~
~ elea~e of Boc-Gly ~ ~Qu ~rom the dicyclohexylamine
salt is accompli~hed by suspending the salt (2 g) in 30 ml o~
a mixture o~ ethyl ether and water(l:l, v~v). Ci~ric acid
(1 Ml is added until all the solid dissolves. The ether
layer is ~eparated, washed with water and then dried over
a~hydrous sodium sulfate. The sodium sulfate is removed by
~iltration and the ether removed by rotary evaporation. T~e
residual oil Ls dried in a vacuum desiccator to constant
weig~t. The yield is 1.45 g (97%). Boc-Gly ~ Leu is added
to peptide-resin following the procedure .or Boc-amino acids
described above.
-47-
Release and deprotection of Pe~tide from ~enzhdrYlamine resln
p uct.
The tosylated benzhydrylamine resin-peptide (2.0 gm)
is treated with 20 ml of Co~3-dried HF in the presence of 2.0
ml of anisole for one hr at 0~ C. After removal of HF
under reduced pressure, the resin is dri.ed overnight under
vacuum. The released, deprotec~ed pep~ide is extracted ~rom
the resin with 60 ml of 7% acetic acid. The filtered extract
is washed with separate 30 ml portions of ethyl ether and
ethyl acetate to remove anisole. The solution is lyophilized
the residue dissolved in 0.2 M acetic acid and lyophilized
again. The crude peptide amide is chromatographed on
Sephadex G-15 in 0.5 M acetic acid. The major component as
determined by Sakaguchi assay is lyophilized to give crude
peptide amide weighing 334 mg (yield 80%). The product is
further purified by par~ition and/or carboxymethylcellulose-
chromatography.
Partition chromatogra~hy on Sephadex G-25.
A column (73 x 2.5 cm) of Sephadex G-25 (ine grade)
is packed in water. The column is e~uilibrated with the lower
pha~ o~ the solvent mix~ure o~ n-butanol:pyridine: 0.1~
aqueous ac~tic acid ~S:3:11, v/v) ~15) and then ~ollowed by
the upper phase o~ ~e s~me solvent sy~tem ~16) at a ~low
ra~e o about 1 cm/min. The peptide (55 mg) is diss~lved in
the upper phase ~5 ml) and chromatographed by elution with
the upper phase. The ~ractions containing peptide are detected
by the quantitati~e Sakaguchi method (17). ~ractions of the
major component are pooled, diluted with an equal ~olume o
water and concentrated by rotorary evaporation under reduced
-48-
pressure, The conce~tr~te is lyophilized to give a white
powder (32 mg (58~)).
EXAMPLE 5
BOC-PHE Y' GLY DICYCLOE~EXYIAMINE SALT
. . . _
This compound can be freed from the dicyclohexylamine
moiety and incorporated i~to a larger peptide by the procedures
illustrated or discussed herein. Alternatively, the Boc group
can be removed by trea~ment with HF as described in the last
section of Example 4. The resulting peudodipeptide can serve
as a surrogate 'or Phe-Gly.
Preparation of 30c-phenYlalaninol
To ~ mixture of 10 ml o~ water and 10 ml of dioxane
are added 3 grams (20 mmol) of phenylalaninol and 20 mmole
of t-butyloxycarbonyl azide and sufficient sodium hydroxide
solution (1 N) to raise the pH to 8Ø The reaction mixture
is stirred for 24 hours after which 5Q ml of ethyl ether
are added, and the mixture cooled to 0C. Solid citric acid
is added until the pH is lowered to 3.5 The aqueous layer
is ex~racted twice with 25 ml o~ ether. The combined ether
extrac~s are washed ~wice with 25 ml portions of 5% citric
acid, ~ollowed by ~hxee washes with satura~ed sodium chloride
solutlon. The organic layer i9 dried over anhydrous sodium
sul~ate. Remo~al o~ ~ol~ent under reduced pressure glves 5.4
g~ams o~ oil. The oil i5 treated with hot hexane to give
4.~ ~rams o~ white solid (needles; 81~ he crude product
is recr~stallized ~rom 100 ml hot hexane containing 10 drops
o~ ether. ~ white solid wi~h melting point 93 to 94C is
obtained (2.8 grams).
-49-
)V~
Pre~aration of 30c-phenylalan~nol Tosylate
To a solution of 2.5 grams (0.01 ~ole) of Boc-
phenylalaninol i~ 10 ml of dry pyridine cooled to -10 is
added 3.8 grams (0.02 mole) of tosyl chloride. This solution
is left in a freezer overnight until white solid precipitate
no longer formed (pyridinium hydrochloride). ~he mixture is
poured into 100 ml each o~ ice and e~her. ~he organic layer
is separated, washed ~hree times each with 5% citric acid
and saturated sodium chloride. The solution is dried over
anhydrous sodium sulfate, and the solvent removed to yive
4.1 grams of white solid, melting point 92 to 93C.
Recrystalli7ation from ether/hexane does not further improve
the melting point.
Preparation of Boc-Phe ~ Gly/DC~A Salt
To 0.24 grams (1.8 mmole) of disodium salt of
mercaptoacetic acid in 2 ml of dry methanol and 5 ml of
dimethylformamide are added 0.6 grams (2.4 mmole) o~ ~oc-
phenylalaninol tosylate. This solutin is kept at room
temperature for five hours after which the methanol is
removed under reduced pressure. The solution is cooled to
-5ac. and ~S mil o~ cooled ethyl ace~ate added. A 2S%
501ution 0~ citric acid is added to lower the pH to 2.
The a~ueous layer is ~xtracted thr~e times with 25 ml o~
~thy~ acetate. Th~ co~bined organic extrac~ are washed once
wi~h 5% Gitric acid and three times with satuxated sodium
chloride~ The ethyl acetate layer is dried over anhydrous
sodium sul~at~ a~d ~he solvent removed under reduced pressure.
The oily residue is dissolved in ethyl acetate and excess
dicyclohexylamine. Addition of anhydrous diethyl ether
results in the preciptitation of the Boc~Phe W Gly~DC~A salt
-50-
~ 6
in 58% yield (0.54 græ~s). This salt is recrystalli~ed from
chloroform ether (4:1) to give 0.4 grams of white solid,
melting point 140 to 141C (43% overall).
EX~LE 6
Preparation or Tablet:s
lO00 g. of Gly ~ Leu and 2000 g. of lactose are
thoroughly mixed together and the whole is passed through a
30 mesh sieve.
A paste is separately prepared with 80 g. of
cornstarch and 3~0 ml. ~ ~ëd water.
The above mixture is well kneaded with the paste
and the mass is passed through a 4 mesh sieve and the
resulting globules are dried at 50C. for 15 hours.
The dried globules are the~ granulated first on a
granulating machine, and passed through a 16 mesh sieve.
The grains are covered with a powdery mixture which is
prepared by blending 30 g. of calcium stearate, 200 g. of
cornstarch and 80 g. of ~alc, and then passed through a 40
mesh sieve.
Tablets each containinq 250 mg. of Gly ~ Leu are
ma~e o~ the above-obtained granules in accordance with
~orlven~ional procedure~.
E 7~E 7
~D~ `
100 g. of the 50dium salt Leu ~ Leu are dissolved
i.n a quanti~y of distilled pyrogen ~ree wa~er speci~ically
prepare~ ~or this purpo~e, and ~ade up to 5 liters. The
solution is made isotonic with addition of a predetermined
-51-
amount of an aqueous solution of physiological salt and
flltered through a millipore bacterial filter.
EXAMPLE 8
Pre~aration of an Aqueous Solutlon for Oral Adminis.ratlon
A .~ixture consisting of:
Gly ~ Ile-----------___________ g-- 20.0
Cane sugar------- ------------- g-- 100.0
Glycerine--~ ---------------ml-- 100. n
Ethyl p-ethoxy~enzoate--------- g-- 1.5
Arti~icial orange essence------ml-- 0.2
Essential oil of orange--------ml-- 1.0
is added to disti.lled water to make up 1000 ml. of the final
volume.
-52-
