(en)A SYTHETIC PRODUCT (PYRO) GLUTAMYL-HISTIDYL-PROLINE AMIDE, HAVING THE SPECIFIC AND UNIQUE ACTIVITIES OF THE THYROTROPIN RELEASING HORMONE OF THE HYPOTHALAMUS GLAND, IS OBTAINED FROM THE TRIPEPTIDE, A-GLUTAMYL-HISTIDYLPROLINE. THE TRIPEPTIDE CAN BE CONVERTED TO THE AMIDE FORM BY REACTING THE DIMETHYLESTER WITH METHANOL AND AMMONIA TO OBTAIN THE (PYRO) GLUTAMYL-HISTIDYL-PROLINE AMIDE.

1.ApplicationNumber: US-3746697D-A
1.PublishNumber: US-3746697-A
2.Date Publish: 19730717
3.Inventor: FOLKERS K,US
ENZMANN F,US

4.Inventor Harmonized: FOLKERS K()
ENZMANN F()

5.Country: US
6.Claims:
(en)A SYTHETIC PRODUCT (PYRO) GLUTAMYL-HISTIDYL-PROLINE AMIDE, HAVING THE SPECIFIC AND UNIQUE ACTIVITIES OF THE THYROTROPIN RELEASING HORMONE OF THE HYPOTHALAMUS GLAND, IS OBTAINED FROM THE TRIPEPTIDE, A-GLUTAMYL-HISTIDYLPROLINE. THE TRIPEPTIDE CAN BE CONVERTED TO THE AMIDE FORM BY REACTING THE DIMETHYLESTER WITH METHANOL AND AMMONIA TO OBTAIN THE (PYRO) GLUTAMYL-HISTIDYL-PROLINE AMIDE.

7.Description:
(en)United States Patent 3,746,697 TRIPEPTIDE HAVING THE ACTIVITY OF THE THYROTROPIN RELEASING HORMONE Karl Folkers, 202 Capri-Lakeway, and Franz H. Enzmann, 1801 S. Lakeshore Blvd., both of Austin, Tex. No Drawing. Filed Sept. 19, 1969, Ser. No. 859,559 Int. Cl. C07c 103/52; C07g 7/00 U.S. Cl. 260-1125 2 Claims ABSTRACT OF THE DISCLOSURE A synthetic product (pyro) glutarnyl-histidyl-proline amide, having the specific and unique activities of the thyrotropin releasing hormone of the hypothalamus gland, is obtained from the tripeptide, a-glutamyl-histidylproline. The tripeptide can be converted to the amide form by reacting the dimethylester with methanol and ammonia to obtain the (pyro) glutamyl-histidyl-proline amide.
BACKGROUND OF THE INVENTION The thyrotropin releasing hormone (TRH) is produced by the hypothalamus gland of mammalian species, including man. It can be extracted from the glands of porcine hypothalami only by enormous effort and at great cost. For example, only 2.8 mg. of the hormone having a purity of about 30% was extracted from the glands of 100,000 pigs as has been reported by A. V. Schally, C. Y. Bowers, T. W. Redding and J. F. Barrett in Biochemistry, Biophysics Research Communications, 1966, at pages 25 and 165. Later, in 1969, only a few milligrams were again extracted from the glands of 165,- 000 pigs as reported by the above authors in the Journal of Biological Chemistry, in press (1969). Since the production of this hormone from animal glands involves great difiiculties and high expense, its extraction for general medical use is impractical.
The thyrotropin releasing hormone, however, is of great value for use in the diagnostic evaluation of the functioning of the anterior pituitary gland. Present medical methods employed for such evaluations have adverse etfects and produce only limited biological responses. Furthermore, because the existing methods are expensive and impractical, physicians and medical researchers have been discouraged from using them.
THE INVENTION It has now been discovered, in accordance with the present invention, that a new, synthetically produced tripeptide derivative provides the same order of activity as the naturally derived thyrotropin releasing hormone obtained from mammals. The newly synthesized tripeptide of the invention is especially useful because of its specificity of action for testing the function of the anterior pituitary gland. Medically, it can be used quickly, easily and accurately and diagnostic evaluation can, therefore, be performed on out-patients as Well as hospital patients. Specifically, the newly synthesized tripeptide of the invention can be administered to a patient intravenously, or subcutaneously and from about -30 minutes later, a blood sample can be taken from the patient and be bioassayed for the appropriate level of the pituitary hormone, particularly the thyrotropin hormone (TSH).
3,746,697 Patented July 17, 1973 "ice In accordance with the present invention, the tripeptide, a-glutamyl-histidyl-proline, hereinafter referred to as G1u- His-Pro, as referred to and reported by A. V. Schally, A. Arimuri, C. Y. Bowers, A. J. Kastin, S. Sarwano, T. W. Redding, Recent Progress in Hormone Research, vol. 24, p. 497, 1968, can be conveniently and easily converted to an amide form by first reacting it with methanol and hydrogen chloride to obtain the dimethylester of the tripeptide, and then reacting the dimethylester with methanol and ammonia to obtain the corresponding (pyro) g1utamyl-histidyl-proline amide, herein referred to as (pyro) Glu-His-Pro (NH These reactions can be illustrated as follows:
Glu-Hls-Pro (I) OH NH N B=J1H GH -CH H, GH -OH, C 0 H CH-C ONHH-C ON NH; CH-CH;
0 H CHaOH l H01 Dlmethylester CH Gln-His-Pro (1) NH N 6: H CIIg-GH; CH CH -GH,
(IJOZOHS JJH-C ONHCJH-C ON IL'H (EH-OH,
U OzCHa CHBOH l NH:
(pyro) Glu-Hls- CH Pro (NH (III) NH N H UH -OH, (3H CH -CH, 30 OH-CONHCH-CON NH CH- H,
0 ONE;
The reaction is effected by dissolving the tripeptide, Glu-His-Pro (I), in anhydrous methanol containing hydrogen chloride and allowing the mixture to stand in a sealed vessel at room temperature for over an hour. The methanol and hydrogen chloride are then removed under vacuum and the residue is dissolved in anhydrous methanol containing ammonia. The reaction of the residue, the dimethylester with ammonia, takes place at room temperature in a sealed vessel over a period of several hours. The resultant product, (pro) Glu-His-Pro (NH (III), is obtained by removing the excess methanol and ammonia under vacuum and subjecting it to chromatography for purification. The initial methylation of the carboxyl groups and the subsequent cyclization of the glutamic acid moiety to the lactam form and the formation of the propline were elfected by synthetic processes generally known to those skilled in the art such as those described by A. F. Beecham, Journal of American Chemical Society, at pages 76, 4615 (1954); D. Coleman, oJurnal of Chemical Society, at page 2294 (1951); and, "I. Shiba, S. Imai, and Kameko, Bulletin of a 3 Chemical Society, Japan, at pages 42 and 244, (1958).
At the time the discovery of the synthetic tripeptide derivative of the invention was made, the structure of thyrotropin releasing hormone was not known, since only limited amounts have been extracted and made available for analysis, and since the extracted, natural homone has a purity of only about 30%. Consequently, it was entirely unpredictable that a hormonally inactive tripeptide would be converted by these reactions of methylation and ammonation to a new product having a hormonal activity indistinguishable from that of the naturally occurring thyrotropin releasing hormone of the hypothalamus.
The synthetic product, (pyro), Glu-His-Pro (NI-I was subjected directly to biological testing for horomnal activity according to the methods described by C. Y. Bowers, T. W. Redding and A. V. Schally, Endocrinology, at pages 77 and 609, (1965); C. Y. Bowers, A. V. Schally, G. A. Reynolds and W. D. Hawley, Endocrinology, pages 81 and 741 (1967); and, C. Y. Bowers and A. V. Schally, Proceedings of NIH Conference on Hypothalamus, Hypophysiotropic Hormones, edited by J. Meitis, Tuscon, Arigona, in press (1969). The responses were determined by the increase of the radioactive iodine I in the blood expressed as the change in the number of counts per minute (Ac.p.m.) two hours after the intravenous injection of the thyrotropin releasing hormone (TRH) and the synthetic preparation. Th'e increase of I is proportional to the amount of thyrotropin hormone (TSH) released from the pituitary gland.
Since the doses of the synthetic preparation represent the weights of the starting material, they are the relative rather than the actual amounts of the synthetic product given. Although the synthetic product was visualized chromatographically its yield was not established.
For example, when tested in a mouse, levels of 6-54 nanograms (billionths of a gram) of the preparation in the mouse increased the level of I in the range of from about Ac.p.m. 670-8,000 as compared with from about Ac.p.m. 140170 for an acid saline solution used as control, it being understood that the amounts employed were relative as described above. On the other hand, levels of 2, 6 and 18 nanograms of porcine TRH increased the level of the I in the range of from about Ac.p.m. 2,000-6,000.
It is significant to note that the synthetic preparation was extremely active in comparison with porcine TRH. Graded responses were obtained when the dosage of the synthetic preparation was increased, and two of the important biological characteristics of natural TRH were observed; viz, (a) the degree of response depended upon the amount of triiodothyronine (T injected; and, (b) incubation of the preparation for minutes at 37 C. in normal human blood plasma inactivated the synthetic preparation.
The synthetic product, (pyro) Glu-His-Pro (NH was also found to be active in vitro. The amount of TSH released from the pituitary into the medium was established by the level of I released from the thyroid gland of mice. Activity was measured as the level of 1 expressed as Ac.p.m., and is proportional to the amount of TSH present in the medium. It was observed that more TSH (Ac.p.m. 2300-3100) was released when 50 nanograms of the (pyro) Glu-His-Pro (NH was added to the medium than in the control (Ac.p.m. 275). It appeared that the T added in vitro or given in vivo, partially or completely inhibited the activity of the synthetic preparation.
A comparison was made of the changes in blood levels of I in mice at various time intervals after intravenous injections of (1) an acid saline control solution, (2) the synthetic (pyro) Glu-His-Pro (NH and (3) porcine and bovine TRH. It was observed that the time-response curves of the active compounds were essentially the same,
but that there was a definite rise after a 60 minute interval, and the levels were even higher at minutes and remained elevated at and minutes.
In the other tests, it was found that the (pyro) Glu- His-Pro (NH elevated the plasma levels of TSH in rats and produced an increase in plasma levels of TSH Within 2 minutes after being intravenously injected into mice. The plasma levels of TSH were observed to be highest at the 10 and 15 minute intervals and remained elevated for 60 minutes, but started to fall at the 120 minute interval. The (pyro) Glu-His-Pro (NH was found to be active in mice whether given intravenously or intraperitoneally.
The following example is set forth to illustrate one method of synthesizing the (pyro) Glu-His-Pro (NHQ).
of the present invention.
EXAMPLE Commercial absolute methanol was employed but was first dried by treating it with magnesium turnings and refluxing it for 2 hours followed by distillation. Anhydrous hydrogen chloride was bubbled through the resultant anhydrous methanol until the mixture contained 5 g. of hydrogen chloride per 100 ml. of methanol (1.25 micromole/ml. hydrogen chloride).
Two milligrams of u-Glu-His-Pro (I) (5 micromoles) was then dissolved in 2 ml. of the anhydrous methanol containing the 1.25 micromole/ ml. hydrogen chloride and the mixture was allowed to stand in a sealed screw cap vial at room temperature for 90 minutes. The methanol and hydrogen chloride were then removed at room temperature under vacuum and over sodium hydroxide in a dessicator. The residue was dissolved in anhydrous methanol and the solvent was removed under vacuum. This residue was then redissolved in anhydrous methanol and the solvent was removed under vacuum several times in order to remove the excess hydrogen chloride.
Next, the dimethylester of Glu-His-Pro hydrochloride was dissolved in 1 ml. of methanol and the mixture was transferred to a screw cap vial. The methanol was removed under vacuum and the residue was then dissolved in 2 ml. of anhydrous methanol with ammonia at -5 C. The mixture was allowed to stand at room temperature for 24 hours in the sealed screw cap vial and, after the excess methanol and ammonia were removed under vacuum at room temperature in a dessicator with sulfuric acid (pyro) Glu-His-Pro (NH was obtained.
The new product (pyro) Glu-His-Pro (NI-I exhibited a positive reaction with the Pauly reageant revealing the presence of the NH group of the histidine moiety. It also exhibited no reaction with the ninhydrin reagent indicating that the amino group of the glutamic acid moiety is cyclized to the lactam form.
In the table set forth below (Table I), there is listed the data obtained for the chromatographic (R,) values from 4 diversified chromatographic solvent systems, each of which involved diiferent commercially available adsorbents and which reveal the chemical mobility and nature of the novel product of the invention. In Table I, the ratios of components in the solvent systems are by volume and the adsorbents are identified by their commercial trademarks.
What is claimed:
'1. A synthetic tripeptide of substantially 100% purity (pyro) glutamyl-histidyl-proline amide, having the structure said synthetic tripeptide having the same hormonal activity as the naturally derived thyrotropin releasing hormone of the hypothalamus gland of mammalian species.
2. A process for preparing (pyro) glutamyl-histidylproline amide, comprising; dissolving, at room temperature, a-glutamyl-histidyl-proline in anhydrous methanol containing hydrogen chloride, the molar ratio of a-glutamyl-histidyl-proline to hydrogen chloride being about 50:25; removing the methanol and hydrogen chloride under vacuum at room temperature to obtain a residue; dissolving said residue in excess anhydrous methanol; removing the solvent under vacuum at room temperature until all excess hydrogen chloride is removed to obtain the dimethylester of a-glutamyl-histidyl-proline hydrogen chloride; dissolving said dimethylester in excess methanol; removing said methanol under vacuum at room temperature to obtain a second residue; dissolving said second residue in anhydrous methanol with 2 mols ammonia at a temperature of about 5 C. to obtain a mixture; sealing said mixture from the atmosphere and permitting it to stand at room temperature for a period of about 24 hours; and, removing excess methanol and ammonia under vacuum at room temperature from said mixture to obtain (pyro) glutamyl-histidyl-proline amide.
References Cited UNITED STATES PATENTS 3,076,797 2/ 1963 Velluz et a1. 260-1125 3,250,759 5/196-6 Bodanszky et a1. 260-112.5
FOREIGN PATENTS 1,331,765 5/ 1963 France 2'60l12.5
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Burgus et al.: C. R. Acad. Sci., Paris, Ser. D, 269, 226- 228 (1969).
Guillemin: Pharmacology of Hormonal Polypeptides and Proteins, Bach et al., ed., Plenum Press, New York (1968), pp. 148-157.
Schally et al.: Biochem. Biophys. Res. Commun. 25, 169 (1966).
Schally et al.: Recent Progress Hormone Research 24, 510-525 (1968).
Shiba et al.: Bull. Chem. Soc., Japan, 31, 244-245 (1958).
Schroder et al.: The Peptides, vol. I, Academic Press, New York (1965), PP- 193-194.
Beecham: J. Amer. Chem. Soc., 76, 4615 (1954).
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ELBERT L. ROBERTS, Primary Examiner US. Cl. X.R. 260l12.5