Compound bearing two, 2,6-diiodophenol-4-YL groups and diagnostic drug for iodine allergy

A compound bearing at least two 2,6-diiodophenyl-4-yl groups represented by formula (IV), particularly a polymer composed of two to eight iodinated tyrosine molecules condensed with each other. This compound is useful as a diagnostic drug for iodine allergy and the diagnostic methods comprises administering the drug to the patient to examine the intracutaneous reaction ##STR1##

This application is a 371 of PCT/JP95/00997, filed May 24, 1995. 
INDUSTRIAL FIELD OF THE INVENTION! 
The present invention relates to a compound bearing at least two 
2,6-diiodophenyl-4-yl groups having diagnostic activity of iodine allergy. 
This compound is useful for predicting adverse effect caused by using 
iodinated X-ray contrast media and other iodine containing drugs. 
DESCRIPTION OF THE PRIOR ART AND BACKGROUND OF THE INVENTION! 
X-ray radiograms of the blood-vessel and the urinary tract were widely used 
today for the purpose of accurate understanding of the condition of 
diseases and better planning therapeutic programs. The X-ray radiograms 
are taken by X-ray irradiation just after administering iodinated 
radiocontrast media by vascular as well as nonvascular routes. 
However, iodine-containing X-ray contrast media are known to cause the 
serious side effects such as shock due to iodine allergy during and after 
their administration. 
Iodine containing drugs other than the X-ray contrast media have been 
developed and their adverse effects depend on iodine allergy are also 
known. 
As described above, there are clinical problems that serious adverse 
effects like shock by iodine allergy might happen during and after the 
administration of iodinated X-ray contrast media. Therefore, the 
development of X-ray contrast media free from such adverse effects is 
desired. However, X-ray contrast media absolutely free from such adverse 
effects have not yet been developed today. 
Thus, a small amount of an X-ray contrast medium is previously administered 
intravenously to a patient before the regular use of the X-ray contrast 
medium, in order to test for hypersensitivity, if any, to the medium. This 
pretest is deficient in reliability and there are examples in which side 
effects are either caused by the pretest amount or in the regular test 
even in the case of a negative result in the pretest, or in which no 
abnormality is caused in the regular test even in the case of a positive 
result in the pretest. 
Thus, presently, there is no established method for predicting any severe 
adverse effects caused by iodine allergy to iodinated.drugs such as 
iodinated X-ray contrast media, and therefore highly reliable agents for 
easily diagnosing iodine allergy are desired. 
It is possible to design in vivo diagnostic compounds of iodine allergy if 
a chemical structure of antigenic determinants is made clear. It has been 
known that immediate type hypersensitivity is provoked by cross-linking 
IgE antibodies by antigen on the surface of mast cells in the skin 
followed by releasing histamine and other biological active substances of 
allergy from the mast cells. Cross-linking of the IgE antibodies is 
possible by the compounds having at least two antigenic determinants in a 
molecule (Immunology page 265, Edited by Ivan M. Roitt, Jonathan Brostoff, 
and David K. Male; Gower Medical publishing Ltd. London, England, 1989). 
Therefore, iodinated proteins produced by reacting iodine to proteins have 
diagnostic activity of iodine allergy. However, these are immunogenic 
because of high molecular weight of these iodinated proteins. Accordingly, 
it is thought undesirable that an administration of the diagnostic agent 
of iodine allergy to a patient is simultaneously to sensitize the patient 
to iodine allergy. 
In order to avoid this problem, we have designed the compounds having 
eliciting antigenicity of iodine allergy but not having sensitizing 
antigenicity. 
EXPLANATION OF THE PRESENT INVENTION! 
We, the inventors of the present invention have studied to obtain agents 
for diagnosing iodine allergy from such a standpoint that the mechanism by 
which adverse effects caused by iodinated drugs such as iodinated X-ray 
contrast media is based on iodine allergy. During our study, however, we 
have experienced a problem that an administration of the diagnostic agent 
of iodine allergy to a patient is simultaneously to sensitize the patient 
to iodine allergy. In order to avoid this problem, we have found that 
compounds having elicitation antigenicity (antigenicity to elicit an 
allergic reaction) but not having sensitization antigenicity (antigenicity 
to induce antibody production or immune lymphocytes) are useful. We have 
studied such compounds to find novel tyrosine derivatives useful as agents 
for diagnosing iodine allergy caused by iodinated drugs such as iodinated 
X-ray contrast media, and have completed the present invention. 
Accordingly, the present invention provides a compound bearing at least two 
2,6-diiodophenol-4-yl groups represented by formula (IV), particularly a 
polymer composed of two to eight iodinated tyrosine molecules condensed 
each other. This compound is useful as a diagnostic drug for iodine 
allergy and the diagnostic method comprises administering the drug to the 
patient to examine the intracutaneous reaction. 
This invention provides compounds represented by the general formula (I) 
which contains at least two 2,6-diiodophenol-4-yl groups represented by 
formula (IV). 
Moreover, the compounds represented by the formulas (II), (III), (V), (VI), 
(VII) or (VIII) are desirable. 
##STR2## 
(In the formula, A indicates an atom of two valences or a radical having 
two valences.) 
##STR3## 
(In the formula (II), X is the same or different and represents an atom or 
radical of one valence, and Y is an atom of two valences or a radical of 
two valences.) 
##STR4## 
(In the formula (III), X is the same or different and represents an atom, 
or radical of one valence. R is the same or different and represents an 
atom of hydrogen or iodine. n is 0 or 1.about.6. R is selected as to 
contain at least two or more radicals represented by formula (IV).) 
##STR5## 
(In the formula (V), R is the same or different and represents an atom of 
hydrogen or iodine. n is 2.about.8 in which R is selected as to contain at 
least two or more radicals represented by formula (IV).) 
##STR6## 
X is preferably amino group, carboxyl group, amino acid group, peptide 
group, hydrogen or hydroxyl group. 
As a substituent, A is preferably --O--, --S--, 
##STR7## 
C.sub.1 .about.C.sub.10 alkylene radicals, C.sub.2 .about.C.sub.10 
alkenylene radicals, --CO--, --CO--NH--, 
##STR8## 
phenylene radical, 
##STR9## 
C.sub.1 .about.C.sub.10 alkylene radicals are methylene, ethylene, 
trimethylene, tetramethylene, pentamethylene, and hexamethylene radicals, 
etc., and also they may be alkylene radicals with branched-chain such as 
ethylidene, isopropylidene, ethylethylene, and propylene radicals. C.sub.2 
.about.C.sub.10 alkenylene radicals are vinylene, propenylene, 
2-butenylene, and 1,3-butadienylene radicals, etc., and they may be 
branched-chain alkylene radicals such as vinylidene, 
4-propyl-2-pentenylene radicals. 
Phenylene radicals are 
##STR10## 
As a substituent, Y is preferably --O--, --S--, 
##STR11## 
C.sub.1 .about.C.sub.10 alkylene radicals, C.sub.2 .about.C.sub.10 
alkenylene radicals, --CO--, --CO--NH--, 
##STR12## 
phenylene radical, 
##STR13## 
C.sub.1 .about.C.sub.10 alkylene radicals are methylene, ethylene, 
trimethylene, tetramethylene, pentamethylene, and hexamethylene radicals, 
etc., and also may be alkylene radicals with branched-chain such as 
ethylidene, isopropylidene, ethylethylene, and propylene radicals. C.sub.2 
.about.C.sub.10 alkenylene radicals are vinylene, propenylene, 
2-butenylene, and 1,3-butadienylene radicals, etc., and also may be 
branched-chain alkylene radicals such as vinylidene, 
4-propyl-2-pentenylene radicals. 
Phenylene radicals are 
##STR14## 
X is --H, --OH, --NH.sub.2, --COOH, halogen, C.sub.1 .about.C.sub.10 alkyl 
radicals, C.sub.2 .about.C.sub.10 alkenyl radicals, C.sub.2 
.about.C.sub.10 alkinyl radicals, amino acid group, peptide group, and 
aryl group. n is 2.about.6 or 2 or 3. C.sub.1 .about.C.sub.10 alkyl 
radicals aremethyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, 
nonyl, and decyl radicals and also may be branched-chain alkyl radicals 
such as 1-methylbutyl and isobutyl radicals. C.sub.2 .about.C.sub.10 
alkenyl radicals are vinyl, allyl, propenyl, 2-butenyl, 1,3-butadienyl and 
2-pentenyl radicals and also may be branched-chain radicals such as 
isopropenyl and 3-methyl-2-butenyl radicals. C.sub.2 .about.C.sub.10 
alkinyl radicals are ethinyl, 2-propinyl and 2-pentine-4-inyl radicals. 
Amino acid groups are aspartic acid, glutamic acid, arginine, histidine, 
lysine, gultamine, asparagine, cysteine, serine, glycine, and 
phenylalanine. 
Moreover, the present invention offers a diagnostic method for iodine 
allergy by administering the compounds described above to the patients to 
examine the intracutaneous reaction. These compounds are injected 
intradermally at doses of 0.5 to 50 .mu.g before the use of iodinated 
drugs. 
The compounds of the present invention which are represented by general 
formulae (I).about.(VIII) are produced by iodination reaction. For 
example, a compound saturated with iodine is obtained by reacting 6 mole 
of iodine molecule to one mole of trityrosine. 
Of the compounds of the present invention, these mentioned below are 
particularly useful agents for diagnosing iodine allergy. 
##STR15## 
Dityrosine derivative represented by the formula (VII) of the present 
invention can be produced by reacting iodine to dityrosine. For example, 
if 4 molecules of iodine are reacted with one mol of dityrosine, the 
compound represented by the formula (VII) may be obtained. 
Of the compounds of the present invention, the compound especially useful 
as diagnostic drug for iodine allergy is shown bellow. 
##STR16## 
The tyrosine derivative of the present invention represented by the 
structural formula (VIII) can be obtained by reacting iodine with 
L-tyrosyl-L-tyrosylphenylalanine. The compound of formula (VIII) can be 
produced if 4 molecules of iodine are reacted with one mole of 
L-tyrosyl-L-tyrosylphenylalanine. 
##STR17## 
The iodination reaction according to the present invention may be carried 
out similarly to general iodination reactions by using iodine potassium 
iodide. 
To diagnose the existence, if any, of iodine allergy by using the agent of 
the present invention, the diagnostic agent is administered to a patient. 
by intradermal injection before the use of iodinated drugs such as X-ray 
contrast media, and then the existence, if any, of iodine allergy is 
diagnosed by the local skin reaction. 
The amount of the diagnostic agent of the present invention to be 
administered is preferably about 0.5 to 50 .mu.g, and more preferably 1 to 
10 .mu.g. 
The iodinated X-ray contrast media are not limited particularly, provided 
that they contain iodine atoms, and may include, for example, non-ionic, 
semi-ionic and ionic iodine-containing X-ray contrast media, such as 
iopamidol, iohexol, ioversol, iomeprol, iotalane, ioxylan, iodixanol, 
ioxaglic acid, and sodium iothalamate. 
In addition, the diagnostic agents of the present invention can be used for 
the diagnosis of iodine allergy including side effects caused by iodinated 
drugs other than iodinated X-ray contrast media. 
Since, in most conventional agents for diagnosing allergy, an antigen 
itself is administered to man, there may be a danger of sensitization 
caused by the antigen administration. 
The present diagnostic compounds have an elicitogenic activity but do not 
have any sensitizing potency. Accordingly, there is no danger of 
sensitization by the administration of the compounds for diagnosis of 
iodine allergy. These facts are illustrated by the following Experimental 
Example 1 (for elicitation antigenicity) and Experimental Example 2 (for 
sensitization antigenicity).

EXPERIMENTAL EXAMPLE 1! 
The elicitation antigenicity of iodinated tyrosine derivatives was studied 
in the bases of the active cutaneous anaphylaxis (ACA) reaction in 
guinea-pigs immunized with iodine. 
Experimental Methods and Experimental Materials 
Animals: Male SPF guinea-pigs of Hartley strain, weighing 300.about.350 g 
were purchased from Charles River Japan Co., Ltd. These were immunized 
after having been acclimated for one week. 
Sensitization: A solution of iodine potassium iodide (Wako Junyaku, special 
grade reagent) and complete Freund's adjuvant (Difco) were mixed in equal 
amounts to form an emulsion. This was administered subcutaneously into the 
back of guinea-pigs at a dose of 2 mg of iodine per animal 3 times at 
intervals of 2 weeks. Active cutaneous anaphylaxis (ACA) was elicited 2 
weeks after the last sensitization injection. 
ACA Induction: 2% Evans blue physiological solution (0.5 ml/animal) was 
administered intravenously to guinea-pigs, and immediately thereafter 0.1 
ml of an elicitation antigen was administered intradermally into the back, 
and after 30 min. the existence of pigment leakage at the intradermally 
injected sites was observed. 
Elicitation antigen: Iodinated dityrosine, iodinated trityrosine, iodinated 
dityrosylphenylalanine were administered intracutaneously at 0.1 ml/site. 
Guinea-pig serum albumin (fraction V: Sigma) was iodinated and used as a 
positive control. Corresponding noniodinated ones were also administered 
intradermally as negative controls. 
Iodination reaction: To a 50 mM boric acid buffer solution (pH 9.5) of 10 
mg/ml of dityrosine, trityrosine, or dityrosylphenylalanine (all produced 
by Sigma Co., Ltd.) or guinea-pig serum albumin (abbreviated hereafter as 
GSA), a 0.5M solution of iodine was added at a ratio of 10:1, and these 
were reacted at 37 C. overnight. The excess iodine in the reaction mixture 
was reduced by adding sodium thiosulfate (Nakarai Chemical Co., Ltd.). For 
purification of the iodinated tyrosine derivatives, the reaction mixture 
was extracted with ethyl acetate and dried with magnesium sulfate, and 
hexane was added to obtain crystals. For purification of the iodinated 
GSA, the reaction mixture was purified by passing it through a Sephadex 
G25 column. 
Results 
Results of ACA reaction elicited by the iodinated tyrosine derivatives in 
guinea-pigs sensitized with iodine were shown in Table 1. 
Table 1 demonstrated that ACA reaction was elicited by intradermal 
injection of each of iodinated trityrosine, iodinated dityrosine and 
iodinated dityrosylphenylalanine in guinea-pigs sensitized with iodine, 
ACA reaction was also elicited by iodinated guinea-pig serum albumin 
(iodinated GSA) used as a positive control, whereas the reaction did not 
occurred by any of iodinated tyrosine derivatives in the non-sensitized 
guinea-pigs. 
Therefore, it is suggested that the iodinated tyrosine derivatives of the 
present invention have the property of eliciting an allergic reaction in 
those animals which have been sensitized with iodine. 
TABLE 1 
______________________________________ 
Elicitation antigenicity of iodinated tyrosine 
derivatives 
Mean diameter of blue spots (mm) 
Elicitation 
Dose of antigen 
Iodine-sensitized 
Non-sensitized 
antigen (.mu.g/site) 
guinea pig guinea-pig 
______________________________________ 
Iodinated 100 11.0 6.0 --* 
trityrosine 
10 8.5 4.0 -- 
1 6.0 -- -- 
Iodinated 100 7.0 5.0 -- 
dityrosine 
10 5.0 -- -- 
1 -- -- -- 
Iodinated 100 8.0 3.0 -- 
dityrosyl- 
10 7.0 -- -- 
phenylalanine 
1 -- -- -- 
Iodinated 100 11 8.5 -- 
GSA 10 7.5** 5.0** -- 
Trityrosine 
1000 -- -- -- 
Dityrosine 
1000 -- -- -- 
Dityrosylphen 
1000 -- -- -- 
ylalanine 
GSA 1000 -- -- -- 
______________________________________ 
*: Negative response 
**: Weak response 
EXPERIMENTAL EXAMPLE 2! 
Sensitization antigenicity of iodinated tyrosine derivative was studied. 
Trityrosine was selected as the tyrosine derivative, and this was iodinated 
to obtain iodinated trityrosine. Guinea-pigs were sensitized with 
iodinated trityrosine. As a positive control, employed was iodinated 
guinea-pig serum albumin (GSA). Guinea-pigs of the control group were 
sensitized with iodinated GSA. Sensitization was examined by active 
systemic anaphylaxis (ASA) reaction and passive cutaneous anaphylaxis 
(PCA) reaction. 
Sensitization: Each of iodinated trityrosine and iodinated GSA was 
emulsified with complete Freund's adjuvant, and injected subcutaneously to 
the back of animals at a dose of 10 mg/animal (2.5 mg, respectively, at 4 
sites), 3 times at intervals of 2 weeks. 
ASA reaction: Iodinated GSA (1 mg/animal) was injected intravenously to the 
animals one week after the last sensitization injection, by which ASA 
reaction was elicited. The animals which indicated symptoms of 
anaphylactic shock were judged as being positive. 
PCA reaction: The blood was taken from each animal, and the serum was 
separated one week after the last sensitization injection. The serum was 
diluted fivefold with physiological saline, and injected intradermally to 
normal guinea-pigs at a dose of 0.1 ml/site. PCA reaction was provoked by 
intravenous injection of iodinated GSA (1 mg/animal) and Evans blue 
solution to the recipient animals 4 hours after the intradermal serum 
injection. PCA reaction was judged as being positive when the diameter of 
blue spot at the site of intradermal injection was 5 mm or larger. 
Results 
The results are shown in Table 2. 
As is obvious from the Table 2, ASA reaction and PCA reaction were positive 
in all animals of the positive control control group, i.e., the iodinated 
guinea-pig serum albumin (iodinated GSA)-sensitized group, while ASA 
reaction and PCA reaction are both negative in all animals of the 
iodinated trityrosine-sensitized group. This means that no anti-iodine 
antibody was produced in the animals sensitized with iodinated 
trityrosine. From this, it is suggested that iodinated trityrosine has no 
sensitization antigenicity. 
TABLE 2 
______________________________________ 
Sensitization Antigenicity of Iodinated Trityrosine 
Number of positive animal/Number of 
sensitized animal 
Sensitized with 
ASA reaction 
PCA reaction 
______________________________________ 
Iodinated 0/5 0/5 
trityrosine 
Iodinated GSA 
5/5 5/5 
______________________________________ 
EXPERIMENTAL EXAMPLE 3! 
The present invention is founded on the finding that an antigenic 
determinant of iodine allergy is diiodotyrosine. It is possible to 
diagnose iodine allergy by skin test using compounds having 2 or more 
residues of diiodotyrosine. 
The finding that an antigenic determinant of iodine allergy is 
diiodotyrosine was discovered as the followings. 
Guinea-pigs were immunized with iodine by injecting subcutaneously 1 mg of 
iodine containing potassium iodide solution treated with complete Freund's 
adjuvant seven times at intervals of 2 weeks, and anti-iodine antiserum 
was obtained from these animals. Antibody titers of the sera were 
determined by enzyme linked immunosorbent assay (ELISA) using 96 well 
microplate coated with iodinated guinea pig serum albumin (I-GSA) ELISA 
inhibition activities of the following compounds were studied. The 
compounds used were potassium iodide, tyrosine and its derivatives such as 
L-tyrosine, monoiodo-L-tyrosine, diiodotyrosine, hexaiodo-L-trityrosine, 
L-hexatyrosine and 12 iodo-L-hexatyrosine, and iodinated X-ray contrast 
media such as amidotrizoic acid, iodamide, ioxaglic acid, iohexol, 
iopamidol and iomeprol. 
ELISA inhibition study was performed as follows. Antibody concentration of 
anti-iodine antiserum gave the maximum optical density in ELISA till one 
hundredfold serum dilution. Accordingly, seven steps of twofold serial 
dilution of anti-iodine antiserum, starting from 50-fold serum dilution, 
were made with physiological saline as a diluent. To each of the serially 
diluted sera, were added equal volume of solution containing each of the 
test compounds described above. After overnight incubation at 4 C, 
antibody activity was determined by ELISA. Phosphate buffered 
physiological saline (PBS) was used as a control. ELISA inhibitory 
activities of the test compounds were determined at the serum dilution 
which gave about 50% of the maximum optical density (OD.sub.415nm =2.6) of 
ELISA of control at serum dilution of one hundredfold. The inhibitory 
activity was calculated by the following calculation formula. 
EQU Inhibition rate=1-Optical density under test compouund/Optical density 
under PBS!.times.100% 
Inhibition rates were shown in Table 3, and inhibitory activity which was 
judged from the inhibition rates was described as + if a compound had 
inhibitory activity and - if a compound did not have inhibitory activity. 
The results were shown in Table 3. 
TABLE 3 
______________________________________ 
ELISA inhibitory activities of compounds on anti-iodine 
antibody 
Concentration 
ELISA inhibitory activity 
Compound of compound 
Inhibition rate (%) 
Judgment 
______________________________________ 
Potassium iodide 
10 mM 0 - 
L-Tyrosine 10 mM -2.6 - 
Monoiodo-L-tyrosine 
10 mM 5.6 - 
Diiodo-L-tyrosine 
10 mM 100 + 
L-Hexatyrosine 
1 mM 0 - 
12 Iodo-L- 1 94.3 + 
hexatyrosine 
Amidotrizoic acid 
10 mM 0.3 - 
Iodamide 10 mM 3.5 + 
Ioxaglic acid 
10 mM -9.4 + 
Iohexol 10 mM -5.0 + 
Iopamidol 10 mM -6.5 + 
Iomeprol 10 mM -1.8 + 
______________________________________ 
In the next, ELISA inhibition study was undertaken to determine the potency 
of ELISA inhibitory activity of active compounds. Serial twofold dilution 
of each solution of active compounds were performed starting from 20 mM of 
diiodo-L-tyrosine, 2 mM of 12 iodo-L-hexatyrosine, and 4 mM of 
hexaiodo-L-trityrosine, respectively, and then equal volume of 50-fold 
diluted anti-iodine antiserum was added to them. The inhibitory activity 
was expressed as the concentration which decreased optical density of 
ELISA to a half (OD.sub.50) of the control. The results were shown in 
Table 4. 
TABLE 4 
______________________________________ 
ELISA inhibitory activity of diiodotyrosine derivatives 
on anti-iodine antibody 
Inhibitory activity 
Compound (OD.sub.50) 
______________________________________ 
Diiodo-L-tyrosine 
5 mM 
Hexaiodo-L-trityrosine 
0.125 mM 
12 iodo-L-hexatyrosine 
0.125 mM 
______________________________________ 
The results shown above indicated that inorganic iodide ion was not an 
antigenic determinant of iodine allergy. It is essential for the antigenic 
determinant to have a chemical structure in which tyrosine residue is 
substituted with 2 iodine atoms. Tyrosine residue which is substituted 
with one iodine atom does not recognized as the antigenic determinant. 
Furthermore, none of ionic and nonionic iodinated X-ray contrast media has 
activity of antigenic determinant of iodine allergy. 
EXAMPLES! 
The following is a further explanation of the present invention using 
examples of preparations, though it is needless to say that the present 
invention is not limited to them. 
Example 1 
##STR18## 
L-Tyr-Tyr-Tyr (250 mg) was added to 20 ml of 50 mM ammonium carbonate 
buffer (pH 9.4), and dissolved by adding 1N NaOH. To the solution,0.5M 
iodine potassium iodide (5.6 ml) and 1N NaOH were added little by little, 
and these were reacted at room temperature for 10 minutes. The non-reacted 
iodine was decomposed with sodium thiosulfate. Afterwards, 1N HCl was 
added to the reaction mixture, by which the mixture was adjusted to have 
pH of 3. The mixture was extracted with ethyl acetate. The extracted ethyl 
acetate layer was collected, dried with magnesium sulfate and then 
filtered. This was concentrated with an evaporator, and n-hexane was added 
to the resulting concentrate. The crystals thus precipitated were taken 
out by filtration and dried (at 50 C, 30 minutes) to obtain 236 mg of the 
above-mentioned compound. 
Decomposition Point: 170 C 
.sup.1 H-NMR (CD.sub.3 OD) .delta. (ppm): 2.81(1H, dd, J=10, 15 Hz), 
2.89(1H, dd, J=9, 15 Hz) 2.95(1H, dd, J=8, 14 Hz), 3.11(1H, dd, J=5, 15 
Hz) 3.13(1H, dd, J=6, 14 Hz), 3.15(1H, dd, J=4, 15 Hz) 4.00(1H, dd, J=4, 9 
Hz), 4.61(1H, dd, J=5, 10 Hz) 4.64(1H, dd, J=6, 8 Hz), 7.70(2H, s), 
7.73(2H, s), 7.74(2H, s) 
MASS (FAB) M+H.sup.+ =1264 
EXAMPLE 2: 
##STR19## 
The same process as in Example 1 was repeated, except that L-Tyr-Tyr (317 
mg) was used instead of L-Try-Try-Try, to obtain 295 mg of the 
above-mentioned compound. 
Decomposition Point: 160 C 
.sup.1 H-NMR (CD.sub.3 OD) .delta. (ppm): 2.91(1H, dd, J=10, 15 Hz), 
2.95(1H, dd, J=9, 14 Hz) 3.15(1H, dd, J=5, 14 Hz), 3.19(1H, dd, J=5, 15 
Hz) 4.03(1H, dd, J=5, 9 Hz), 4.60(1H, dd, J=4, 9 Hz) 7.69(2H, s), 7.76(2H, 
s) 
MASS (FAB) M+H.sup.+ =849 
EXAMPLE 3 
##STR20## 
The same process as in Example 1 was repeated, except that L-Tyr-Tyr-Phe 
(40 mg) was used instead of L-Tyr-Tyr-Tyr, to obtain 43 mg of the 
above-mentioned compound. 
Decomposition Point: 110 C 
.sup.1 H-NMR (CD.sub.3 OD) .delta. (ppm): 2.77(1H, dd, J=10, 14 Hz), 
2.81(1H, dd, J=9, 15 Hz) 3.08(1H, dd, J=8, 14 Hz), 3.10(1H, dd, J=4, 15 
Hz) 3.26(1H, dd, J=5, 14 Hz), 3.98(1H, dd, J=4, 9 Hz) 4.65(1H, dd, J=5, 10 
Hz), 4.74(1H, dd, J=5, 8 Hz) 7.3(5H, m),7.71(2H, s), 7.74(2H,s) 
MASS (FAB) M+H.sup.+ =996 
EXAMPLE 4 
##STR21## 
The same process as in Example 1 was repeated, except that 
L-Tyr-Tyr-Tyr-Tyr-Tyr-Tyr (20 mg) was used instead of L-Tyr-Tyr-Tyr, to 
obtain 34 mg of the above-mentioned compound. 
MASS (FAB) M+H.sup.+ =2509