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[Des-Gly-NH210, pro-ethylamide9]-LH-RH: A highly potent analog of luteinizing hormone releasing hormone
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
164, 488-489
(1973)
COMMUNICATION [Des-Gly-NH:‘,
Pro-ethylamideg]-LH-RH: Hormone
A Highly Releasing
Potent
Analog
of Luteinizing
Hormone
A new analog of luteinizing hormone releasing presence of ova in the oviduct was made on the hormone (LH-RH), [Des-Gly-NH? , Pro-ethylnext day and then the results were compared with amide9]-LH-RH was synthesized and LH-RH those of synthetic LH-RH. activity was also evaluated in vivo and in vitro. For more detailed evaluation, the analog was This analog was extremely potent, approximately incubated with hemisected male rat pituitaries@) five times as active as LH-RH, in the ovulationand the media was assayed for LH, by the bioassay inducing assay. This is the first example of a synof Parlow(9) and the radioimmunoassay of Nisthetic LH-RH analog exhibiting a higher potency than the natural hormone. TABLE; I Since the elucidation of the structure of porcine BIOLOQICAL ACTIVITY OF [Des-Gly-NH:‘, (1) and ovine (2) hypothalamic luteinizing horProethylamideg]-LH-RH mone releasing hormone(LH-RH), many syntheses of LH-RH analogs have been reported from Dosage” LH-RH [Des-Gly-NH:‘, a number of laboratories (3, 4), however, none of (ng/lOO g body wt) Ratb Rabbitc Pro~~?R~de’lthe synthetic analogs reported has exhibited an activity superior to that of the parent peptide. Rat Rabbit We now report the synthesis and biological activPro-ethylamidegl-LH-RH, ity of [Des-Gly-NH?, Ovulation-inducing activity an analog of LH-RH in which the glycine amide at 2.5 O/4 O/4 C-terminal is replaced by ethylamine. 5 O/4 4/g For the synthesis of this nonapeptide analog a 10 l/4 o/10 4/4 key intermediate, H-Leu-Arg(NOz)-Pro-NH-CHz20 4/3 l/10 7/7 CHI, was synthesized by the stepwise method and 30 5/10 the intermediate was coupled with pGlu-His-Trp40 6110 Ser-Tyr-Gly-OH(4) by the dicyclohexylcarbodi50 o/10 9/10 imide/N-hydroxy-5-norbornene-2,3-dicarboximide 80 3/4 414 method (5) to yield the protected nonapeptide loo 2110 414 amide. This protected nonapeptide amide was then 150 5/10 deblocked by the hydrogen fluoride method(6). 200 7/10 313 4/4 The resulting crude peptide was purified by col300 10/14 umn chromatography on Amberlite XAD-2 and on 400 lO/lO carboxymethyl cellulose: [~I]~~D - 66.2” (C = 0.5 in 5% acetic acid). The amino acid composition of an In Vitro assays acid hydrolysate was: Hiso.gs, Arg0.98, Gluo.ss, 250-300 (‘%) LH-released 100 (%I’ Sera.ga, Pror.00, Gly1.00, Leur.oo, Tyrr.oo, N&250-575 (%) FSH-release” 100 (%I’ = 1.05. Thin-layer chroC&L 1.10, Tyr/Trp(uv) matography and paper electrophoresis of the 5 Actual peptide contents of all the preparapurified material indicated that the peptide was measured by quantitative tions are 84-86%, homogeneous. amino acid analyses and uv analyses. The ovulation-inducing activity of t,his nonab See Ref. 4. peptide amide was measured in adult Spraguec See Ref. 7. Dawley diestrous rats in which the injection of d The bioassay of Parlow (9) and the radiosample was made subcutaneously at 1430 (Yamaimmunoassay of Niswender et al. (10). zaki et al., in preparation), or in constant estrus e The bioassay of Steelman-Pohley (11) and rabbits in which the sample was injected intrathe radioimmunoassay (NIAMD-Rat-FSH-Kit). f Accepted as 100%. venously (7). In both cases examination for the 468 Copyright All rights
@ 1973 by Academic Press, of reproduction in any form
Inc. reserved.
489
COMMUNICATION wender et aZ.(lO) and for FSH by the bioassay of Steelman and Pohley(l1) and the radioimmunoassay employing the NIAMD-Rat-FSH-Kit. The data on the biological activities of the nonapeptide amide are summarized in Table I. Further details of the synthesis and in viva and in vilro biological assessments will be published elsewhere together with the syntheses and biological activities of other related nonapeptide analogs. The results clearly indicate that the nonapeptide ethylamide exhibits approximately five times greater activity for the induction of ovulation than LH-RH itself and the analog possesses over 2.5 times the potency of LH-RH in release of both LH and FSH in vitro. This is the first example of a synthetic LH-RH analog exhibiting a higher potency than the natural releasing hormone. We thank Drs. K. Shimamoto, E. Ohmura and K. Morita of Takeda Chemical Industries, Ltd., and Dr. W. Close of Abbott Laboratories for their encouragement throughout this work. &I. FUJINO S. SHINAGA~A I. YAMIZAKI S. KOBAYASHI M. OBAYASHI T. FUKUDA R. NAKAYAMA
Central Research Division, Takeda Chemical Industries, Osaka, Japan
Ltd. WILFRID RIEMOSD
Division of Antibiotics and Natural Abbott Laboratories, North Chicago, Illinois 60064 Received October 10, 1972
F. WHITE H. R.IPPEL Products,
REFERENCES 1. MATSUO, H., B~BA, ARIMURA, A., AND
Biochem. Biophys.
Y., NAIR, R. M. G., SCHALLY, A. V. (1971)
Res. Commun. 43,1334.
2. BURGUS, R., BUTCHER, M., AMOSS, M., LING, N., MONAH~N, M., RIVIER, J., FELLOWS, R., BLXKWELL, R., VALE, W., AND GUILLEMIN,
R.. (1972) Proc. Nat. Acad. Sci. (USA), 69, 278. 3. STIEVI~RTSOS, H., CHANG, J. K., BOGENTOFT, C., CUI~RIE, B. L., FOLKERS, K., AND BOWEHS, C. Y. (1971) Biochem. Biophys. Res. Commun. 44, 1566; KIMURA, T., KISHIDA, Y., Kus.~MA, T., ABD SAKAKIBARA,
S. (1972). Proceeding of the 9th Symposium on Peptide Chemistry in Japan (Yanaihara, N., ed.), p. 90, Protein Research Foundation Press, Osaka; YANAIHARA, N., SAKAGaMI, M., KANEKO, T., SAITO, S., ABE, K., NAGATA, N., AXD OKA, H. (1972) Proceeding of the 9th Symposium on Peptide Chemistry in Japan (Yanaihara, N., ed.), p. 96, Protein Research Foundation Press, Osaka; MONAHAx, MIX;,
M. W., RIVIER, J., VALE, W., GUILLER., AND BURGUS, R. (1972) Biochem. 47, 551; CHANG, Biophys. Res. Commun. J. K., WILLIAMS, R. II., HUMPHRIES, A. J., JOHANSSON, N., FOLKERS, K., AND BOWERS,
C. Y.
(1972) Biochem.
Biophys.
Res. Com-
mun. 47, 727; HITIER, J., MOXAHAN, iM., VOLE, W., GRANT, G., AMOS~, M., BLOCKWELL, R., GUILLEMIN, R., AND BURGUS, Ii. (1972) Chimia 26, 300; SCHALLY, A. V., ARIMURA, A., CARTER, W. H., REDDING, T. W., GEIGER, R., K~NIG, W., WI~SMAN, H., JAEGER, G., S.\NDOW, J., YANAIHARA, N., YAN~IH~RA, C., HASHIMOTO, T., AND S~KAGAMI, M. (1972) Biochem. Biophys. Res.
Commun. 48, 366. M., KOBAYASHI, 4. FUJINO, FUKUD.~, T., SHINAGA~A, NAKAYAMA,
R., WHITE,
S., Ons~as~r, M., S., YAMAZAKI, I., W. F., ASD RIPPEL, Biophys. Res. Com-
R. H. (1972) Biochem. mun. (in press). 5. Fun-o, M., KOBAYaSHI, S., FUKUD~, T., OBAYASHI, M., AND SHINAGA~.~, P. (1972) Proceedings of the 10th Symposium on Peptide Chemistry in Japan (Noguchi, J., ed.) Protein Research Foundation Press, Osaka, in press. S., SHIMONISHI, Y., KISHID.4, Y., 6. SAKAKIBARA, OKAD.4, M., AND SUGIH.4R.4, H. (1967) Bull. Chem. Sot. Japan 40,2164. 7. WHITE, W. F., HEDLUND, M. T., RIPPEL, R. II., ARNOLD, W., AXD FLOURET, G. R. (1972) J. Med. Chem. (in press). 8. MITTER, J. C., AND MEITES, J. (1964) Proc. Sot. Exp. Biol. Med. 117, 309. 9. PARLOW, A. F. (1961) in Human Pituitary Gonadotropins (Albert, A., ed.), p. 300, Charles C Thomas, Springfield, Illinois. 10. NISWENDER, CT. D., MIDGLEY, A. R., JR., MONROE, S. E., AND REICHERT, L. E. (1968) Proc. Sot. Exp. Biol. Med. 128,807. 11. STEELMAN, S. L., AND POHLEY, F. M. (1953) Endocrinology 63, 604.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
164, 488-489
(1973)
COMMUNICATION [Des-Gly-NH:‘,
Pro-ethylamideg]-LH-RH: Hormone
A Highly Releasing
Potent
Analog
of Luteinizing
Hormone
A new analog of luteinizing hormone releasing presence of ova in the oviduct was made on the hormone (LH-RH), [Des-Gly-NH? , Pro-ethylnext day and then the results were compared with amide9]-LH-RH was synthesized and LH-RH those of synthetic LH-RH. activity was also evaluated in vivo and in vitro. For more detailed evaluation, the analog was This analog was extremely potent, approximately incubated with hemisected male rat pituitaries@) five times as active as LH-RH, in the ovulationand the media was assayed for LH, by the bioassay inducing assay. This is the first example of a synof Parlow(9) and the radioimmunoassay of Nisthetic LH-RH analog exhibiting a higher potency than the natural hormone. TABLE; I Since the elucidation of the structure of porcine BIOLOQICAL ACTIVITY OF [Des-Gly-NH:‘, (1) and ovine (2) hypothalamic luteinizing horProethylamideg]-LH-RH mone releasing hormone(LH-RH), many syntheses of LH-RH analogs have been reported from Dosage” LH-RH [Des-Gly-NH:‘, a number of laboratories (3, 4), however, none of (ng/lOO g body wt) Ratb Rabbitc Pro~~?R~de’lthe synthetic analogs reported has exhibited an activity superior to that of the parent peptide. Rat Rabbit We now report the synthesis and biological activPro-ethylamidegl-LH-RH, ity of [Des-Gly-NH?, Ovulation-inducing activity an analog of LH-RH in which the glycine amide at 2.5 O/4 O/4 C-terminal is replaced by ethylamine. 5 O/4 4/g For the synthesis of this nonapeptide analog a 10 l/4 o/10 4/4 key intermediate, H-Leu-Arg(NOz)-Pro-NH-CHz20 4/3 l/10 7/7 CHI, was synthesized by the stepwise method and 30 5/10 the intermediate was coupled with pGlu-His-Trp40 6110 Ser-Tyr-Gly-OH(4) by the dicyclohexylcarbodi50 o/10 9/10 imide/N-hydroxy-5-norbornene-2,3-dicarboximide 80 3/4 414 method (5) to yield the protected nonapeptide loo 2110 414 amide. This protected nonapeptide amide was then 150 5/10 deblocked by the hydrogen fluoride method(6). 200 7/10 313 4/4 The resulting crude peptide was purified by col300 10/14 umn chromatography on Amberlite XAD-2 and on 400 lO/lO carboxymethyl cellulose: [~I]~~D - 66.2” (C = 0.5 in 5% acetic acid). The amino acid composition of an In Vitro assays acid hydrolysate was: Hiso.gs, Arg0.98, Gluo.ss, 250-300 (‘%) LH-released 100 (%I’ Sera.ga, Pror.00, Gly1.00, Leur.oo, Tyrr.oo, N&250-575 (%) FSH-release” 100 (%I’ = 1.05. Thin-layer chroC&L 1.10, Tyr/Trp(uv) matography and paper electrophoresis of the 5 Actual peptide contents of all the preparapurified material indicated that the peptide was measured by quantitative tions are 84-86%, homogeneous. amino acid analyses and uv analyses. The ovulation-inducing activity of t,his nonab See Ref. 4. peptide amide was measured in adult Spraguec See Ref. 7. Dawley diestrous rats in which the injection of d The bioassay of Parlow (9) and the radiosample was made subcutaneously at 1430 (Yamaimmunoassay of Niswender et al. (10). zaki et al., in preparation), or in constant estrus e The bioassay of Steelman-Pohley (11) and rabbits in which the sample was injected intrathe radioimmunoassay (NIAMD-Rat-FSH-Kit). f Accepted as 100%. venously (7). In both cases examination for the 468 Copyright All rights
@ 1973 by Academic Press, of reproduction in any form
Inc. reserved.
489
COMMUNICATION wender et aZ.(lO) and for FSH by the bioassay of Steelman and Pohley(l1) and the radioimmunoassay employing the NIAMD-Rat-FSH-Kit. The data on the biological activities of the nonapeptide amide are summarized in Table I. Further details of the synthesis and in viva and in vilro biological assessments will be published elsewhere together with the syntheses and biological activities of other related nonapeptide analogs. The results clearly indicate that the nonapeptide ethylamide exhibits approximately five times greater activity for the induction of ovulation than LH-RH itself and the analog possesses over 2.5 times the potency of LH-RH in release of both LH and FSH in vitro. This is the first example of a synthetic LH-RH analog exhibiting a higher potency than the natural releasing hormone. We thank Drs. K. Shimamoto, E. Ohmura and K. Morita of Takeda Chemical Industries, Ltd., and Dr. W. Close of Abbott Laboratories for their encouragement throughout this work. &I. FUJINO S. SHINAGA~A I. YAMIZAKI S. KOBAYASHI M. OBAYASHI T. FUKUDA R. NAKAYAMA
Central Research Division, Takeda Chemical Industries, Osaka, Japan
Ltd. WILFRID RIEMOSD
Division of Antibiotics and Natural Abbott Laboratories, North Chicago, Illinois 60064 Received October 10, 1972
F. WHITE H. R.IPPEL Products,
REFERENCES 1. MATSUO, H., B~BA, ARIMURA, A., AND
Biochem. Biophys.
Y., NAIR, R. M. G., SCHALLY, A. V. (1971)
Res. Commun. 43,1334.
2. BURGUS, R., BUTCHER, M., AMOSS, M., LING, N., MONAH~N, M., RIVIER, J., FELLOWS, R., BLXKWELL, R., VALE, W., AND GUILLEMIN,
R.. (1972) Proc. Nat. Acad. Sci. (USA), 69, 278. 3. STIEVI~RTSOS, H., CHANG, J. K., BOGENTOFT, C., CUI~RIE, B. L., FOLKERS, K., AND BOWEHS, C. Y. (1971) Biochem. Biophys. Res. Commun. 44, 1566; KIMURA, T., KISHIDA, Y., Kus.~MA, T., ABD SAKAKIBARA,
S. (1972). Proceeding of the 9th Symposium on Peptide Chemistry in Japan (Yanaihara, N., ed.), p. 90, Protein Research Foundation Press, Osaka; YANAIHARA, N., SAKAGaMI, M., KANEKO, T., SAITO, S., ABE, K., NAGATA, N., AXD OKA, H. (1972) Proceeding of the 9th Symposium on Peptide Chemistry in Japan (Yanaihara, N., ed.), p. 96, Protein Research Foundation Press, Osaka; MONAHAx, MIX;,
M. W., RIVIER, J., VALE, W., GUILLER., AND BURGUS, R. (1972) Biochem. 47, 551; CHANG, Biophys. Res. Commun. J. K., WILLIAMS, R. II., HUMPHRIES, A. J., JOHANSSON, N., FOLKERS, K., AND BOWERS,
C. Y.
(1972) Biochem.
Biophys.
Res. Com-
mun. 47, 727; HITIER, J., MOXAHAN, iM., VOLE, W., GRANT, G., AMOS~, M., BLOCKWELL, R., GUILLEMIN, R., AND BURGUS, Ii. (1972) Chimia 26, 300; SCHALLY, A. V., ARIMURA, A., CARTER, W. H., REDDING, T. W., GEIGER, R., K~NIG, W., WI~SMAN, H., JAEGER, G., S.\NDOW, J., YANAIHARA, N., YAN~IH~RA, C., HASHIMOTO, T., AND S~KAGAMI, M. (1972) Biochem. Biophys. Res.
Commun. 48, 366. M., KOBAYASHI, 4. FUJINO, FUKUD.~, T., SHINAGA~A, NAKAYAMA,
R., WHITE,
S., Ons~as~r, M., S., YAMAZAKI, I., W. F., ASD RIPPEL, Biophys. Res. Com-
R. H. (1972) Biochem. mun. (in press). 5. Fun-o, M., KOBAYaSHI, S., FUKUD~, T., OBAYASHI, M., AND SHINAGA~.~, P. (1972) Proceedings of the 10th Symposium on Peptide Chemistry in Japan (Noguchi, J., ed.) Protein Research Foundation Press, Osaka, in press. S., SHIMONISHI, Y., KISHID.4, Y., 6. SAKAKIBARA, OKAD.4, M., AND SUGIH.4R.4, H. (1967) Bull. Chem. Sot. Japan 40,2164. 7. WHITE, W. F., HEDLUND, M. T., RIPPEL, R. II., ARNOLD, W., AXD FLOURET, G. R. (1972) J. Med. Chem. (in press). 8. MITTER, J. C., AND MEITES, J. (1964) Proc. Sot. Exp. Biol. Med. 117, 309. 9. PARLOW, A. F. (1961) in Human Pituitary Gonadotropins (Albert, A., ed.), p. 300, Charles C Thomas, Springfield, Illinois. 10. NISWENDER, CT. D., MIDGLEY, A. R., JR., MONROE, S. E., AND REICHERT, L. E. (1968) Proc. Sot. Exp. Biol. Med. 128,807. 11. STEELMAN, S. L., AND POHLEY, F. M. (1953) Endocrinology 63, 604.